Publications

UNLABELLED: Natural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching. IMPORTANCE: In favorable environments, species may face space and nutrient limits due to overcrowding. Bacteria provide an excellent model for analyzing principles underlying overcrowding and regulation of density in nature, since their population dynamics can be easily and accurately assessed under controlled conditions. We describe a newly discovered mechanism for survival of a bacterial population during overcrowding. When competing with sibling colonies, Paenibacillus dendritiformis produces a lethal protein (Slf) that kills cells at the interface of encroaching colonies. Slf also induces a small proportion of the cells to switch from motile, rod-shaped cells to nonmotile, Slf-resistant, vegetative cocci. When crowding is reduced and nutrients are no longer limiting, the bacteria produce a signal that induces cocci to switch back to motile rods, allowing the population to spread. Genes encoding components of this phenotypic switching pathway are widespread among bacterial species, suggesting that this survival mechanism is not unique to P. dendritiformis.

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron. It transports the catechol siderophores vibriobactin, which it synthesizes and secretes, and enterobactin. These siderophores are transported across the inner membrane by one of two periplasmic binding protein-dependent ABC transporters, VctPDGC or ViuPDGC. We show here that one of these inner membrane transport systems, VctPDGC, also promotes iron acquisition in the absence of siderophores. Plasmids carrying the vctPDGC genes stimulated growth in both rich and minimal media of a Shigella flexneri mutant that produces no siderophores. vctPDGC also stimulated the growth of an Escherichia coli enterobactin biosynthetic mutant in low iron medium, and this effect did not require feoB, tonB or aroB. A tyrosine to phenylalanine substitution in the periplasmic binding protein VctP did not alter enterobactin transport, but eliminated growth stimulation in the absence of a siderophore. These data suggest that the VctPDGC system has the capacity to transport both catechol siderophores and a siderophore-free iron ligand. We also show that VctPDGC is the previously unidentified siderophore-independent iron transporter in V. cholerae, and this appears to complete the list of iron transport systems in V. cholerae.

Shigella flexneri is a facultative intracellular pathogen that invades and disrupts the colonic epithelium. In order to thrive in the host, S. flexneri must adapt to environmental conditions in the gut and within the eukaryotic cytosol, including variability in the available carbon sources and other nutrients. We examined the roles of the carbon consumption regulators CsrA and Cra in a cell culture model of S. flexneri virulence. CsrA is an activator of glycolysis and a repressor of gluconeogenesis, and a csrA mutant had decreased attachment and invasion of cultured cells. Conversely, Cra represses glycolysis and activates gluconeogenesis, and the cra mutant had an increase in both attachment and invasion compared to the wild-type strain. Both mutants were defective in plaque formation. The importance of the glycolytic pathway in invasion and plaque formation was confirmed by testing the effect of a mutation in the glycolysis gene pfkA. The pfkA mutant was noninvasive and had cell surface alterations as indicated by decreased sensitivity to SDS and an altered lipopolysaccharide profile. The loss of invasion by the csrA and pfkA mutants was due to decreased expression of the S. flexneri virulence factor regulators virF and virB, resulting in decreased production of Shigella invasion plasmid antigens (Ipa). These data indicate that regulation of carbon metabolism and expression of the glycolysis gene pfkA are critical for synthesis of the virulence gene regulators VirF and VirB, and both the glycolytic and gluconeogenic pathways influence steps in S. flexneri invasion and plaque formation.

Sibling Paenibacillus dendritiformis bacterial colonies grown on low-nutrient agar medium mutually inhibit growth through secretion of a lethal factor. Analysis of secretions reveals the presence of subtilisin (a protease) and a 12 kDa protein, termed sibling lethal factor (Slf). Purified subtilisin promotes the growth and expansion of P. dendritiformis colonies, whereas Slf is lethal and lyses P. dendritiformis cells in culture. Slf is encoded by a gene belonging to a large family of bacterial genes of unknown function, and the gene is predicted to encode a protein of approximately 20 kDa, termed dendritiformis sibling bacteriocin. The 20 kDa recombinant protein was produced and found to be inactive, but exposure to subtilisin resulted in cleavage to the active, 12 kDa form. The experimental results, combined with mathematical modeling, show that subtilisin serves to regulate growth of the colony. Below a threshold concentration, subtilisin promotes colony growth and expansion. However, once it exceeds a threshold, as occurs at the interface between competing colonies, Slf is then secreted into the medium to rapidly reduce cell density by lysis of the bacterial cells. The presence of genes encoding homologs of dendritiformis sibling bacteriocin in other bacterial species suggests that this mechanism for self-regulation of colony growth might not be limited to P. dendritiformis.

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.

The sit-encoded iron transport system is present within pathogenicity islands in all Shigella spp. and some pathogenic Escherichia coli strains. The islands contain numerous insertion elements and sequences with homology to bacteriophage genes. The Shigella flexneri sit genes can be lost as a result of deletion within the island. The formation of deletions was dependent upon RecA and occurred at relatively high frequency. This suggests that the sit region is inherently unstable, yet sit genes are maintained in all of the clinical isolates tested. Characterization of the sitABCD genes in S. flexneri indicates that they encode a ferrous iron transport system, although the genes are induced aerobically. The sit genes provide a competitive advantage to S. flexneri growing within epithelial cells, and a sitA mutant is outcompeted by the wild type in cultured epithelial cells. The Sit system is also required for virulence in a mouse lung model. The sitA mutant was able to infect the mice and induce a protective immune response but was avirulent compared to its wild-type parent strain.

Most research on growing bacterial colonies on agar plates has concerned the effect of genetic or morphotype variation. Some studies have indicated that there is a correlation between microscopic bacterial motion and macroscopic colonial expansion, especially for swarming strains, but no measurements have been obtained for a single strain to relate the microscopic scale to the macroscopic scale. We examined here a single strain (Paenibacillus dendritiformis type T; tip splitting) to determine both the macroscopic growth of colonies and the microscopic bacterial motion within the colonies. Our multiscale measurements for a variety of growth conditions revealed that motion on the microscopic scale and colonial growth are largely independent. Instead, the growth of the colony is strongly affected by the availability of a surfactant that reduces surface tension.

Bacterial-sensing circuits may be triggered by molecules originating from the environment (e.g., nutrients and chemoattractants). Bacteria also actively probe the environment for information by releasing molecular probes to measure conditions beyond the cell surface: a process known as telesensing. Perceiving the environment beyond is achieved by sensing environmentally induced changes in those probes, as occurs when a siderophore chelates an iron atom or a quorum-sensing signal is inactivated by a specific enzyme or adsorbent. This information, captured by chemical and physical changes induced in specifically produced molecules transiting through the environment, enables bacteria to mount a contextually appropriate response.

Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.

Invasion and plaque formation in epithelial monolayers are routinely used to assess the virulence of Shigella flexneri, a causative agent of dysentery. A modified plaque assay was developed to identify factors contributing to the virulence of S. flexneri under the anaerobic conditions present in the colon. This assay demonstrated the importance of the ferrous iron transport system Feo, as well as the global transcription factors Fur, ArcA, and Fnr, for Shigella plaque formation in anoxic environments. Transcriptional analyses of S. flexneri iron transport genes indicated that anaerobic conditions activated feoABC while repressing genes encoding two other iron transport systems, the ABC transporter Sit and the Iuc/Iut aerobactin siderophore synthesis and transport system. The anaerobic transcription factors ArcA and Fnr activated expression of feoABC, while ArcA repressed iucABCD iutA. Transcription of fur, encoding the iron-responsive transcriptional repressor of bacterial iron acquisition, was also repressed anaerobically in an ArcA-dependent manner.

Clinical isolates of enterohaemorrhagic Escherichia coli, both O157 and non-O157 serotypes, were investigated for siderophore production, for growth promotion by haem and esculetin in iron-restricted conditions, for production of enterohaemolysin and esculin hydrolase, and for the presence of the chuA and ehx genes by PCR. As expected, all the strains produced enterobactin, but the prevalence of other factors varied among the serovars tested. None of the O157 and O26 strains produced aerobactin or "colibactin", whereas among other enterohaemorrhagic E. coli non-O157 serovars the frequencies of aerobactin and "colibactin" production were similar to those of commensal E. coli strains. The ability to use ferric esculetin for growth in iron-limited media was markedly more prevalent among non-O157 serovars and less prevalent among O157 strains compared with commensal E. coli strains. Almost all O157, O26 and O103 strains expressed enterohaemolysin, compared with only 50% of other non-O157 strains. Similarly, almost all O157 and O26 strains utilised haem as a host iron source; the frequency of haem use by other non-O157 strains was generally lower and variable among serovars, such that none of the O103:H2 isolates tested used haem as an iron source. The gene chuA, which encodes the haem transport protein ChuA and which is prevalent in O157:H7 strains, was only rarely noted among non-O157 serovars of enterohaemorrhagic E. coli, even among isolates that could use haem as an iron source. Overall our data demonstrate that O157:H7 and non-O157 serovars, in particular O26:H(-)/H11 and O103:H2, use distinctly different strategies for obtaining iron, and suggest two evolutionary distinct lines of enterhaemorrhagic E. coli.

A Shigella flexneri degP mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the degP mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The degP mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the degP phenotype was not limited to IcsA surface presentation. The plaque defect of the degP mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA. S. flexneri skp and surA mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by S. flexneri.

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.

Regulation of bacterial gene expression by small RNA (sRNA) molecules is an increasingly recognized phenomenon but one that is not yet fully understood. We show that the sRNA RyhB suppresses several virulence-associated phenotypes of Shigella dysenteriae, a causative agent of bacillary dysentery in humans. The virulence genes repressed by S. dysenteriae RyhB include those encoding the type III secretion apparatus, its secreted effectors, and specific chaperones. Suppression of Shigella virulence occurs via RyhB-dependent repression of the transcriptional activator VirB, leading to reduced expression of genes within the VirB regulon. Efficient repression of virB is mediated by a single-stranded region of RyhB that is distinct from the region required for repression of Shigella sodB. Regulation of virB by RyhB implicates iron as an environmental factor contributing to the complex regulation of Shigella virulence determinants.

Vibrio cholerae has multiple iron acquisition systems, including TonB-dependent transport of heme and of the catechol siderophore vibriobactin. Strains defective in both of these systems grow well in laboratory media and in the infant mouse intestine, indicating the presence of additional iron acquisition systems. Previously uncharacterized potential iron transport systems, including a homologue of the ferrous transporter Feo and a periplasmic binding protein-dependent ATP binding cassette (ABC) transport system, termed Fbp, were identified in the V. cholerae genome sequence. Clones encoding either the Feo or the Fbp system exhibited characteristics of iron transporters: both repressed the expression of lacZ cloned under the control of a Fur-regulated promoter in Escherichia coli and also conferred growth on a Shigella flexneri mutant that has a severe defect in iron transport. Two other ABC transporters were also evaluated but were negative by these assays. Transport of radioactive iron by the Feo system into the S. flexneri iron transport mutant was stimulated by the reducing agent ascorbate, consistent with Feo functioning as a ferrous transporter. Conversely, ascorbate inhibited transport by the Fbp system, suggesting that it transports ferric iron. The growth of V. cholerae strains carrying mutations in one or more of the potential iron transport genes indicated that both Feo and Fbp contribute to iron acquisition. However, a mutant defective in the vibriobactin, Fbp, and Feo systems was not attenuated in a suckling mouse model, suggesting that at least one other iron transport system can be used in vivo.

Hfq plays an important role in cellular physiology by regulating the expression of several genes. Hfq synthesis in Escherichia coli is subject to auto-repression at translational level. Studies with Shigella flexneri show that hfq transcription is regulated by a pleiotropic regulator, DksA. Comparison of gene expression profiles of wild type and dksA mutant S. flexneri determined that hfq expression was reduced in the dksA mutant. As DksA is required for stress resistance and plaque formation in cultured cell monolayers, a measure of virulence, we assessed the role of Hfq in the dksA virulence phenotype. Expression of hfq in the dksA mutant restored plaque formation, and an hfq mutant failed to form plaques. Thus, DksA plays a role in regulating hfq gene expression and this regulation is important for S. flexneri virulence. In an in vitro transcription assay, addition of DksA increased transcription of hfq and this effect was greatest with one of the known hfq promoters. Addition of ppGpp, a stringent response molecule, along with DksA in the in vitro transcription assay resulted in a further increase in transcription of hfq, indicating that DksA is required for maximal transcription of hfq during both exponential and stringent response growth conditions.

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.

Most inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.

Vibrio cholerae encodes a small RNA with homology to Escherichia coli RyhB. Like E. coli ryhB, V. cholerae ryhB is negatively regulated by iron and Fur and is required for repression of genes encoding the superoxide dismutase SodB and multiple tricarboxylic acid cycle enzymes. However, V. cholerae RyhB is considerably longer (>200 nucleotides) than the E. coli RNA (90 nucleotides), and it regulates the expression of a variety of genes that are not known to be regulated by RyhB in E. coli, including genes involved in motility, chemotaxis, and biofilm formation. A mutant with a deletion in ryhB had reduced chemotactic motility in low-iron medium and was unable to form wild-type biofilms. The defect in biofilm formation was suppressed by growing the mutant in the presence of excess iron or succinate. The wild-type strain showed reduced biofilm formation in iron-deficient medium, further supporting a role for iron in normal biofilm formation. The ryhB mutant was not defective for colonization in a mouse model and appeared to be at a slight advantage when competing with the wild-type parental strain. Other genes whose expression was influenced by RyhB included those encoding the outer membrane porins OmpT and OmpU, several iron transport systems, and proteins containing heme or iron-sulfur clusters. These data indicate that V. cholerae RyhB has diverse functions, ranging from iron homeostasis to the regulation of biofilm formation.

Shigella flexneri requires iron for survival, and the genes for iron uptake and homeostasis are regulated by the Fur protein. Microarrays were used to identify genes regulated by Fur and to study the physiological effects of iron availability in S. flexneri. These assays showed that the expression of genes involved in iron acquisition and acid response was induced by low-iron availability and by inactivation of fur. A fur null mutant was acid sensitive in media at pH 2.5, and acid sensitivity was also observed in the wild-type strain grown under iron-limiting conditions. Acid resistance of the fur mutant in minimal medium was restored by addition of glutamate during acid challenge, indicating that the glutamate-dependent acid resistance system was not defective. Inactivation of ryhB, a small regulatory RNA whose expression is repressed by Fur, restored acid resistance in the fur mutant, while overexpressing ryhB increased acid sensitivity in the wild-type strain. RyhB-regulated genes were identified by microarray analysis. The expression of one of the RyhB-repressed genes, ydeP, which encodes a putative oxidoreductase, suppressed acid sensitivity in the fur mutant. Furthermore, an S. flexneri ydeP mutant was defective for both glutamate-independent and glutamate-dependent acid resistance. The repression of ydeP by RyhB may be indirect, as real time polymerase chain reaction (PCR) experiments indicated that RyhB negatively regulates evgA, which encodes an activator of ydeP. These results demonstrate that the acid sensitivity defect of the S. flexneri fur mutant is due to repression of ydeP by RyhB, most likely via repression of evgA.