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The marine animal pathogen Vibrio harveyi and the human pathogen Vibrio cholerae are aquatic bacteria that engage in a process of cell-cell communication called quorum sensing (QS). Autoinducer (AI)-2 is derived from S-adenosylmethionine in three enzymatic steps. First, S-adenosylmethionine serves as a methyl donor for many biochemical processes, and these methyltransferase-dependent reactions yield S-adenosylhomocysteine. Second, S-adenosylhomocysteine is metabolized to adenine and S-ribosylhomocysteine by the enzyme Pfs, and third, S-ribosylhomocysteine is the substrate for the LuxS enzyme. In mixed species consortia, other microbes also have the potential to alter AI-2 levels, and other classes of AIs are clearly manipulated, but the authors have restricted the discussion to AI-2 and how that pertains to Vibrio QS. Appropriate and distinct responses to potentially different communities are possible because of signal integration in the Vibrio circuits. Channel proteins LsrC and LsrD mediate the delivery of the ligand across the membrane. LsrA is an ATPase that supplies the energy required for transport. Rapid Lsr-dependent transport of R-THMF into the cell occurs at high cell densities. Recent studies in V. harveyi show that it possesses five qrr genes, like its closest Vibrio relatives. Examination of their functions reveals that, in stark contrast to V. cholerae, in V. harveyi the quorum-regulatory RNAs (Qrr) sRNAs act additively to control luxR mRNA levels.

Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics., IMPORTANCE Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.

Biofilms promote attachment of Vibrio cholerae in aquatic ecosystems and aid in transmission. Intracellular c-di-GMP levels that control biofilm development positively correlate with expression of Qrr sRNAs, which are transcribed when quorum sensing (QS) autoinducer levels are low. The Qrr sRNAs base-pair with and repress translation of hapR encoding the QS 'master regulator', hence increased c-di-GMP and biofilm development at low density were believed to be solely a consequence of Qrr/hapR pairing. We show that Qrr sRNAs also base-pair with and activate translation of the mRNA of a diguanylate cyclase (DGC), Vca0939; relieving an inhibitory structure in vca0939 that occludes the ribosome binding site. A nucleotide substitution in vca0939 disrupted sRNA/mRNA base-pairing and prevented vca0939 translation, while a compensating Qrr sRNA substitution restored pairing and Vca0939 levels. Qrr-dependent DGC activation led to c-di-GMP accumulation and biofilm development in V. cholerae. This represents the first description of (1) a DGC post-transcriptionally activated by direct pairing with an Hfq-dependent sRNA, and (2) control of a V. cholerae QS phenotype, independent of HapR. Thus, direct interactions of the same sRNAs with two mRNAs promote c-di-GMP-dependent biofilm formation by complementary mechanisms in V. cholerae; by negatively regulating HapR, and positively regulating the DGC Vca0939.

In this work we consider nano-scale communication using bacterial populations as transceivers. We demonstrate using a microfluidic test-bed and a population of genetically engineered Escherichia coli bacteria serving as the communication receiver that a simple modulation like on-off keying (OOK) is indeed achievable, but suffers from very poor data-rates. We explore an alternative communication strategy called time elapse communication (TEC) that uses the time period between signals to encode information. We identify the severe limitations of TEC under practical non-zero error conditions in the target environment, and propose an advanced communication strategy called smart time elapse communication (TEC-SMART) that achieves over a 10× improvement in data-rate over OOK.

Sediment stratigraphy and diatom succession were studied in an 80.5-cm core taken from the deepest part of Third Sister Lake, a small kettle hole in a recently urbanized landscape of southeastern Michigan. Alternating light clay and dark organic bands documented sporadic inputs of clay from outside the basin during rain events, rather than annual laminations. Urban construction activity also disrupted the inflow stream bed and facilitated transport of clay into the lake to generate non-rhythmic banding in the lake's deep hole. Diatom analysis revealed dramatic changes in predominant taxa with sediment depth verifying the non-annual nature of the sediment bands. Observation of halophilic diatom taxa also documented effects of human activity such as road salting on this small, urban lake.

A recombinant endotoxin neutralizing protein was evaluated for its ability to ameliorate the effects of Escherichia coli sepsis in rats.Prospective, controlled animal trial.Hospital animal research laboratory.Wistar rats, treated with gentamicin 1 hr after challenge with intraperitoneal E. coli O18ac.The animals received a recombinant endotoxin neutralizing protein, in doses of 5, 25, or 50 mg/kg, either 30 or 60 mins after challenge; controls received saline.Geometric mean serum endotoxin concentrations in endotoxin neutralizing protein-treated animals did not differ from control animals. Tumor necrosis factor concentrations in animals treated with endotoxin neutralizing protein 30 mins after challenge were significantly lower than controls. Animals treated with 25 or 50 mg/kg of endotoxin neutralizing protein 30 mins after E. coli challenge had significant improvements in survival compared with controls. Animals treated with 50 mg/kg of endotoxin neutralizing protein 60 mins after E. coli challenge had significant improvements in survival compared with controls.Endotoxin neutralizing protein significantly reduces mortality from Gram-negative sepsis in an antibiotic-treatment model of E. coli peritonitis and bacteremia in rats, mediated by a neutralization of the biological effects of endotoxin.

Competence for genetic transformation in Vibrio cholerae is triggered by chitin-induced transcription factor TfoX and quorum sensing (QS) regulator HapR. Transformation requires expression of ComEA, described as a DNA receptor in other competent bacteria. A screen for mutants that poorly expressed a comEA-luciferase fusion identified cytR, encoding the nucleoside scavenging cytidine repressor, previously shown in V. cholerae to be a biofilm repressor and positively regulated by TfoX, but not linked to transformation. A ΔcytR mutant was non-transformable and defective in expression of comEA and additional TfoX-induced genes, including pilA (transformation pseudopilus) and chiA-1 (chitinase). In Escherichia coli, 'anti-activation' of nucleoside metabolism genes, via protein-protein interactions between critical residues in CytR and CRP (cAMP receptor protein), is disrupted by exogenous cytidine. Amino acid substitutions of the corresponding V. cholerae CytR residues impaired expression of comEA, pilA and chiA-1, and halted DNA uptake; while exogenous cytidine hampered comEA expression levels and prevented transformation. Our results support a speculative model that when V. cholerae reaches high density on chitin, CytR-CRP interactions 'anti-activate' multiple genes, including a possible factor that negatively controls DNA uptake. Thus, a nucleoside scavenging mechanism couples nutrient stress and cell-cell signalling to natural transformation in V. cholerae as described in other bacterial pathogens.

Vibrio cholerae is the waterborne bacterium responsible for worldwide outbreaks of the acute, potentially fatal cholera diarrhea. The primary factors this human pathogen uses to cause the disease are controlled by a complex regulatory program linking extracellular signaling inputs to changes in expression of several critical virulence genes. Recently it has been uncovered that many non-coding regulatory sRNAs are important components of the V. cholerae virulence regulon. Most of these sRNAs appear to require the RNA-binding protein, Hfq, to interact with and alter the expression of target genes, while a few sRNAs appear to function by an Hfq-independent mechanism. Direct base-pairing between the sRNAs and putative target mRNAs has been shown in a few cases but the extent of each sRNAs regulon is not fully known. Genetic and biochemical methods, coupled with computational and genomics approaches, are being used to validate known sRNAs and also to identify many additional putative sRNAs that may play a role in the pathogenic lifestyle of V. cholerae.

Vibrio cholerae, the causative agent of cholera and a natural inhabitant of aquatic environments, regulates numerous behaviors using a quorum-sensing (QS) system conserved among many members of the marine genus Vibrio. The Vibrio QS response is mediated by two extracellular autoinducer (AI) molecules: CAI-I, which is produced only by Vibrios, and AI-2, which is produced by many bacteria. In marine biofilms on chitinous surfaces, QS-proficient V. cholerae become naturally competent to take up extracellular DNA. Because the direct role of AIs in this environmental behavior had not been determined, we sought to define the contribution of CAI-1 and AI-2 in controlling transcription of the competence gene, comEA, and in DNA uptake. In this study we demonstrated that comEA transcription and the horizontal acquisition of DNA by V. cholerae are induced in response to purified CAI-1 and AI-2, and also by autoinducers derived from other Vibrios co-cultured with V. cholerae within a mixed-species biofilm. These results suggest that autoinducer communication within a consortium may promote DNA exchange among Vibrios, perhaps contributing to the evolution of these bacterial pathogens.

Vibrio cholerae quorum sensing controls expression of four redundant sRNAs, Qrr1-4. The Qrr sRNAs are predicted to alter the translation of several mRNAs, including, hapR, which encodes a transcription factor that controls genes for virulence factors, biofilm formation, protease production and DNA uptake. Each Qrr contains a 21 nucleotide region absolutely conserved among pathogenic Vibrios, and predicted to base pair with mRNA targets, like hapR, aided by the RNA chaperone Hfq. This molecular mechanism was not experimentally tested previously, and we provide here both in vivo and in vitro evidence to validate this model. In Escherichia coli, Qrr expression repressed a HapR-GFP translational fusion, and a specific nucleotide substitution in the 21 nucleotide region eliminated HapR control, while a compensatory mutation in hapR restored it. In V. cholerae, the identical mutations also deregulated HapR-dependent gene expression and corresponding QS phenotypes by altering HapR protein levels. We calculated in vitro binding affinities of a Qrr/hapR complex and show that Hfq stabilizes complex formation. Finally, the Qrr mutation with in vivo defects also prevented Qrr/hapR binding, while the compensatory hapR mutation restored binding. These results demonstrate that the V. cholerae QS response is mediated by base pairing interactions between Qrr sRNAs and hapR mRNA.

The two-component quorum sensing (QS) system, first described in the marine bacterium Vibrio harveyi and evolutionarily conserved among members of the genus Vibrio, has been best studied in the human pathogen Vibrio cholerae (1, 2). In the V. cholerae QS system, the response to the accumulation of extracellular autoinducers triggers a signaling cascade resulting in the transcription of four small regulatory RNAs (sRNAs). Our results support the model that the QS sRNAs bind to the 5' untranslated region of multiple mRNAs and alter the fate of one in a positive manner and several others in a negative manner. This mechanism ensures the proper timing of the QS response, which includes the expression of traits critical for virulence and for the formation of biofilms (2-6).

The two-component quorum sensing (QS) system, first described in the marine bacterium Vibrio harveyi and evolutionarily conserved among members of the genus Vibrio, has been best studied in the human pathogen Vibrio cholerae (1, 2). In the V. cholerae QS system, the response to the accumulation of extracellular autoinducers triggers a signaling cascade resulting in the transcription of four small regulatory RNAs (sRNAs). Our results support the model that the QS sRNAs bind to the 5' untranslated region of multiple mRNAs and alter the fate of one in a positive manner and several others in a negative manner. This mechanism ensures the proper timing of the QS response, which includes the expression of traits critical for virulence and for the formation of biofilms (2-6).

Quorum sensing (QS), or cell-cell communication in bacteria, is achieved through the production and subsequent response to the accumulation of extracellular signal molecules called autoinducers (AIs). To identify AI-regulated target genes in Vibrio cholerae El Tor ( V. cholerae El ), the strain responsible for the current cholera pandemic, luciferase expression was assayed in an AI − strain carrying a random lux transcriptional reporter library in the presence and absence of exogenously added AIs. Twenty-three genes were identified and shown to require the QS transcription factor, HapR, for their regulation. Several of the QS-dependent target genes, annotated as encoding hypothetical proteins, in fact encode HD-GYP proteins, phosphodiesterases that degrade the intracellular second messenger cyclic dimeric GMP (c-di-GMP), which is important for controlling biofilm formation. Indeed, overexpression of a representative QS-activated HD-GYP protein in V. cholerae El reduced the intracellular concentration of c-di-GMP, which in turn decreased exopolysaccharide production and biofilm formation. The V. cholerae classical biotype ( V. cholerae Cl ), which caused previous cholera pandemics and is HapR − , controls c-di-GMP levels and biofilm formation by the VieA signaling pathway. We show that the VieA pathway is dispensable for biofilm formation in V. cholerae El but that restoring HapR in V. cholerae Cl reestablishes QS-dependent repression of exopolysaccharide production. Thus, different pandemic strains of V. cholerae modulate c-di-GMP levels and control biofilm formation in response to distinct sensory pathways.

The facultative pathogen Vibrio cholerae transitions between its human host and aquatic reservoirs where it colonizes chitinous surfaces. Growth on chitin induces expression of chitin utilization genes, genes involved in DNA uptake by natural transformation, and a type VI secretion system that allows contact-dependent killing of neighboring bacteria. We have previously shown that the transcription factor CytR, thought to primarily regulate the pyrimidine nucleoside scavenging response, is required for natural competence in V. cholerae. Through high-throughput RNA sequencing (RNA-seq), we show that CytR positively regulates the majority of competence genes, the three type VI secretion operons, and the four known or predicted chitinases. We used transcriptional reporters and phenotypic analysis to determine the individual contributions of quorum sensing, which is controlled by the transcription factors HapR and QstR; chitin utilization that is mediated by TfoX; and pyrimidine starvation that is orchestrated by CytR, toward each of these processes. We find that in V. cholerae, CytR is a global regulator of multiple behaviors affecting fitness and adaptability in the environment.

Pathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae. To persist in the environment, the microbe must be proficient at both replication and transmission. In laboratory cultures, as nutrients become scarce a stringent response-like pathway coordinates exit from the exponential growth phase with induction of traits correlated with virulence, including motility. A screen for mutants that express the flagellin gene poorly identified five activators of virulence: LetA/LetS, a two-component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA of Salmonella; the stationary-phase sigma factor RpoS; the flagellar sigma factor FliA; and a new locus, letE. Unlike wild type, post-exponential-phase letA and letS mutants were not motile, cytotoxic, sodium sensitive or proficient at infecting macrophages. L. pneumophila also required fliA to become motile, cytotoxic and to infect macrophages efficiently and letE to express sodium sensitivity and maximal motility and cytotoxicity. When induced to express RelA, all of the strains exited the exponential phase, but only wild type converted to the fully virulent form. In contrast, intracellular replication was independent of letA, letS, letE or fliA. Together, the data indicate that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating L. pneumophila to a transmissible form.

▪ Abstract Legionella pneumophila first commanded attention in 1976, when investigators from the Centers for Disease Control and Prevention identified it as the culprit in a massive outbreak of pneumonia that struck individuals attending an American Legion convention ( 84 ). It is now clear that this gram-negative bacterium flourishes naturally in fresh water as a parasite of amoebae, but it can also replicate within alveolar macrophages. L. pneumophila pathogenesis is discussed using the following model as a framework. When ingested by phagocytes, stationary-phase L. pneumophila bacteria establish phagosomes which are completely isolated from the endosomal pathway but are surrounded by endoplasmic reticulum. Within this protected vacuole, L. pneumophila converts to a replicative form that is acid tolerant but no longer expresses several virulence traits, including factors that block membrane fusion. As a consequence, the pathogen vacuoles merge with lysosomes, which provide a nutrient-rich replication niche. Once the amino acid supply is depleted, progeny accumulate the second messenger guanosine 3′,5′-bispyrophosphate (ppGpp), which coordinates entry into the stationary phase with expression of traits that promote transmission to a new phagocyte. A number of factors contribute to L. pneumophila virulence, including type II and type IV secretion systems, a pore-forming toxin, type IV pili, flagella, and numerous other factors currently under investigation. Because of its resemblance to certain aspects of Mycobacterium, Toxoplasma, Leishmania, and Coxiella pathogenesis, a detailed description of the mechanism used by L. pneumophila to manipulate and exploit phagocyte membrane traffic may suggest novel strategies for treating a variety of infectious diseases. Knowledge of L. pneumophila ecology may also inform efforts to combat the emergence of new opportunistic macrophage pathogens.

Legionella pneumophila survives in aquatic environments, but replicates within amoebae or the alveolar macrophages of immunocompromised individuals. Here, the signal transduction pathway that co-ordinates L. pneumophila virulence expression in response to amino acid depletion was investigated. To facilitate kinetic and genetic studies, a phenotypic reporter of virulence was engineered by fusing flaA promoter sequences to a gene encoding green fluorescent protein. When subjected to amino acid depletion, L. pneumophila accumulated ppGpp and converted from a replicative to a virulent state, as judged by motility and sodium sensitivity. ppGpp appeared to initiate this response, as L. pneumophila induced to express the Escherichia coli RelA ppGpp synthetase independently of nutrient depletion accumulated ppGpp, exited the exponential growth phase and expressed flaAgfp, motility, sodium sensitivity, cytotoxicity and infectivity, five traits correlated with virulence. Although coincident with the stationary phase, L. pneumophila virulence expression appeared to require an additional factor: mutant Lp120 accumulated ppGpp and acquired two stationary phase traits but none of six virulence phenotypes analysed. We propose that, when nutrients are limiting, ppGpp acts as an alarmone, triggering the expression of multiple traits that enable L. pneumophila to escape its spent host, to survive and disperse in the environment and to re-establish a protected intracellular replication niche.

OBJECTIVES Limulus anti-lipopolysaccharide factor, an 11.8-kilodalton peptide isolated from amebocytes of Limulus polyphemus inhibits the biologic activities of endotoxin in vitro, including gelation of Limulus amebocyte lysate. A recombinant version of Limulus anti-lipopolysaccharide factor, termed endotoxin neutralizing protein, has now been expressed in yeast. Endotoxin-neutralizing protein was evaluated for its potential prophylactic and therapeutic effects in rabbits challenged with Escherichia coli endotoxin. DESIGN Controlled animal trial. SETTING Animal research laboratory. SUBJECTS A total of 112 New Zealand white rabbits were studied. INTERVENTIONS Rabbits were challenged with an LD80 dose of E. coli endotoxin (100 micrograms/kg); control animals (n = 52) were treated with saline solution at the time of endotoxin challenge; experimental animals received endotoxin-neutralizing protein 2.5 mg/kg prechallenge (n = 20), 5.0 mg/kg prechallenge (n = 20), or 5.0 mg/kg 30 mins postchallenge (n = 20).