- Volume 155, Issue 11, 2009

Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.

The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB-like genes, mreB, mbl and mreBH, the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreBB. licheniformis (mreBBl ) or mreBC. perfringens (mreBCp ) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCDBl could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod-shaped cells. In contrast, expression of mreBCDCp was not sufficient to confer a rod shape to B. subtilis ΔmreBCD, indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis.

OmpR has been demonstrated to negatively regulate the expression of the flagellar master operon flhDC in a wide variety of bacterial species. Here we report the positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis. A σ 70-dependent promoter was identified by primer extension analysis and an active region with two conserved OmpR-binding sites around the flhDC promoter was confirmed. To confirm the regulation of flhDC expression by OmpR, flhDC as well as the downstream flagellar genes fliA, flgD, flgA, flgM, fliC and flaA were fused to lacZ, and decreased expression of all these genes in an ompR mutant (ΔompR) was detected. Furthermore, ΔompR was defective in bacterial motility and flagella synthesis. This defect was due to the low level of expression of flhDC in ΔompR since overproduction of FlhDC in ΔompR restored bacterial motility. The importance of two conserved OmpR-binding sites around the flhDC promoter region in the regulation of flhDC expression by OmpR was demonstrated by the fact that mutation of either one or both sites significantly decreased the promoter activity in the wild-type but not in ΔompR. The binding of OmpR to these two sites was also demonstrated by DNA mobility shift assay. The possible mechanism underlying this positive regulation in Y. pseudotuberculosis is discussed. To our knowledge, this is the first report to demonstrate that OmpR positively regulates flhDC expression.

DNA replication in eukaryotes is a highly conserved process marked by the licensing of multiple origins, with pre-replication complex assembly in G1 phase, followed by the onset of replication at these origins in S phase. The two strands replicate by different mechanisms, and DNA synthesis is brought about by the activity of the replicative DNA polymerases Pol δ and Pol ϵ. Proliferating cell nuclear antigen (PCNA) augments the processivity of these polymerases by serving as a DNA sliding clamp protein. This study reports the cloning of PCNA from the protozoan Leishmania donovani, which is the causative agent of the systemic disease visceral leishmaniasis. PCNA was demonstrated to be robustly expressed in actively proliferating L. donovani promastigotes. We found that the protein was present primarily in the nucleus throughout the cell cycle, and it was found in both proliferating procyclic and metacyclic promastigotes. However, levels of expression of PCNA varied through cell cycle progression, with maximum expression evident in G1 and S phases. The subnuclear pattern of expression of PCNA differed in different stages of the cell cycle; it formed distinct subnuclear foci in S phase, while it was distributed in a more diffuse pattern in G2/M phase and post-mitotic phase cells. These subnuclear foci are the sites of active DNA replication, suggesting that replication factories exist in Leishmania, as they do in higher eukaryotes, thus opening avenues for investigating other Leishmania proteins that are involved in DNA replication as part of these replication factories.

Incidences of bacterial foodborne illness caused by ingestion of fresh produce are rising. Instead of this being due to incidental contamination, the animal pathogen Salmonella enterica utilizes specific molecular mechanisms to attach to and colonize plants. This work characterizes two S. enterica genes of unknown function: a putative periplasmic protein, STM0278, and a putative protein with a hydrolase in the C-terminus, STM0650. STM0278 and STM0650 are important for seedling colonization but appear to have different roles during the process of colonization. Mutants of either STM0278 or STM0650 showed reduced colonization of alfalfa seedlings at 24 h, and the STM0278 mutant also showed reduced colonization at 48 h. Both genes were expressed in planta at 4 h following inoculation of 3-day-old seedlings and at 72 h after seed inoculation. This suggests that the role of STM0650 in seedling colonization is less important later in the process or is duplicated by other mechanisms. Mutants of STM0278 and STM0650 were defective in swarming. The STM0278 mutant failed to swarm in 24 h, while swarming of the STM0650 mutant was delayed. Addition of surfactant restored swarming of the STM0278 mutant, suggesting that STM0278 is involved in surfactant or osmotic agent production or deployment. Alfalfa seed exudates as the sole nutrient source were capable of perpetuating S. enterica swarming. Sequence analysis revealed sequences homologous to STM0278 and STM0650 in plant-associated bacteria, but none in Escherichia coli. Phylogenetic analysis of STM0650 showed similar sequences from diverse classes of plant-associated bacteria. Bacteria that preferentially colonize roots, including S. enterica, may use a similar hydrolase for swarming or biofilm production on plants. Multicellular behaviours by S. enterica appear central to plant colonization. S. enterica genes involved in plant colonization and survival outside of a host are most likely among the ‘function unknown’ genes of this bacterium.

Mycoplasmas belonging to the Mycoplasma mycoides phylogenetic cluster are all important ruminant pathogens that are genetically closely related but differ in terms of severity and prevalence of the associated diseases. They are distributed among six taxa, the description of which has recently been amended. In the present study, DNA fragments that diverge between the type strains of three taxa were enriched using suppression subtractive hybridization. Of the three taxa, two were representative of the well-established species M. mycoides and M. capricolum, while the third one, Mycoplasma sp. bovine group 7 (Mbg7), has only recently been proposed as a separate species, Mycoplasma leachii. Specific DNA fragments were further characterized by sequencing and used as markers to assess the genetic diversity within and between taxa. The data indicate that the selected markers are unequally distributed within their own taxon but also across taxa. The patterns observed suggest the occurrence of a genetic continuum of strains within the M. mycoides cluster that may compromise the boundaries between taxa and, in turn, diagnosis outcomes. For Mbg7, the overall nature and distribution of the markers indicate a rather homogeneous group that is distinct from the M. capricolum and M. mycoides species and might be considered as a genomic chimera between these two species.

When grown with vaporized alkylphenols such as p-cresol as the sole carbon and energy source, several isolated Rhodococcus strains formed growth structures like miniature mushrooms, termed here specialized aerial architectures (SAA), that reached sizes of up to 0.8 mm in height. Microscopic examination allowed us to view the distinct developmental stages during the formation of SAA from a selected strain, Rhodococcus sp. KL96. Initially, mounds consisting of long rod cells arose from a lawn of cells, and then highly branched structures were formed from the mounds. During the secondary stage of development, branching began after long rod cells grew outward and twisted longitudinally, serving as growth points, and the cells at the base of the mound became short rods that supported upward growth. Cells in the highly fluffy structures were eventually converted, via reductive division, into structures that resembled cocci, with a diameter of approximately 0.5 μm, that were arranged in chains. Most cells inside the SAA underwent a phase variation in order to form wrinkled colonies from cells that originally formed smooth colonies. Approximately 2 months was needed for complete development of the SAA, and viable cells were recovered from SAA that were incubated for more than a year. An extracellular polymeric matrix layer and lipid bodies appeared to play an important role in structural integrity and as a metabolic energy source, respectively. To our knowledge, similar formation of aerial structures for the purpose of substrate utilization has not been reported previously for Gram-positive bacteria.

Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in the presence of low concentrations of oxygen. Microarray analysis revealed alteration in gene expression in the presence of 6 % oxygen. During the exponential growth phase, 96 genes were upregulated and 79 genes were downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative stress protection were upregulated, including alkyl hydroperoxide reductase (ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant changes in gene expression of proteins that mediate oxidative metabolism, such as cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The expression of genes encoding formate uptake transporter (PG0209) and formate tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that formate metabolism plays a major role under aerobic conditions. The concomitant reduction of expression of a gene encoding the lactate transporter PG1340 suggests decreased utilization of this nutrient. The concentrations of both formate and lactate were assessed in culture supernatants and cells, and they were in agreement with the results obtained at the transcriptional level. Also, genes encoding gingipain protease secretion/maturation regulator (porR) and protease transporter (porT) had reduced expression in the presence of oxygen, which also correlated with reduced protease activities under aerobic conditions. In addition, metal transport was affected, and while iron-uptake genes such as the genes encoding the haemin uptake locus (hmu) were downregulated, expression of manganese transporter genes, such as feoB2, was elevated in the presence of oxygen. Finally, genes encoding putative regulatory proteins such as extracellular function (ECF) sigma factors as well as small proteins had elevated expression levels in the presence of oxygen. As P. gingivalis is distantly related to the well-studied model organism Escherichia coli, results from our work may provide further understanding of oxygen metabolism and protection in other related bacteria belonging to the phylum Bacteroidetes.

Polymorphonuclear neutrophilic leukocytes (PMNs) play a central role in innate immunity, where they dominate the response to infections, in particular in the cystic fibrosis lung. PMNs are phagocytic cells that produce a wide range of antimicrobial agents aimed at killing invading bacteria. However, the opportunistic pathogen Pseudomonas aeruginosa can evade destruction by PMNs and thus cause persistent infections. In this study, we show that biofilm cells of P. aeruginosa recognize the presence of attracted PMNs and direct this information to their fellow bacteria through the quorum sensing (QS) signalling system. The bacteria respond to the presence of PMNs by upregulating synthesis of a number of QS-controlled virulence determinants including rhamnolipids, all of which are able to cripple and eliminate cells of the host defence. Our in vitro and in vivo analyses support a ‘launch a shield’ model by which rhamnolipids surround the biofilm bacteria and on contact eliminate incoming PMNs. Our data strengthen the view that cross-kingdom communication plays a key role in P. aeruginosa recognition and evasion of the host defence.

muxA-muxB-muxC-opmB (formerly PA2528-PA2527-PA2526-opmB), encoding a putative resistance nodulation cell division (RND)-type multidrug efflux pump system, was cloned from Pseudomonas aeruginosa PAO1. Introduction of muxABC-opmB into P. aeruginosa YM64, a drug-hypersusceptible strain, led to elevated MICs of aztreonam, macrolides, novobiocin and tetracycline. Since muxB and muxC, both of which encode RND components, were essential for function, MuxABC-OpmB is thought to be a drug efflux pump with four components. One novobiocin-resistant mutant, PMX725, isolated from P. aeruginosa PMX7 showed elevated resistance not only to novobiocin but also to aztreonam, macrolides and tetracycline. Increased mRNA expression of muxABC-opmB was observed in the mutant PMX725 compared with the parental strain. Sequencing analysis revealed that a single-nucleotide insertion had occurred in the deduced promoter region for muxABC-opmB in PMX725. In this study, we have characterized the last RND-type multidrug efflux pump predicted from the genome sequence in P. aeruginosa.

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout ΔahyRI mutant. Protease production was substantially reduced in the ΔahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the ΔahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the ΔahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the ΔahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the ΔahyRI/ahyR+ complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

Isolated in vitro more than half a century ago, the H37Rv strain of Mycobacterium tuberculosis still remains the strain of choice for the majority of laboratories conducting in vivo studies of TB pathogenesis. In this report we reveal that H37Rv is highly prone to losing the ability to synthesize the cell wall lipid phthiocerol dimycocerosate (PDIM) during extended periods of in vitro culture. In addition, H37Rv stocks that have been held in vitro for even a short length of time should be thought of as a heterogeneous population of PDIM-positive and PDIM-negative cell types. We demonstrate that after weekly subculture of PDIM-positive isolates over a period of 20 weeks, the proportion of PDIM-negative cells rises above 30 %. That PDIM biosynthesis is negatively selected in vitro is evident from the broad range of mutation types we observe within cultures originating from a single PDIM-positive parental clone. Moreover, the appearance of these multiple mutation types coupled with an enhanced growth rate of PDIM-negative bacteria ensures that ‘PDIM-less’ clones rapidly dominate in vitro cultures. It has been known for almost a decade that strains of M. tuberculosis that lack PDIM are severely attenuated during in vivo infection. Therefore, the loss of PDIM raises a very serious issue in regard to the interpretation of putative virulence factors where heterogeneous parental cultures are potentially being compared in vivo to recombinant clones isolated within a PDIM-negative background. It is essential that researchers undertaking in vivo virulence studies confirm the presence of PDIM within all recombinant clones and the parental strains they are derived from.

Shiga-toxigenic Escherichia coli (STEC) colonizing the bowel are exposed to a variety of short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, produced by gut microflora. However, the total concentrations and relative amounts of SCFAs in the lumen vary with intestinal niche. Here we report that conditions simulating SCFA concentrations present in the human gut trigger expression of the iha gene, which encodes an adherence-conferring outer-membrane protein of pathogenic E. coli. We show that growth under conditions simulating colonic, but not ileal, SCFA concentrations increases iha expression in three tested STEC strains, with the strongest expression detected in LEE-negative STEC O113:H21 strain 98NK2. Expression of iha is known to be subject to Fur-mediated iron repression in O157:H7 STEC, and the same occurs in 98NK2. However, exogenous iron did not repress iha expression in the presence of colonic SCFAs in either 98NK2 or the O157:H7 strain EDL933. Moreover, exposure to the iron chelator 2,2′-dipyridyl caused no further enhancement of iha expression over that induced by colonic SCFAs. These findings indicate that SCFAs regulate iha expression in STEC independently of iron. Increased expression of iha under colonic but not ileal SCFA conditions possibly may contribute to preferential colonization of the human colon by STEC.