SUMMARY Species compositions of gut microbiomes impact host health [1–3], but the processes determining these compositions are largely unknown. An unexplained observation is that gut species composition varies widely between individuals but is largely stable over time within individuals [4, 5]. Stochastic factors during establishment may drive these alternative stable states (colonized versus non-colonized) [6, 7], which can influence susceptibility to pathogens such as Clostridium difficile. Here we sought to quantify and model the dose-response, dynamics, and stability of bacterial colonization in the fruit fly (Drosophila melanogaster) gut. Our precise, high-throughput technique revealed stable between-host variation in colonization when individual germ-free flies were fed their own natural commensals (including the probiotic Lactobacillus plantarum). Some flies were colonized while others remained germ-free even at extremely high bacterial doses. Thus, alternative stable states of colonization exist even in this low complexity model of host-microbe interactions. These alternative states are driven by a fundamental asymmetry between the inoculum population and the stably colonized population that is mediated by spatial localization and a population bottleneck, which makes stochastic effects important by lowering the effective population size. Prior colonization with other bacteria reduced the chances of subsequent colonization, thus increasing the stability of higher diversity guts. Therefore, stable gut diversity may be driven by inherently stochastic processes, which has important implications for combatting infectious diseases and for stably establishing probiotics in the gut.

SummaryThe Pseudomonas aeruginosa Wsp signal transduction system produces cyclic-di-GMP (c-di-GMP), an intracellular messenger that stimulates biofilm formation and suppresses motility. The Wsp system is homologous to chemotaxis systems and includes a membrane-bound receptor protein, WspA, and a response regulator GGDEF protein, WspR, that catalyses c-di-GMP synthesis when phosphorylated. We found that the subcellular distributions of fluorescent protein-tagged WspA and WspR differed markedly from their chemotaxis counterparts. WspA-YFP formed patches in cells whereas WspR-YFP was dispersed when unphosphorylated and formed bright cytoplasmic clusters when phosphorylated. WspR formed clusters in cells of a DwspF mutant, a genetic background that causes constitutive phosphorylation of WspR, but was dispersed in cells of a wspA mutant, a genetic background necessary for WspR phosphorylation. In addition, WspR mutated at Asp70, its predicted site of phosphorylation, did not form clusters. C-di-GMP synthesis was not required for cluster formation. WspR-YFP was dispersed in liquid-grown wild-type cells, but formed clusters that sometimes appeared and disappeared over the course of a few minutes in cells grown on an agar surface. Our results suggest that the compartmentalized production of c-di-GMP in response to a stimulus associated with growth on a surface is an important functional characteristic of the Wsp system.

SummaryVibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose-sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co-regulated protein and an RTX toxin-like membrane fusion transporter, as well as potential modifiers of cell surface molecules ( nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H-NS, a CsgD-like repressor and an AraC-like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.

WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated.

Vibrio parahaemolyticus, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP) colony morphologies. Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies and that opacity is controlled by the Vibrio harveyi LuxR-type transcriptional activator OpaR. CPS has also been shown to be regulated by the scrABC signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein. The present study identifies cps genes and examines their regulation. Transposon insertions in the cps locus, which contains 11 genes, abolished opacity. Such mutants failed to produce CPS and were defective in pellicle formation in microtiter wells and in a biofilm attachment assay. Reporter fusions to cpsA, the first gene in the locus, showed ϳ10-fold-enhanced transcription in the OP (opaR ؉ ) strain compared to a TR (⌬opaR) strain. Two additional transcriptional regulators were discovered. One potential activator, CpsR, participates in the scrABC GGDEF-EAL-signaling pathway; CpsR was required for the increased cps expression observed in scrA ⌬opaR strains. CpsR, which contains a conserved module found in members of the AAA؉ superfamily of ATP-interacting proteins, is homologous to Vibrio cholerae VpsR; however, unlike VpsR, CpsR was not essential for cps expression. CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a repressor. Mutants with cpsS defects have greatly elevated cps transcription; their high level of cpsA expression was CpsR dependent in ⌻R strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative regulators modulates CPS production in V. parahaemolyticus.Vibrio parahaemolyticus is a ubiquitous marine bacterium and human pathogen that can be isolated as a free-living bacterium and from a variety of animate and inanimate surfaces (24,25,29). V. parahaemolyticus strains retrieved from these natural sources display heterogeneity in colony morphology. The phase variation is slow and can be reversible, such that opaque (OP) and translucent (TR) clonal dimorphism is generally observed (30). The OP colony is small, mounded, and has a sticky character when touched with a toothpick. The TR colony is large, flat, and not sticky. Colony opacity correlates with increased capsular polysaccharide (CPS) production (18). One locus has been identified as controlling opacity (30). It encodes the transcriptional regulator, OpaR, which is homologous to Vibrio harveyi LuxR. Expression of opaR using an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible vector converts the TR colony to opaque, and mutation of opaR causes an OP strain to become translucent. Multiple mechanisms for this phase variation appear to exist. One mechanism involves spontaneous alterations in the opaR locus (30); many, but not all, TR strains can be converted to OP by using the opaR expression clone.In addition to the opaR locus, the scr locus is also known to aff...