- Volume 158, Issue 8, 2012

Geobacillus kaustophilus HTA426, a thermophilic Bacillus-related species, utilizes some inositol stereoisomers, including myo-, d-chiro- and scyllo-inositols (MI, DCI and SI), as sole carbon sources. Within its genome are three paralogous genes that possibly encode inositol dehydrogenase. These genes are located in tandem within a large gene cluster containing an almost complete set of iol genes homologous to genes involved in inositol catabolism in Bacillus subtilis. Each of the three plausible inositol dehydrogenases was purified as a His6-tag fusion. The enzymes exhibited thermophilic activity, each with its own characteristic specificity for the inositol stereoisomers and cofactors. Northern blot and primer extension analyses revealed that the three enzymes were encoded by the same 5 kb polycistronic transcript and were induced simultaneously in the presence of MI. HTA426 was subjected to ethyl methanesulfonate (EMS) mutagenesis to isolate a mutant strain, PS8, which was not able to utilize MI. In PS8, inositol dehydrogenase activity was abolished along with the 5 kb transcript, suggesting that any of the three enzymes supports MI-dependent growth. Analysis of metabolites in HTA426 cells grown in the presence of MI revealed that substantial amounts of DCI and SI appeared intracellularly during the stationary phase, while only MI was present in PS8 cells, suggesting that interconversion of inositol stereoisomers may involve these three enzymes.

In Azotobacter vinelandii the two-component GacS/GacA system is required for synthesis of polyhydroxybutyrate (PHB) and of the exopolysaccharide alginate. The RsmA protein was shown to interact with the alginate biosynthetic algD mRNA, acting as a translational repressor, and GacA was found to activate transcription of the rsmZ1 and rsmZ2 genes that encode small RNAs interacting with RsmA to counteract its repressor activity. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR. This study shows that GacA is required for transcription of one rsmY and seven rsmZ1–rsmZ7 genes present in the A. vinelandii genome, and that inactivation of rsmA results in increased PHB production. Transcriptional and translational phbR–gusA gene fusions were used to show that the gacA mutation negatively affected the expression of the phbR gene at the translational level. We also demonstrated an in vitro interaction of RsmA with RNAs corresponding to phbB and phbR mRNA leaders, and showed that the stability of phbR and phbB mRNAs is increased in the rsmA mutant. Taken together these results indicate that in A. vinelandii, RsmA post-transcriptionally represses the expression of PhbR.

Bacterial gene regulation is controlled by complex regulatory cascades which integrate input environmental signals and adapt specific and adequate output cellular responses. These complex networks are far from being elucidated, in particular in Pseudomonas aeruginosa. In the present study, we developed bacterial two-hybrid genome fragment libraries of the P. aeruginosa PAO1 strain to identify potential partners involved in the HptB/HsbR/HsbA pathway. This powerful tool, validated by the interaction previously described between HsbR and HsbA proteins, allowed us to demonstrate that the HsbR response regulator dimerizes through its PP2C/ATPase C-terminal effector domain, an observation further confirmed by pull-down experiments. This will also allow us to identify further new partners in this cascade.

The Min system plays an important role in ensuring that cell division occurs at mid-cell in rod-shaped bacteria. In Escherichia coli, pole-to-pole oscillation of the Min proteins specifically inhibits polar septation. This system also prevents polar division in Bacillus subtilis during vegetative growth; however, the Min proteins do not oscillate in this organism. The Min system of B. subtilis plays a distinct role during sporulation, a process of differentiation which begins with an asymmetrical cell division. Here, we show that oscillation of the E. coli Min proteins can be reproduced following their introduction into B. subtilis cells. Further, we present evidence that the oscillatory behaviour of the Min system inhibits sporulation. We propose that an alternative Min system mechanism avoiding oscillation is evolutionarily important because oscillation of the Min system is incompatible with efficient asymmetrical septum formation and sporulation.

The RecG enzyme, a superfamily 2 helicase, is present in nearly all bacteria. Here we report for the first time that the recG gene is also present in the genomes of most vascular plants as well as in green algae, but is not found in other eukaryotes or archaea. The precise function of RecG is poorly understood, although ample evidence shows that it plays critical roles in DNA repair, recombination and replication. We further demonstrate that Mycobacterium tuberculosis RecG (RecGMtb) DNA binding activity had a broad substrate specificity, whereas it only unwound branched-DNA substrates such as Holliday junctions (HJs), replication forks, D-loops and R-loops, with a strong preference for the HJ as a helicase substrate. In addition, RecGMtb preferentially bound relatively long (≥40 nt) ssDNA, exhibiting a higher affinity for the homopolymeric nucleotides poly(dT), poly(dG) and poly(dC) than for poly(dA). RecGMtb helicase activity was supported by hydrolysis of ATP or dATP in the presence of Mg2+, Mn2+, Cu2+ or Fe2+. Like its Escherichia coli orthologue, RecGMtb is also a strictly DNA-dependent ATPase.

In Enterococcus faecalis, production of guanosine tetraphosphate/guanosine pentaphosphate [(p)ppGpp], the effector molecule of the stringent response, is controlled by the bifunctional synthetase/hydrolase RelA and the monofunctional synthetase RelQ. Previously, the (p)ppGpp profiles of strains lacking relA, relQ or both genes indicated that RelA is the primary enzyme responsible for (p)ppGpp synthesis under stress conditions, while the contributions of RelQ to the stringent response and cell homeostasis remained elusive. Here, survival within the mouse-derived macrophage cell line J774A.1 and killing of Galleria mellonella supported initial evidence that virulence was attenuated in the (p)ppGpp0 ΔrelAΔrelQ strain but not in the ΔrelA or ΔrelQ strains. We performed, for the first time to our knowledge, global transcriptome analysis in a documented (p)ppGpp0 Gram-positive bacterium and provided the first insights into the role of a Gram-positive monofunctional (p)ppGpp synthetase in transcriptional regulation. Transcription profiling after mupirocin treatment confirmed that RelA is the major enzyme responsible for the (p)ppGpp-mediated transcriptional repression of genes associated with macromolecular biosynthesis, but also revealed that RelQ is required for full and timely stringent response induction. The delayed transcriptional response of ΔrelQ could not be correlated with reduced or slower production of (p)ppGpp, in part because RelA-dependent (p)ppGpp accumulation occurred very rapidly. Comparisons of the transcriptional responses of ΔrelA or ΔrelAΔrelQ strains with the parent strain under starvation conditions revealed upregulation of operons involved in energy metabolism in the (p)ppGpp0 strain. Thus, while ΔrelA and ΔrelAΔrelQ cannot use (p)ppGpp to sense and respond to stresses, fitness of ΔrelAΔrelQ may be further impaired due to an unbalanced metabolism.

The bacterial pathogen Vibrio cholerae requires colonizination of the human small intestine to cause cholera. The anaerobic and slightly acidic conditions predominating there enhance toxicity of low copper concentrations and create a selective environment for bacteria with evolved detoxifying mechanisms. We reported previously that the VCA0260, VCA0261 and VC2216 gene products were synthesized only in V. cholerae grown in microaerobiosis or anaerobiosis. Here we show that ORFs VCA0261 and VCA0260 are actually combined into a single gene encoding a 18.7 kDa protein. Bioinformatic analyses linked this protein and the VC2216 gene product to copper tolerance. Following the approach of predict-mutate and test, we describe for the first time, to our knowledge, the copper tolerance systems operating in V. cholerae. Copper susceptibility analyses of mutants in VCA0261–0260, VC2216 or in the putative copper-tolerance-related VC2215 (copA ATPase) and VC0974 (cueR), under aerobic and anaerobic growth, revealed that CopA represents the main tolerance system under both conditions. The VC2216-encoded periplasmic protein contributes to resistance only under anaerobiosis in a CopA-functional background. The locus tag VCA0261–0260 encodes a copper-inducible, CueR-dependent, periplasmic protein, which mediates tolerance in aerobiosis, but under anaerobiosis its role is only evident in CopA knock-out mutants. None of the genes involved in copper homeostasis were required for V. cholerae virulence or colonization in the mouse model. We conclude that copper tolerance in V. cholerae, which lacks orthologues of the periplasmic copper tolerance proteins CueO, CusCFBA and CueP, involves CopA and CueR proteins along with the periplasmic Cot (VCA0261–0260) and CopG (VC2216) V. cholerae homologues.

A single polar flagellum and motility are potential virulence factors of Vibrio vulnificus, a foodborne pathogen. In the present study, the functions of FlhF and regulatory characteristics of the flhF expression of V. vulnificus were investigated. A deletion mutation in flhF abolished motility, flagella formation and flagellin synthesis, and introduction of flhF in trans complemented the defects. The flhF mutant revealed decreased expression of the class III and IV flagella genes, indicating that FlhF is a key regulator for the flagellar biogenesis of V. vulnificus. The influence of global regulatory proteins on the expression of flhF was examined and SmcR, a LuxR homologue, was found to downregulate flhF expression at the transcriptional level. SmcR represses flhF expression only in the stationary phase of growth and exerts its effects by directly binding to the flhF promoter region. Finally, an SmcR binding site, centred at 22.5 bp upstream of the transcription start site, was identified by a DNase I protection assay. The combined results demonstrate that a quorum sensing master regulator SmcR influences the motility and flagellar biogenesis of V. vulnificus through modulating the expression of FlhF in a growth-phase-dependent manner.

Purine nucleotides are either synthesized de novo from 5-phosphoribosyl-1-pyrophosphate (PRPP) or salvaged from the environment. In Lactococcus lactis, transcription of the de novo synthesis operons, purCSQLF and purDEK, has genetically been shown to be activated by the PurR protein when bound to a conserved PurBox motif present on the DNA at a fixed distance from the promoter −10 element. PurR contains a PRPP-binding site, and activation occurs when the intracellular PRPP pool is high as a consequence of low exogenous purine nucleotide pools. By an iterative approach of bioinformatics searches and motif optimization, 21 PurR-regulated genes were identified and used in a redefinition of the PurBox consensus sequence. In the process a new motif, the double-PurBox, which is present in a number of promoters and contains two partly overlapping PurBox motifs, was established. Transcriptional fusions were used to analyse wild-type promoters and promoters with inactivating PurBox mutations to confirm the relevance of the PurBox motifs as PurR-binding sites. The promoters of several operons were shown to be devoid of any −35 sequence, and found to be completely dependent on PurR-mediated activation. This suggests that binding of the PurR protein to the PurBox takes over the role of the −35 sequence. The study has expanded the PurR regulon to include promoters in nucleotide metabolism, C1 compound metabolism, phosphonate transport, pyrophosphatase activity, (p)ppGpp metabolism, and translation-related functions. Of special interest is the presence of PurBox motifs in rrn promoters, suggesting a novel connection between nucleotide availability and the translational machinery.

We determined the ability of Vibrio parahaemolyticus to utilize enterobactin (Ent) as a xenosiderophore. Homology searches of the V. parahaemolyticus genomic sequence revealed the presence of genes that are homologous to the V. cholerae ferric Ent utilization genes, which consist of the iron-repressible outer-membrane protein genes irgA and vctA, and the ATP-binding cassette transport system operon vctPDGC. Moreover, the irgB and vctR genes, which encode transcriptional regulators, were also found immediately upstream of irgA and vctA, respectively. Growth assays of V. parahaemolyticus indicated that both irgA and vctA mutants grew well in the presence of Ent under iron-limiting conditions, whereas both the irgA/vctA double mutant and the vctPDGC mutant barely grew under the same conditions. In addition, growth assays of three isogenic tonB mutants demonstrated that the TonB2 system, and to a lesser extent the TonB1 system, can provide energy for both IrgA and VctA to transport ferric Ent. SDS-PAGE analysis showed that expression of both IrgA and VctA was enhanced by the presence of Ent. Complementation of the irgB and vctR mutants with their respective genes resulted in the increased expression of IrgA and VctA, respectively. Finally, reverse transcriptase-quantitative PCR revealed that transcription of the Ent utilization system genes is iron-regulated, and that transcription of irgA and vctA under iron-limiting conditions is further activated by proteins encoded by irgB and vctR, respectively, together with Ent.

The application of toxic triphenylmethane dyes such as crystal violet (CV) in various industrial processes leads to large amounts of dye-contaminated sludges that need to be detoxified. Specific bacteria residing in wastewater treatment plants (WWTPs) are able to degrade triphenylmethane dyes. The objective of this work was to gain insights into the genetic background of bacterial strains capable of CV degradation. Three bacterial strains isolated from a municipal WWTP harboured IncP-1β plasmids mediating resistance to and decolorization of CV. These isolates were assigned to the genera Comamonas and Delftia. The CV-resistance plasmid pKV29 from Delftia sp. KV29 was completely sequenced. In addition, nucleotide sequences of the accessory regions involved in conferring CV resistance were determined for plasmids pKV11 and pKV36 from the other two isolates. Plasmid pKV29 contains typical IncP-1β backbone modules that are highly similar to those of previously sequenced IncP-1β plasmids that confer antibiotic resistance, degradative capabilities or mercury resistance. The accessory regions located between the conjugative transfer (tra) and mating pair formation modules (trb) of all three plasmids analysed share common modules and include a triphenylmethane reductase gene, tmr, that is responsible for decolorization of CV. Moreover, these accessory regions encode other enzymes that are dispensable for CV degradation and hence are involved in so-far-unknown metabolic pathways. Analysis of plasmid-mediated degradation of CV in Escherichia coli by ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight MS revealed that leuco crystal violet was the first degradation product. Michler’s ketone and 4-dimethylaminobenzaldehyde appeared as secondary degradation metabolites. Enzymes encoded in the E. coli chromosome seem to be responsible for cleavage of leuco crystal violet. Plasmid-mediated degradation of triphenylmethane dyes such as CV is an option for the biotechnological treatment of sludges contaminated with these dyes.

Streptococcus gordonii, a normal inhabitant of the human oral cavity, is a potential live vaccine vehicle. Several pathogen-associated molecular patterns from S. gordonii that are recognized by antigen-presenting cells have recently been identified. In this study, we have identified that the cell-wall-anchored proteins SspA and SspB are immunostimulatory components of S. gordonii. SspA and SspB are members of the antigen I/II family of proteins widely expressed by viridans oral streptococci. The results showed that the mutant (OB219) lacking SspA and SspB had a reduced ability to induce cytokine/chemokine production in epithelial cells and bone-marrow-derived dendritic cells as compared with the parent strain (DL1). Purified SspA induced interleukin-6 and monocyte chemotatic protein-1 production from human lung epithelial A549 cells. The induction could be inhibited by a function-blocking anti-β1 integrin mAb and the purified SspA could bind to β1 integrin precoated on microtitre plates, suggesting that the induction was effected by SspA–β1 integrin interactions. The role of SspA and SspB in innate immunity was further demonstrated in a mouse intranasal challenge experiment, which showed that the clearance of OB219, the recruitment of neutrophils (as indicated by myeloperoxidase activity), and chemokine and cytokine production in the lungs of OB219-inoculated mice were delayed or reduced as compared with the DL1-inoculated mice. In addition to the above, S. gordonii OB219 was more sensitive to polymyxin, nisin and histatin-5 than DL1, suggesting that SspA and SspB also play a role in susceptibility to cationic antimicrobial peptides. Collectively, the results indicate that SspA and SspB are immunostimulatory components of S. gordonii and play an important role in modulating the host’s innate immunity.

Although much is known about the signals that trigger transcription of virulence genes in plant pathogens, their prevalence and timing during infection are still unknown. In this work, we address these questions by analysing expression of the main pathogenicity determinants in the bacterial pathogen Ralstonia solanacearum. We set up a quantitative, non-invasive luminescent reporter to monitor in planta transcription from single promoters in the bacterial chromosome. We show that the new reporter provides a real-time measure of promoter output in vivo – either after re-isolation of pathogens from infected plants or directly in situ – and confirm that the promoter controlling exopolysaccharide (EPS) synthesis is active in bacteria growing in the xylem. We also provide evidence that hrpB, the master regulator of type III secretion system (T3SS) genes, is transcribed in symptomatic plants. Quantitative RT-PCR assays demonstrate that hrpB and type III effector transcripts are abundant at late stages of plant infection, suggesting that their function is required throughout disease. Our results challenge the widespread view in R. solanacearum pathogenicity that the T3SS, and thus injection of effector proteins, is only active to manipulate plant defences at the first stages of infection, and that its expression is turned down when bacteria reach high cell densities and EPS synthesis starts.

Haemophilus parasuis is a porcine respiratory pathogen, well known as the aetiological agent of Glässer’s disease. H. parasuis comprises strains of different virulence, but the virulence factors of this bacterium are not well defined. A neuraminidase activity has been previously detected in H. parasuis, but the role of sialylation in the virulence of this bacterium has not been studied. To explore the relationship between sialic acid (Neu5Ac) and virulence, we assessed the distribution of genes involved in sialic acid metabolism in 21 H. parasuis strains from different clinical origins (including nasal and systemic isolates). The neuraminidase gene nanH, together with CMP-Neu5Ac synthetase and sialyltransferase genes neuA, siaB and lsgB, were included in the study. Neuraminidase activity was found to be common in H. parasuis isolates, and the nanH gene from 12 isolates was expressed in Escherichia coli and further characterized. Sequence analysis showed that the NanH predicted protein contained the motifs characteristic of the catalytic site of sialidases. While an association between the presence of nanH and the different origins of the strains was not detected, the lsgB gene was predominantly present in the systemic isolates, and was not amplified from any of the nasal isolates tested. Analysis of the lipooligosaccharide (LOS) from reference strains Nagasaki (virulent, lsgB +) and SW114 (non-virulent, lsgB −) showed the presence of sialic acid in the LOS from the Nagasaki strain, supporting the role of sialylation in the virulence of this bacterial pathogen. Further studies are needed to clarify the role of sialic acid in the pathogenicity of H. parasuis.

The virulence profiles of Pseudomonas aeruginosa quorum-sensing (QS) mutants were assessed in Drosophila melanogaster feeding and nicking infection models. Functional RhlIR and LasIR QS systems were required for killing in the fly feeding infection model but were not essential in the fly nicking infection model. Mixed infections between PAO1 and strains harbouring mutations in lasR, rhlI and lasI rhlI resulted in increased lethality in the fly feeding model compared with either isolate alone. These results suggested that the parental strain could cooperate with QS mutants in the Drosophila feeding infection model. Finally, the mixed infection between PAO1 and an rhlR mutant resulted in spiteful behaviour and reduced pathogenicity of the mixed culture.

Streptococcus mutans, a causative agent of dental caries in humans, adapts to changing environmental conditions, such as pH, in order to survive and cause disease in the oral cavity. Previously, we have shown that S. mutans increases the proportion of monounsaturated membrane fatty acids as part of its acid-adaptive strategy. Membrane lipids function as carriers of membrane fatty acids and therefore it was hypothesized that lipid backbones themselves could participate in the acid adaptation process. Lipids have been shown to protect other bacterial species from rapid changes in their environment, such as shifts in osmolality and the need for long-term survival. In the present study, we have determined the contribution of cardiolipin (CL) to acid resistance in S. mutans. Two ORFs have been identified in the S. mutans genome that encode presumptive synthetic enzymes for the acidic phospholipids: phosphatidylglycerol (PG) synthase (pgsA, SMU.2151c) and CL synthase (cls, SMU.988), which is responsible for condensing two molecules of PG to create CL. A deletion mutant of the presumptive cls gene was created using PCR-mediated cloning; however, attempts to delete pgsA were unsuccessful, indicating that pgsA may be essential. Loss of the presumptive cls gene resulted in the inability of the mutant strain to produce CL, indicating that SMU.988 encodes CL synthase. The defect in cls rendered the mutant acid sensitive, indicating that CL is required for acid adaptation in S. mutans. Addition of exogenous CL to the mutant strain alleviated acid sensitivity. MS indicated that S. mutans could assimilate exogenous CL into the membrane, halting endogenous CL incorporation. This phenomenon was not due to repression, as a cls gene transcriptional reporter fusion exhibited elevated activity when cells were supplemented with exogenous CL. Lipid analysis, via MS, indicated that CL is a reservoir for monounsaturated fatty acids in S. mutans. We demonstrated that the cls mutant exhibits elevated F-ATPase activity but it is nevertheless unable to maintain the normal membrane proton gradient, indicating cytoplasmic acidification. We conclude that the control of lipid backbone synthesis is part of the acid-adaptive repertoire of S. mutans.