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Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus
Abstract
A high-frequency transducing element for erythromycin resistance in Staphylococcus aureus has been found. This element, P11de, is apparently the result of a recombinational event between a temperate phage, P11, and a penicillinase plasmid, γ; it lacks substantial sections both of the plasmid and of the bacteriophage. The phage moiety is cryptic, conferring neither lysogeny nor superinfection immunity upon host cells carrying it; helper phage is required for the production of transducing particles but not for the transduction process. Demonstrable phage-related properties include reactivation of ultraviolet-inactivated homologous phage and rescue of temperature-sensitive phage mutants.The plasmic moiety includes an erythromycin resistance marker, and the mc region, which is responsible for plasmid compatibility, is involved in a specific host-plasmid interaction and is required for plasmid replication; all the other known plasmid markers are missing. The autonomous replication of P11de as well as its intracellular behavior vis-à-vis prophages and other plasmids appear to be governed by its mc determinant. In these respects P11de is similar to the parental γ plasmid. This plasmid control, however, is superseded by active P11, which induces P11de to multiply extensively.P11de can recombine with wild-type plasmids; the resulting recombinants are not transduced at high frequency, nor do they reactivate UV inactivated phage particles. Based on these findings, a possible genetic structure for P11de is presented and compared with maps of naturally occurring plasmids.
... Almost all clinical isolates of S. aureus can be shown to harbor at least one temperate prophage, and frequently several; for example, strain 8325 is known to harbor three (148). There seems to be only one reported naturally occurring strain from which no phage could be obtained, i.e., strain 1030 (143). It is possible to cure strains of their prophages by, e.g., ultraviolet (UV) irradiation or treatment with mitomycin C. Strain RN 450 is a derivative of strain 8325 that has lost all three prophages (146). ...
... Downloaded from phages arise by incorporation of particular chromosomal or plasmid genes into the phage geneset. Novick has constructed an agent of this type in S. aureus by a recombination event between a phage and a penicillinase plasmid (143). The resultant element is a plasmid since it is isolable as covalently closed circular DNA (182), and, although it has lost regions of both the phage and plasmid, some phage and plasmid functions are retained (143). ...
... Novick has constructed an agent of this type in S. aureus by a recombination event between a phage and a penicillinase plasmid (143). The resultant element is a plasmid since it is isolable as covalently closed circular DNA (182), and, although it has lost regions of both the phage and plasmid, some phage and plasmid functions are retained (143). Novick and Morse (148) injected pairs of staphylococcal cultures, one of which contained this element, into mice, and demonstrated that antibiotic resistance (in this case resistance to erythromycin) could be transferred between staphylococci experimentally in vivo. ...
... In bacteria, incompatibility appears to be a fundamental property of replicons in that it is universally exhibited by plasmids isogenic for autonomy functions. Thus, various F-merogenotes are incompatible with one another (46,63,231) as are derivatives of an R(f) factor (102), a Col factor (186,198), and a staphylococcal plasmid (192,194,195). ...
... There are at least two different mechanisms involved in their formation: that of aberrant excision of integrated prophages (31; Fig. 6), and that of rare direct recombination between phages and bacterial genes that are ordinarily unlinked. Such Hft derivatives of a number of generalized transducing phages [P22 (61), P1 (161), e (141), and P11 (194)] have been isolated. The bacterial genes most commonly involved are derived from extrachromosomal elements, a fact which may reflect a more direct evolutionary relationship between generalized transducing phages and extrachromosomal elements than between these phages and the chromosomes of their host bacteria. ...
... A somewhat artificial example of alternating replication control has been observed in staphylococcus (194) with an Hft element. This element, PI Ide, originated as a recombinant between two heterogenic replicons: a generalized transducing phage, P11, and a plasmid, PI2,%. ...
... host cell lysis as well as its own transcription (3,4). Upon induction, the prophage produces 10 the lytic regulator protein Cro, which is encoded divergent to the cI repressor gene, binds to 11 the cI promoter, and stabilizes the lytic cycle by blocking transcription from the PRM promoter 12 driving cI transcription (3). ...
... host cell lysis as well as its own transcription (3,4). Upon induction, the prophage produces 10 the lytic regulator protein Cro, which is encoded divergent to the cI repressor gene, binds to 11 the cI promoter, and stabilizes the lytic cycle by blocking transcription from the PRM promoter 12 driving cI transcription (3). Expression levels of CI and Cro are finely balanced to ensure tight 13 repression of the phage lytic cycle while at the same time enabling its rapid activation upon 14 sensing the correct stimulus. ...
Bacteriophages (phages) are the predominant biological entities on the planet and play an important role in the spread of bacterial virulence, pathogenicity, and antimicrobial resistance. After infection, temperate phages can integrate in the bacterial chromosome thanks to the expression of the prophage-encoded CI master repressor. Upon SOS induction, and promoted by RecA*, CI auto-cleaves generating two fragments, one containing the N-terminal domain (NTD), which retains strong DNA-binding capacity, and other corresponding to the C-terminal part of the protein. However, it is unknown how the CI NTD is removed, a process that is essential to allow prophage induction. Here we identify for the first time that the specific interaction of the ClpX protease with the CI NTD repressor fragment is essential and sufficient for prophage activation after SOS-mediated CI autocleavage, defining the final stage in the prophage induction cascade. Our results provide unexpected roles for the bacterial protease ClpX in phage biology.
... A selection of daughter strains of the 475 NCTC8325 lineage and how they were generated. RN25 (also called 8325-3) was generated 476 from NCTC8325 following UV curing of prophages 11 and 12(23). RN450 (also called 8325-477 4) was generated from RN25 by a second round of UV exposure, to cure prophage 13(23). ...
... RN25 (also called 8325-3) was generated 476 from NCTC8325 following UV curing of prophages 11 and 12(23). RN450 (also called 8325-477 4) was generated from RN25 by a second round of UV exposure, to cure prophage 13(23). ...
The Type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified in Staphylococcus aureus , EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identical esaG and tsaI immunity genes are encoded in all S. aureus strains. To investigate the evolution of esaG repertoires, we analysed the sequences of the tandem esaG genes and their encoded proteins. We identified three blocks of high sequence similarity shared by all esaG genes and identified evidence of extensive recombination events between esaG paralogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss and expansion of esaG genes in S. aureus genomes and we identified evidence of such events during evolution of strains in clonal complex 8. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence similarity locate to the 5’ and 3’ end of tipC genes, and we found strong evidence for recombination between tipC paralogues encoded by Streptococcus mitis BCC08. By contrast, we found only a single homology block across tsaI genes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.
DATA SUMMARY
All sequence data for strains used in this study are available on NCBI under BioProject PRJNA789916. Sequences from the NCTC3000 project are available on NCBI under BioProject PRJEB6403. Supplementary data 2 and all custom scripts are available on Github: https://github.com/GM110Z/Garret-et-al.-recombination-paper .
IMPACT STATEMENT
The type VII secretion system (T7SS) in firmicutes secretes polymorphic toxins that target other bacteria. To protect from the action of these toxins, bacteria carry multiple paralogous copies of immunity protein-encoding genes that are sequence-related but non-identical. To date, little is known about how T7 immunity gene families evolve. In this study we analysed a cluster of EsaG-encoding genes in Staphylococcus aureus which are found at the ess/T7 secretion locus and provide immunity against the T7 secreted nuclease toxin, EsaD. We identified three homology blocks covering esaG genes and their downstream intergenic regions, which are separated by two variable regions. We have shown that recombination can occur between these homology blocks, leading to loss or expansion of esaG genes at this locus. Using a historical dataset of closely related S. aureus strains from clonal complex 8, we identified several independent recombination events leading to changes in the esaG repertoire. We further showed that similar events are observed for an immunity protein encoded by Group B Streptococcus spp. suggesting that recombination plays a broader role in the evolution of T7SS immunity-encoding genes. We speculate that gain and loss of T7 immunity genes is weighed in response to environmental pressure and metabolic burden.
... Phages are key mobile genetic elements that contribute to the emergence of novel pathogenic bacterial strains through the transfer of virulence and fitness factors via HGT. The phenomenon of phage-mediated transfer of bacterial DNA is known as transduction and is crucial for bacterial biology, diversity, and evolution (Saeed et al., 2021). of a naturally occurring strain of S. aureus (NCTC8325), which expresses SaPIbov5 genomic elements, and is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from E. coli (Novick, 1967;Kreiswirth et al., 1983). S. aureus inocula was grown overnight at 37°C for plasmid replication (Novick, 1991). ...
S TAPHYLOCOCCUS aureus can enter the bloodstream, leading to health complications such as sepsis, arthritis, endocarditis, and pneumonia. This study screened for the impact of six replication genes of cos phage 12 (φ12) on the evolution and transfer of cos S. aureus pathogenicity islands (SaPIbov5). An overnight culture of the S. aureus strain RN4220 diluted with fresh TSB was used in the bacteriophage φ12 titering assay. Phage-point mutagenesis was achieved using the pMAD vector, which facilitates homologous recombination in a two-step process. Finally, the transduction SaPI titering assays also utilized the S. aureus strain RN4220, which was then mixed with CaCl 2 and fresh TSB. The study showed that ORF11, ORF12, and VirE mutants did not lyse or produce phage particles after titering into the recipient RN4220 strains. While ORF26, ORF10, and ORF04 mutants generated detectable plaques, the mutations may have an effect on phage replication or packaging after tittering. For instance, complemented φ12 ORF26, ORF10, and ORF04 mutant strains had fully restored phage titers. The experiment found that cos phages facilitate inter and intra-generically transfer of cosSaPIs. It was also established that φ12 transduces SaPIbov5. However, φ12 mutants in VirE, ORF12, and ORF11 did not show SaPI mobilization because of their effects on infective phage particles and lack of replication. The complemented φ12 VirE, ORF12, and ORF11 mutants had partly restored phage titers when they were expressed in the recipient and donor strains. Mutations in ORF26, 0RF10, and ORFO4 decrease the φ12's ability to transfer SaPIbov5. Generally, this study found a new mechanism that facilitates the transfer of SaPI and cos genes. While the VirE, ORF12, and ORF11 affect packaging and are necessary for replication and phage biology
... The bacterium utilized in this study is Staphylococcus aureus strain RN6390 [82], known to cause consistent endophthalmitis when injected into mice eyes [10,21,83]. The strain is routinely cultured in Tryptic Soy medium (TSA or TSB; Sigma, St. Louis, MO, USA) at 37 °C. ...
Bacterial endophthalmitis is a severe complication of eye surgeries that can lead to vision loss. Current treatment involves intravitreal antibiotic injections that control bacterial growth but not inflammation. To identify newer therapeutic targets to promote inflammation resolution in endophthalmitis, we recently employed an untargeted metabolomics approach. This led to the discovery that the levels of S-nitroso-l-glutathione (GSNO) were significantly reduced in an experimental murine Staphylococcus aureus (SA) endophthalmitis model. In this study, we tested the hypothesis whether GSNO supplementation via different routes (oral, intravitreal) provides protection during bacterial endophthalmitis. Our results show that prophylactic administration of GSNO via intravitreal injections ameliorated SA endophthalmitis. Therapeutically, oral administration of GSNO was found to be most effective in reducing intraocular inflammation and bacterial burden. Moreover, oral GSNO treatment synergized with intravitreal antibiotic injections in reducing the severity of endophthalmitis. Furthermore, in vitro experiments using cultured human retinal Muller glia and retinal pigment epithelial (RPE) cells showed that GSNO treatment reduced SA-induced inflammatory mediators and cell death. Notably, both in-vivo and ex-vivo data showed that GSNO strengthened the outer blood-retinal barrier during endophthalmitis. Collectively, our study demonstrates GSNO as a potential therapeutic agent for the treatment of intraocular infections due to its dual anti-inflammatory and cytoprotective properties.
Graphical abstract
... The bacterial strains used in this study were methicillin susceptible Staphylococcus aureus (MSSA) 8325-4 [27], methicillin resistant Staphylococcus aureus (MRSA) BH1CC [28], Listeria monocytogenes EGD-e [29], Pseudomonas aeruginosa PA01 [30] and Vibrio parahaemolyticus RIMD2210633 [31]. All bacterial strains were cultivated in brain heart infusion (BHI) (Oxoid) media, except V. parahaemolyticus (BHI + 3% NaCl), and grown at 37 • C. ...
Snake venoms possess a range of pharmacological and toxicological activities. Here we evaluated the antibacterial and anti-biofilm activity against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) of venoms from the Samar spitting cobra Naja samarensis and the Puff adder Bitis arietans. Both venoms prevented biofilm production by pathogenic S. aureus in a growth-independent manner, with the B. arietans venom being most potent. Fractionation showed the active molecule to be heat-labile and >10 kDa in size. Proteomic profiles of N. samarensis venom revealed neurotoxins and cytotoxins, as well as an abundance of serine proteases and three-finger toxins, while serine proteases, metalloproteinases and C-lectin types were abundant in B. arietans venom. These enzymes may have evolved to prevent bacteria colonising the snake venom gland. From a biomedical biotechnology perspective, they have valuable potential for anti-virulence therapy to fight antibiotic resistant microbes.
... S. aureus 6850∆hla (kindly provided by L. Tuchscherr de Hauschopp, Institute of Medical Microbiology, University Hospital of Jena, Germany) and wild-type 6850 [42] were used as assay control strains to test for detection of α-hemolysin secretion. The rsbU-repaired SH1000 (SigB-functional) [43], the wild-type 8325-4 (SigB-deficient, 11 bp deletion in rsbU gene) [44] and the isogenic mutant SH1000∆sigB (SigB-deficient) [4], as well as strain 6850 (SigB-functional) [42] and its isogenic ∆sigB mutant (SigB-deficient) [45], were used as SigB-functional and SigB-deficient reference strains to determine S. aureus carotenoid pigmentation. S. aureus SH1000 and SH1000∆sigB were included as assay control and reference for mRNA expression studies of asp23 (RT-qPCR). ...
Phenotypic adaptation has been associated with persistent, therapy-resistant Staphylococcus aureus infections. Recently, we described within-host evolution towards a Sigma factor B (SigB)-deficient phenotype in a non-human host, a naturally infected dairy cow with chronic, persistent mastitis. However, to our knowledge, the prevalence of SigB deficiency among clinical S. aureus isolates remains unknown. In this study, we screened a collection of bovine mastitis isolates for phenotypic traits typical for SigB deficiency: decreased carotenoid pigmentation, increased proteolysis, secretion of α-hemolysin and exoproteins. Overall, 8 out of 77 (10.4%) isolates of our bovine mastitis collection exhibited the SigB-deficient phenotype. These isolates were assigned to various clonal complexes (CC8, CC9, CC97, CC151, CC3666). We further demonstrated a strong positive correlation between asp23-expression (a marker of SigB activity) and carotenoid pigmentation (r = 0.6359, p = 0.0008), underlining the role of pigmentation as a valuable predictor of the functional status of SigB. Sequencing of the sigB operon (mazEF-rsbUVW-sigB) indicated the phosphatase domain of the RsbU protein as a primary target of mutations leading to SigB deficiency. Indeed, by exchanging single nucleotides in rsbU, we could either induce SigB deficiency or restore the SigB phenotype, demonstrating the pivotal role of RsbU for SigB functionality. The data presented highlight the clinical relevance of SigB deficiency, and future studies are needed to exploit its role in staphylococcal infections.
... The bacterial strains used in this study were methicillin susceptible Staphylococcus aureus (MSSA) 8325-4 [27], methicillin resistant Staphylococcus aureus (MRSA) BH1 -CC [28], Listeria monocytogenes EGD-e [29], Pseudomonas aeruginosa PA01 [30] and Vibrio parahaemolyticus RIMD2210633 [31]. All bacterial strains were cultivated in brain heart infusion (BHI) (Oxoid) media, except V. parahaemolyticus (BHI + 3% NaCl), and grown at 37°C. ...
... Prophages induce insensitivity of S. aureus to kayviruses. Prophageless S. aureus strains ISP8 and 1039 were lysogenized by prophages 11, 29, 42E, 47, 53, 71, 77, 80a, 83A, 84, 85, or 96 and tested for susceptibility to lytic kayviruses 812, 812a, and K. The integration of prophages 47, 53, and 80a resulted in a resistant phenotype to the lytic phages 812 and K. Phage 53 was used for the lysogenization of multiple strains, including methicillinresistant S. aureus (MRSA) USA300 (Table 1), which led to the establishment of a resistant phenotype to phages 812 and K independently of the genetic background of the lysogens. ...
ABSTRACT Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated Pdp Sau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdp Sau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted Pdp Sau protein structure shows the presence of a membrane-binding a-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of Pdp Sau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding Pdp Sau , and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins.
IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staph-ylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.
... The bacterial strains and plasmids used in this study are listed in Supplementary Table 1. Standard strain S. aureus FK300, a rsbU-repaired (Giachino et al., 2001) derivative of NCTC8325-4 (Novick, 1967;Herbert et al., 2010), was used in a functional study of the role of icaB. DNA restriction systemdeficient S. aureus RN4220 (Kreiswirth et al., 1983) was used as the initial recipient for manipulation of recombinant plasmids. ...
Biofilms are microbial communities of cells embedded in a matrix of extracellular polymeric substances generated and adhering to each other or to a surface. Cell aggregates formed in the absence of a surface and floating pellicles that form biofilms at the air-liquid interface are also considered to be a type of biofilm. Staphylococcus aureus is a well-known cause of biofilm infections and high-molecular-weight polysaccharides, poly-N-acetylglucosamine (PNAG) is a main constituent of the biofilm. An icaADBC operon comprises major machinery to synthesize and extracellularly secrete PNAG. Extracellular PNAG is partially deacetylated by IcaB deacetylase, and the positively charged PNAG hence interacts with negatively charged cell surface to form the major component of biofilm. We previously reported a new regulator of biofilm (Rob) and demonstrated that Rob binds to a unique 5-bp motif, TATTT, present in intergenic region between icaADBC operon and its repressor gene icaR in Yu et al. The deletion of the 5-bp motif induces excessive adherent biofilm formation. The real function of the 5-bp motif is still unknown. In an attempt to isolate the 5-bp motif deletion mutant, we isolated several non-adherent mutants. They grew normally in turbid broth shaking culture but immediately auto-aggregated upon weak vortexing and sedimented as a lump resulting in a clear supernatant. Whole genome sequencing of the mutants identified they all carried mutations in icaB in addition to deletion of the 5-bp motif. Purification and molecular characterization of auto-aggregating factor in the culture supernatant of the mutant identified that the factor was a massively produced non-deacetylated PNAG. Therefore, we created a double deficient strain of biofilm inhibitory factors (5-bp motif, icaR, rob) and icaB to confirm the aggregation phenomenon. This peculiar phenomenon was only observed in Δ5bpΔicaB double mutant but not in ΔicaR ΔicaB or ΔrobΔicaB mutant. This study explains large amount of extracellularly produced non-deacetylated PNAG by Δ5bpΔicaB double mutation induced rapid auto-aggregation of S. aureus cells by vortexing. This phenomenon indicated that Staphylococcus aureus may form biofilms that do not adhere to solid surfaces and we propose this as a new mechanism of non-adherent biofilm formation of S. aureus.
... NCTC8325-4 is a derivative of a clinical isolate NCTC8325 obtained by curing the three prophages Φ11, Φ12, and Φ13 [2]. Therefore, both NCTC8325-4 and RN4220 lack the three prophages. ...
Staphylococcus aureus RN4220 has been extensively used by staphylococcal researchers as an intermediate strain for genetic manipulation due to its ability to accept foreign DNA. Despite its wide use in laboratories, its complete genome is not available. In this study, we used a hybrid genome assembly approach using minION long reads and Illumina short reads to sequence the complete genome of S. aureus RN4220. The comparative analysis of the annotated complete genome showed the presence of 39 genes fragmented in the previous assembly, many of which were located near the repeat regions. Using RNA-Seq reads, we showed that a higher number of reads could be mapped to the complete genome than the draft genome and the gene expression profile obtained using the complete genome also differs from that obtained from the draft genome. Furthermore, by comparative transcriptomic analysis, we showed the correlation between expression levels of staphyloxanthin biosynthetic genes and the production of yellow pigment. This study highlighted the importance of long reads in completing microbial genomes, especially those possessing repetitive elements.
... Wild type S. aureus 8325-4 [27], and mutant strains DU1090 (hla) [28], LES12 (aur), LES17 (sspB), LES22 (sspABC) and LES27 (scpA) [29] were used. ...
Infection by Staphylococcus aureus is the leading cause of infective endocarditis (IE). Activation of platelets by this pathogen results in their aggregation and thrombus formation which are considered to be important steps in the development and pathogenesis of IE. Here, we show that a secreted cysteine protease, staphopain A, activates human platelets and induces their aggregation. The culture supernatant of a scpA mutant deficient in staphopain A production was reduced in its ability to trigger platelet aggregation. The platelet agonist activity of purified staphopain A was inhibited by staphostatin A, a specific inhibitor, thus implicating its protease activity in the agonism. In whole blood, using concentrations of staphopain A that were otherwise insufficient to induce platelet aggregation, increased binding to collagen and thrombus formation was observed. Using antagonists specific to protease-activated receptors 1 and 4, we demonstrate their role in mediating staphopain A induced platelet activation.
... Since we also observed that genes involved in DNA repair, such as recA and uvrA, were upregulated in S. aureus mixed species macrocolonies, we hypothesized that prophage induction contributes to E. coli-mediated killing of S. aureus as recently suggested for some S. aureus prophage-expressing strains [32]. However, when we tested the prophage-cured S. aureus strain RN450 [33] in the mixed species macrocolony assay, we observed that it was also readily killed by E. coli (Fig 1B), indicating that prophage induction was not the mechanism by which E. coli-induced death in S. aureus in this setting. ...
Wound infections are often polymicrobial in nature, biofilm associated and therefore tolerant to antibiotic therapy, and associated with delayed healing. Escherichia coli and Staphylococcus aureus are among the most frequently cultured pathogens from wound infections. However, little is known about the frequency or consequence of E . coli and S . aureus polymicrobial interactions during wound infections. Here we show that E . coli kills Staphylococci, including S . aureus , both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. Colibactin biosynthesis is encoded by the pks locus, which we identified in nearly 30% of human E . coli wound infection isolates. While it is not clear how colibactin is released from E . coli or how it penetrates target cells, we found that the colibactin intermediate N-myristoyl-D-Asn (NMDA) disrupts the S . aureus membrane. We also show that the BarA-UvrY two component system (TCS) senses the environment created during E . coli and S . aureus mixed species interaction, leading to upregulation of pks island genes. Further, we show that BarA-UvrY acts via the carbon storage global regulatory (Csr) system to control pks expression. Together, our data demonstrate the role of colibactin in interspecies competition and show that it is regulated by BarA-UvrY TCS during interspecies competition.
... The donor strain for conjugation, E. coli WM3064, was grown in LB broth or LB agar supplemented with 2,6-diaminopimelic acid (DAP) to a final concentration of 0.3 mM. Vibrio anguillarum 90- 11-287 (52) (53) was cultured in BHI broth (Thermo Scientific, CM1135) or in basal agar with 1% (wt/vol) peptone (BD Biosciences, 211677) and 0.4% (wt/vol) glucose. ...
The marine bacterium Photobacterium galatheae S2753 produces a group of cyclodepsipeptides, called solonamides, which impede the virulence but not the survival of Staphylococcus aureus. In addition to their invaluable antivirulence activity, little is known about the biosynthesis and physiological function of solonamides in the native producer. This study generated a solonamide-deficient (Dsol) mutant by in-frame deletion of the sol gene, thereby identifying the core gene for solonamide biosynthesis. By annotation from antiSMASH, the biosynthetic pathway of solonamides in S2753 was also proposed. Mass spectrometry analysis of cell extracts found that deficiency of solonamide production influenced the production of a group of unknown compounds but otherwise did not alter the overall secondary metabolite profile. Physiological comparison between Dsol and wild-type S2753 demonstrated that growth dynamics and biofilm formation of both strains were similar; however, the Dsol mutant displayed reduced motility rings compared to the wild type. Reintroduction of sol restored solonamide production and motility to the mutant, indicating that solonamides influence the motility behavior of P. galatheae S2753. Proteomic analysis of the Dsol and wild-type strains found that eliminating solonamides influenced many cellular processes, including swimming-related proteins and proteins adjusting the cellular cyclic di-GMP concentration. In conclusion, our results revealed the biosynthetic pathway of solonamides and their ecological benefits to P. galatheae S2753 by enhancing motility, likely by altering the motile physiology.
... To that end, S. aureus RN4220 transformed with plasmids pCN51 (Ery r ) or pCN57 (Ery r , gfp,) were used as the donor in transduction assays. Transducing phage f 11 (45,46) (kindly provided by L. Marrafini, Rockefeller University) was used as a positive control for these assays. Plasmid transductions were selected by antibiotic resistance and confirmed by the detection of fluorescence using an Olympus MVX10 binocular scope. ...
To the best of our knowledge, this set of sequenced bacteriophages is the largest one in South America. Our report describes for the first time the utilization of MultiTwin software to analyze the relationship between phage protein families.
... after treating by desiccation stress S. aureus NCTC8325 was clinically isolated from a sepsis patient and is often used as a model strain for laboratory research (Novick, 1967). However, there were few reports on the tolerance of S. aureus to desiccation stress, or no report on the comparison of the survival rate of foodborne S. aureus and model strains NCTC8325 under desiccation stress. ...
Staphylococcus aureus (S. aureus) is a momentous factor affecting food safety. It can survive under long-term desiccation stress and contaminate foods that have intermediate to low water activities. However, the specific molecular mechanisms by which it survives and persists under low water activity stress are often overlooked. In this study, transcriptome analysis was applied to investigate the effect of desiccation stress on gene expression of S. aureus RMSA24, a food-borne S. aureus strain that was isolated from a raw milk sample. Results of transcriptome analysis showed that the mRNA levels of genes related to capsular polysaccharides (CPs) synthesis were significantly upregulated after desiccation treatment, which was further confirmed by real-time reverse transcription PCR assays. Furthermore, the results of colony count experiments demonstrated that the survival of CPs mutant was decreased compared with the wild type strain. And the biofilm formation ability of CPs mutant showed no difference compared with that of wild type according to biofilm formation assays. Those results indicated that CPs mutant decrease the resistance to desiccation in S. aureus RMSA24 via a biofilm-independent pathway. This study provides new evidence regarding the mechanisms of desiccation resistance of food-borne S. aureus and contributes to the prevention of food contamination caused by this bacterium.
... Bacterial Isolates, Drugs, and Mice S. aureus RN450, ATCC6538, methicillin-resistant S. aureus (MRSA) USA300, and vancomycin-resistant S. aureus (VRSA) Mu50 were wild-type standard strains (Novick, 1967;Garrido et al., 1982;Hiramatsu et al., 1997;Diep et al., 2006). The other three MRSA isolates, BN226, BN295, and BN508, were clinical wild-type pathogens. ...
Given the increasing prevalence of Staphylococcus aureus antibiotic resistance, there is an urgent need to repurpose approved drugs with known pharmacology and toxicology as an alternative therapeutic strategy. We have reported that the sustained monotherapy of auranofin (AUR) inevitably resulted in reduced susceptibility or even the emergence of resistance to AUR in S. aureus. However, whether drug combination could increase antibacterial activity while preventing AUR resistance is still unknown. Here, we focused on the important role of AUR combined with phenethyl isothiocyanate (PEITC) in skin infection and determined the synergistic antimicrobial effect on S. aureus by using checkerboard assays and time-kill kinetics analysis. This synergistic antimicrobial activity correlated with increased reactive oxygen species (ROS) generation, disruption of bacterial cell structure, and inhibition of biofilm formation. We also showed that AUR synergized with PEITC effectively restored the susceptibility to AUR via regulating thioredoxin reductase (TrxR) and rescued mice from subcutaneous abscesses through eliminating S. aureus pathogens, including methicillin-resistant S. aureus (MRSA). Collectively, our study indicated that the AUR and PEITC combination had a synergistic antimicrobial impact on S. aureus in vitro and in vivo. These results suggest that AUR and PEITC treatment may be a promising option for S. aureus infection.
... Bacteriophage <(>11 was first identified as a prophage of S. aureus in 1967 (Novick, 1967) and is now one of the most genetically characterised of all staphylococcal phages. c|>l 1 is a temperate, generalised transducing phage whose NCTC 8325 lysogenic for bacteriophage <j>l 1 (Jayaswal et al., 1990 andWang et al., 1991). ...
Lysostaphin from S. simulans was cloned in expression vector pET21a and expressed and purified in E .coli. A spot test and a broth dilution assay indicated that the minimum inhibitory concentration of lysostaphin required to kill EMRSA-16 was 40 nM. Lysostaphin consists of an endopeptidase and a trargeting domain the former of which codes for catalytic activity while the latter is responsible for substrate specificity. In order to find out whether the targeting domain of lysostaphin is an individually functional domain, it was cloned in vector pET21d and expressed in E. coli. The purified protein was assayed against EMRSA-16 in the presence of mature lysostaphin and it was found that the targeting domain alone can protect EMRSA-16 cells. This further indicated the potential of the targeting domain of lysostaphin to be used in future domainswapping studies with other proteins. Random PCR mutagenesis was used to identify putative active site residues in the C-terminal targeting domain of lysostaphin. One mutation was isolated, where a phenylalanine was replaced by a serine at position 172 of the mature protein. Sequence alignment with other lysostaphin homologues indicated the presence of three more conserved amino acids, two tyrosines at positions 203 and 226 and a tryptophan at position 214. Site-directed mutagenesis was employed to mutate all conserved residues to alanine and FI 72 to tyrosine to distinguish between important from unimportant sites. All six mutants (F172S, F172A, F172Y, Y203A, Y226A and W214A) were cloned and their proteins expressed and purified in E. coli. Their activity was assayed in an agar diffusion assay, a broth dilution assay, turbidimetrically and in a FRET assay. The results indicated that mutants F172S, F172Y, Y203A and Y226A remained bacteriolytic while mutants F172A and W214A had lost most of their activity, suggesting their significance in the activity of lysostaphin. Finally, a reversion experiment carried out with FI72A confirmed the importance of phenylalanine at position 172 of the mature protein.
... However, when we tested the prophage-cured S. aureus strain RN450 in the mixed species macrocolony assay (Novick 1967), we observed that it was also readily killed by E. coli ( Figure 1A), indicating that prophage induction was not the only mechanism by which E. ...
Wound infections are often polymicrobial in nature and are associated with poor disease prognoses. Escherichia coli and Staphylococcus aureus are among the top five most cultured pathogens from wound infections. However, little is known about the polymicrobial interactions between E. coli and S. aureus during wound infections. In this study, we show that E. coli kills S. aureus both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. We also show that the BarA-UvrY two component system (TCS) is a novel regulator of the pks island , which acts through the carbon storage global regulatory (Csr) system. Together, our data demonstrate the role of colibactin in inter-species competition and show that it is regulated by BarA-UvrY TCS, a previously uncharacterized regulator of the pks island.
... Hume et al. (2020) reported that staphopain A secreted by S. aureus promotes the bacterial adhesion in a mouse model of keratitis using the clinical isolate Staph 38 recovered from a human corneal ulcer but not with the NCTC 8325 strain initially isolated from a human corneal ulcer. In our study, all the clinical isolates included had roughly the same capability to invade corneal epithelial cells than the 8325-4 strain, which is indeed the 8325 strain curated of its three prophages for laboratory use (Novick, 1967). Further studies are needed to unravel how staphopain A could be involved in S. aureus keratitis in humans. ...
Objectives
Staphylococcus aureus is one of the main causes of bacterial keratitis in humans. This study was aimed at investigating the mechanisms of S. aureus adhesion to the human corneal epithelium involved during the initial stage of infectious keratitis.
Methods
Human corneas stored in a specific active storage machine that restores a normal pluristratified epithelium were used to assess S. aureus adhesion level to intact and injured tissues using immunostaining. S. aureus adhesion to immobilized fibronectin was measured in microtiter plate. Internalization of S. aureus clinical isolates recovered from keratitis was assessed on human corneal epithelial HCE-2 cells.
Results
Superficial corneal injury unmasked fibronectin molecules expressed within the human corneal epithelium. S. aureus adhesion level was increased by 117-fold in the area of injured epithelium (p < 0.0001). The deletion of staphylococcal fnbA/B genes decreased by 71% the adhesion level to immobilized fibronectin (p < 0.001). The deletion of fnbA/B genes and the incubation of the corneas with anti-fibronectin blocking antibodies prior to the infection significantly reduced the S. aureus adhesion level to injured corneal epithelium (p < 0.001). Finally, S. aureus clinical isolates triggered its internalization in human corneal epithelial cells as efficiently as the 8325-4 wt.
Conclusion
S. aureus was almost unable to bind the intact corneal epithelium, whereas a superficial epithelial injury of the corneal epithelium strongly increased S. aureus adhesion, which is mainly driven by the interaction between staphylococcal fibronectin-binding proteins and unmasked fibronectin molecules located underneath the most superficial layer of the corneal epithelium.
... Induction of endogenous prophages can lead to transduction of host DNA, and often leads to mobilization of other resident MGEs 4,5 . The common S. aureus lab strain NCTC 8325 has three prophages: ϕ11, ϕ12, and ϕ13 6,7 . ...
Staphylococcus aureus pathogenicity islands (SaPIs) are molecular parasites that hijack helper phages for their transfer. SaPIbov5, the prototypical member of a family of cos type SaPIs, redirects the assembly of ϕ12 helper capsids from prolate to isometric. This size and shape shift is dependent on the SaPIbov5-encoded protein Ccm, a homolog of the ϕ12 capsid protein (CP). Using cryo-electron microscopy, we have determined structures of prolate ϕ12 procapsids and isometric SaPIbov5 procapsids. ϕ12 procapsids have icosahedral end caps with Tend = 4 architecture and a Tmid = 14 cylindrical midsection, whereas SaPIbov5 procapsids have T = 4 icosahedral architecture. We built atomic models for CP and Ccm, and show that Ccm occupies the pentameric capsomers in the isometric SaPIbov5 procapsids, suggesting that preferential incorporation of Ccm pentamers prevents the cylindrical midsection from forming. Our results highlight that pirate elements have evolved diverse mechanisms to suppress phage multiplication, including the acquisition of phage capsid protein homologs. Phage-inducible chromosomal islands (PICIs) are a group of mobile genetic elements that hijack the replication and assembly machinery of helper bacteriophages. Here the authors describe a mechanism by which a group of PICIs from Staphylococcus aureus re-direct the assembly pathway of their helpers using a capsid protein homolog.
... To compare UV inactivation kinetics within the same species, two culture-collection strains belonging to S. aureus were also included in the study. One strain was naturally aggregating when reaching late exponential phase (S. aureus 8325-4, wild type strain NCTC 8325, cured of phages φ11, φ12 and φ13) (Haaber et al., 2012;Novick, 1967), while the other was non-aggregating (S. aureus Newman, clinical isolate ATCC 25904) (Duthie and Lorenz, 1952;Haaber et al., 2012). According to Haaber et al. (2012), the difference in the aggregation level between these two strains derives from the icaADBC operon (present in S. aureus 8325-4), the expression of which is induced in the exponential growth phase and leads to the production of Polysaccharide Intercellular Adhesin (PIA). ...
Reconditioning of food processing water streams for reuse is an increasingly common water management practice in the food industry and UV disinfection is often employed as part of the water treatment. Several factors may impact the effect of UV radiation. Here, we aim to assess the impact of cell aggregation on UV inactivation kinetics and investigate if UV exposure induces aggregation. Three strains, isolated from food-processing water reuse lines (Raoultella ornithinolytica, Pseudomonas brenneri, Rothia mucilaginosa) and both an aggregating and a non-aggregating strain of Staphylococcus aureus were exposed to UVC light at 255 nm using UV LED equipment. Total Viable Count and phase-contrast microscopy, coupled with image analysis, were used to compare the UV inactivation kinetics with the average particle size for a range of UV doses. Tailing effect, seen as a strong reduction in inactivation rate, was observed for all strains at higher UV doses (industrial strains ≥ 50 or 120 mJ/cm², S. aureus strains ≥ 40 or 60 mJ/cm²). The naturally aggregating strains were more UV tolerant, both within and between species. When aggregates of S. aureus were broken, UV tolerance decreased. For the processing water isolates, the lowest applied UV dose (25 mJ/cm²) significantly increased the average particle size. Application of higher UV doses obtained with longer exposure times did not further increase the particle size compared with untreated samples. For the S. aureus strains, however, no consistent change in average particle size was observed due to UV. Our results demonstrate that aggregating strains have a higher degree of protection and that UV radiation induces aggregation in some, but not all bacteria. A better understanding of the mechanisms governing microbial aggregation and survival during UV treatment could help to improve UV applications and predictions of microbial inactivation.
... Previously described prophage curing methods, both for Gram-negative and Grampositive bacteria, mostly relied on promoting the phage loss with the use of DNA-damaging reagents and the activation of the SOS-response and thus potentially causing a variety of genomic mutations in the bacterial genome, which might result in further variation of the phenotypes [18,19]. Here, we introduce a simple, non-SOS based, highly efficient prophage curing method in P. aeruginosa, using the Gram-negative popular counter-selection marker sacB [20]. ...
Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker's temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage-cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this method can support the research and growing interest in prophage biology in P. aeruginosa as well as additional Gram-negative bacteria.
Staphylococcus aureus produces various hemolysins regulated by the Agr-QS system, except β-hemolysin encoded by the gene hlb . A classical laboratory S. aureus strain RN4220 displays only the β-hemolysin phenotype. It was suspected that the 8A mutation at the end of its agrA gene delayed the expressions of hla and RNAIII , then failed to express α- and δ-hemolysins. However, hla gene expression was detected at the later culture time without α-hemolysin phenotype, the reason for such a phenotype has not been clearly understood. We created hlb knockout and complementary mutants via homologous recombination in RN4220 and NRS049, two strains that normally produce β-hemolysin and carry agrA mutation. We found interestingly that the presence or absence of α-hemolysin phenotype in such strains depended on the expression of β-hemolysin instead of agrA mutations, which only inhibited δ-hemolysin expression. The hemolysis phenotype was verified by the Christie-Atkinson-Munch-Peterson (CAMP) test. Quantitative reverse transcription PCR was carried out to evaluate the relative gene expressions of hlb , hla , and RNAIII . The construction of mutants did not affect the agrA mutation status. We demonstrate that the absence of α-hemolysin in S. aureus RN4220 and NRS049 strains is attributed to their production of β-hemolysin instead of agrA mutation. Our findings broaden the understanding of the molecular mechanisms that control hemolysin expression in S. aureus that is crucial for the development of new therapeutic strategies to combat S. aureus infections.
IMPORTANCE
α-Hemolysin is a critical virulence factor in Staphylococcus aureus and its expression is largely controlled by the Agr-QS system. Nonetheless, the hemolysis phenotype and the regulation of the Agr-QS system in S. aureus still hold many mysteries. Our study finds that it is the expression of β- hemolysin rather than the agrA mutation that inhibits the function of the α-hemolysin in an important S. aureus strain RN4220 and a clinical strain presents a similar phenotype, which clarifies the misunderstood hemolytic phenotype and mechanism of S. aureus . Our findings highlight the interactions among different toxins and their biological roles, combined with QS system regulation, which is ultimately the true underlying cause of its virulence. This emphasizes the importance of considering the collaborative action of various factors in the infection process caused by this significant human pathogen.
Endogenous transporters protect Staphylococcus aureus against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on S. aureus survival following exposure to pyocyanin (PYO) of Pseudomonas aeruginosa, using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced S. aureus susceptibility to pyocyanin killing. The MICPYO of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a ΔnorA mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in norA expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using P. aeruginosa showed that ΔnorA mutant was less susceptible to killing by PYO-deficient mutants PA14phzM and PA14phzS than to killing by PA14. NorA overexpression led to reduced killing by all tested P. aeruginosa. We evaluated the NorA–PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both S. aureus (CF-SA) and P. aeruginosa (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and norA transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low norA transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.
We have optimized a clustered regularly interspaced short palindromic repeat (CRISPR) interference system to facilitate gene knockdown in the Gram-positive bacterial pathogen Staphylococcus aureus . Our approach used a CRISPRi system derived from Streptococcus pyogenes, which involves the co-expression of the dcas9 gene encoding a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In our system, dcas9 is expressed from a single copy in the chromosome of methicillin-resistant S. aureus strains COL or JE2, under the control of a tightly regulated promoter, inducible by anhydrotetracycline. The sgRNAs are expressed from a replicative plasmid under the control of a constitutively active promoter. This system enables efficient, inducible, knockdown of both essential and non-essential genes. Using this approach, we constructed the Lisbon CRISPRi Mutant Library comprising 261 strains, in the JE2 background, containing sgRNAs targeting 200 essential genes/operons. This library facilitates the study of the function of essential S. aureus genes and is complementary to the Nebraska Transposon Mutant Library, which consists of nearly 2,000 strains, each carrying a transposon insertion within a non-essential gene. The availability of these two libraries will facilitate the study of S. aureus pathogenesis and biology.
IMPORTANCE
Staphylococcus aureus is an important clinical pathogen that causes a high number of antibiotic-resistant infections. The study of S. aureus biology, and particularly of the function of essential proteins, is of particular importance to develop new approaches to combat this pathogen. We have optimized a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system that allows efficient targeting of essential S. aureus genes. Furthermore, we have used that system to construct a library comprising 261 strains, which allows the depletion of essential proteins encoded by 200 genes/operons. This library, which we have named Lisbon CRISPRi Mutant Library, should facilitate the study of S. aureus pathogenesis and biology.
Invertebrates can provide a valuable alternative to traditional vertebrate animal models for studying bacterial and fungal infections. This study aimed to establish the larvae of the coleoptera Tenebrio molitor (mealworm) as an in vivo model for evaluating virulence and horizontal gene transfer between Staphylococcus spp. After identifying the best conditions for rearing T. molitor, larvae were infected with different Staphylococcus species, resulting in dose-dependent killing curves. All species tested killed the insects at higher doses, with S. nepalensis and S. aureus being the most and least virulent, respectively. However, only S. nepalensis was able to kill more than 50% of larvae 72 h post-infection at a low amount of 105 CFU. Staphylococcus infection also stimulated an increase in the concentration of hemocytes present in the hemolymph, which was proportional to the virulence. To investigate T. molitor's suitability as an in vivo model for plasmid transfer studies, we used S. aureus strains as donor and recipient of a plasmid containing the gentamicin resistance gene aac(6')-aph(2″). By inoculating larvae with non-lethal doses of each, we observed conjugation, and obtained transconjugant colonies with a frequency of 1.6 × 10-5 per donor cell. This study demonstrates the potential of T. molitor larvae as a reliable and cost-effective model for analyzing the virulence of Staphylococcus and, for the first time, an optimal environment for the plasmid transfer between S. aureus carrying antimicrobial resistance genes.
Aim:
A promising approach for the development of next generation antimicrobials is to shift their target from causing bacterial death to inhibiting virulence. Marine sponges are an excellent potential source of bioactive anti-virulence molecules (AVM). We screened fractions prepared from 26 samples of Irish coastal sponges for anti-biofilm activity against clinically relevant pathogens.
Methods and results:
Fifteen fractions from eight sponge species inhibited biofilm of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) and/or Listeria monocytogenes without causing growth inhibition. GC-MS analyses of Mycale contarenii fractions revealed the presence of myristic acid and oleic acid. These fatty acids repressed transcription of the fibronectin-binding protein fnbA and fnbB genes and the polysaccharide intercellular adhesin icaADBC operon, required for MRSA and MSSA biofilm formation, respectively.
Conclusions:
This study illustrates the potential of AVM from Irish coastal sponges to specifically target bacterial virulence phenotypes, in this case repression of biofilm formation via decreased transcription of biofilm-associated genes in MSSA and MRSA.
In this study, we introduced a Raman detection technique based on a combination of functionalized magnetic beads and surface-enhanced Raman scattering (SERS) tags to develop a rapid and sensitive strategy for the detection of Staphylococcus aureus (S. aureus), a typical foodborne pathogen. Polyethylene glycol (PEG) and bovine serum albumin (BSA) dual-mediated teicoplanin functionalized magnetic beads (TEI-BPBs) were prepared for separation of target bacteria. SERS tags were used to immobilize antibodies on gold surfaces with bifunctional linker proteins to ensure specific recognition of S. aureus. Under optimal conditions, the combination of TEI-BPBs and SERS tags showed reliable performance, exhibiting good capture efficiency even in the presence of 106 CFU mL-1 of non-target bacteria. The SERS tag provided an effective hot spot for subsequent Raman detection, presenting good linearity in the range of 102-107 CFU mL-1. Good performance has also been shown in detecting target bacteria in milk samples, where it has a recovery of 95.5-101.3%. Thus, the highly sensitive Raman detection technique combined with TEI-BPBs capture probes and SERS tags is a promising method for the detection of foodborne pathogens in food or clinical samples.
Most microbes share their environmental niches with very different forms of life thereby engaging in specialised relationships to enable their persistence. The bacterium Bacillus cereus occurs ubiquitously in the environment with certain strain backgrounds causing foodborne and opportunistic infections in humans. The emetic lineage of B. cereus is capable of producing the toxin cereulide, which evokes emetic illnesses. Although food products favouring the accumulation of cereulide are known, the ecological role of cereulide and the environmental niche of emetic B. cereus remain elusive. To better understand the ecology of cereulide-producing B. cereus, we systematically assayed the toxicological spectrum of cereulide on a variety of organisms belonging to different kingdoms. As cereulide is a potassium ionophore, we further tested the effect of environmental potassium levels on the action of cereulide. We found that adverse effects of cereulide exposure are species-specific, which can be exacerbated with increased environmental potassium. Additionally, we demonstrate that cereulide is produced within an insect cadaver indicating its potential ecological function for a saprophytic lifestyle. Collectively, distinct cereulide susceptibilities of other organisms may reflect its role in enabling competitive niche specialization of emetic B. cereus.
Human pathogenic bacteria circulating in the bloodstream need to find a way to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, in particular to fibronectin (Fn). Here, we analysed the general role of EC-expressed Fn for bacterial adhesion. For this, we evaluated the expression levels of ECM coding genes in different ECs, revealing that Fn is the highest expressed gene and thereby, it is highly abundant in the ECM environment of ECs. The role of Fn as a mediator in bacterial cell-host adhesion was evaluated in adhesion assays of Acinetobacter baumannii, Bartonella henselae, Borrelia burgdorferi, and Staphylococcus aureus to ECs. The assays demonstrated that bacteria colocalised with Fn fibres, as observed by confocal laser scanning microscopy. Fn removal from the ECM environment (FN1 knockout ECs) diminished bacterial adherence to ECs in both static and dynamic adhesion assays to varying extents, as evaluated via absolute quantification using qPCR. Interactions between adhesins and Fn might represent the crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection.
Staphylococcus aureus is a common colonizer of the human gut and in doing so it must be able to resist the actions of the host's innate defences. Bile salts are a class of molecules that possess potent antibacterial activity that control growth. Bacteria that colonize and survive in that niche must be able to resist the action of bile salts, but the mechanisms by which S. aureus does so are poorly understood. Here we show that FadB is a bile-induced oxidoreductase which mediates bile salt resistance and when heterologously expressed in Escherichia coli renders them resistant. Deletion of fadB attenuated survival of S. aureus in a model of the human distal colon.
The biocide triclosan is used extensively in both household and hospital settings. The chronic exposure to the biocide occurring in individuals that use triclosan-containing products results in low levels of triclosan present in the human body that has been linked to induction of antibiotic tolerance and altered biofilm formation. Here we aimed to unravel the molecular mechanisms involved in triclosan induced antibiotic tolerance and biofilm formation in Staphylococcus aureus . Triclosan treatment prior to planktonic exposure to bactericidal antibiotics resulted in 1,000 fold higher viable cell counts compared to non-pretreated cultures. Triclosan pretreatment also protected S. aureus biofilms against otherwise lethal doses of antibiotics as shown by live/dead cell staining and viable cell counting. Triclosan mediated antibiotic tolerance in planktonic and biofilm cultures required an active stringent response because a pppGpp ⁰ strain was not protected from antibiotic killing. Incubation of S. aureus with triclosan also altered biofilm structure due to SarA-mediated overproduction of the polysaccharide intercellular adhesin (PIA) in the biofilm matrix. Thus, physiologically relevant concentrations of triclosan can trigger (p)ppGpp dependent antibiotic tolerance as well as SarA dependent biofilm formation.
Importance
The prevalent bacterium Staphylococcus aureus infects skin lesions and indwelling devices, and this can cause sepsis with 33% mortality. Intrinsic to this is the formation of co-ordinated communities (biofilms) protected by a polysaccharide coat. S. aureus is increasingly difficult to eradicate due to its antibiotic resistance. Protection against Methicillin Resistant S. aureus (MRSA) includes pre-hospital admission washing with products containing biocides. The biocide triclosan is the predominant antibacterial compound in sewage in Ontario due to its use in household and hospital settings. Levels of triclosan accumulate with exposure in humans. The significance of our research is in identifying the mechanisms triggered by exposure of S. aureus to physiological levels of triclosan that go on to raise the tolerance of S. aureus to antibiotics and promote the formation of biofilms. This understanding will inform future criteria used to determine effective antimicrobial treatments.
RN6390 is a commonly used laboratory strain of Staphylococcus aureus derived from NCTC8325. In this study, we sequenced the RN6390 genome and compared it to available genome sequences for NCTC8325. We confirmed that three prophages, Φ11, Φ12 and Φ13, which are present in NCTC8325 are absent from the genome of RN6390, consistent with the successive curing events leading to the generation of this strain. However, we noted that a separate prophage is present in RN6390 that is not found in NCTC8325. Two separate genome sequences have been deposited for the parental strain, NCTC8325. Analysis revealed several differences between these sequences, in particular, between the copy number of esaG genes, which encode immunity proteins to the type VII secreted anti-bacterial toxin, EsaD. Single nucleotide polymorphisms were also detected in ribosomal RNA genes and genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM) between the two NCTC8325 sequences. Comparing each NCTC8325 sequence to other strains in the RN6390 lineage confirmed that sequence assembly errors in the earlier NCTC8325 sequence are the most likely explanation for most of the differences observed.
Staphylococcus aureus is increasingly recognized as a facultative intracellular pathogen, although the significance and pervasiveness of its intracellular lifestyle remain controversial. Here, we applied fluorescence microscopy-based infection assays and automated image analysis to profile the interaction of 191 S. aureus isolates from patients with bone/joint infections, bacteremia, and infective endocarditis, with four host cell types, at five times post-infection. This multiparametric analysis revealed that almost all isolates are internalized and that a large fraction replicate and persist within host cells, presenting distinct infection profiles in non-professional vs. professional phagocytes. Phenotypic clustering highlighted interesting sub-groups, including one comprising isolates exhibiting high intracellular replication and inducing delayed host death in vitro and in vivo. These isolates are deficient for the cysteine protease staphopain A. This study establishes S. aureus intracellular lifestyle as a prevalent feature of infection, with potential implications for the effective treatment of staphylococcal infections. Staphylococcus aureus is increasingly recognized as a facultative intracellular pathogen, but it is unclear whether the intracellular lifestyle is a general feature or is restricted to some isolates. Here, Rodrigues Lopes et al. profile the interaction of 191 clinical isolates with four host cell types over time, showing that almost all isolates are internalized and that a large fraction replicate and persist within host cells.
Aims
Staphylococcus aureus is a major cause of septic arthritis, and in vitro studies suggest α haemolysin (Hla) is responsible for chondrocyte death. We used an in vivo murine joint model to compare inoculation with wild type S. aureus 8325-4 with a Hla-deficient strain DU1090 on chondrocyte viability, tissue histology, and joint biomechanics. The aim was to compare the actions of S. aureus Hla alone with those of the animal’s immune response to infection.
Methods
Adult male C57Bl/6 mice (n = 75) were randomized into three groups to receive 1.0 to 1.4 × 10 ⁷ colony-forming units (CFUs)/ml of 8325-4, DU1090, or saline into the right stifle joint. Chondrocyte death was assessed by confocal microscopy. Histological changes to inoculated joints were graded for inflammatory responses along with gait, weight changes, and limb swelling.
Results
Chondrocyte death was greater with 8325-4 (96.2% (SD 5.5%); p < 0.001) than DU1090 (28.9% (SD 16.0%); p = 0.009) and both were higher than controls (3.8% (SD 1.2%)). Histology revealed cartilage/bone damage with 8325-4 or DU1090 compared to controls (p = 0.010). Both infected groups lost weight (p = 0.006 for both) and experienced limb swelling (p = 0.043 and p = 0.018, respectively). Joints inoculated with bacteria showed significant alterations in gait cycle with a decreased stance phase, increased swing phase, and a corresponding decrease in swing speed.
Conclusion
Murine joints inoculated with Hla-producing 8325-4 experienced significantly more chondrocyte death than those with DU1090, which lack the toxin. This was despite similar immune responses, indicating that Hla was the major cause of chondrocyte death. Hla-deficient DU1090 also elevated chondrocyte death compared to controls, suggesting a smaller additional deleterious role of the immune system on cartilage. Cite this article: Bone Joint Res 2022;11(9):669–678.
In the time of antimicrobial resistance, phage therapy is frequently suggested as a possible solution for such difficult-to-treat infections. Vancomycin-intermediate Staphylococcus aureus (VISA) remains a relatively rare yet increasing occurrence in the clinic for which phage therapy may be an option. However, the data presented herein suggest a potential cross-resistance mechanism to phage following vancomycin exposure in VISA strains. When comparing genetically similar strains differing in their susceptibility to vancomycin, those with intermediate levels of vancomycin resistance displayed decreased sensitivity to phage in solid and liquid assays. Serial passaging with vancomycin induced both reduced vancomycin susceptibility and phage sensitivity. As a consequence, the process of phage infection was shown to be interrupted after DNA ejection from adsorbed phage but prior to phage DNA replication, as demonstrated through adsorption assays, lysostaphin sensitivity assays, electron microscopy, and quantitative PCR (qPCR). At a time when phage products are being used for experimental treatments and tested in clinical trials, it is important to understand possible interference between mechanisms underlying antibiotic and phage resistance in order to design effective therapeutic regimens.
Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.
Mupirocin induced expression of genes encoding efflux pumps NorA and MepA as well as a YFP fluorescence reporter of NorA. Mupirocin exposure also produced reduced susceptibility to pump substrates ciprofloxacin and chlorhexidine, a change that was dependent on intact norA and mepA , respectively.
Bacterial pathogens commonly carry prophages that express virulence factors, and human strains of Staphylococcus aureus carry Sa3int phages, which promote immune evasion. Recently, however, these phages have been found in livestock-associated, methicillin-resistant S. aureus (LA-MRSA). This is surprising, as LA-MRSA strains contain a mutated primary bacterial integration site, which likely explains why the rare integration events that do occur mostly happen at alternative locations. Using deep sequencing, we show that after initial integration at secondary sites, Sa3int phages adapt through nucleotide changes in their attachment sequences to increase homology with alternative bacterial attachment sites. Importantly, this homology significantly enhances integrations in new rounds of infections. We propose that promiscuity of the phage-encoded tyrosine recombinase is responsible for establishment of Sa3int phages in LA-MRSA. Our results demonstrate that phages can adopt extensive population heterogeneity, leading to establishment in strains lacking bona fide integration sites. Ultimately, their presence may increase virulence and zoonotic potential of pathogens with major implications for human health.
IMPORTANCE A growing number of humans are being infected by antibiotic resistant Staphylococcus aureus originating from livestock. The preference of S. aureus for humans or animals is in part determined by factors encoded by viruses (phages) that reside in the bacterial genome. Here, we reveal a process by which phages adapt to and become integrated in new strains of S. aureus lacking the preferred phage integration site. We propose that this is due to the relaxed specificity of a phage-encoded enzyme called recombinase. As this recombinase is used by many other phages, our results might have implications for a broader range of phages. Importantly, the adaptation described here enables S. aureus to jump between host organisms and increases its zoonotic threat.
Antimicrobial resistance (AMR) is a complex threat to human health and, to date, it represents a hot topic in drug discovery. The use of non-antibiotic molecules to block resistance mechanisms is a powerful alternative to the identification of new antibiotics. Bacterial efflux pumps exert the early step of AMR development, allowing the bacteria to grow in presence of sub-inhibitory drug concentration and develop more specific resistance mechanisms. Thus, efflux pump inhibitors (EPIs) offer a great opportunity to fight AMR, potentially restoring antibiotic activity. Based on our experience in designing and synthesizing novel EPIs, herein, we retrieved information around quinoline and indole derivatives reported in literature on this topic. Thus, our aim was to collect all data around these promising classes of EPIs in order to delineate a comprehensive structure–activity relationship (SAR) around each core for different microbes. With this review article, we aim to help future research in the field in the discovery of new microbial EPIs with improved activity and a better safety profile.
For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert.
The emergence of daptomycin-resistant (DAP-R) Staphylococcus aureus strains has become a global problem. Point mutations in mprF are the main cause of daptomycin (DAP) treatment failure. However, the impact of these specific point-mutations in methicillin-resistant S. aureus (MRSA) strains associated with DAP resistance and the “see-saw effect” of distinct beta-lactams remains unclear. In this study, we used three series of clinical MRSA strains with three distinct mutated mprF alleles from clone complexes (CC) 5 and 59 to explore the “see-saw effect” and the combination effect of DAP plus beta-lactams. Through construction of mprF deletion and complementation strains of SA268, we determined that mprF -S295A, mprF -S337L and one novel mutation of mprF- I348del within the bifunctional domain lead to DAP resistance. Compared with wild-type mprF cloned from a DAP-susceptible (DAP-S) strain, these three mprF mutations conferred the “see-saw effect” to distinct beta-lactams in the SA268Δ mprF strains and mutated- mprF (I348del and S337L) did not alter the cell surface positive charge ( P > 0.05). The susceptibility to beta-lactams increased significantly in DAP-R CC59 strains and the “see-saw effect” was found to be associated with distinct mutated mprF alleles and the category of beta-lactams. The synergistic activity of DAP plus oxacillin was detected in all DAP-R MRSA strains. Continued progress in understanding the mechanism of restoring susceptibility to beta-lactam antibiotics mediated by the mprF mutation and its impact on beta-lactam combination therapy will provide fundamental insights into treatment of MRSA infections.
Bacteriophage ɸ13 belongs to a group of phages that carries virulence factors and colonizes human strains of Staphylococcus aureus. Knowledge about the mechanism behind the genetic switch that decides between lysogeny (prophage integration) or lysis (bacterial killing) is important for combating S. aureus infection. Using the switch region from ɸ13, we have created reporter plasmids that mimic the decision phase and can detect prophage-inducing conditions.
Abstract
Temperate phages are bacterial viruses that after infection either reside integrated into a bacterial genome as prophages forming lysogens or multiply in a lytic lifecycle. The decision between lifestyles is determined by a switch involving a phage-encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here, we investigate the switch of phage ɸ13 from the human pathogen Staphylococcus aureus. ɸ13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the ɸ13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high-affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from ɸ13 with the lytic promoter fused to lacZ into S. aureus and Bacillus subtilis. Analysis of β-galactosidase expression indicated that decision frequency is independent of host factors. The white “lysogenic” phenotype, which relies on the expression of cI, could be switched to a stable blue “lytic” phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage ɸ13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus.
Staphylococcus epidermidis is a leading opportunistic pathogen causing nosocomial infections that is notable for its ability to form a biofilm and for its high rates of antibiotic resistance. It serves as a reservoir of multiple antimicrobial resistance genes that spread among the staphylococcal population by horizontal gene transfer such as transduction. While phage-mediated transduction is well studied in Staphylococcus aureus, S. epidermidis transducing phages have not been described in detail yet. Here, we report the characteristics of four phages, 27, 48, 456, and 459, previously used for S. epidermidis phage typing, and the newly isolated phage E72, from a clinical S. epidermidis strain. The phages, classified in the family Siphoviridae and genus Phietavirus, exhibited an S. epidermidis-specific host range, and together they infected 49% of the 35 strains tested. A whole-genome comparison revealed evolutionary relatedness to transducing S. aureus phietaviruses. In accordance with this, all the tested phages were capable of transduction with high frequencies up to 10⁻⁴ among S. epidermidis strains from different clonal complexes. Plasmids with sizes from 4 to 19 kb encoding resistance to streptomycin, tetracycline, and chloramphenicol were transferred. We provide here the first evidence of a phage-inducible chromosomal island transfer in S. epidermidis. Similarly to S. aureus pathogenicity islands, the transfer was accompanied by phage capsid remodeling; however, the interfering protein encoded by the island was distinct. Our findings underline the role of S. epidermidis temperate phages in the evolution of S. epidermidis strains by horizontal gene transfer, which can also be utilized for S. epidermidis genetic studies.
IMPORTANCE
Multidrug-resistant strains of S. epidermidis emerge in both nosocomial and livestock environments as the most important pathogens among coagulase-negative staphylococcal species. The study of transduction by phages is essential to understanding how virulence and antimicrobial resistance genes spread in originally commensal bacterial populations. In this work, we provide a detailed description of transducing S. epidermidis phages. The high transduction frequencies of antimicrobial resistance plasmids and the first evidence of chromosomal island transfer emphasize the decisive role of S. epidermidis phages in attaining a higher pathogenic potential of host strains. To date, such importance has been attributed only to S. aureus phages, not to those of coagulase-negative staphylococci. This study also proved that the described transducing bacteriophages represent valuable genetic modification tools in S. epidermidis strains where other methods for gene transfer fail.
Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.
We have devised a rapid plate method for the detection and isolation of lysogenic phages and have used the procedure to screen 15 strains of Staphylococcus aureus. This method should prove applicable to a wide variety of bacterial species and should be especially valuable for those pathogens for which no phage-typing system is available.
Seventy-five mutants with alterations in penicillinase formation were isolated from a strain of Staphylococcus aureus inducible for penicillinase. The mutants fell into three main categories on the basis of penicillinase activity and inducibility: (i) microinducible mutants which formed decreased amounts of penicillinase but retained the property of inducibility; (ii) penicillinase-negative variants which produced no detectable penicillinase and which showed no effect of inoculum size on penicillin resistance; (iii) strains with a wide range of penicillinase activities that produced the enzyme constitutively. Treatment of the wild-type strain with ethylmethane sulphonate increased the frequency of occurrence of microinducible and constitutive mutants but did not alter the incidence of the penicillinase-negative variants which were present in all cultures at a frequency of about 10-3. Representative mutants of each class were examined for ability to revert to wild type and to give wild-type recombinants in transductional crosses. The constitutive strains and the microinducible strains behaved like point mutants in that they reverted and in that they gave wild-type recombinants. The penicillinase-negative mutants, however, behaved differently in that they were not observed to revert nor did they give wild-type recombinants in crosses, either with one another or with microinducible or constitutive mutants. A naturally occurring penicillinase-negative strain of S. aureus behaved similarly to the penicillinase-negative mutants in these respects. The possibility that the penicillinase region in S. aureus is associated with a plasmid and thus inherited extrachromosomally is considered and discussed. The properties of the penicillinase-negative variants could be explained as resulting from the loss of such a plasmid. Consistent with the plasmid hypothesis is the finding that ultraviolet irradiation of transducing phage produced an exponential decline of transducing titre for penicillinase; against it is the failure of acridine orange to increase the frequency of the penicillinase-negative variants.
Novick, Richard P. (The Rockefeller Institute, New York, N.Y.), and Mark H. Richmond. Nature and interactions of the genetic elements governing penicillinase synthesis in Staphylococcus aureus. J. Bacteriol.
90
467–480. 1965.—It has been found previously that penicillinase-producing strains of Staphylococcus aureus each harbor an extrachromosomal element, or plasmid, which apparently carries all the genetic information necessary for penicillinase synthesis. These plasmids behave in a manner consistent with their being small chromosomelike structures in that they comprise linkage groups containing several markers and in that they undergo such genetic events as mutation, segregation, and recombination. There is currently no evidence for conjugal cell-cell transfer of the plasmids or for a state of stable integration into the staphylococcal chromosome. A certain amount of variability has been encountered among the penicillinase plasmids harbored by different staphylococcal strains. It has been found that: (i) there are at least three molecular variants of the enzyme itself; (ii) most, but not all, of the penicillinase plasmids carry a genetic determinant of resistance to mercuric ion; (iii) plasmids carried by a very small number of the strains bear a determinant of resistance to erythromycin; (iv) the plasmids determine the fraction of penicillinase excreted into the medium during growth, and this also varies from strain to strain. The penicillinase plasmids appear to behave as integral genetic structures. The entire known linkage group is transduced intact, and is occasionally lost completely as a spontaneous occurrence during the growth of the organisms. Rarely, the plasmid markers dissociate during transduction, resulting in transduced clones which have inherited only a part of the plasmid linkage group. Similarly, dissociation occurs as a spontaneous event during normal growth, also resulting in rare clones which appear to have lost one or more but not all of the plasmid markers. When crosses are performed between two plasmid-harboring strains, a plasmid heterozygote is formed. In most cases, this persists for only one or a few cell divisions before segregating, with or without the formation of recombinant plasmids. In two instances thus far observed, the heterozygote persists as a stable plasmid heterodiploid, in which the continued presence of both plasmids can be readily demonstrated. The fate of the heterozygote, i.e., early segregation or persistence as a heterodiploid, depends upon which particular pair of plasmids is involved in the cross. This observation has led to the hypothesis of a plasmid-linked determinant of plasmid compatibility. A pair of plasmids is considered compatible if it can form a stable heterodiploid, incompatible if it cannot.
A “cryptic” λ prophage has been detected in an apparently cured strain of K12 (λ). This strain, called U173, is normally sensitive to λ mutants, to other phages related to λ and to rII mutants of T4. Induction with ultraviolet light has no effect either in producing active phages, or in promoting lysis of the culture. Rescue of λ wild-type alleles has been possible upon infection of this strain with certain λ mutants. The extension of the “cryptic” fragment covers about one-half or more of the map of λ.
This chapter presents a historical account of the known episomes other than phage as well as some instances of possibly similar elements in higher organisms. Most of the episomes of bacteria fall into one of two classes—namely, temperate bacteriophages and transfer factors that can pass from cell to cell during conjugation independently of the bulk of the bacterial genome. Some transfer factors play a causative role in the conjugation process itself, such as the fertility factor of Escherichia coli K12. The episomic nature of temperate bacteriophage was established earlier than that of the transfer factors. A large amount of chemical and genetic work has converged to form a picture of intracellular bacteriophage growth, which is satisfying at a certain level. Much of the work on episomes and gene pick-up depends on the formation of partially diploid strains. The discovery of such strains antedates the findings with episomes, and there are some cases in which no known episome is implicated. Episomes consist of genetic material and frequently affect the intercellular transfer of other genetic material. The transfer of one episome by another reflects a physical connection of the two, so that at least during the time of transfer one episome behaves as part of the other.
Data from crosses between strains of Escherichia coli K12 lysogenic for λ prophages suitably marked show three facts that bear on the problem of structural relationships between prophage and host bacterium. A marked increase can be observed in recombination between the prophage markers when one performs a selection for crossovers between alleles of gal. This increase permits a test of linearity of prophage markers relative to bacterial markers. The results of the test show that the two structures are linearly assembled. A marker mi can be mapped at a position different from the one found by phage crosses. The meaning of this last fact is discussed in relation to the main problem of structural organization.
Kameda, Mitsuo (Gunma University, Maebashi, Japan), Kenji Harada, Mitsue Suzuki, and Susumu Mitsuhashi. Drug resistance of enteric bacteria. V. High frequency of transduction of R factors with bacteriophage epsilon. J. Bacteriol.
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1174–1181. 1965.—In the transduction of R factors with phage ε15, a lysate capable of transducing the markers for (TC) or (CM.SM.SA) resistance at high frequency was obtained. The transducing agent is a defective element called ε15dR23 which lacks certain functions of phage ε15. After lysogenization with normal ε15 phage and ultraviolet (UV) induction, strains carrying the ε15dR23 element produce lysates which have a high frequency of transduction (HFT) on group E1Salmonella. Lytic lysates prepared on phage ε15 sensitive strain with the ε15dR23 element have a low frequency of transduction (LFT). Lytic growth of phage ε34 on an ε15dR23 strain or UV induction of an ε34 lysogenic strain containing ε15dR23 results in LFT lysates on group E2Salmonella. On UV induction, group E2Salmonella (ε15 lysogens) with the ε15dR23 element give lysates which are HFT on group E1Salmonella but are LFT when tested on group E2Salmonella. In all instances, the production of drug-resistant transductants requires infection of the cell with only a single ε15dR23 element. It appears that the resistance region of the R factor has replaced that portion of phage genome which is essential for vegetative replication and superinfection immunity. The ε15dR23 element does not contain the genetic determinants of the R factor responsible for transmissibility, inhibition of F mating, and interference between two R factors.
Cotransduction analysis by means of bacteriophage P1 has been used to study the relation between the bacterial genetic markers gal and bio and the prophage λ. The order of a series of markers of the λ prophage with respect to the bacterial gal locus has now been found. This marker order is a circular permutation of the order present in vegetative phage λ, in agreement with Campbell's hypothesis (1962). The frequency of cotransduction of gal and bio is drastically reduced when both donor and recipient strains are lysogenic either for prophage λ or prophage 82, an indication that both λ and 82 are located between the gal and bio markers. Additional evidence is presented suggesting that prophage 434 and the λ-434 hybrid may also be located between gal and bio.
It was not possible to find the order of the λ prophage markers with respect to the bio marker; hence, conclusive evidence for a linear insertion of the prophage into the bacterial chromosome could not be obtained.
The presence of a lambdoid prophage appears to affect the early steps of transduction by phage P1. The possible nature of this effect is discussed.
Bromouracil-labelled Escherichia coli thy− was infected with a virulent mutant of phage P1kc and incubated in a medium containing thymine until lysis occurred. The analysis of the phage yield by CsCl density-gradient centrifugation showed that the transducing particles carrying various chromosomal genes have a density and band profile similar to those prepared on bromouracil-labelled bacteria in a medium containing bromouracil. When the phage particles were prepared on the bromouracil-labelled bacteria in a medium containing thymine and 32PO4, radioactivity was not found in fractions which contained transducing particles. These reults indicate that most of the transducing particles lack phage genome and carry only fragments of the bacterial chromosome existing at the time of infection.The results show interruption of the replication of bacterial chromosome by phage infection. The replication of λ prophage and F′ lac was also interrupted by infection. On the other hand, the replication of an R factor was not arrested.In contrast to the behaviour of the virulent mutant, the original P1kc phage did not arrest the replication of bacterial chromosome.When the virulent mutant was grown on [3H]thymidine-labelled bacteria in a medium containing 32PO4, infective particles and transducing particles could be differentially labelled with 32P and 3H, respectively. The results of the analysis of the phage particles showed that the transducing particles comprised 0·3% of the total phage particles.Physical properties of DNA's of infective and transducing particles were studied. The density of P1 DNA is 1·706 g cm−3, which corresponds to a guanine–cytosine content of 46%. The molecular weights of DNA's of infective and transducing particles are the same, namely, 6 × 107 daltons. Homology between PI DNA and E. coli DNA is rare.
Transduction of the lac+ property by phage P1 among strains of Escherichia coli K12 and Shigella dysenteriae Sh has been studied. When E. coli strains are used both as donors and recipients, transduction gives stable lac+ transductants (transduction by integration). Transduction from E. coli lac+ donors to strain Sh gives mainly unstable, lacv transductants, in which the lac+ genes are apparently associated with some phage P1 genes in a defective prophage, called P1 dl (transduction by lysogenization). A variety of P1 dl elements is postulated to account for the properties of different Sh lacv transductant strains. Some of these strains produce phage lysates that transduce the lac+ property at high frequency (HFT lysates), probably because of a high content of phage particles carrying the defective phage element P1 dl. A different type of P1 dl element, called P1 dlw, has been obtained by using HFT lysates from Sh lacv strains to transduce the lac+ property in a strain of E. coli that appears to have a deletion of the lac genetic region.The results of lac+ transduction by various types of lysates to various recipient strains have been compared, using both untreated and ultraviolet-irradiated lysates. The results are interpreted by assuming that the transducing particles contain genetic elements with various combinations of phage genes and of bacterial genes, and that the transduction type that is obtained, by integration or by lysogenization, depends both on the ability of the transducing elements to lysogenize and on the degree of genetic homology between the donor and the recipient of the transduced bacterial genes. High genetic homology favors integration; low genetic homology hinders integration and allows detection of transductants carrying the transducing element as prophage.
Mahler, I. (Brandeis University, Waltham, Mass.), M. Cahoon, and J. Marmur. Bacillus subtilis deoxyribonucleic acid transfer in PBS2 transduction. J. Bacteriol.
87
1423–1428. 1964.—Lysates of the general transducing bacteriophage PBS2 grown on Bacillus subtilis SB19 were fractionated by preparative CsCl density-gradient centrifugation. Five distinct and separate bands which varied in their ability to transduce three nutritional markers were obtained by this procedure. Deoxyribonucleic acid (DNA) samples prepared from unfractionated lysates and from each of the separate bands were examined by analytical CsCl density-gradient centrifugation. In addition to a major band (density, 1.723 g/cc) identified as PBS2 DNA, a satellite band of lighter density was detectable in the nucleic acids obtained from whole lysates and those bands that possessed transducing activity. The biological activity of the purified nucleic acids, determined by transformation experiments, was found to reside in the light satellite band (1.703 g/cc) characteristic of B. subtilis DNA. In view of the correlation between general transducing ability of phage particles and the presence of bacterial DNA which appears not to be physically associated with the phage DNA, it is suggested that transduction by bacteriophage PBS2 does not depend upon areas of homology between the phage genome and the host chromosome but resides in specific particles which have incorporated segments of bacterial DNA.
Kondo, Eiko (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Drug resistance of enteric bacteria. IV. Active transducing phage P1 CM produced by the combination of R factor with phage P1. J. Bacteriol.
88
1266–1276. 1964.—During an investigation of the transduction of R factors with phage P1, a phage lysate capable of transducing the character of chloramphenicol resistance (CMr) in extremely high frequency was obtained. The transduction of the CMr character with the lysate was consistently accompanied by lysogenization with the phage used for transduction. This lysate exhibits no beneficial effect with normal P1, and no effect is produced by decreasing the multiplicity of infection. A single infection with the phage allows the formation of plaques as well as CMr lysogenic cells at the center of the plaque. Both the transducing and plaque-forming activities of the lysate were lost by neutralization with anti-P1 phage serum, and its absorption to the host bacteria was enhanced by the addition of Ca⁺⁺. Thus, it was concluded that a derivative of P1 phage (P1 CM) was isolated which had not only the ability to transduce the CMr character but also the capacity to form plaques; i.e., the CMr gene of R factor is specifically associated with the genome of phage P1. No detectable differences were noted between P1 CM and normal P1 phage in density-gradient analyses in CsCl, in stability of lysogenization, in ability to transduce chromosomal markers, and in the mode of induction from lysogenic cells by ultraviolet irradiation. The instance of transduction of the CMr character described here may also be considered as an example of lysogenic conversion, in the sense that the alteration in CMr character is inseparable from lysogenicity.
IN bacteria certain genetic determinants which may be gained or lost can multiply without incorporation in the bacterial chromosome. Some such plasmids1, the class of episomes2, may become reversibly integrated into the chromosome; for others, including the transmissible drug-resistance (R) factors3, integration has not been unequivocally demonstrated. We report observations on the transmission of an R factor and of colicine factors by conjugation and transduction in Salmonella typhimurium, and show that R factor genetic material may become attached to the bacterial chromosome-but only by becoming associated with an episome, prophage P22.
Richmond, Mark H. (National Institute for Medical Research, Mill Hill, London, England). Dominance of the inducible state in strains of Staphylococcus aureus containing two distinct penicillinase plasmids. J. Bacteriol.
90
370–374. 1965.—The phenotypic expression of penicillinase synthesis is studied in a strain of S. aureus containing two distinct plasmids, each carrying the penicillinase structural gene and its associated control (inducibility) gene. One plasmid in the heterodiploid carries the genes responsible for the normal, fully inducible synthesis of a structural mutant of type A penicillinase, and the second plasmid carries the genes required for constitutive synthesis of C-type enzyme. The heterodiploid is phenotypically fully inducible, indicating that the inducible state dominates the constitutive in penicillinase synthesis. The heterodiploid synthesizes approximately equal quantities of A- and C-type enzyme when fully induced. Parental types are obtained from the heterodiploid by spontaneous segregation at a frequency of about 1 per 1,000 divisions of each character. Recombination between the two plasmids in such a persistent heterodiploid is a rare event.
Fine genetic structure as revealed by transduction
- Clowes
Specialized transduction of the tryp gene by the temperate phage ∅80
- Matsushiro
Trans. duction des marqueurs galactose par les bacteriophages tempérés 82 et 434 d'Escherichia coli
- Reissig