- Volume 162, Issue 10, 2016

During passage through the gastrointestinal tract, enterohaemorrhagic Escherichia coli (EHEC) encounters numerous stresses, each producing unique antimicrobial conditions. Beyond surviving these stresses, EHEC may also use them as cues about the local microenvironment to modulate its virulence. Of particular interest is how exposure to changing concentrations of short-chain fatty acids (SCFAs) associated with passage through the small and large intestines affects EHEC virulence, as well as flagella expression and motility specifically. In this study, we investigate the impact of exposure to SCFA mixes simulating concentrations and compositions within the small and large intestines on EHEC flagella expression and function. Using a combination of DNA microarray, quantitative real-time PCR, immunoblot analysis, flow cytometry and motility assays, we show that there is a marked, significant upregulation of flagellar genes, the flagellar protein, FliC, and motility when EHEC is exposed to SCFA mixes representative of the small intestine. By contrast, when EHEC is exposed to SCFA mixes representative of the large intestine, there is a significant downregulation of flagellar genes, FliC and motility. Our results demonstrate that EHEC modulates flagella expression and motility in response to SCFAs, with differential responses associated with SCFA mixes typical of the small and large intestines. This research contributes to our understanding of how EHEC senses and responds to host environmental signals and the mechanisms it uses to successfully infect the human host. Significantly, it also suggests that EHEC is using this key gastrointestinal chemical signpost to cue changes in flagella expression and motility in different locations within the host intestinal tract.

Colletotrichum acutatum is a major fungal pathogen of fruit crops, which causes severe yield losses in strawberry production. A potential key factor in plant–pathogen interactions is fungal sesquiterpenoids which have mycotoxic and phytotoxic activities. The first committed step in sesquiterpenoid biosynthesis is performed by sesquiterpene synthases (TPS). Only a few TPSs have been functionally characterized from filamentous fungi and none from the genus Colletotrichum. Despite being an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical levels. The terpenoid biochemistry in Coll. acutatum strain SA 0-1 was studied and one Coll. acutatum TPS (CaTPS) was successfully cloned and characterized in yeast. CaTPS catalyses the biosynthesis of multiple sesquiterpenoids. The two major products are β-caryophyllene and an unidentified sesquiterpenoid along with α-humulene as one of the minor sesquiterpenoid products. These products were also secreted by the fungus in strawberry fruit medium along with several other sesquiterpenoids indicating other TPSs are active during in vitro growth. β-Caryophyllene and α-humulene are known cytotoxic products important for ecological interactions and are produced by SA 0-1. Interestingly, a gene expression analysis using quantitative real-time PCR revealed a significant increase in expression of CaTPS during strawberry fruit infection, thus indicating that it could be involved in fruit infection. This is, we believe, the first characterization of TPS in Colletotrichum spp. and terpenoid profiles of Coll. acutatum, which could facilitate studies on the role of terpenoids in the ecology of Coll. acutatum.

Mycobacterium tuberculosis (Mtb) is one of the world’s most successful pathogens. Millions of new cases of tuberculosis occur each year, emphasizing the need for better methods of treatment. The design of novel therapeutics is dependent on our understanding of factors that are essential for pathogenesis. Many bacterial pathogens use pili and other adhesins to mediate pathogenesis. The recently identified Mycobacterium tuberculosis pilus (MTP) and the hypothetical, widely conserved Flp pilus have been speculated to be important for Mtb virulence based on in vitro studies and homology to other pili, respectively. However, the roles for these pili during infection have yet to be tested. We addressed this gap in knowledge and found that neither MTP nor the hypothetical Flp pilus is required for Mtb survival in mouse models of infection, although MTP can contribute to biofilm formation and subsequent isoniazid tolerance. However, differences in mtp expression did affect lesion architecture in infected lungs. Deletion of mtp did not correlate with loss of cell-associated extracellular structures as visualized by transmission electron microscopy in Mtb Erdman and HN878 strains, suggesting that the phenotypes of the mtp mutants were not due to defects in production of extracellular structures. These findings highlight the importance of testing the virulence of adhesion mutants in animal models to assess the contribution of the adhesin to infection. This study also underscores the need for further investigation into additional strategies that Mtb may use to adhere to its host so that we may understand how this pathogen invades, colonizes and disseminates.

Despite its presence in most bacteria, yqgF remains one of only 13 essential genes of unknown function in Escherichia coli. Predictions of YqgF function often derive from sequence similarity to RuvC, the canonical Holliday junction resolvase. To clarify its role, we deleted yqgF from a bacterium where it is not essential, Acinetobacter baylyi ADP1. Loss of yqgF impaired growth and increased the frequency of transformation and allelic replacement (TAR). When E. coli yqgF was inserted in place of its A. baylyi chromosomal orthologue, wild-type growth and TAR were restored. Functional similarities of yqgF in both gamma-proteobacteria were further supported by defective 16S rRNA processing by the A. baylyi mutant, an effect previously shown in E. coli for a temperature-sensitive yqgF allele. However, our data question the validity of deducing YqgF function strictly by comparison to RuvC. A. baylyi studies indicated that YqgF and RuvC can function in opposition to one another. Relative to the wild type, the ΔyqgF mutant had increased TAR frequency and increased resistance to nalidixic acid, a DNA-damaging agent. In contrast, deletion of ruvC decreased TAR frequency and lowered resistance to nalidixic acid. YqgF, but not RuvC, appears to increase bacterial susceptibility to DNA damage, including UV radiation. Nevertheless, the effects of yqgF on growth and TAR frequency were found to depend on amino acids analogous to catalytically required residues of RuvC. This new heterologous system should facilitate future yqgF investigation by exploiting the viability of A. baylyi yqgF mutants. In addition, bioinformatic analysis showed that a non-essential gene immediately upstream of yqgF in A. baylyi and E. coli (yqgE) is similarly positioned in most gamma- and beta-proteobacteria. A small overlap in the coding sequences of these adjacent genes is typical. This conserved genetic arrangement raises the possibility of a functional partnership between yqgE and yqgF.

Dps family proteins have the ferroxidase activity that contributes to oxidative stress resistance. In addition, a part of Dps family proteins including Escherichia coli Dps and Staphylococcus aureus MrgA (metallo regulon gene A) bind DNA and induce the structural change of the nucleoid. We previously showed that a mutated MrgA with reduced ferroxidase activity was unable to contribute to the hydrogen peroxide (H2O2) and UV resistance in S. aureus, suggesting that the nucleoid clumping by MrgA is not sufficient for the resistance. However, it remained elusive whether the nucleoid clumping is dispensable for the resistance. Here, we aimed to clarify this question by employing the E. coli Dps lacking DNA-binding activity, DpsΔ18. Staphylococcal nucleoid was clumped by E. coli Dps, but not by DpsΔ18. H2O2 stress assay indicated that Dps and DpsΔ18 restored the reduced susceptibility of S. aureus ΔmrgA. Thus, we concluded that the staphylococcal nucleoid clumping is dispensable for the Dps-mediated H2O2 resistance. In contrast, Dps was unable to complement S. aureus ΔmrgA in the UV resistance, suggesting the MrgA function that cannot be compensated for by E. coli Dps.

We analysed the response of the model bacterium Lactococcus lactis to abrupt depletion of glucose after several generations of exponential growth. Glucose depletion resulted in a drastic drop in the energy charge accompanied by an extremely low GTP level and an almost total arrest of protein synthesis. Strikingly, the cell prioritized the continued synthesis of a few proteins, of which the ribosomal dimerization factor YfiA was the most highly expressed. Transcriptome analysis showed no immediate decrease in total mRNA levels despite the lowered nucleotide pools and only marginally increased levels of the yfiA transcript. Severe up-regulation of genes in the FruR, CcpA, ArgR and AhrC regulons were consistent with a downshift in carbon and energy source. Based upon the results, we suggest that transcription proceeded long enough to record the transcriptome changes from activation of the FruR, CcpA, ArgR and AhrC regulons, while protein synthesis stopped due to an extremely low GTP concentration emerging a few minutes after glucose depletion. The yfiA deletion mutant exhibited a longer lag phase upon replenishment of glucose and a faster death rate after prolonged starvation supporting that YfiA-mediated ribosomal dimerization is important for keeping long-term starved cells viable and competent for growth initiation.

σ factors are single subunit general transcription factors that reversibly bind core RNA polymerase and mediate gene-specific transcription in bacteria. Previously, an atypical two-subunit σ factor was identified that activates transcription from a group of related promoters in Bacillus subtilis. Both of the subunits, named SigO and RsoA, share primary sequence similarity with the canonical σ70 family of σ factors and interact with each other and with RNA polymerase subunits. Here we show that the σ70 region 2.3-like segment of RsoA is unexpectedly sufficient for interaction with the amino-terminus of SigO and the β′ subunit. A mutational analysis of RsoA identified aromatic residues conserved amongst all RsoA homologues, and often amongst canonical σ factors, that are particularly important for the SigO–RsoA interaction. In a canonical σ factor, region 2.3 amino acids bind non-template strand DNA, trapping the promoter in a single-stranded state required for initiation of transcription. Accordingly, we speculate that RsoA region 2.3 protein-binding activity likely arose from a motif that, at least in its ancestral protein, participated in DNA-binding interactions.

OxyR proteins are LysR-type transcriptional regulators, which play an important role in responding to oxidative stress in bacteria. Azospirillum brasilense Sp7 harbours two copies of OxyR. The inactivation of the oxyR1, the gene organized divergently to ahpC in A. brasilense Sp7, led to an increased tolerance to alkyl hydroperoxides, which was corroborated by an increase in alkyl hydroperoxide reductase (AhpC) activity, enhanced expression of ahpC :lacZ fusion and increased synthesis of AhpC protein in the oxyR1::km mutant. The upstream region of ahpC promoter harboured a putative OxyR binding site, T-N11-A. Mutation of T, A or both in the T-N11-Amotif caused derepression of ahpC in A. brasilense suggesting that T-N11-A might be the binding site for a negative regulator. Retardation of the electrophoretic mobility of the T-N11-A motif harbouring oxyR1-ahpC intergenic DNA by recombinant OxyR1, under reducing as well as oxidizing conditions, indicated that OxyR1 acts as a negative regulator of ahpC in A. brasilense. Sequence of the promoter of ahpC, predicted on the basis of transcriptional start site, and an enhanced expression of ahpC: lacZ fusion in chrR2::km mutant background suggested that ahpC promoter was RpoE2 dependent. Thus, this study shows that in A. brasilense Sp7, ahpC expression is regulated negatively by OxyR1 but is regulated positively by RpoE2, an oxidative-stress-responsive sigma factor. It also shows that OxyR1 regulates the expression RpoE1, which is known to play an important role during photooxidative stress in A. brasilense.