The pathogenic actinobacterium Rhodococcus equi: what's in a name?

A key regulatory decision for many bacteria is the switch between biofilm formation and motile dispersal, and this dynamic is well illustrated in the light‐organ symbiosis between the bioluminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid. Biofilm formation mediated by the syp gene cluster helps V. fischeri transition from a dispersed planktonic lifestyle to a robust aggregate on the surface of the nascent symbiotic organ. However, the bacteria must then swim to pores and down into the deeper crypt tissues that they ultimately colonize. A number of positive and negative regulators control syp expression and biofilm formation, but until recently the environmental inputs controlling this clash between opposing regulatory mechanisms have been unclear. Thompson et al. have now shown that Syp‐mediated biofilms can be repressed by a well‐known host‐derived molecule: nitric oxide. This regulation is accomplished by the NO sensor HnoX exerting control over the biofilm regulator HahK. The discoveries reported here by Thompson et al. cast new light on a critical early stage of symbiotic initiation in the V. fischeri‐squid model symbiosis, and more broadly it adds to a growing understanding of the role(s) that NO and HnoX play in biofilm regulation by many bacteria.     

Roles of the Structural Symbiosis Polysaccharide (syp) Genes in Host Colonization,Biofilm Formation,and Polysaccharide Biosynthesis in Vibrio fischeri

The symbiosis polysaccharide locus, syp, is required for Vibrio fischeri to form a symbiotic association with the squid Euprymna scolopes. It is also required for biofilm formation induced by the unlinked regulator RscS. The syp locus includes 18 genes that can be classified into four groups based on putative function: 4 genes encode putative regulators, 6 encode glycosyltransferases, 2 encode export proteins, and the remaining 6 encode proteins with other functions, including polysaccharide modification. To understand the roles of each of the 14 structural syp genes in colonization and biofilm formation, we generated nonpolar in-frame deletions of each gene. All of the deletion mutants exhibited defects in their ability to colonize juvenile squid, although the impact of the loss of SypB or SypI was modest. Consistent with their requirement for colonization, most of the structural genes were also required for RscS-induced biofilm formation. In particular, the production of wrinkled colonies, pellicles, and the matrix on the colony surface was eliminated or severely decreased in all mutants except for the sypB and sypI mutants; in contrast, only a subset of genes appeared to play a role in attachment to glass. Finally, immunoblotting data suggested that the structural Syp proteins are involved in polysaccharide production and/or export. These results provide important insights into the requirements for the syp genes under different environmental conditions and thus lay the groundwork for a more complete understanding of the matrix produced by V. fischeri to enhance cell-cell interactions and promote symbiotic colonization.     

LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV

Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid’s light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.     

Arabinose Induces Pellicle Formation by Vibrio fischeri

Biofilms are multicellular communities of bacteria attached to a surface and embedded in a protective matrix. In many cases, the signals that induce biofilm formation are unknown. Here, we report that biofilm formation by the marine bacterium Vibrio fischeri can be induced by the addition of arabinose to LBS (Luria-Bertani-salt), a tryptone-based medium. Growth of cells in the presence of 0.2% arabinose, but not other sugars, induced the production of a pellicle at the air/liquid interfaces of static cultures. V. fischeri failed to grow on arabinose as the sole carbon source, suggesting that pellicle production did not occur as a result of increased growth, but experiments using the acid/base indicator phenol red suggested that V. fischeri may partially metabolize arabinose. Pellicle production was independent of the syp polysaccharide locus but was altered upon disruption of the bcs cellulose locus. Through a screen for mutants defective for pellicle production, we found that loss of motility disrupted the formation of the arabinose-induced pellicle. Among the ∼20 mutants that retained motility were strains with insertions in a putative msh pilus locus and a strain with a defect in yidK, which is involved in galactose catabolism. Mutants with the msh gene disrupted grew poorly in the presence of arabinose, while the yidK mutant appeared to be “blind” to the presence of arabinose. Finally, arabinose impaired symbiotic colonization by V. fischeri. This work thus identifies a novel signal and new pathways involved in control of biofilm formation by V. fischeri.     

The response regulator SypE controls biofilm formation and colonization through phosphorylation of the syp‐encoded regulator SypA in Vibrio fischeri

Bacteria utilize multiple regulatory systems to modulate gene expression in response to environmental changes, including two‐component signalling systems and partner‐switching networks. We recently identified a novel regulatory protein, SypE, that combines features of both signalling systems. SypE contains a central response regulator receiver domain flanked by putative kinase and phosphatase effector domains with similarity to partner‐switching proteins. SypE was previously shown to exert dual control over biofilm formation through the opposing activities of its terminal effector domains. Here, we demonstrate that SypE controls biofilms in Vibrio fischeri by regulating the activity of SypA, a STAS (sulphate transporter and anti‐sigma antagonist) domain protein. Using biochemical and genetic approaches, we determined that SypE both phosphorylates and dephosphorylates SypA, and that phosphorylation inhibits SypA's activity. Furthermore, we found that biofilm formation and symbiotic colonization required active, unphosphorylated SypA, and thus SypA phosphorylation corresponded with a loss of biofilms and impaired host colonization. Finally, expression of a non‐phosphorylatable mutant of SypA suppressed both the biofilm and symbiosis defects of a constitutively inhibitory SypE mutant strain. This study demonstrates that regulation of SypA activity by SypE is a critical mechanism by which V. fischeri controls biofilm development and symbiotic colonization.     

The alternative oxidase (AOX) gene in Vibrio fischeri is controlled by NsrR and upregulated in response to nitric oxide

Alternative oxidase (AOX) is a respiratory oxidase found in certain eukaryotes and bacteria; however, its role in bacterial physiology is unclear. Exploiting the genetic tractability of the bacterium Vibrio fischeri, we explore the regulation of aox expression and AOX function. Using quantitative PCR and reporter assays, we demonstrate that aox expression is induced in the presence of nitric oxide (NO), and that the NO‐responsive regulatory protein NsrR mediates the response. We have identified key amino acid residues important for NsrR function and experimentally confirmed a bioinformatically predicted NsrR binding site upstream of aox. Microrespirometry demonstrated that oxygen consumption by V. fischeri CydAB quinol oxidase is inhibited by NO treatment, whereas oxygen consumption by AOX is less sensitive to NO. NADH oxidation assays using inverted membrane vesicles confirmed that NO directly inhibits CydAB, and that AOX is resistant to NO inhibition. These results indicate a role for V. fischeri AOX in aerobic respiration during NO stress.     

Roles of Vibrio fischeri and Nonsymbiotic Bacteria in the Dynamics of Mucus Secretion during Symbiont Colonization of the Euprymna scolopes Light Organ

During light organ colonization of the squid Euprymna scolopes by Vibrio fischeri, host-derived mucus provides a surface upon which environmental V. fischeri forms a biofilm and aggregates prior to colonization. In this study we defined the temporal and spatial characteristics of this process. Although permanent colonization is specific to certain strains of V. fischeri, confocal microscopy analyses revealed that light organ crypt spaces took up nonspecific bacteria and particles that were less than 2 μm in diameter during the first hour after hatching. However, within 2 h after inoculation, these cells or particles were not detectable, and further entry by nonspecific bacteria or particles appeared to be blocked. Exposure to environmental gram-negative or -positive bacteria or bacterial peptidoglycan caused the cells of the organ's superficial ciliated epithelium to release dense mucin stores at 1 to 2 h after hatching that were used to form the substrate upon which V. fischeri formed a biofilm and aggregated. Whereas the uncolonized organ surface continued to shed mucus, within 48 h of symbiont colonization mucus shedding ceased and the formation of bacterial aggregations was no longer observed. Eliminating the symbiont from the crypts with antibiotics restored the ability of the ciliated fields to secrete mucus and aggregate bacteria. While colonization by V. fischeri inhibited mucus secretion by the surface epithelium, secretion of host-derived mucus was induced in the crypt spaces. Together, these data indicate that although initiation of mucus secretion from the superficial epithelium is nonspecific, the inhibition of mucus secretion in these cells and the concomitant induction of secretion in the crypt cells are specific to natural colonization by V. fischeri.     

Salinity and Temperature Effects on Physiological Responses of <Emphasis Type="Italic">Vibrio fischeri</Emphasis> from Diverse Ecological Niches

Vibrio fischeri is a bioluminescent bacterial symbiont of sepiolid squids (Cephalopoda: Sepiolidae) and monocentrid fishes (Actinopterygii: Monocentridae). V. fischeri exhibit competitive dominance within the allopatrically distributed squid genus Euprymna, which have led to the evolution of V. fischeri host specialists. In contrast, the host genus Sepiola contains sympatric species that is thought to have given rise to V. fischeri that have evolved as host generalists. Given that these ecological lifestyles may have a direct effect upon the growth spectrum and survival limits in contrasting environments, optimal growth ranges were obtained for numerous V. fischeri isolates from both free-living and host environments. Upper and lower limits of growth were observed in sodium chloride concentrations ranging from 0.0% to 9.0%. Sepiola symbiotic isolates possessed the least variation in growth throughout the entire salinity gradient, whereas isolates from Euprymna were the least uniform at <2.0% NaCl. V. fischeri fish symbionts (CG101 and MJ101) and all free-living strains were the most dissimilar at >5.0% NaCl. Growth kinetics of symbiotic V. fischeri strains were also measured under a range of salinity and temperature combinations. Symbiotic V. fischeri ES114 and ET101 exhibited a synergistic effect for salinity and temperature, where significant differences in growth rates due to salinity existed only at low temperatures. Thus, abiotic factors such as temperature and salinity have differential effects between free-living and symbiotic strains of V. fischeri, which may alter colonization efficiency prior to infection.     

Identification of a Novel Matrix Protein That Promotes Biofilm Maturation in Vibrio fischeri

Bacteria form communities, termed biofilms, in which cells adhere to each other within a matrix, typically comprised of polysaccharides, proteins, and extracellular DNA. Biofilm formation by the marine bacterium Vibrio fischeri requires the Syp polysaccharide, but the involvement of matrix proteins is as yet unknown. Here we identified three genes, termed bmpA, -B, and -C (biofilm maturation protein), with overlapping functions in biofilm maturation. A triple bmpABC mutant, but not single or double mutants, was defective in producing wrinkled colonies, a form of biofilm. Surprisingly, the triple mutant was competent to form pellicles, another biofilm phenotype, but they generally lacked a three-dimensional architecture. Transmission electron microscopy revealed that the extracellular matrix of the bmp mutant contained electron-dense, thread-like structures that were also present in the wild type but lacking in syp mutant strains. We hypothesized that the bmp mutant produces the Syp polysaccharide but fails to produce/export a distinct matrix component. Indeed, a mixture of the bmp and syp mutants produced a wrinkled colony. Finally, BmpA could be detected in cell-free supernatants from disrupted pellicles. Thus, this work identifies a new matrix protein necessary for biofilm maturation by V. fischeri and, based on the conservation of bmp, potentially other microbes.     

Ecological Diversification of Vibrio fischeri Serially Passaged for 500 Generations in Novel Squid Host Euprymna tasmanica

Vibrio fischeri isolated from Euprymna scolopes (Cephalopoda: Sepiolidae) was used to create 24 lines that were serially passaged through the non-native host Euprymna tasmanica for 500 generations. These derived lines were characterized for biofilm formation, swarming motility, carbon source utilization, and in vitro bioluminescence. Phenotypic assays were compared between “ES” (E. scolopes) and “ET” (E. tasmanica) V. fischeri wild isolates to determine if convergent evolution was apparent between E. tasmanica evolved lines and ET V. fischeri. Ecological diversification was observed in utilization of most carbon sources examined. Convergent evolution was evident in motility, biofilm formation, and select carbon sources displaying hyperpolymorphic usage in V. fischeri. Convergence in bioluminescence (a 2.5-fold increase in brightness) was collectively evident in the derived lines relative to the ancestor. However, dramatic changes in other properties—time points and cell densities of first light emission and maximal light output and emergence of a lag phase in growth curves of derived lines—suggest that increased light intensity per se was not the only important factor. Convergent evolution implies that gnotobiotic squid light organs subject colonizing V. fischeri to similar selection pressures. Adaptation to novel hosts appears to involve flexible microbial metabolism, establishment of biofilm and swarmer V. fischeri ecotypes, and complex changes in bioluminescence. Our data demonstrate that numerous alternate fitness optima or peaks are available to V. fischeri in host adaptive landscapes, where novel host squids serve as habitat islands. Thus, V. fischeri founder flushes occur during the initiation of light organ colonization that ultimately trigger founder effect diversification.     

Population Dynamics of Vibrio fischeri during Infection of Euprymna scolopes

The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.     

Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont

Cells acclimate to fluctuating environments by utilizing sensory circuits. One common sensory pathway used by bacteria is two‐component signaling (TCS), composed of an environmental sensor [the sensor kinase (SK)] and a cognate, intracellular effector [the response regulator (RR)]. The squid symbiont Vibrio fischeri uses an elaborate TCS phosphorelay containing a hybrid SK, RscS, and two RRs, SypE and SypG, to control biofilm formation and host colonization. Here, we found that another hybrid SK, SypF, was essential for biofilms by functioning downstream of RscS to directly control SypE and SypG. Surprisingly, although wild‐type SypF functioned as an SK in vitro, this activity was dispensable for colonization. In fact, only a single non‐enzymatic domain within SypF, the HPt domain, was critical in vivo. Remarkably, this domain within SypF interacted with RscS to permit a bypass of RscS‘s own HPt domain and SypF‘s enzymatic function. This represents the first in vivo example of a functional SK that exploits the enzymatic activity of another SK, an adaptation that demonstrates the elegant plasticity in the arrangement of TCS regulators.