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Curing bacterial cells of lysogenic viruses by using UCB indicator plates
BioTechniques
... On these plates, the pseudolysogens appear dark green whereas non-lysogens are yellow. This method can be used also to " cure " the pseudolysogens by streaking a dark green colony on green plates and choosing a yellow segregant (Berget and Chidambaram, 1989; Bochner, 1984) lyse, so the portion of the streak after the phage will be spotty and dark green (Jeff, 2005). Another method for detecting lysogenic phages is by using the mutagen, Mitomycin C. ...
... If the bacterial cells were non-lysogenic, it was expected that light green or yellow colonies would to be produced. If the cells had unstable lysogens, small dark green colonies were to be produced (Hughes and Maloy, 2007; Berget and Chidambaram, 1989; Bochner, 1984). ...
... Dark green, small colonies after 24 hours represent the presence of unstable lysogenic bacterial cells. Yellow or light green colonies are evidence of the presence of stable or non-lysogenic bacterial cells (Bochner, 1984; Berget and Chidambaram, 1988). Isolate C5A contained a mixture of both light and dark green colonies. ...
Production of numerous biotechnologically-important products such as threonine is based on cultivation of bacterial cultures. Infection of these bacterial cultures by bacteriophages has a detrimental effect in the production of these bioproducts. Despite this, most people controlling these bioprocesses do not recognize the early signs of bacteriophage infection. SA Bioproducts (Ply) Ltd was no exception and has suffered tremendous loss of production time after bacteriophages infected threonine producing E. coli strain B. This study was aimed at developing assays to control and prevent bacteriophage infection at this company. These included determining the source of phages by monitoring the process plant environment, optimising the detection and enumeration methods so as to monitor the levels of bacteriophages in the environment, identification of bacteriophages in order to determine the number of bacteriophages capable of infection threonine producing E. coli strain B, treatment and of phages, and possible prevention of phage infection. Adam's DAL method was very efficient at detecting phages in the samples collected at various areas (sumps, odour scrubber, process water, and soil) around the plant for 16 weeks. High levels of phages were found in the sumps and this was identified as the source of infection. Samples collected were grouped together according to their source. The samples were enriched and purified in order to characterise them. The prevalent phage in all samples was identified as a T1-like phage. Bacterial strains that grew on the plate in the presence of phages were assumed to be resistant to phages or contained lysogenic phages which would explain the new lytic cycles that were observed whenever these resistant strains were used for production. UV light, green v indicator plates, and a mutagen (Mitomycin C) were used to detect Iysogens. Mitomycin C at 1 IJg/ml was found to be most effective in detecting lysogenic phages. This was shown by new plaque forming units that were visible on the DAL plates. Temperature (heat), chemicals, and inhibitors (vitamins) were investigated as strategies for prevention and treatment of bacteriophage infection. Bacteriophage samples were exposed to 70, 80, 100, and 120°C. At these temperatures pfu counts in the samples were reduced significantly. At 120°C there was a complete inactivation of bacteriophages within 30 minutes. Chemicals investigated such as sodium hydroxide and Albrom 100T were capable of complete deactivation of bacteriophages at a very low concentration (0.1%). Therefore, these chemicals can be used to clean the plant area and sumps. Vitamins C, K and E solutions were investigated to determine their inhibitory effect on bacteriophages. Vitamin C, K and E reduced pfu counts by 3, 2, and 4 logs, respectively. Therefore vitamin C and E solutions were mixed and to determine if mixing them would enhance their inactivation capabilities. This resulted in a reduction greater than 9 logs of phage in the sample (from 7.7 x 109 to 3 pfu/ml). The host bacterium was also exposed to this mixture to determine effect of the vitamin mixture on its growth. It was found that there was no effect exerted by this mixture on the host bacteria. This proved to be an ideal mixture for combating phages during fermentation. However, vitamin E is not cost effective for co-feeding in 200 m' fermenters, and therefore vitamin C solution was a cost-effective alternative. It was concluded that bacteriophage contaminated bioprocessing plant should be properly cleaned using a combination of heat and chemicals. Bacteriophage infection should be prevented by employing inhibitors. Submitted in partial fulfillment of the academic requirements for the degree of Master of Technology: Biotechnology, 2009.
... We demonstrate here its first use in P1 phage transduction experiments using E. coli cells. When temperate phages are present in cells, a colony formed from these cells will have a pH that differs from that of uninfected cells as a result of pH lowering through the lysis of pseudo-lysogenic cells [36]. This property has been exploited in the EBU plate assay to directly visualize colonies containing Evans blue stained pseudo-lysogenic cells (Fig. 2d). ...
... f The growth of ΔmotAB10 and ΔmotAB14 strains in 96 well-plate reader containing LB medium with various concentrations of chloramphenicol (17 μg/mL to 68 μg/mL). The results show that ΔmotAB14 strain containing the extraneous insertion grew at a higher concentration of chloramphenicol (51 μg/mL) than the normal concentration (34 μg/mL), while the ΔmotAB10 did not grow at 51 μg/mL of chloramphenicol The temperate phage-infected cells verified by the cross-streak agar experiment were tested using the EBU plate assay (Methods section IIE): they exhibited exclusively dark green colonies (Fig. 2e), which we associate with infection by temperate phages that results in a change in cellular pH [36]. Conversely, the cells verified to be free of phages displayed exclusively pale green colonies (Fig. 2f ). ...
Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome.
Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check.
The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.
... Bacteriophage P22 HT105/1 int Ϫ was used for generalized transduction of the aroA::Tn10 marker from serotype Typhimurium strain CL1509 into RAK1 or AJB75 (10). Transductants were routinely purified from contaminating phage by streaking the strain twice for single colonies on Evans blue uridine plates (16). Subsequently, strains were tested in a cross streak for P22 sensitivity. ...
... For single infection experiments, groups of 12 BALB/c mice were inoculated with 10 9 CFU of either CL1509 (aroA::Tn10-tet r ) or RAK7 (aroA::Tn10-tet r shdA::Cm r ). The presence of inoculum strain in fecal pellets was determined on 29 days during the first 79 days postinoculation (days 1 to 16,18,21,24,27,31,34,37,44,51,58,65,72, and 79 postinoculation). Approximately 20 mg of fresh fecal pellets were resuspended in PBS (pH 7.4), and bacteria were enumerated on LB agar plates containing tetracycline (20 g/ml) or tetracycline (20 g/ml) and chloramphenicol (50 g/ml). ...
Little is known about factors which enable Salmonellaserotypes to circulate within populations of livestock and domestic fowl. We have identified a DNA region which is present
inSalmonella serotypes commonly isolated from livestock and domestic fowl (S. enterica subspecies I) but absent from reptile-associated Salmonella serotypes (S. bongori and S. enterica subspecies II to VII). This DNA region was cloned from Salmonella serotype Typhimurium and sequence analysis revealed the presence of a 6,105-bp open reading frame, designated shdA, whose product's deduced amino acid sequence displayed homology to that of AIDA-I from diarrheagenicEscherichia coli, MisL of serotype Typhimurium, and IcsA ofShigella flexneri. The shdA gene was located adjacent to xseA at 52 min, in a 30-kb DNA region which is not present in Escherichia coli K-12. A serotype Typhimurium shdA mutant was shed with the feces in reduced numbers and for a shorter period of time compared to its isogenic parent. A possible
role for the shdA gene during the expansion in host range of S. enterica subspecies I to include warm-blooded vertebrates is discussed.
... For transduction, 0.1 mL of serial 10-fold dilutions of phage lysate were mixed with 0.1 mL of recipient overnight culture, incubated at room temperature for 1 hour, and plated on LB agar plates containing the appropriate antibiotics. Single colonies were streaked on Evans Blue-Uranine (EBU) agar plates (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 2.5 g/L glucose, 15 g/L agar, 5 g/L K 2 HPO 4 , 12.5 mg/L Evans Blue, and 25 mg fluorescein sodium salt) (77). White colonies were evaluated for phage resistance using a cross streak with P22 H5. ...
Salmonella enterica serovar Typhimurium induces intestinal inflammation to create a niche that fosters the outgrowth of the pathogen over the gut microbiota. Under inflammatory conditions, Salmonella utilizes terminal electron acceptors generated as byproducts of intestinal inflammation to generate cellular energy through respiration. However, the electron donating reactions in these electron transport chains are poorly understood. Here, we investigated how formate utilization through the respiratory formate dehydrogenase-N (FdnGHI) and formate dehydrogenase-O (FdoGHI) contribute to gut colonization of Salmonella . Both enzymes fulfilled redundant roles in enhancing fitness in a mouse model of Salmonella -induced colitis, and coupled to tetrathionate, nitrate, and oxygen respiration. The formic acid utilized by Salmonella during infection was generated by its own pyruvate-formate lyase as well as the gut microbiota. Transcription of formate dehydrogenases and pyruvate-formate lyase was significantly higher in bacteria residing in the mucus layer compared to the lumen. Furthermore, formate utilization conferred a more pronounced fitness advantage in the mucus, indicating that formate production and degradation occurred predominantly in the mucus layer. Our results provide new insights into how Salmonella adapts its energy metabolism to the local microenvironment in the gut.
IMPORTANCE
Bacterial pathogens must not only evade immune responses but also adapt their metabolism to successfully colonize their host. The microenvironments encountered by enteric pathogens differ based on anatomical location, such as small versus large intestine, spatial stratification by host factors, such as mucus layer and antimicrobial peptides, and distinct commensal microbial communities that inhabit these microenvironments. Our understanding of how Salmonella populations adapt its metabolism to different environments in the gut is incomplete. In the current study, we discovered that Salmonella utilizes formate as an electron donor to support respiration, and that formate oxidation predominantly occurs in the mucus layer. Our experiments suggest that spatially distinct Salmonella populations in the mucus layer and the lumen differ in their energy metabolism. Our findings enhance our understanding of the spatial nature of microbial metabolism and may have implications for other enteric pathogens as well as commensal host-associated microbial communities.
... EBU plates contain pH indicators that are light green at neutral pH but turn dark green/blue at low pH. Lysis of P22-infected cells lowers the pH of the medium (Bochner 1984). Infection foci can then be visualized as dark green-colored plaques on a light green background (Fig. 1A). ...
Genomic engineering of Escherichia coli and Salmonella often requires introducing plasmids into strains obtained during the intermediate stages of the process. Such strains are typically transformed only once, making the preparation of large batches of competent cells for storage purposes unnecessary. Here, we describe a simple scaled-down procedure for transforming E. coli or Salmonella with plasmid DNA that uses as little as 2 mL of culture.
... Markers were moved between Salmonella strains using P22 transductions. Transductants were purified on EBU plates [56] and clones were confirmed by PCR and sequencing. Antibiotic resistance markers were excised using Flp recombinase expressed from the pcp20 vector as described previously [55]. ...
RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.
... suggested that lysis and recovery of live bacteria in solid media should be possible. To test this hypothesis, we induced lysis of colonies grown on EBU plates, which are normally used for detection of pseudolysogens in P22 transduction protocols 9 . This medium contains dyes that stain colonies containing lysed bacteria. ...
Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. During functional screenings of enzymes, the ability of the substrate to enter the surrogate host or the ability of this bacterium to export heterologous extracellular enzymes may hamper the technique. Here we have used an inducible autolysis system that lyses bacteria thus releasing its content in both, liquid and solid cultures, in response to anhydrotetracycline. The lytic cluster is tightly regulated to prevent impaired bacterial growth in absence of the inducer and produced very efficient though not complete bacterial lysis upon induction, which allowed the recovery of live bacteria. The system can be used in combination with specialised fosmids and E. coli strains that maximize transcription of metagenomic DNA. Our results show that colony-lysis on plates allows detection of an endogenous intracellular amylase activity naturally present in E. coli and clearly increased detection of clones coding for cellulase activities in a metagenomic screening, while allowing recovery of survivor positive clones from the lysed colonies in all cases. Therefore, this tool represents an important step towards the effective access to the extraordinary potential of the uncultivated bacteria genetic resources.
... Salmonella enterica serovar Typhimurium (S. Typhimurium) BRD509 strain [13] was kindly provided by Dr. D. Xu, University of Glasgow, Glasgow, U.K. To generate the S. Typhimurium BRD509 strain expressing the 2W1S epitope (EAWGALANWAVDSA) (strain SPN555), P22 generalized transduction [14,15] was performed using as a donor an S. Typhimurium SL1344 strain expressing 2W1S [16], which was a kind gift from Dr. M. Jenkins, University of Minnesota, Minneapolis, MN. Salmonella were cultured overnight in Luria-Bertani (LB) broth (Difco, BD Diagnostics, Sparks, MD) and diluted in PBS after estimation of bacterial concentration using a spectrophotometer. ...
In immunocompetent individuals, non-typhoidal Salmonella serovars (NTS) are associated with gastroenteritis, however, there is currently an epidemic of NTS bloodstream infections in sub-Saharan Africa. Plasmodium falciparum malaria is an important risk factor for invasive NTS bloodstream in African children. Here we investigated whether a live, attenuated Salmonella vaccine could be protective in mice, in the setting of concurrent malaria. Surprisingly, mice acutely infected with the nonlethal malaria parasite Plasmodium yoelii 17XNL exhibited a profound loss of protective immunity to NTS, but vaccine-mediated protection was restored after resolution of malaria. Absence of protective immunity during acute malaria correlated with maintenance of antibodies to NTS, but a marked reduction in effector capability of Salmonella-specific CD4 and CD8 T cells. Further, increased expression of the inhibitory molecule PD1 was identified on memory CD4 T cells induced by vaccination. Blockade of IL-10 restored protection against S. Typhimurium, without restoring CD4 T cell effector function. Simultaneous blockade of CTLA-4, LAG3, and PDL1 restored IFN-γ production by vaccine-induced memory CD4 T cells but was not sufficient to restore protection. Together, these data demonstrate that malaria parasite infection induces a temporary loss of an established adaptive immune response via multiple mechanisms, and suggest that in the setting of acute malaria, protection against NTS mediated by live vaccines may be interrupted.
... Typically, P22 lysates were used at a 1:50 dilution, mixed with aliquots from overnight cultures of recipient bacteria in a 1:2 ratio, and incubated for 30 min at 37°C prior to being plated on selective media. Transductant colonies were purified by two sequential passages on selective plates and verified to be free of phage by streaking on Evans Blue Uranine plates (Bochner, 1984). ...
GcvB is a conserved 200 nucleotide RNA that downregulates several genes involved in amino acid uptake or biosynthesis in bacteria. The physiological role of GcvB action is not entirely clear, but it is likely aimed at balancing of nutritional resources under fast growth conditions. GcvB inhibits translation of target messenger RNAs by pairing with sequences inside or upstream of ribosome binding sites. In the present study, characterization of a novel GcvB-regulated locus revealed some unique features in the mode of functioning of this regulatory RNA. We found that GcvB represses yifK - a highly conserved locus encoding a putative amino acid transporter - by targeting a translational enhancer element. Two ACA motifs within the target sequence are the main determinants of the enhancer activity. Replacing either of these motifs with random triplets caused up to a 10-fold decrease in yifK expression regardless of the GcvB allele (deleted or suitably modified to recognize the mutated target). It thus appears that GcvB effectiveness as a regulator results from countering the enhancer activity. When the enhancer is removed, GcvB action no longer constitutes a rate-limiting factor for yifK expression. Overall, this study is relevant not only to a better understanding of GcvB function but it also provides insight into an elusive aspect of the translation initiation process. Besides the GcvB control, the yifK locus is regulated at the transcriptional level by the leucine responsive regulator Lrp, and by HdfR (YifA) a poorly known transcriptional regulator, that appears to require the product of the adjacent, divergently oriented gene, yifE, for expression or activity. Transcription initiating at the yifK promoter extends into the adjacent argX-hisR-leuT-proM tRNA operon yielding an unusual primary transcript which both a messenger RNA and a tRNA precursor. This chimeric RNA si rapidly processed by RNAse E.
... Chrome azurol S (CAS) plates used to assay for siderophore production were prepared according to Schwyn and Nielands (44). Evans Blue-uranine plates were prepared according to Bochner (6). ...
... P22 HTint was used for generalized transduction of transposon insertions into a different genetic background. Transductants were routinely streaked on Evans blue-uranine plates to detect phage contamination before use in further experiments (7). Quantification of -galactosidase activity was performed as described elsewhere (27). ...
Pathogenic bacteria are able to grow in various niches in the host. These niches differ in the types of available sources of iron, an essential nutrient for bacterial growth. For example, under anaerobic conditions found in the intestine, soluble iron(II) may be available for bacterial growth. An additional iron source is provided by host compounds such as heme orig- inating from dead epithelial cells which are constantly shed from the tips of villi. Once bacteria manage to penetrate the intestinal barrier, they face components of the host's iron- withholding defense (37). In the blood and extracellularfluid, iron is withheld from invading microorganisms by the high- affinity iron(III)-binding proteins lactoferrin and transferrin. In response to infection, the host reduces the serum iron to about 30% of its normal levels, making it even harder for microbes to obtain this nutrient (37). Some pathogens are able to grow in an intracellular location, where they again encoun- ter dramatically different conditions. It is not known what iron sources are available to bacteria growing intracellularly. How do bacteria obtain iron under all of these different conditions? Because it passes through all of these compart- ments during murine typhoid, Salmonella typhimurium is a good model with which to address this question. Several genes involved in iron uptake, such as genes encoding functions in- volved in biosynthesis and uptake of the siderophore enter- obactin, have been identified in S. typhimurium (36). In addi- tion to enterobactin,S. typhimuriumis able to utilize a variety of other siderophores which it does not produce, including ferrioxamine and ferrichrome (26). The outer membrane re- ceptor genes for these siderophores,foxAandsidK, have been identified recently (36). The receptor for enterobactin, as well as the otherS. typhimuriumsiderophore receptors, belongs to a family of proteins whose transport activity depends on the function of TonB (2, 18, 32). These TonB-dependent uptake systems have in common that they mediate uptake of iron(III) complexes. Recently, a new type of iron uptake system has been found in Escherichia coli, a close relative of S. typhimu- rium.Thissystem,encodedbythefeoABgenes,pumpsiron(II) throughthecytoplasmicmembrane(22).Sinceiron(II)isread- ily soluble and can enter the periplasmic space by diffusion through porins, specialized outer membrane receptors are not necessary for its uptake. To assess the roles of these iron uptake systems, we con- structedS. typhimuriumstrains carrying mutations intonBand feoB. The effects of these mutations on bacterial growth were tested, singly and in combination, by using in vitro assays and the murine model of typhoid fever.
... Solid medium for sucrose counterselection was prepared as previously described, except sucrose was at 10% (Blomfield et al., 1993). EBU plates were prepared as has been described previously (Bochner, 1984). ...
Plasmid-encoded fimbriae (Pef) expressed by Salmonella typhimurium mediate adhesion to mouse intestinal epithelium. The pef operon shares features with the Escherichia coli pyelonephritis-associated pilus (pap) operon, which is under methylation-dependent transcriptional regulation. These features include conserved DNA GATC box sites in the upstream regulatory region as well as homologues of the PapI and PapB regulatory proteins. Unlike Pap fimbriae, which are expressed in a variety of laboratory media, Pef fimbriae were expressed only in acidic, rich broth under standing culture conditions. Analysis of S. typhimurium grown under these conditions indicated that Pef production was regulated by a phase variation mechanism, in which the bacterial population was skewed between fimbrial expression (phase ON) and non-expression (phase OFF) states. Leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam) were required for pef transcription. In contrast, the histone-like protein (H-NS) and the stationary-phase sigma factor (RpoS) repressed pef transcription. Methylation of the pef GATC II site appeared to be required for pef fimbrial expression based on analysis of a GCTC II mutant that did not express Pef fimbriae. Analysis of the DNA methylation states of pef GATC sites indicated that, under acidic growth conditions, which induced Pef production, most GATC I sites were non-methylated, whereas GATC II and GATC X were predominantly methylated. The methylation protection at GATC I and GATC II was dependent upon Lrp and was modulated by PefI. Together, these results indicate that Pef production is regulated by DNA methylation, which is the first example of methylation-dependent gene regulation outside of E. coli.
... The artB13::MudJ allele (31), which directs constitutive expression of -galactosidase, was introduced into S. enterica serovar Typhimurium 8916 by transduction using a bacteriophage P22HTint lysate from Salmonella 4574 harboring this allele, resulting in strain 9930. Transductants with kanamycin-resistant and -galactosidase phenotypes were selected and grown on Evans blue uridine agar plates to confirm that the transductants were phage free and not P22 lysogens (6). The invAC genes were deleted in S. Typhimurium 8916 by allelic exchange using the suicide vector pYA4141 to generate 11406. ...
Tuberculosis remains a global health threat, and there is dire need to develop a vaccine that is safe and efficacious and
confers long-lasting protection. In this study, we constructed recombinant attenuated Salmonella vaccine (RASV) strains with plasmids expressing fusion proteins consisting of the 80 amino-terminal amino acids of the type
3 secretion system effector SopE of Salmonella and the Mycobacterium tuberculosis antigens early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10). We demonstrated
that the SopE-mycobacterial antigen fusion proteins were translocated into the cytoplasm of INT-407 cells in cell culture
assays. Oral immunization of mice with RASV strains synthesizing SopE–ESAT-6–CFP-10 fusion proteins resulted in significant
protection of the mice against aerosol challenge with M. tuberculosis H37Rv that was similar to the protection afforded by immunization with Mycobacterium bovis bacillus Calmette-Guérin (BCG) administered subcutaneously. In addition, oral immunization with the RASV strains specifying
these mycobacterial antigens elicited production of significant antibody titers to ESAT-6 and production of ESAT-6- or CFP-10-specific
gamma interferon (IFN-γ)-secreting and tumor necrosis factor alpha (TNF-α)-secreting splenocytes.
... Typically, P22 lysates were used at a 1:50 dilution, mixed with aliquots from overnight cultures of recipient bacteria in a 1:2 ratio, and incubated for 30 min at 37uC prior to being plated on selective media. Transductant colonies were purified by two sequential passages on selective plates and verified to be free of phage by streaking on Evans Blue Uranine plates [40] . Chromosomal engineering was carried out by the l Red recombination method414243 as previously described [20]. ...
Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.
... Maps of each deletion-insertion mutation present in 9373 and its derivative 9447 were described previously for ⌬pmi-2426, ⌬(gmd-fcl)-26, and ⌬relA198::araC P BAD lacI TT (39); ⌬P fur81 ::TT araC P BAD fur, ⌬P crp527 ::TT araC P BAD crp, ⌬araE25, and ⌬araBAD23 (14); ⌬P murA7 ::TT araC P BAD murA and ⌬endA2311 (35); and ⌬asd21::TT araC P BAD c2 (24). Evans blue uridine agar plates were used to confirm that transductants were phage free and not P22 lysogens (3). MacConkey agar plates supplemented with 1% maltose were used to confirm the phenotype of the cyclic AMP receptor protein gene (crp) mutants (10). ...
A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication
(pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing
the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ΔasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain χ9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers,
respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/Pcro) (PR), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC PBAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of β-lactamase,
and cloned into pYA4534 under the control of the Ptrc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain χ9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against
lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.
... After mutagenesis, all mutations were P22-transduced to a clean background using phage P22 HT105/1 int-201. EBU plates were used to select for nonlysogens (Bochner, 1984). P22-transduction was also used to combine mutations for the generation of double mutants. ...
StpA is a paralogue of the nucleoid-associated protein H-NS that is conserved in a range of enteric bacteria and had no known function in Salmonella Typhimurium. We show that 5% of the Salmonella genome is regulated by StpA, which contrasts with the situation in Escherichia coli where deletion of stpA only had minor effects on gene expression. The StpA-dependent genes of S. Typhimurium are a specific subset of the H-NS regulon that are predominantly under the positive control of sigma(38) (RpoS), CRP-cAMP and PhoP. Regulation by StpA varied with growth phase; StpA controlled sigma(38) levels at mid-exponential phase by preventing inappropriate activation of sigma(38) during rapid bacterial growth. In contrast, StpA only activated the CRP-cAMP regulon during late exponential phase. ChIP-chip analysis revealed that StpA binds to PhoP-dependent genes but not to most genes of the CRP-cAMP and sigma(38) regulons. In fact, StpA indirectly regulates sigma(38)-dependent genes by enhancing sigma(38) turnover by repressing the anti-adaptor protein rssC. We discovered that StpA is essential for the dynamic regulation of sigma(38) in response to increased glucose levels. Our findings identify StpA as a novel growth phase-specific regulator that plays an important physiological role by linking sigma(38) levels to nutrient availability.
... The addition of 10 mM EGTA [ethylene glycol-bis(3-aminoethyl ether)-N,N,N',N'tetraacetic acid] to the replica plates greatly increased the number of phage-sensitive mutants recovered from the screening process by preventing the readsorption of phage to the cells. Green plates were used for the isolation of phagefree transductants (2,9). Transductants were tested for sensitivity to phage P22 infection by cross-streaking against the P22 clear plaque mutant H-5 (9). ...
The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.
... Chrome azurol S (CAS) plates used to assay for siderophore production were prepared according to Schwyn and Nielands (44). Evans Blue-uranine plates were prepared according to Bochner (6). ...
In order to identify genes belonging to the Fur regulon of Salmonella typhimurium, a bank of 10,000 independent S. typhimurium MudJ insertion mutants was screened for lacZ fusions regulated by the iron response regulator Fur. In parallel, a plasmid gene bank of S. typhimurium consisting of 10,000 independent clones was screened for Fur-regulated promoters or iron binding proteins by the Fur titration assay (FURTA). Fur-regulated MudJ insertions and Fur-regulated promoters were mapped. In addition, iron-regulated promoter activities of transcriptional fusions from MudJ insertions and FURTA-positive clones were quantified. The nucleotide sequences of 11 FURTA-positive plasmids and of short fragments of DNA flanking three MudJ insertions were determined. By these methods we identified 14 Fur-regulated genes of S. typhimurium. For 11 of these genes, Fur-regulated homologs have been described in Escherichia coli or Yersinia enterocolitica, including fhuA,fhuB,fepA,fes,fepD,p43,entB,fur ,foxA,hemP, and fhuE. In addition, we identified three genes with homologs in other bacteria which have not previously been shown to be Fur regulated.
... Luria-Bertani (LB) broth (64) was used for all experiments, with the addition of 1.5% agar for solid medium. EBU medium (7) was used to identify pseudolysogen-free transductants. Penicillin (250 g/ml), kanamycin (50 g/ml), tetracycline (15 g/ml), chloramphenicol (20 g/ml), L-(ϩ)-arabinose (0.1%), isopropylthio--D-galactoside (IPTG) (1 mM), or 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) (50 g/ml) was used as a supplement as needed. ...
sigma S (RpoS)-regulated lacZ transcriptional fusions in Salmonella typhimurium were identified from a MudJ transposon library by placing the rpoS gene under the control of the araBAD promoter and detecting lacZ expression in the presence or absence of arabinose supplementation. Western blot (immunoblot) analysis of bacteria carrying PBAD::rpoS demonstrated arabinose-dependent rpoS expression during all phases of growth. sigma S-dependent gene expression of individual gene fusions was confirmed by P22-mediated transduction of the MudJ insertions into wild-type or rpoS backgrounds. Analysis of six insertions revealed the known sigma S-regulated gene otsA, as well as five novel loci. Each of these genes is maximally expressed in stationary phase, and all but one show evidence of cyclic AMP receptor protein-dependent repression during logarithmic growth which is relieved in stationary phase. For these genes, as well as for the sigma S-regulated spvB plasmid virulence gene, a combination of rpoS overexpression and crp inactivation can result in high-level expression during logarithmic growth. The approach used to identify sigma S-regulated genes in this study provides a general method for the identification of genes controlled by trans-acting regulatory factors.
... P22 HTint was used for generalized transduction of transposon insertions into a different genetic background. Transductants were routinely streaked on Evans blue-uranine plates to detect phage contamination before use in further experiments (7). Quantification of -galactosidase activity was performed as described elsewhere (27). ...
We examined the role of iron(II) and iron(III) uptake, mediated by FeoB and TonB, respectively, in infection of the mouse by Salmonella typhimurium. The S. typhimurium feoB gene, encoding a homolog of an Escherichia coli cytoplasmic membrane iron(II) permease, was cloned, and a mutant was generated by allelic exchange. In addition, an S. typhimurium tonB mutant was constructed. Together these two mutations inactivate all known iron uptake systems of S. typhimurium. We examined the abilities of these mutants to grow in vitro and in different compartments of the host. Mutants in feoB were outcompeted by the wild type during mixed colonization of the mouse intestine, but the feoB mutation did not attenuate S. typhimurium for oral or intraperitoneal infection of mice. The tonB mutation attenuated S. typhimurium for infection of mice by the intragastric route but not the intraperitoneal route, and the mutant was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes than the wild type. These results indicate that TonB-mediated iron uptake contributes to colonization of the Peyer's patches and mesenteric lymph nodes but not the liver and spleen of the mouse. The tonB feoB double mutant, given intraperitoneally, was able to infect the liver and spleen at wild-type doses, indicating that additional iron acquisition systems are used during growth at systemic sites of infection.
... Transductions were done with P22 HTlO5/1 int-220, a highfrequency generalized transducing phage that cannot form stable lysogens. Green plates (Davis et al., 1980) and EBU plates (Bochner, 1984) were used to screen for phage-free transductants. Sensitivity of strains to phage was verified by cross-streaking against a P22 c2 mutant. ...
Wild-type Salmonella typhimurium expresses three proline transport systems: a high-affinity proline transport system encoded by the putP gene, and two glycine betaine transport systems with a lower affinity for proline encoded by the proP and proU genes. Although proline uptake by the ProP and ProU transport systems is sufficient to supplement a proline auxotroph, it is not efficient enough to allow proline utilization as a sole source of carbon or nitrogen. Thus, the PutP transport system is required for utilization of proline as a carbon or nitrogen source. In this study, an overexpression suppressor, designated proY, which allows proline utilization in a putP genetic background and does not require the function of any of the known proline transport systems, was cloned and characterized. The suppressor gene, designated proY, maps at 8 min on the S. typhimurium linkage map, distant from any of the other characterized proline transport genes. The DNA sequence of the proY gene predicts that it encodes a hydrophobic integral membrane protein, with strong similarity to a family of amino acid transporters. The suppressor phenotype requires either a multicopy done of the proY+ gene or both a single copy of the proY+ gene and a proZ mutation. These results indicate that the proY gene is the structural gene for a cryptic proline transporter that is silent unless overexpressed on a multicopy plasmid or activated by a proZ mutation.
... ampicillin 50 mg ml 21 . Phage-induced bacterial lysis on agar plates was monitored using`Green' medium [ (Chan et al., 1972) 0.8% bacto tryptone, 0.1% yeast extract (w/v), 0.5% NaCl (w/v), 6.7 g l 21 glucose, 630 mg l 21 Alizarin yellow (GG), 66 mg l 21 Methyl blue] or EBU medium [ (Bochner, 1984) 1% bacto tryptone (w/v), 0.5% Difco yeast extract (w/ v), 0.5% NaCl (w/v) 2.5 g l 21 glucose, 0.5% K 2 HPO 4 (w/v), 12.5 mg l 21 Evans blue, 25 mg l 21 uranine (sodium fluorescein)]. Liquid cultures were grown in New Brunswick gyratory shakers, and growth was monitored by measuring the optical density (OD) at 600 nm with a Milton±Roy Spectronic 301 spectrophotometer. ...
Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.
... Bacteria were grown in Luria-Bertani (LB) medium or M9 minimal medium with 0.2% glucose (DeGroote et al., 1997) at 37°C supplemented with 250 mg ml -1 penicillin, 50 mg ml -1 kanamycin, 40 mg ml -1 chloramphenicol, 100 mg ml -1 streptomycin or 100 mg ml -1 trimethoprim from Sigma as appropriate. Xylose-lysine-deoxycholate medium (Difco) was used to differentiate exconjugant Salmonella strains from Escherichia coli donors, Evans blue uranine medium was used to identify pseudolysogen-free transductants (Bochner, 1984), and LB with the omission of NaCl and the addition of 6% sucrose was used at 30°C for allelic exchange procedures (Blomfield et al., 1991). ...
Bacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors sigmaS (sigma38, RpoS) and sigmaH (sigma32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor sigmaE (sigma24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the sigmaE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking sigmaE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both sigmaE and sigmaS is non-viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary-phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of sigmaE-deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the sigmaE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.
... After mutagenesis, the mutation was P22-transduced to a clean background using phage P22 HT105/1 int- 201. EBU plates were used to select for non-lysogens [35]. The mutation was verified by PCR using primers external to the mutation. ...
Histone-like nucleoid structuring protein (H-NS) is a modular protein that is associated with the bacterial nucleoid. We used chromatin immunoprecipitation to determine the binding sites of H-NS and RNA polymerase on the Salmonella enterica serovar Typhimurium chromosome. We found that H-NS does not bind to actively transcribed genes and does not co-localize with RNA polymerase. This shows that H-NS principally silences gene expression by restricting the access of RNA polymerase to the DNA. H-NS had previously been shown to preferentially bind to curved DNA in vitro. In fact, at the genomic level we discovered that the level of H-NS binding correlates better with the AT-content of DNA. This is likely to have evolutionary consequences because we show that H-NS binds to many Salmonella genes acquired by lateral gene transfer, and functions as a gene silencer. The removal of H-NS from the cell causes un-controlled expression of several Salmonella pathogenicity islands, and we demonstrate that this has deleterious consequences for bacterial fitness. Our discovery of this novel role for H-NS may have implications for the acquisition of foreign genes by enteric bacteria.
... Generalized transducing phages P22 HT int-105 and KB1 int were used to generate lysates of S. typhimurium as previously described (Miller, 1972). Transductants were streaked for single colonies on Evans blue Uridine (EBU) agar (Bochner, 1984 ) and phage-free colonies were crossstreaked against P22 H5 (for P22 HT int-105), or KB1 int (for KB1) to confirm phage sensitivity. Mud-Cam insertion mutants of S. typhimurium strain AJB4 were isolated using the method of Hughes and Roth (1988). ...
The Salmonella enterica serotype Typhimurium (S. typhimurium) genome contains a large repertoire of putative fimbrial operons that remain poorly characterized because they are not expressed in vitro. In this study, insertions that induced expression of the putative stdABCD fimbrial operon were identified from a random bank of transposon mutants by screening with immuno-magnetic particles for ligand expression (SIMPLE). Transposon insertions upstream of csgC and lrhA or within dam, setB and STM4463 (renamed rosE) resulted in expression of StdA and its assembly into fimbrial filaments on the cell surface. RosE is a novel negative regulator of Std fimbrial expression as indicated by its repression of a std::lacZ reporter construct and by binding of the purified protein to a DNA region upstream of the stdA start codon. Expression of Std fimbriae in the rosE mutant resulted in increased attachment of S. typhimurium to human colonic epithelial cell lines (T-84 and CaCo-2). A rosE mutant exhibited a reduced ability to compete with virulent S. typhimurium for colonization of murine organs, while no defect was observed when both competing strains carried a stdAB deletion. These data suggest that a tight control of Std fimbrial expression mediated by RosE is required during host pathogen interaction.
Following ingestion, Salmonella must adhere to and colonize the intestinal epithelium of the host in order to establish infection. S. typhimurium synthesize several appendages that are believed to mediate attachment. These include type 1 fimbriae, plasmid-encoded (PE) fimbriae, long polar (LP) fimbriae, and thin aggregative fimbriae. However, the precise roles of these putative adhesins remain unclear, due to conflicting data in the literature. We constructed strains lacking four different fimbriae including type 1 fimbriae, PE fimbriae, LP fimbriae, and thin aggregative fimbriae, as well as strains lacking each fimbriae alone. In cell culture adhesion assays, these mutants adhered to several mammalian cell lines as well as wild-type S. typhimurium. These strains were also screened for virulence in mice, and all strains were virulent or nearly as virulent as their wild-type parents. In contrast, When a strain lacking four fimbriae was screened for virulence in chicks, it was found to be highly attenuated. This suggests a role for either type 1 fimbriae, PE fimbriae, LP fimbriae or thin aggregative fimbriae or a combination of thease fimbriae in the colonization of chicks. It also suggests that differences exist with respect to the surface structures that mediate attachment of Salmonella in chicks as compared with mice.
This chapter describes methods for mutagenesis of the Salmonella typhimurium genome and potential methods for screening a bank of mutants and genetic analysis of interesting variants. The chapter focuses on frequently used systems for transposon mutagenesis and variant selection. Variant selection describes in vitro cell culture assays designed to enrich a pool of mutants for those with defects in genes related to pathogenesis. Although the chapter focuses on methods developed in S. typhimurium, many of these are also applicable to other Salmonella serovars. Variants that have alterations in a defined region of the genome have been created in two independent ways: plasmid curing and construction of hybrids between different Salmonella strains. The breakthrough in creating large numbers of defined mutants that are easy to analyze came with the introduction of transposon mutagenesis. These mobile genetic elements insert more or less randomly in the genome, thus disrupting the function of the gene into which they have inserted. Transposons that are used for mutagenesis contain selectable markers. This important feature allows transfer of the mutation into a clean background, ensuring that an observed phenotype can be attributed to a single mutation.
Salmonella enterica serotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgK mutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljB mutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3alpha and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.
Transduction is the process in which bacterial DNA is transferred from one bacterial cell to another by means of a phage particle. There are two types of transduction, generalized transduction and specialized transduction. In this chapter two of the best-studied systems - Escherichia coli-phage P1, and Salmonella enterica-phage P22 - are discussed from theoretical and practical perspectives.
A class of mutations that suppress the recombination defects of recB mutants in Salmonella enterica serovar Typhimurium strain LT2 activates the normally silent recET module of the Gifsy-1 prophage. Allele sbcE21 is a 794-bp deletion within the immunity region of the prophage. Concomitant with activating recET, sbcE21 stimulates Gifsy-1 excision, resulting in unstable suppression. Early studies found both recB suppression and its instability to depend on the presence of the related Gifsy-2 prophage elsewhere in the chromosome. In
cells lacking Gifsy-2, the sbcE21 allele became stable but no longer corrected recB defects. Here, we show that a single Gifsy-2 gene is required for Gifsy-1 recET activation in the sbcE21 background. This gene encodes GtgR, the Gifsy-2 repressor. Significantly, the sbcE21 deletion has one end point within the corresponding gene in the Gifsy-1 genome, gogR, which in strain LT2 is a perfect duplicate of gtgR. The deletion truncates gogR and places the Gifsy-1 left operon, including the recET and xis genes, under the control of the gogR promoter. The ability of GtgR to trans-activate this promoter therefore implies that GtgR and GogR normally activate the transcription of their own genes. Consistent
with the symmetry of the system, a similar deletion in Gifsy-2 results in a Gifsy-1-dependent sbc phenotype (sbcF24). Two additional Gifsy-1 deletions (sbcE23 and sbcE25) were characterized, as well. The latter causes all but the last codon of the gogR gene to fuse, in frame, to the second half of recE. The resulting hybrid protein appears to function as both a transcriptional regulator and a recombination enzyme.
The enteric bacterium Salmonella typhimurium utilizes 1,2-propanediol as a sole carbon and energy source during aerobic growth, but only when the cells are also provided with cobalamin as a nutritional supplement. This metabolism is mediated by the cobalamin-dependent propanediol dehydratase enzyme pathway. Thirty-three insertion mutants were isolated that lacked the ability to utilize propanediol, but retained the ability to degrade propionate. This phenotype is consistent with specific blocks in one or more steps of the propanediol dehydratase pathway. Enzyme assays confirmed that propanediol dehydratase activity was absent in some of the mutants. Thus, the affected genes were designated pdu (for defects in propanediol utilization). Seventeen mutants carried pdu::lac operon fusions, and these fusions were induced by propanediol in the culture medium. All of the pdu mutations were located in a single region (41 map units) on the S. typhimurium chromosome between the his (histidine biosynthesis) and branch I cob (cobalamin biosynthesis) operons. They were shown to be P22-cotransducible with a branch I cob marker at a mean frequency of 12%. Mutants that carried deletions of the genetic material between his and cob also failed to utilize propanediol as a sole carbon source. Based upon the formation of duplications and deletions between different pairs of his::MudA and pdu::MudA insertions, the pdu genes were transcribed in a clockwise direction relative to the S. typhimurium genetic map.
The ability to bind Congo red (Crb+) is associated with virulence of Shigella flexneri and is encoded by a large, 220-kilobase plasmid. We cloned fragments of this plasmid to isolate the sequences encoding Congo red binding, to determine the degree of conservation of these sequences among S. flexneri strains, and to study the molecular basis for loss of the Crb+ phenotype. At least two separate BamHI fragments cloned into plasmid vectors encode Congo red binding in E. coli or S. flexneri. One Crb+ clone, pTKS2, contains a copy of IS1 adjacent to the crb sequences. IS1 appears to be responsible for deletions leading to loss of Congo red binding in this clone. In addition, this clone was found to integrate into the chromosome at relatively high frequency. Integration resulted in loss of the Crb+ phenotype. A second clone, pTKS15, which has only limited homology to pTKS2, also encodes Congo red binding. The Crb+ phenotype of transformants carrying pTKS15 was detected at 37 degrees C but not at 30 degrees C, and thus it resembles Congo red binding in wild-type S. flexneri. HindIII digests of plasmid DNA from 10 different S. flexneri strains were hybridized to both of these Crb+ clones and to an IS1 probe. More than one fragment hybridized to pTKS2 or pTKS15. In general, the sizes of these fragments were the same in S. flexneri strains of different serotypes, indicating conservation of these sequences. Three of five copies of IS1 were also found on the large S. flexneri plasmids. Two of the copies were on fragments of the same size in each strain. Analysis of Crb- derivatives of the 10 strains indicated that, although IS1 may be closely linked to crb sequences on the 220-kilobase plasmid, it is not responsible for the majority of deletions of this plasmid associated with loss of Congo red binding.
Doses of 0.1 to 1.0 micrograms/ml of mitomycin C induced cell lysis of six of eight strains of Actinobacillus actinomycetemcomitans tested. Infectious phages were induced from ATCC strains 43717, 29524, 33384, and 43719; non-plaque-forming, possibly defective phages were induced from ATCC strains 29522 and 29523. No phages were detected in strain FDC 651 or ATCC strain 43718. No correlation between lysogeny and leukotoxin production or serotype of the strains could be established. Gel electrophoresis of phage DNAs indicated that the induced phages were of three types, based on size. By electron microscopy, the phages were found to belong to either morphotype A1 or morphotype B1; no other morphotypes were observed. Curing experiments led to the isolation of nonlysogenic derivatives of two strains, which supported plaque formation by the phages they originally carried. On the basis of our results, lysogeny appears to be widespread in A. actinomycetemcomitans.
During its life cycle, Salmonella typhimurium is exposed to a variety of acidic conditions. Survival in the acidic environments within the host may require the adaptive acid tolerance response (ATR), which is characterized by the induction of several Salmonella proteins upon exposure to mildly acidic conditions. These induced proteins protect the bacterium from death under severe acid challenge. The goal of this study was to examine the role of ATR in Salmonella pathogenesis. Initially, we observed that differences exist between the virulent S. typhimurium strains and the laboratory S. typhimurium strain LT2 with respect to their ATR. Mutations affecting the ATR of S. typhimurium LT2, including atrB, atrC (polA), atrD, atbR, and fur, were crossed into virulent Salmonella strains, and the resultant transductants were screened for virulence in mice and acid sensitivity. Surprisingly, with the exception of the fur mutation, none of the muatations had a major effect on acid resistance or virulence in the pathogenic strains. The fur mutants showed a 1-to 3-log increase in the 50% lethal dose; however, the magnitude of its effect was dependent on the strain background. Strains containing two or three different atr mutations were constructed, and these were also examined for acid sensitivity and virulence. The double and triple mutants that contained an atrC mutation no longer displayed an ATR. Those mutants which were more acid sensitive were also highly attenuated, suggesting a strong correlation between the ability to mount and ATR and virulence in S. typhimurium. Comparison of the ability of the various atr single, double, and triple mutants to survive within macrophages showed that strains containing an atrC mutation survived much less than the wild type in bone marrow-derived macrophages. No difference in survival within J774 macrophage like cells were detected.
Previous studies have shown that inactivation of the MutS or MutL mismatch repair enzymes increases the efficiency of homeologous recombination between Escherichia coli and Salmonella typhimurium and between S. typhimurium and Salmonella typhi. However, even in mutants defective for mismatch repair the recombination frequencies are 10(2)- to 10(3)-fold less than observed during homologous recombination between a donor and recipient of the same species. In addition, the length of DNA exchanged during transduction between S. typhimurium and S. typhi is less than in transductions between strains of S. typhimurium. In homeologous transductions, mutations in the recD gene increased the frequency of transduction and the length of DNA exchanged. Furthermore, in mutS recD double mutants the frequency of homeologous recombination was nearly as high as that seen during homologous recombination. The phenotypes of the mutants indicate that the gene products of mutS and recD act independently. Because S. typhimurium and S. typhi are approximately 98-99% identical at the DNA sequence level, the inhibition of recombination is probably not due to a failure of RecA to initiate strand exchange. Instead, these results suggest that mismatches act at a subsequent step, possibly by slowing the rate of branch migration. Slowing the rate of branch migration may stimulate helicase proteins to unwind rather than extend the heteroduplex and leave uncomplexed donor DNA susceptible to further degradation by RecBCD exonuclease.
Periplasmic Cu, Zn-cofactored superoxide dismutase (SodC) protects Gram-negative bacteria from exogenous oxidative damage. The virulent Salmonella typhimurium strain ATCC 14028s has been found to contain two discrete periplasmic Cu, Zn-SOD enzymes that are only 57% identical at the amino acid level. SodCI is carried by a cryptic bacteriophage, and SodCII is closely related to the Cu, Zn-superoxide dismutase of Escherichia coli. All Salmonella serotypes appear to carry the sodCII locus, but the phage-associated sodCI gene is found only in certain strains belonging to the most highly pathogenic serotypes. Expression of either sodC locus appears to be enhanced during stationary phase, but only sodCII is regulated by the alternative sigma factor sigmas (RpoS). Mutants lacking both sodC genes are less lethal for mice than mutants possessing either sodC locus alone, indicating that both Cu, Zn-SOD enzymes contribute to Salmonella pathogenicity. The evolutionary acquisition of an additional sodC gene has contributed to the enhanced virulence of selected Salmonella strains.
Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.
The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells. Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen. Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g. 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy. Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy. Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches. In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments. The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells. This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.
The shdA gene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of the Salmonella enterica serotype Typhimurium chromosome. In addition to shdA, the CS54 island of Salmonella serotype Typhimurium strain LT2 contains four open reading frames designated ratA, ratB, sivI, and sivH. DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution
within the genus Salmonella. Homologues of shdA and ratB were detected only in serotypes of Salmonella enterica subsp. I. In contrast, sequences hybridizing with ratA, sivI, and sivH were present in S. enterica subsp. II and S. bongori in addition to S. enterica subsp. I. Deletion of the ratA and sivI genes did not alter the ability of Salmonella serotype Typhimurium to colonize the organs of mice. Insertional inactivation of the sivH gene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum,
mesenteric lymph nodes, and spleen. Deletion of the shdA gene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser
degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in the
ratB gene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen.
The shdA and ratB deletion strains exhibited a shedding defect in mice, whereas the sivH deletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is
required for efficient fecal shedding in mice.
Environmental shedding of genetically manipulated microorganisms is an issue impeding the development of new live vaccines.
We have investigated the immunogenicity of a number of novel Salmonella enterica serotype Typhimurium oral vaccine candidates that express the fragment C (TetC) component of tetanus toxin and harbor combinations
of additional mutations in genes shdA, misL, and ratB that contribute to the persistence of serotype Typhimurium's colonization of the intestine. Serotype Typhimurium aroA (TetC) derivatives harboring additional mutations in either shdA or misL or combinations of these mutations exhibited a marked decrease in shedding of the vaccine strain in the feces of orally vaccinated
mice. However, equivalent levels of anti-TetC and anti-Salmonella lipopolysaccharide immunoglobulin G (IgG), IgG1, IgG2a, and IgA were detected in sera of the vaccinated but not of the control
mice. Cellular immune responses to TetC were detected in all vaccinated mice, regardless of the presence of the additional
mutations in shdA or misL. Further, immunization with serotype Typhimurium aroA candidate vaccines harboring shdA and misL afforded complete protection against challenge with a virulent strain of serotype Typhimurium.
Genomes of members of the family Enterobacteriaceae contain large repertoires of putative fimbrial operons. Since many of these operons are poorly expressed in vitro, a convenient
method for inducing elaboration of the encoded fimbriae would greatly facilitate their functional characterization. Here we
describe a new technique for identifying fimbriated bacteria from a library of transposon mutants by screening with immunomagnetic
particles for ligand expression (SIMPLE). The SIMPLE method was applied to identify the T-POP mutants of Salmonella enterica serotype Typhimurium carrying on their surfaces filaments composed of PefA, the major subunit product of a fimbrial operon
(pef) that is not expressed during growth in Luria-Bertani broth. Four such mutants were identified from a library of 24,000 mutants,
each of which carried a T-POP insertion within the hns gene, which encodes a global silencer of horizontally acquired genes. Our data suggest that the SIMPLE method is an effective
approach for isolating fimbriated bacteria, which can be readily applied to fimbrial operons identified by whole-genome sequencing.
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