TABLE 2 - uploaded by Heimo Strohmaier
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We have cloned and sequenced a cluster of six open reading frames containing gene kdsA from Escherichia coli K-12. The gene encodes 3-deoxy-D-manno-octulosonate 8-phosphate synthetase (KDO-8-phosphate synthetase), which catalyzes formation of 3-deoxy-D-manno-octulosonic acid (KDO), an essential component of enterobacterial lipopolysaccharide. We ha...
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... order to be able to assess the extent of mRNA degradation, mRNA stability measurements were car- ried out at three different time points of the growth curve, i.e., at the early and mid-log phases and at the beginning of the stationary phase. The results are shown in Table 2. At each time point rifampin was added to a culture of plasmid-free E. coli 5K cells, and total RNA was prepared at different time intervals as indicated in Table 2, after addition of the antibi- otic. ...
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... results are shown in Table 2. At each time point rifampin was added to a culture of plasmid-free E. coli 5K cells, and total RNA was prepared at different time intervals as indicated in Table 2, after addition of the antibi- otic. Ten micrograms of each RNA sample was separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed with the same radioactive DNA fragment as used for the Northern blot analysis (Fig. 3). ...
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... larly, primer extension reactions gave detectable signals only with RNA isolated at early growth phases (data not shown). Since no changes in mRNA stability could be detected during the growth phase (Table 2), these data indicate that the regu- lation is due to changes in the rate of transcription. ...
Citations
... Furthermore, in EVs from the E. coli M19736 strain, the proteins from the LPS biosynthesis pathway were found (Supplementary Table 2), and 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA) is responsible for linking lipid A and core oligosaccharides through the synthesis of other proteins (Hussein et al., 2023). The experiments of Strohmaier et al. (1995) demonstrate that KdsA undergoes growth phase-dependent regulation at the transcriptional level, which again supports the idea that the growth phase is related to the selection of cargo in EV. Also, this protein increases following polymyxin B treatment (Hussein et al., 2023) which supports the idea that treatment with each antibiotic could collaborate in the selection of load in EV. ...
There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, or aadB genes. Escherichia coli M19736 acquired bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-E. coli M19736 acquired sequentially bla CTX-M-15 and bla NDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid’s maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-E. coli M19736 strains. Interestingly, EVs from the evolved XDR-E. coli M19736 harbored bla CTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.
... Instead, a novel S60L mutation in uncharacterized protein YcaR was identified. Located within the KDO2-lipid A biosynthesis gene cluster and co-transcribed with kdsB [43], ycaR could serve as a novel candidate gene that potentially influences colistin resistance. Additionally, an interrogation of 84 publicly available Klebsiella spp. ...
... Although the pmrA pseudogene causes an increase in the level of polymyxin B susceptibility by reducing cationic groups on the LPS [19], the isolates in our study containing both the pmrA pseudogene and the altered pmrB gene had a high level of colistin resistance, suggesting that the loss of pmrA gene function may have promoted a different lipid A modification pathway. We also identified a novel mutation in protein YcaR, which is part of the KDO2-lipid A biosynthetic pathway [43], in the K. pneumoniae isolates from hospitalized patients. However, further genomic analyses of this point mutation are required to determine whether this mutation is present in other colistin-resistant strains without the mcr gene. ...
Background
Colistin is one of the last resort therapeutic options for treating carbapenemase-producing Enterobacterales, which are resistant to a broad range of beta-lactam antibiotics. However, the increased use of colistin in clinical and livestock farming settings in Thailand and China, has led to the inevitable emergence of colistin resistance. To better understand the rise of colistin-resistant strains in each of these settings, we characterized colistin-resistant Enterobacterales isolated from farmers, swine, and hospitalized patients in Thailand.
Methods
Enterobacterales were isolated from 149 stool samples or rectal swabs collected from farmers, pigs, and hospitalized patients in Thailand between November 2014–December 2017. Confirmed colistin-resistant isolates were sequenced. Genomic analyses included species identification, multilocus sequence typing, and detection of antimicrobial resistance determinants and plasmids.
Results
The overall colistin-resistant Enterobacterales colonization rate was 26.2% ( n = 39/149). The plasmid-mediated colistin-resistance gene ( mcr ) was detected in all 25 Escherichia coli isolates and 9 of 14 (64.3%) Klebsiella spp. isolates. Five novel mcr allelic variants were also identified: mcr -2.3, mcr -3.21, mcr -3.22, mcr -3.23, and mcr -3.24, that were only detected in E. coli and Klebsiella spp. isolates from farmed pigs.
Conclusion
Our data confirmed the presence of colistin-resistance genes in combination with extended spectrum beta-lactamase genes in bacterial isolates from farmers, swine, and patients in Thailand. Differences between the colistin-resistance mechanisms of Escherichia coli and Klebsiella pneumoniae in hospitalized patients were observed, as expected. Additionally, we identified mobile colistin-resistance mcr -1.1 genes from swine and patient isolates belonging to plasmids of the same incompatibility group. This supported the possibility that horizontal transmission of bacterial strains or plasmid-mediated colistin-resistance genes occurs between humans and swine.
... The 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA), phosphoheptose isomerase (GmhA), and bi-functional ADP-Lglycero-D-manno-heptose synthase (HldE) were significantly upregulated in the OMVs of polymyxin-susceptible ATCC 700721 following polymyxin B treatment (log 2 FC = 4.0, 9.2, and 1.3, respectively; Fig. 2; Table S1), whereas in the polymyxin-resistant OMVs, only 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA) was significantly increased following polymyxin B treatment (Table S1). The 2-dehydro-3-deoxyphosphooctonate aldolase is responsible for linking lipid A and core oligosaccharides through synthesis of Kdo (30). In most Gram-negative bacteria, bi-functional ADP-L-glycero-D-manno-heptose synthase and phosphoheptose isomerase proteins are involved in the formation of a key component of the LPS core domain as well as enhancing the bacterial tolerance toward cell-wall-damaging agents (31,32). ...
Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria serve as transporters for the delivery of cargo such as virulence and antibiotic resistance factors. OMVs play a key role in the defense against membrane-targeting antibiotics such as the polymyxin B. Herein, we conducted comparative proteomics of OMVs from paired Klebsiella pneumoniae ATCC 700721 polymyxin-susceptible (polymyxin B MIC = 0.5 mg/L) and an extremely resistant (polymyxin B MIC ≥128 mg/L), following exposure to 2 mg/L of polymyxin B. Comparative profiling of the OMV subproteome of each strain revealed proteins from multiple perturbed pathways, particularly in the polymyxin-susceptible strain, including outer membrane assembly (lipopolysaccharide, O-antigen, and peptidoglycan biosynthesis), cationic antimicrobial peptide resistance, β-lactam resistance, and quorum sensing. In the polymyxin-susceptible strain, polymyxin B treatment reduced the expression of OMV proteins in the pathways related to adhesion, virulence, and the cell envelope stress responses, whereas, in the polymyxin-resistant strain, the proteins involved in LPS biosynthesis, RNA degradation, and nucleotide excision repair were significantly overexpressed in response to polymyxin B treatment. Intriguingly, the key polymyxin resistance enzymes 4-amino-4-deoxy-l-arabinose transferase and the PhoPQ two-component protein kinase were significantly downregulated in the OMVs of the polymyxin-susceptible strain. Additionally, a significant reduction in class A β-lactamase proteins was observed following polymyxin B treatment in the OMVs of both strains, particularly the OMVs of the polymyxin-susceptible strain. These findings shed new light on the OMV subproteome of extremely polymyxin resistant K. pneumoniae, which putatively may serve as active decoys to make the outer membrane more impervious to polymyxin attack. IMPORTANCE OMVs can help bacteria to fight antibiotics not only by spreading antibiotic resistance genes but also by acting as protective armor against antibiotics. By employing proteomics, we found that OMVs have a potential role in shielding K. pneumoniae and acting as decoys to polymyxin attack, through declining the export of proteins (e.g., 4-amino-4-deoxy-l-arabinose transferase) involved in polymyxin resistance. Furthermore, polymyxin B treatment of both strains leads to shedding of the OMVs with perturbed proteins involved in outer membrane remodeling (e.g., LPS biosynthesis) as well as pathogenic potential of K. pneumoniae (e.g., quorum sensing). The problematic extended spectrum beta-lactamases SHV and TEM were significantly reduced in both strains, suggesting that polymyxin B may act as a potentiator to sensitize the bacterium to β-lactam antibiotics. This study highlights the importance of OMVs as "molecular mules" for the intercellular transmission and delivery of resistance and cellular repair factors in the bacterial response to polymyxins.
... This gene is homologous to Arabidopsis At1g53000 and is a candidate to the Csleth mutant. Strohmaier et al. (1995) found two Escherichia coli regulatory genes, kdsA and kdsB, which are involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide and expressed in the growth phase regulated primarily at the transcriptional level in Escherichia coli K-12. In other crop, Royo et al. (2000) first discovered the maize root development-related gene ZmCKS, which is involved in the synthesis of corn CMP-KDO enzymes. ...
Seed germination plays an important role in the initial stage of plant growth. However, few related studies focused on lethality after seed germination in plants. In this study, we identified an Ethyl methanesulfonate (EMS) mutagenesis mutant Csleth with abnormal seed germination in cucumber (Cucumis sativus L.). The radicle of the Csleth mutant grew slowly and detached from the cotyledon until 14 d after sowing. Genetic analysis showed that the mutant phenotype of Csleth was controlled by a single recessive gene. MutMap⁺ and Kompetitive Allele Specific PCR (KASP) genotyping results demonstrated that Csa3G104930 encoding 3-deoxy-manno-octulosonate cytidylyltransferase (CsKDO) was the candidate gene of the Csleth mutant. The transition mutation of aspartate occurred in Csa3G104930 co-segregated with the phenotyping data. CsKDO was highly expressed in male flowers in wild type cucumbers. Subcellular localization results showed that CsKDO was located in the nucleus. Overall, these results suggest CsKDO regulates lethality during seed germination in cucumber.
... WP_003812536.1 protein is encode by dapD gene and is involved in the process of L-lysine biosynthesis inside aminoacid biosynthesis (UniProt, 2019). The protein WP_003813703.1 is encode by kdsA gene, which has important functions in the carbohydrate synthesis pathway and the lipopolysaccharide synthesis pathway, resulting in the formation of keto-3-deoxy-D-manno-octulosonic acid, which is a key sugar-acid in the biogenesis of the bacterial outer membrane (Jensen et al., 2002;Strohmaier et al., 1995;UniProt, 2019). All these proteins perform roles in vital bacterial processes, and then changes in these processes can compromise bacterial integrity. ...
Pertussis is a highly contagious respiratory disease caused by Bordetella pertussis, a Gram-negative bacterium described over a century ago. Despite broad vaccine coverage and treatment options, the disease is remerging as a public health problem especially in infants and older children. Recent data indicate re-emergence of the disease is related to bacterial resistance to immune defences and decreased vaccine effectiveness, which obviously suggests the need of new effective vaccines and drugs. In an attempt to contribute with solutions to this great challenge, bioinformatics tools were used to genetically comprehend the species of these bacteria and predict new vaccines and drug targets. In fact, approaches were used to analysis genomic plasticity, gene synteny and species similarities between the 20 genomes of Bordetella pertussis already available. Furthermore, it was conducted reverse vaccinology and docking analysis to identify proteins with potential to become vaccine and drug targets, respectively. The analyses showed the 20 genomes belongs to a homogeneous group that has preserved most of the genes over time. Besides that, were found genomics islands and good proteins to be candidates for vaccine and drugs. Taken together, these results suggests new possibilities that may be useful to develop new vaccines and drugs that will help the prevention and treatment strategies of pertussis disease caused by these Bordetella strains.
Communicated by Ramaswamy H. Sarma
... Phosphoheptose isomerase (encoded by gmhA) and ADP-L-glycero-D-manno-heptose-6-epimerase (encoded by hldD) catalyze the biosynthesis of the heptose precursors. On the other hand, 2-dehydro-3-deoxyphosphooctonate aldolase (encoded by kdsA) is responsible for the synthesis of the Kdo, which links lipid A and core oligosaccharides (Strohmaier et al., 1995). Lipid A is the innermost leaflet of the outer membrane. ...
Klebsiella pneumoniae is an opportunistic bacterial pathogen leading to life-threatening nosocomial infections. Emergence of highly resistant strains poses a major challenge in the management of the infections by healthcare-associated K. pneumoniae isolates. Thus, despite intensive efforts, the current treatment strategies remain insufficient to eradicate such infections. Failure of the conventional infection-prevention and treatment efforts explicitly indicates the requirement of new therapeutic approaches. This prompted us to systematically analyze the K. pneumoniae metabolism to investigate drug targets. Genome-scale metabolic networks (GMNs) facilitating the systematic analysis of the metabolism are promising platforms. Thus, we used a GMN of K. pneumoniae MGH 78578 to determine putative targets through gene- and metabolite-centric approaches. To develop more realistic infection models, we performed the bacterial growth simulations within different host-mimicking media, using an improved biomass formation reaction. We selected more suitable targets based on several property-based prioritization procedures. KdsA was identified as the high-ranked putative target satisfying most of the target prioritization criteria specified under the gene-centric approach. Through a structure-based virtual screening protocol, we identified potential KdsA inhibitors. In addition, the metabolite-centric approach extended the drug target list based on synthetic lethality. This revealed the importance of combined metabolic analyses for a better understanding of the metabolism. To our knowledge, this is the first comprehensive effort on the investigation of the K. pneumoniae metabolism for drug target prediction through the constraint-based analysis of its GMN in conjunction with several bioinformatic approaches. This study can guide the researchers for the future drug designs by providing initial findings regarding crucial components of the Klebsiella metabolism.
... In fact, loss of fatty acids by the outer leaflet of OM as an effect of both degradation of phospholipids and loss of LPS would require an increase of the synthesis of fatty acids. However, kdsA gene, coding for the enzyme involved in the synthesis of 3-deoxy-D-mannooctulosonic acid (Kdo) of the LPS oligosaccharide core, 33,34 was apparently in contrast with the above gene expression profile, as its transcription level was not significantly altered compared to untreated cells. Despite this, no conflict is suggested with the synthesis of the LPS induced by the film, as the control of Kdo availability does not strictly correlate with that of the kdsA gene transcription as evidenced in several bacterial strains. ...
... In E. coli K-12, a half-life of enzyme KdsA higher than that of the relative transcript was described. 33 Salmonella typhimurium was able to synthesize LPS even in absence of Kdo synthesis, probably using a pool of cellular components. 56 One may therefore speculate that during the treatment phase, the greater stability of KdsA enzyme and/or the use of a reserve of cellular elements would be sufficient to support, at least for a short time, a more modest increase in the synthesis of LPS than required during the growth phase in the rich medium. ...
In this study we use E. coli as a model to investigate the antimicrobial mechanism of a film made of a copolymer based on mPEG, MMA and DMAEMA and whose surface is active towards Gram-negative and Gram-positive bacteria. The polymer contains not quaternized amino groups able to generate a charged surface by protonation when in contact with water. For the purpose, we adopted a dual strategy based on the analysis of cell damage caused by contact with the polymer surface, and on the evaluation of the cell response to the surface toxic action. The lithic effect on the protoplasts of E. coli showed that polymer surface can affect the structure of cytoplasmic membranes, while assays of calcein leakage from LUVs at different phospholipids composition indicated that action on membranes does not need a functionally active cell. On the other hand, the significant increase in sensitivity to actinomycin D demonstrates that polymer interferes also with the structure of the outer membrane, modifying its permeability. The study on gene expression, based on the analysis of the transcripts in a temporal window where the contact with the polymer is not lethal and the damage is reversible, showed that some key genes of the synthesis and maintenance of OM structure (fabR, fadR, fabA, waaA, waaC, kdsA, pldA, pagP), as well as regulators of cellular response to oxidative stress (soxS), are more expressed when bacteria are exposed to polymer surface. All together these results identified the outer membrane as the main cellular target of antimicrobial surface and indicated a specific cellular response to damage, providing more information on the antimicrobial mechanism. In perspective, data here reported could play a pivotal role in a microbial growth control strategy based not only on the structural improvements of the materials, but also on the possibility of intervening on the cellular pathways involved in the contrast reaction to these and other polymers with similar mechanisms.
... One of them, KdsB, is involved in LPS biosynthesis, whereas the other one, KpsU, is necessary for capsular polysaccharide synthesis [123, 124]. Both enzymes catalyse the same reaction, but they are differentially regulated by temperature [124] and growth rate [125]. Structural data reveal that KpsU [126, 127] and KdsB [128] are highly conserved and their active site configuration and overall chemistry catalysed is similar to DNA/RNA polymerases [128]. ...
Lipopolysaccharide (LPS), an integral component of the outer membrane in Gram-negative bacteria, consists of lipid A, core oligosaccharide (core), and O-specific polysaccharide or O antigen (OAg). LPS protects Gram-negative bacteria from environmental chemical and physical stress and is also recognized by the innate immune system upon infection. LPS biosynthesis, export and assembly require a large number of enzymes and structural proteins encoded by numerous genes. Both the lipid A and core are assembled on the cytoplasmic side of the inner membrane and translocated across the inner membrane. The OAg is independently assembled in a separate pathway, also translocated to the periplasmic side of the cell membrane, and ligated to the lipid A-core. Newly formed LPS is then shuttled across the periplasm by a complex multiprotein pathway, which also mediates the insertion of LPS into the outer leaflet of the outer membrane. This chapter discusses current mechanistic understanding of the synthesis and assembly of the LPS molecule.
... Due to its effect on SirC expression and the ability to suppress sirA mutant phenotypes, SirB is considered to be a putative transcriptional regulator, although it does not belong to any known class of transcription factors. In E. coli, the sirB locus is nonessential [109], and no function has been reported. However, the putative operon in which the locus is situated is well characterized and contains genes involved in basic cellular functions, such as protein synthesis and the biosynthesis of LPS109110111. ...
... In E. coli, the sirB locus is nonessential [109], and no function has been reported. However, the putative operon in which the locus is situated is well characterized and contains genes involved in basic cellular functions, such as protein synthesis and the biosynthesis of LPS109110111. To our knowledge, SirB has not been reported to regulate the other genes in the operon. ...
Soft rot disease is economically one of the most devastating bacterial diseases affecting plants worldwide. In this study, we present novel insights into the phylogeny and virulence of the soft rot model Pectobacterium sp. SCC3193, which was isolated from a diseased potato stem in Finland in the early 1980s. Genomic approaches, including proteome and genome comparisons of all sequenced soft rot bacteria, revealed that SCC3193, previously included in the species Pectobacterium carotovorum, can now be more accurately classified as Pectobacterium wasabiae. Together with the recently revised phylogeny of a few P. carotovorum strains and an increasing number of studies on P. wasabiae, our work indicates that P. wasabiae has been unnoticed but present in potato fields worldwide. A combination of genomic approaches and in planta experiments identified features that separate SCC3193 and other P. wasabiae strains from the rest of soft rot bacteria, such as the absence of a type III secretion system that contributes to virulence of other soft rot species. Experimentally established virulence determinants include the putative transcriptional regulator SirB, two partially redundant type VI secretion systems and two horizontally acquired clusters (Vic1 and Vic2), which contain predicted virulence genes. Genome comparison also revealed other interesting traits that may be related to life in planta or other specific environmental conditions. These traits include a predicted benzoic acid/salicylic acid carboxyl methyltransferase of eukaryotic origin. The novelties found in this work indicate that soft rot bacteria have a reservoir of unknown traits that may be utilized in the poorly understood latent stage in planta. The genomic approaches and the comparison of the model strain SCC3193 to other sequenced Pectobacterium strains, including the type strain of P. wasabiae, provides a solid basis for further investigation of the virulence, distribution and phylogeny of soft rot bacteria and, potentially, other bacteria as well.
... Similar high-level expression of toxins during late-log and stationary phase growth-related time points was observed in other bacteria (Thompson et al., 2003; Wagner et al., 2003; Snyder et al., 2004). Distinct from cards gene transcription, we observed high-level expression of adherence-and stress-related genes during late-log and stationary stage growth of M. pneumoniae, and similar growth dependent expression patterns of adherence-and stress-related genes have been observed in other bacteria (Strohmaier et al., 1995; Xu and Johnson, 1995; Nicholson et al., 2003; Thompson et al., 2003). Earlier, we reported that CARDS toxin was a surfaceassociated , trypsin-sensitive surfactant protein A binding 68 kDa protein (Kannan et al., 2005). ...
Mycoplasma pneumoniae causes acute and chronic respiratory infections, including tracheobronchitis and community acquired pneumonia, and is linked to asthma and an array of extra-pulmonary disorders. Recently, we identified an ADP-ribosylating and vacuolating toxin of M. pneumoniae, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we analysed CARDS toxin gene (annotated mpn372) transcription and identified its promoter. We also compared CARDS toxin mRNA and protein profiles in M. pneumoniae during distinct in vitro growth phases. CARDS toxin mRNA expression was maximal, but at low levels, during early exponential growth and declined sharply during mid-to-late log growth phases, which was in direct contrast to other mycoplasma genes examined. Between 7% and 10% of CARDS toxin was localized to the mycoplasma membrane at mid-exponential growth, which was reinforced by immunogold electron microscopy. No CARDS toxin was released into the medium. Upon M. pneumoniae infection of mammalian cells, increased expression of CARDS toxin mRNA was observed when compared with SP-4 broth-grown cultures. Further, confocal immunofluorescence microscopy revealed that M. pneumoniae readily expressed CARDS toxin during infection of differentiated normal human bronchial epithelial cells. Analysis of M. pneumoniae-infected mouse lung tissue revealed high expression of CARDS toxin per mycoplasma cell when compared with M. pneumoniae cells grown in SP-4 medium alone. Taken together, these studies indicate that CARDS toxin expression is carefully controlled by environmental cues that influence its transcription and translation. Further, the acceleration of CARDS toxin synthesis and accumulation in vivo is consistent with its role as a bona fide virulence determinant.
