Figure - available from: Journal of Bacteriology
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The effects of Δ1 to Δ3 mutations on the β-galactosidase produced by a FimB-LacZ fusion. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods.
Source publication
The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate
that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-...
Citations
... The Escherichia coli micA gene was the most cited bacterial antisense gene with 25 related articles in PubMed (Table S7). This gene stands out for being a post-transcriptional regulator of several genes [140][141][142] and for acting in the mechanisms of virulence [143]. A vaccine produced with micA-derived OMVs (outer membrane vesicles) protected mice against Salmonella typhimurium [143]. ...
Initially, natural antisense transcripts (NATs, natRNAs, or asRNAs) were considered repressors; however, their functions in gene regulation are diverse. Positive, negative, or neutral correlations to the cognate gene expression have been noted. Although the first studies were published about 50 years ago, there is still much to be investigated regarding antisense transcripts in plants. A systematic review of scientific publications available in the Web of Science databases was conducted to contextualize how the studying of antisense transcripts has been addressed. Studies were classified considering three categories: “Natural antisense” (208), artificial antisense used in “Genetic Engineering” (797), or “Natural antisense and Genetic Engineering”-related publications (96). A similar string was used for a systematic search in the NCBI Gene database. Of the 1132 antisense sequences found for plants, only 0.8% were cited in PubMed and had antisense information confirmed. This value was the lowest when compared to fungi (2.9%), bacteria (2.3%), and mice (54.1%). Finally, we present an update for the cis-NATs identified in Saccharum spp. Of the 1413 antisense transcripts found in different experiments, 25 showed concordant expressions, 22 were discordant, 1264 did not correlate with the cognate genes, and 102 presented variable results depending on the experiment.
... In contrast to effect of the three genes mentioned above, rpoS mutant cells demonstrated additive SDS sensitivity when combined with rfaH and ydgH mutations (Fig. 7A, right panel). As rfaH is required for efficient LPS biosynthesis (48)(49)(50)(51), it is likely that rfaH mutants have OM permeability unrelated to the pathway affected by RpoS. YdgH is predicted to be a periplasmic protein, and its levels have been suggested to be downregulated by MicA, an sRNA that increases upon entry to stationary phase but is transcriptionally regulated by sigma E, the envelope stress sigma factor (52)(53)(54)(55). ...
Importance:
Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When depleted of an essential nutrient, a program of gene expression is induced providing cross protection against many stresses. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase further strengthening the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negatives that may be in a growth-limited state.
Escherichia coli (E. coli) is a gram negative rod shape bacterium that is part of the natural intestinal microflora of humans and animals. However E. coli is also an opportunistic pathogen and uropathogenic E. coli (UPEC) is the causative agent for majority of urinary tract infections. Most E. coli strains are motile and studies on E. coli motility is mainly confined to flagella-mediated swimming in a liquid medium and swarming on a semi-solid surface. The importance of motility on the virulence of uropathogenic E. coli is poorly understood. The objective of the current research is to understand various motility mechanisms observed in non-pathogenic and uropathogenic E. coli strains. This study identified three forms of motility in E. coli: a fast, nonpattern-forming, flagella-mediated motility, a slow pattern-forming type I fimbriae-mediated motility, and a flagella- and fimbriae-independent form of motility that was specific to W3110- LR strain. In a glucose-supplemented motility medium, fimbriae-mediated motility is favored in non-pathogenic strains derived from E. coli K-12 due to lack of activation of flagella from low levels of cyclic-AMP. Several non-pathogenic E.coli strains that displayed fast motility carried an insertion element in the flhDC promoter region and IS elements upregulate flagellar gene expression. Occasionally, slow-moving strains acquired the insertion element and began to exhibit flagella-mediated fast motility. In contrast to these results, three uropathogenic E. coli strains exhibited fast, flagella-mediated surface motility, and these strains did not have an insertion in flhDC promoter. Glucose prevented flagella-dependent swimming for the nonpathogenic strains, but did not prevent swimming for the pathogenic strains: we propose that regulation of cyclic-AMP synthesis, which is required for flagella synthesis is different in uropathogenic strains. To address the lack of knowledge about type I fimbriae mediated motility, we investigated the regulation and metabolic requirements associated with it. The deletion of regulators such as FimZ, H-NS, HdfR, IHF, OmpR, DksA and RcsB inhibited fimbriae-mediated surface motility. Analysis of regulator mutants indicated a possible mutually exclusive synthesis of flagella and fimbriae in the bacterial cell. In non-pathogenic E. coli strains glucose degradation via the Embden-Meyerhof-Parnas pathway and an oxidative TCA cycle facilitated fimbriae-mediated motility. However, uropathogenic strain UTI89 did not appear to require glycolysis but was completely dependent on TCA cycle for energy generation during flagella-mediated surface motility.
Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli . By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we mapped the topology of the protein. Using these data, we experimentally validated a structural model of YqjA generated using evolutionary covariance, which consists of an α-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggest that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.
Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake, and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we have performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli. By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we have mapped the topology of the protein. Using these data, we have experimentally validated a structural model of YqjA generated using evolutionary co-variance, which consists of an a-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggests that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.
The antiterminator RfaH is required for the expression of lipopolysaccharide (LPS), capsule, hemolysin, exotoxin, hemin uptake receptor, and F pilus. Since these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here we inactivated the rfaH gene of Yersinia enterocolitica O:3 to study its role in this enteropathogen. RNA-sequencing of the wild type and ΔrfaH strain transcriptomes revealed that RfaH acts as a highly specific regulator that enhances the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core but does not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the ΔrfaH strain was very similar to that of an O-antigen negative mutant. This indicated that the some changes seen in the ΔrfaH strain such as the genes involved in outer membrane homeostasis or in the stress response associated Cpx pathway were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the ΔrfaH strain cell surface resulted in attenuated stress response and lower resistance to compounds such as sodium dodecyl sulfate and polymyxin B. On the other hand, the ΔrfaH strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and outer core of LPS in Yersinia enterocolitica O:3. It may be speculated that RfaH might have in vivo role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.
