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Volume 163,
Issue 4,
2017
Volume 163, Issue 4, 2017

- Review
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Temporal upregulation of host surface receptors provides a window of opportunity for bacterial adhesion and disease
More LessHost surface receptors provide bacteria with a foothold from which to attach, colonize and, in some cases, invade tissue and elicit human disease. In this review, we discuss several key host receptors and cognate adhesins that function in bacterial pathogenesis. In particular, we examine the elevated expression of host surface receptors such as CEACAM-1, CEACAM-6, ICAM-1 and PAFR in response to specific stimuli. We explore how upregulated receptors, in turn, expose the host to a range of bacterial infections in the respiratory tract. It is apparent that exploitation of receptor induction for bacterial adherence is not unique to one body system, but is also observed in the central nervous, gastrointestinal and urogenital systems. Prokaryotic pathogens which utilize this mechanism for their infectivity include Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. A number of approaches have been used, in both in vitro and in vivo experimental models, to inhibit bacterial attachment to temporally expressed host receptors. Some of these novel strategies may advance future targeted interventions for the prevention and treatment of bacterial disease.
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- Biotechnology
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Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis
Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.
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Enhancing ethanol yields through d-xylose and l-arabinose co-fermentation after construction of a novel high efficient l-arabinose-fermenting Saccharomyces cerevisiae strain
More LessLignocellulose contains two pentose sugars, l-arabinose and d-xylose, neither of which is naturally fermented by first generation (1G) ethanol-producing Saccharomyces cerevisiae yeast. Since these sugars are inaccessible to 1G yeast, a significant percentage of the total carbon in bioethanol production from plant residues, which are used in second generation (2G) ethanol production, remains unused. Recombinant Saccharomyces cerevisiae strains capable of fermenting d-xylose are available on the market; however, there are few examples of l-arabinose-fermenting yeasts, and commercially, there are no strains capable of fermenting both d-xylose and l-arabinose because of metabolic incompatibilities when both metabolic pathways are expressed in the same cell. To attempt to solve this problem we have tested d-xylose and l-arabinose co-fermentation. To find efficient alternative l-arabinose utilization pathways to the few existing ones, we have used stringent methodology to screen for new genes (metabolic and transporter functions) to facilitate l-arabinose fermentation in recombinant yeast. We demonstrate the feasibility of this approach in a successfully constructed yeast strain capable of using l-arabinose as the sole carbon source and capable of fully transforming it to ethanol, reaching the maximum theoretical fermentation yield (0.43 g g−1). We demonstrate that efficient co-fermentation of d-xylose and l-arabinose is feasible using two different co-cultured strains, and observed no fermentation delays, yield drops or accumulation of undesired byproducts. In this study we have identified a technically efficient strategy to enhance ethanol yields by 10 % in 2G plants in a process based on C5 sugar co-fermentation.
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Butanol is cytotoxic to Lactococcus lactis while ethanol and hexanol are cytostatic
More LessLactic acid bacteria currently used extensively by the dairy industry have a superior tolerance towards short-chain alcohols, which makes them interesting targets for use in future bio-refineries. The mechanism underlying the alcohol tolerance of lactic acid bacteria has so far received little attention. In the present study, the physiological alcohol stress response of Lactococcus lactis subsp. cremoris MG1363 towards the primary, even-chain alcohols ethanol, butanol and hexanol, was characterized. The alcohol tolerance of L. lactis was found to be comparable to those reported for highly alcohol-resistant lactic acid bacteria. Combined results from alcohol survival rate, live/dead staining, and a novel usage of the β-galactosidase assay, revealed that while high concentrations of ethanol and hexanol were cytostatic to L. lactis, high concentrations of butanol were cytotoxic, causing irreparable damages to the cell membrane.
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Synthetic temperature-inducible lethal gene circuits in Escherichia coli
More LessTemperature sensitivity is often used as a way to attenuate micro-organisms to convert them into live vaccines. In this work, we explore the use of temperature-sensitive (TS) genetic circuits that express lethal genes as a widely applicable approach to TS attenuation. We tested different combinations of TS repressors and cognate promoters controlling the expression of genes encoding restriction endonucleases inserted at four different non-essential sites in the Escherichia coli chromosome. We found that the presence of the restriction endonuclease genes did not affect the viability of the host strains at the permissive temperature, but that expression of the genes at elevated temperatures killed the strains to varying extents. The chromosomal insertion site of the lethal cassettes affected their functionality, and insertion at one site, ycgH, rendered them ineffective at inducing death at high temperature. Induction of a TS circuit in a growing culture led to a reduced cell mass and a reduction of the number of cells that could exclude a dye that indicated viability. Incubation of cells carrying a TS lethal gene circuit initially grown at low temperature and then suspended in phosphate buffered saline at high temperature led to about 100-fold loss of cell viability per day, compared to a minimal loss of viability for the parental strain. Strains carrying either one or two TS lethal circuits could generate mutants that survived at high temperature. These mutants included complete deletions of the lethal gene circuits.
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- Environmental Biology
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Pb2+ tolerance by Frankia sp. strain EAN1pec involves surface-binding
More LessSeveral Frankia strains have been shown to be lead-resistant. The mechanism of lead resistance was investigated for Frankia sp. strain EAN1pec. Analysis of the cultures by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDAX) and Fourier transforming infrared spectroscopy (FTIR) demonstrated that Frankia sp. strain EAN1pec undergoes surface modifications and binds high quantities of Pb +2 . Both labelled and unlabelled shotgun proteomics approaches were used to determine changes in Frankia sp. strain EAN1pec protein expression in response to lead and zinc. Pb 2+ specifically induced changes in exopolysaccharides, the stringent response, and the phosphate (pho) regulon. Two metal transporters (a Cu2+-ATPase and cation diffusion facilitator), as well as several hypothetical transporters, were also upregulated and may be involved in metal export. The exported Pb2+ may be precipitated at the cell surface by an upregulated polyphosphate kinase, undecaprenyl diphosphate synthase and inorganic diphosphatase. A variety of metal chaperones for ensuring correct cofactor placement were also upregulated with both Pb+2 and Zn+2 stress. Thus, this Pb+2 resistance mechanism is similar to other characterized systems. The cumulative interplay of these many mechanisms may explain the extraordinary resilience of Frankia sp. strain EAN1pec to Pb+2. A potential transcription factor (DUF156) binding site was identified in association with several proteins identified as upregulated with heavy metals. This site was also discovered, for the first time, in thousands of other organisms across two kingdoms.
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- Genomics and Systems Biology
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Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c
Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1- and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.
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- Host-microbe Interaction
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A chitinase is required for Xylella fastidiosa colonization of its insect and plant hosts
More LessXylella fastidiosa colonizes the xylem network of host plant species as well as the foregut of its required insect vectors to ensure efficient propagation. Disease management strategies remain inefficient due to a limited comprehension of the mechanisms governing both insect and plant colonization. It was previously shown that X. fastidiosa has a functional chitinase (ChiA), and that chitin likely serves as a carbon source for this bacterium. We expand on that research, showing that a chiA mutant strain is unable to grow on chitin as the sole carbon source. Quantitative PCR assays allowed us to detect bacterial cells in the foregut of vectors after pathogen acquisition; populations of the wild-type and complemented mutant strain were both significantly larger than the chiA mutant strain 10 days, but not 3 days, post acquisition. These results indicate that adhesion of the chiA mutant strain to vectors may not be impaired, but that cell multiplication is limited. The mutant was also affected in its transmission by vectors to plants. In addition, the chiA mutant strain was unable to colonize host plants, suggesting that the enzyme has other substrates associated with plant colonization. Lastly, ChiA requires other X. fastidiosa protein(s) for its in vitro chitinolytic activity. The observation that the chiA mutant strain is not able to colonize plants warrants future attention to be paid to the substrates for this enzyme.
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Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts
Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown that virulence of X. bovienii–Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS+ strains generally outcompeted the less virulent T6SS− strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.
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Volatile organic compounds produced by a soil-isolate, Bacillus subtilis FA26 induce adverse ultra-structural changes to the cells of Clavibacter michiganensis ssp. sepedonicus, the causal agent of bacterial ring rot of potato
Rhizobacterial volatile organic compounds (VOCs) play an important role in the suppression of soil-borne phytopathogens. In this study, the VOCs produced by a soil-isolate, Bacillus subtilis FA26, were evaluated in vitro for their antibacterial activity against Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. The VOCs emitted by FA26 inhibited the growth of Cms significantly compared with the control. Scanning and transmission electron microscopy analyses revealed distorted colony morphology and a wide range of abnormalities in Cms cells exposed to the VOCs of FA26. Varying the inoculation strategy and inoculum size showed that the production and activity of the antibacterial VOCs of FA26 were dependent on the culture conditions. Headspace solid-phase microextraction/gas chromatography–mass spectrometry analyses revealed that FA26 produced 11 VOCs. Four VOCs (benzaldehyde, nonanal, benzothiazole and acetophenone) were associated with the antibacterial activity against Cms. The results suggested that the VOCs produced by FA26 could control the causal agent of bacterial ring rot of potato. This information will increase our understanding of the microbial interactions mediated by VOCs in nature and aid the development of safer strategies for controlling plant disease.
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- Physiology and Metabolism
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Vacuolar H+-ATPase subunit Vma1p functions as the molecular ligand in the vacuole-targeting fungicidal activity of polymyxin B
Polymyxin B (PMB) is a cationic cyclic peptide that can selectively inhibit the growth of Gram-negative bacteria by disrupting the outer membrane permeability barrier through binding to lipopolysaccharide (LPS). Here, a fluorescent PMB derivative (PMB-Ds) was applied to visually confirm the vacuole as a direct lethal target of PMB against fungal cells, which lack LPS. PMB-Ds could be visualized in the normal rounded vacuolar membrane of Saccharomyces cerevisiae cells, suggesting the presence of a molecular ligand assisting the vacuole-targeting mobilization of the peptide in the organism. Vma1p, a cytoplasmic subunit constituent of the yeast vacuolar-type ATPase, was identified as one of the PMB-binding proteins by means of mass spectrometry. Mutant cells carrying a deletion of Vma1p but not those with deletions in two separate PMB-binding proteins were shown to be resistant to the vacuolar membrane disruptive action of PMB. Furthermore, the mutant cells were resistant to PMB lethality even when treated with PMB in combination with allicin, an allyl sulfur compound, which can selectively enhance the vacuole-targeting fungicidal activity of the peptide. In contrast, the parent cells were not made resistant to the vacuolar membrane disruptive action of PMB even if cells were pre-treated with bafilomycin A1, a specific inhibitor of the yeast vacuolar-type H+-ATPase. However, the parent cells were rendered more resistant to PMB consequent to Vma1p-GFP localization in the cytoplasm. These findings suggested a role for Vma1p in the vacuole-targeting fungicidal activity of PMB comparable to that of LPS in the outer membrane of Gram-negative bacteria.
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- Regulation
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Unravelling the biosynthesis of pyriculol in the rice blast fungus Magnaporthe oryzae
Pyriculol was isolated from the rice blast fungus Magnaporthe oryzae and found to induce lesion formation on rice leaves. These findings suggest that it could be involved in virulence. The gene MoPKS19 was identified to encode a polyketide synthase essential for the production of the polyketide pyriculol in the rice blast fungus M. oryzae. The transcript abundance of MoPKS19 correlates with the biosynthesis rate of pyriculol in a time-dependent manner. Furthermore, gene inactivation of MoPKS19 resulted in a mutant unable to produce pyriculol, pyriculariol and their dihydro derivatives. Inactivation of a putative oxidase-encoding gene MoC19OXR1, which was found to be located in the genome close to MoPKS19, resulted in a mutant exclusively producing dihydropyriculol and dihydropyriculariol. By contrast, overexpression of MoC19OXR1 resulted in a mutant strain only producing pyriculol. The MoPKS19 cluster, furthermore, comprises two transcription factors MoC19TRF1 and MoC19TRF2, which were both found individually to act as negative regulators repressing gene expression of MoPKS19. Additionally, extracts of ΔMopks19 and ΔMoC19oxr1 made from axenic cultures failed to induce lesions on rice leaves compared to extracts of the wild-type strain. Consequently, pyriculol and its isomer pyriculariol appear to be the only lesion-inducing secondary metabolites produced by M. oryzae wild-type (MoWT) under these culture conditions. Interestingly, the mutants unable to produce pyriculol and pyriculariol were as pathogenic as MoWT, demonstrating that pyriculol is not required for infection.
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Environment-directed activation of the Escherichia coli flhDC operon by transposons
More LessThe flagellar system in Escherichia coli K12 is expressed under the control of the flhDC-encoded master regulator FlhDC. Transposition of insertion sequence (IS) elements to the upstream flhDC promoter region up-regulates transcription of this operon, resulting in a more rapid motility. Wang and Wood (ISME J 2011;5:1517–1525) provided evidence that insertion of IS5 into upstream activating sites occurs at higher rates in semi-solid agar media in which swarming behaviour is allowed as compared with liquid or solid media where swarming cannot occur. We confirm this conclusion and show that three IS elements, IS1, IS3 and IS5, transpose to multiple upstream sites within a 370 bp region of the flhDC operon control region. Hot spots for IS insertion correlate with positions of stress-induced DNA duplex destabilization (SIDD). We show that IS insertion occurs at maximal rates in 0.24 % agar, with rates decreasing dramatically with increasing or decreasing agar concentrations. In mixed cultures, we show that these mutations preferentially arise from the wild-type parent at frequencies of up to 3×10−3 cell−1 day−1 when the inoculated parental and co-existing IS-activated mutant cells are entering the stationary growth phase. We rigorously show that the apparent increased mutation frequencies cannot be accounted for by increased swimming or by increased growth under the selective conditions used. Thus, our data are consistent with the possibility that appropriate environmental conditions, namely those that permit but hinder flagellar rotation, result in the activation of a mutational pathway that involves IS element insertion upstream of the flhDC operon.
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RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti
The ribonucleases (RNases) E and J play major roles in E. coli and Bacillus subtilis, respectively, and co-exist in Sinorhizobium meliloti. We analysed S. meliloti 2011 mutants with mini-Tn5 insertions in the corresponding genes rne and rnj and found many overlapping effects. We observed similar changes in mRNA levels, including lower mRNA levels of the motility and chemotaxis related genes flaA, flgB and cheR and higher levels of ndvA (important for glucan export). The acyl-homoserine lactone (AHL) levels were also higher during exponential growth in both RNase mutants, despite no increase in the expression of the sinI AHL synthase gene. Furthermore, several RNAs from both mutants migrated aberrantly in denaturing gels at 300 V but not under stronger denaturing conditions at 1300 V. The similarities between the two mutants could be explained by increased levels of the key methyl donor S-adenosylmethionine (SAM), since this may result in faster AHL synthesis leading to higher AHL accumulation as well as in uncontrolled methylation of macromolecules including RNA, which may strengthen RNA secondary structures. Indeed, we found that in both mutants the N 6-methyladenosine content was increased almost threefold and the SAM level was increased at least sevenfold. Complementation by induced ectopic expression of the respective RNase restored the AHL and SAM levels in each of the mutants. In summary, our data show that both RNase E and RNase J are needed for SAM homeostasis in S. meliloti.
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Regulation of the Escherichia coli ydhY-T operon in the presence of alternative electron acceptors
More LessThe Escherichia coli K-12 ydhY-T operon, coding for a predicted oxidoreductase complex, is activated under anaerobic conditions and repressed in the presence of nitrate or nitrite. Anaerobic activation is mediated by the transcription factor FNR, and nitrate/nitrite repression is mediated by NarXL and NarQP. In vitro transcription reactions revealed that the DNA upstream of ydhY-T contains sufficient information for RNA polymerase alone to initiate transcription from five locations. FNR severely inhibited synthesis of two of these transcripts (located upstream of, and within, the FNR binding site) and activated the FNR-dependent promoter previously identified in vivo. Enhanced expression of ydhY-T in an hns mutant was consistent with the location of ydhY-T within a promoter island and the FNR-independent transcription observed in vitro. FNR-dependent transcription in vitro was decreased in the presence of NarL~P. DNaseI footprinting indicated that FNR and NarL~P simultaneously bound at the ydhY-T promoter region and that NarL~P-mediated repression was due to occupation of the 7-2-7 site located downstream of the FNR-dependent promoter. Expression of ydhY-T during the anaerobic growth cycle was repressed when nitrate was present but less so in the presence of nitrite. In vivo transcription measurements indicated that the alternative electron acceptors, DMSO and fumarate, could also lower ydhY-T expression, whereas trimethylamine-N-oxide (TMAO) permitted high expression. Therefore, expression of ydhY-T is subject to complex regulation in response to electron acceptor availability that involves at least three transcription factors, FNR (anaerobic activation), NarL~P (nitrate repression) and H-NS (repression in the absence of an antagonist; e.g. FNR).
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Mutual interaction enables the mycobacterial plasmid pAL5000 origin binding protein RepB to recruit RepA, the plasmid replicase, to the origin
More LessThe Mycobacterium fortuitum plasmid, pAL5000, is the most-studied member of a family of plasmids that are found in Actinobacteria. Its replication is brought about by the combined action of two plasmid-encoded replication proteins, RepA and RepB. RepB has earlier been shown to be a sigma factor homologue that possesses origin-binding activity. The mechanism by which RepA functions, and its relationship with RepB, if any, has not been explored yet. In this study, we show that RepA shares a common catalytic domain, with proteins belonging to the primase-polymerase and DNA polymerase X families. We demonstrate that RepA is functionally a DNA polymerase and that mutations that alter two conserved aspartic acid residues present within the catalytic core lead to inactivation of plasmid replication. Replication of pAL5000 was shown not to depend on the host primase, and thus it is most likely that RepA is responsible for the priming act. We further demonstrate that RepA and RepB function as a pair and that the functional cooperation between the two requires physical contact. The C-terminal domain of RepA, which is structurally a helical bundle, is responsible for unwinding the origin in a site-specific manner and also for the establishment of contacts with RepB. The results presented show that RepB functions by recruiting RepA to the origin in much the same way as sigma factors recruit RNA polymerase core enzyme to promoters.
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Genome amplification and promoter mutation expand the range of csgD-dependent biofilm responses in an STEC population
More LessExpression of the major biofilm components of E. coli, curli fimbriae and cellulose, requires the CsgD transcription factor. A complex regulatory network allows environmental control of csgD transcription and biofilm formation. However, most clinical serotype O157 : H7 strains contain prophage insertions in the csgD regulator, mlrA, or mutations in other regulators that restrict csgD expression. These barriers can be circumvented by certain compensating mutations that restore higher csgD expression. One mechanism is via csgD promoter mutations that switch sigma factor utilization. Biofilm-forming variants utilizing RpoD rather than RpoS have been identified in glycerol freezer stocks of the non-biofilm-forming food-borne outbreak strain, ATCC 43894. In this study we used whole genome sequencing and RNA-seq to study genotypic and transcriptomic differences between those strains. In addition to defining the consequences of the csgD promoter switch and identifying new csgD-controlled genes, we discovered a region of genome amplification in our laboratory stock of 43894 (designated 43894OW) that contributed to the regulation of csgD-dependent properties.
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