We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.Toxigenic strains of the Gram-negative bacterium Vibrio cholerae cause a life-threatening diarrheal disease that can kill its victims within hours of the onset of symptoms. The disease is endemic in the Indian subcontinent and continues to reemerge elsewhere in Asia, Africa, and the Americas with the incidence estimated to exceed 5 million cases each year (1-3). Although over 180 serogroups of V. cholerae exist, only the O1 and O139 serovars are known to cause epidemic cholera. The O1 serogroup can be divided further into two biotypes, classical and El Tor, based on various biochemical and phenotypic differences (4). Since 1817, seven cholera pandemics have occurred, of which the classical biotype is responsible for at least the fifth (1881-1896) and the sixth . The El Tor biotype is responsible for the seventh and current pandemic (1961-present). Toxigenic strains of O139 serovar appeared in India and Bangladesh in late 1992 as the first non-O1 serovar to cause epidemic cholera (5, 6). These strains are closely related and probably derived from toxigenic El Tor O1 strains (7). The factors that allowed El Tor biotype and O139 serotype to overtake the classical strains and establish pandemics remain a mystery.Work focusing on the pathogenesis of V. cholerae led to the identification of several key virulence factors, including the cholera toxin responsible for the diarrhea and the toxincoregulated pilus essential for colonizing the human small intestine (8, 9). Several groups tried to construct live, attenuated V. cholerae vaccines by deleting the genes for cholera toxin and several other putative accessory toxins (10-16). However, many of these strains remain reactogenic in human subjects, causing diarrhea or other significant symptoms (17).The virulence factors responsible for the residual reactogenicity remain to be identified.Bacterial genomics offers an opportunity to identify undiscov...

The Hgt4 protein of Candida albicans (orf19.5962) is orthologous to the Snf3 and Rgt2 glucose sensors of Saccharomyces cerevisiae that govern sugar acquisition by regulating the expression of genes encoding hexose transporters. We found that HGT4 is required for glucose induction of the expression of HGT12, HXT10, and HGT7, which encode apparent hexose transporters in C. albicans. An hgt4⌬ mutant is defective for growth on fermentable sugars, which is consistent with the idea that Hgt4 is a sensor of glucose and similar sugars. Hgt4 appears to be sensitive to glucose levels similar to those in human serum (ϳ5 mM). HGT4 expression is repressed by high levels of glucose, which is consistent with the idea that it encodes a high-affinity sugar sensor. Glucose sensing through Hgt4 affects the yeast-to-hyphal morphological switch of C. albicans cells: hgt4⌬ mutants are hypofilamented, and a constitutively signaling form of Hgt4 confers hyperfilamentation of cells. The hgt4⌬ mutant is less virulent than wild-type cells in a mouse model of disseminated candidiasis. These results suggest that Hgt4 is a high-affinity glucose sensor that contributes to the virulence of C. albicans.Candida albicans is responsible for most yeast infections in humans. In addition to causing ordinary vaginal and oral (thrush) infections, this fungus is a serious threat to immunocompromised patients, for whom disseminated, invasive candidiasis is common and often fatal (31, 56). The fungus is normally maintained as a harmless commensal on skin, on mucosa, and in the gut (23) but becomes an opportunistic pathogen upon entry into the bloodstream of immunocompromised (particularly neutropenic) individuals (25,45).

Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. One class, called type IV, are defined as having homology to the conjugal transfer systems of naturally occurring plasmids. It has been proposed that pathogens with type IV secretion systems have acquired and adapted the conjugal transfer systems of plasmids and now use them to export toxins. Several well-characterized intracellular pathogens, including Legionella pneumophila, Coxiella burnetii, Brucella abortus, and Rickettsia prowazekii, contain type IV systems which are known or suspected to be of critical importance in their ability to cause disease. Specifically, these systems are believed to be the key factors determining intracellular fate, and thus the ability to replicate and cause disease.

Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ⌬dotU ⌬icmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ⌬dotU ⌬icmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

The ability of Legionella pneumophila to grow and cause disease in the host is completely dependent on a type IV secretion system known as the Dot/Icm complex. This membrane-spanning apparatus translocates effector molecules into host cells in a process that is poorly understood but that is known to require the putative ATPase DotB. One possible role for DotB is suggested by its similarity to the PilT family of proteins, which mediate pilus retraction. To better understand the molecular behavior of DotB, we have purified the protein and shown that it forms stable homohexameric rings and hydrolyzes ATP with a specific activity of 6.4 nmol of ATP/min/mg of protein. ATPase activity is critical to the function of DotB, as alteration of the conserved Walker box lysine residue resulted in a mutant protein, DotB K162Q, which failed to bind or hydrolyze ATP and which could not complement a ⌬dotB strain for intracellular growth in macrophages. Consistent with the ability of DotB to interact with itself, the dotBK162Q allele exhibited transdominance over wild-type dotB, providing the first example of such a mutation in L. pneumophila. Finally, the DotB K162Q mutant protein had a significantly enhanced membrane localization in L. pneumophila compared to wild-type DotB, suggesting a relationship between nucleotide binding and membrane association. These results are consistent with a model in which DotB cycles between the cytoplasm and the Dot/Icm complex at the membrane, where it hydrolyzes nucleotides to provide energy to the complex.Legionella pneumophila, an intracellular bacterial pathogen, causes Legionnaires' disease by replicating inside alveolar macrophages (13). This ability is dependent on a set of genes called dot or icm, which encode a specialized type IV secretion system (32, 37). The type IV secretion system family is composed of both classical plasmid transfer systems and a related protein export apparatus used by a wide range of pathogens to export toxins (reviewed in reference 4). For L. pneumophila, the Dot/Icm complex is believed to translocate numerous effector molecules into host cells in order to modify the endocytic pathway. Thus far, two translocated proteins, LidA and RalF, have been identified (6,19). RalF is an ADP-ribosylating factor that functions to recruit the macrophage protein ARF1 to L. pneumophila-containing phagosomes, while the function of LidA remains unknown. Because neither of these is absolutely required for L. pneumophila intracellular growth, additional Dot/Icm-secreted proteins are believed to exist (6).The Dot/Icm apparatus is a macromolecular complex that comprises a large number of membrane proteins and several proteins predicted to have cytoplasmic localization. One such cytoplasmic protein, DotB, contains conserved nucleotide binding domains present in many ATPases, as well as domains specific to ATPases found in bacterial type II and type IV secretion systems (23,27,39). Though DotB is known to be a critical component of the Dot/Icm complex (37, 38), its molecular function i...

Consortia-based approaches are a promising avenue towards efficient bioprocessing. However, many complex microbial interactions dictate community dynamics and stability. The rumen of large herbivores harbors a network of biomass-degrading fungi and bacteria, as well as archaea and protozoa that work together to degrade lignocellulose, yet the microbial interactions responsible for consortia stability and activity remain uncharacterized. In this work, we demonstrate a novel enrichment-based isolation method selecting for a minimal biomass-degrading community containing anaerobic fungi, methanogenic archaea, and bacteria. The enriched culture displayed an increase of up to 2.1 times the growth rate and 1.9 times the fermentation gas produced by the isolated fungus from the enriched culture alone. Metagenomic sequencing revealed functional compartmentalization of the community spread across anaerobic fungi (Piromyces), bacteria (Sphaerochaeta), and methanogens (Methanosphaera and Methanocorpusculum). The minimal consortium enabled more complete degradation of biomass, including 2 hemicelluloses like xylan and pectin and a wider range of sugar utilization like xylose and galacturonate. Complementing the "top-down" enrichment analysis, a synthetic rumen system was formed from the "bottom-up" with isolated fungi (Piromyces finnis or Neocallimastix californiae) and methanogens (Methanobacterium bryantii) where only the hydrogenotrophic connection was preserved between members. These synthetic consortia also showed improved growth rate and degradation compared to fungi alone, yet lack the temporal stability seen in native consortia, remaining in culture together for fewer than 10 transfers on average. Taken together, these two complementary approaches both resulted in productive microbial consortia with faster growth rates and wider substrate uptake than mono-cultured fungi.

Although many bacteria are known to be naturally competent for DNA uptake, this ability varies dramatically between species and even within a single species, some isolates display high levels of competence while others seem to be completely nontransformable. Surprisingly, many nontransformable bacterial strains appear to encode components necessary for DNA uptake. We believe that many such strains are actually competent but that this ability has been overlooked because standard laboratory conditions are inappropriate for competence induction. For example, most strains of the gram-negative bacterium Legionella pneumophila are not competent under normal laboratory conditions of aerobic growth at 37°C. However, it was previously reported that microaerophilic growth at 37°C allows L. pneumophila serogroup 1 strain AA100 to be naturally transformed. Here we report that another L. pneumophila serogroup 1 strain, Lp02, can also be transformed under these conditions. Moreover, Lp02 can be induced to high levels of competence by a second set of conditions, aerobic growth at 30°C. In contrast to Lp02, AA100 is only minimally transformable at 30°C, indicating that Lp02 is hypercompetent under these conditions. To identify potential causes of hypercompetence, we isolated mutants of AA100 that exhibited enhanced DNA uptake. Characterization of these mutants revealed two genes, proQ and comR, that are involved in regulating competence in L. pneumophila. This approach, involving the isolation of hypercompetent mutants, shows great promise as a method for identifying natural transformation in bacterial species previously thought to be nontransformable.