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Eric V Stabb

Publications and source records attributed to Eric V Stabb.

13 recordsLinked to original sources
PubMed2025

Mutant gltS alleles enable a Vibrio fischeri D-glutamate auxotroph to grow with lower requirements for exogenous D-glutamate.

D-glu is a key component of peptidoglycan (PG) and is essential for growth in most bacteria. To assess constraints on PG evolution and bacterial requirements for D-glu, we sought to artificially evolve PG biosynthesis, leading to either replacement of D-glu in the PG peptide or alternative pathways to D-glu incorporation. We previously found that suppression of D-glu auxotrophy in a murI racD mutant of Vibrio fischeri grown on lysogeny broth salts (LBS) medium was rare but could be accomplished by mutation of bsrF, with restoration of wild-type PG structure. Here, we selected nine additional prototrophic suppressors of the same murI racD mutant from 1010 colony-forming units (CFU) plated on LBS supplemented with ~2.7 mM D-gln. Each suppressor had a mutation in gltS, which encodes a putative sodium:glutamate symporter. Increased copy numbers of mutant gltS alleles enabled growth on unsupplemented LBS and resulted in PG containing D-glu. Examination of media components suggests that D-gln supplementation had inadvertently added ~14 μM D-glu, and that LBS itself contains ~1.4 μM D-glu. The mutations in gltS enabled growth with similarly low D-glu concentrations, but also increased sensitivity to homocysteic acid, suggesting more promiscuous transport. Surprisingly, we discovered that expression of mutant gltS in the auxotroph leads to incorporation of lysine into PG, in addition to canonical D-glu. When seawater is supplemented with D-glu, this V. fischeri mutant still colonized Euprymna scolopes and triggered PG-induced morphogenesis. Our results shed light on glutamate transport, highlight trade-offs in GltS structure and function, and reveal an unusual PG modification.IMPORTANCED-glu is an important building block in the peptidoglycan (PG) component of the bacterial cell wall, and its endogenous production is considered essential in most bacteria, even when grown in complex media. In Vibrio fischeri, in trans expression of mutant GltS symporters allows D-glu auxotrophic strains to grow on lysogeny broth salts (LBS) medium without exogenous D-glu, although there is a fitness trade-off of increased sensitivity to homocysteic acid. Our finding that LBS contains sufficient D-glu to support robust growth highlights the undervalued importance of D-amino acid transport and the ubiquity of D-amino acids. Moreover, the discovery of D-lysine in the PG peptide is an unusual PG modification that warrants further study.

Aliivibrio fischeri↗
PubMed2006

Beyond quorum sensing: the complexities of prokaryotic parliamentary procedures.

Bacterial quorum-sensing regulatory systems can be summarized in a simple model wherein an autoinducer molecule accumulates in cultures and stimulates regulatory changes in gene expression upon reaching a critical threshold concentration. Although quorum sensing was originally thought to be an isolated phenomenon governing the regulation of a handful of processes in only a few bacteria, it is now considered to be a widespread mechanism for coordinating bacterial gene expression. Over decades of research, investigations of autoinducer-mediated regulation have revealed that these systems are far more complicated than originally appreciated, and such discoveries have accelerated recently with the application of molecular and genomic tools. The focus of this review is to highlight recent advances describing complexities that go beyond the simple model of quorum sensing. These complexities include the regulation of autoinducer production and degradation, the presence of multiple quorum-sensing systems in individual bacteria that regulate diverse genes, often in coordination with other regulatory elements, and the influence of interorganismal interactions on quorum sensing.

4-Butyrolactone↗
PubMed2006

Photolyase confers resistance to UV light but does not contribute to the symbiotic benefit of bioluminescence in Vibrio fischeri ES114.

Recent reports suggest that the selective advantage of bioluminescence for bacteria is mediated by light-dependent stimulation of photolyase to repair DNA lesions. Despite evidence for this model, photolyase mutants have not been characterized in a naturally bioluminescent bacterium, nor has this hypothesis been tested in bioluminescent bacteria under natural conditions. We have now characterized the photolyase encoded by phr in the bioluminescent bacterium Vibrio fischeri ES114. Consistent with Phr possessing photolyase activity, phr conferred light-dependent resistance to UV light. However, upon comparing ES114 to a phr mutant and a dark Delta luxCDABEG mutant, we found that bioluminescence did not detectably affect photolyase-mediated resistance to UV light. Addition of the light-stimulating autoinducer N-3-oxo-hexanoyl homoserine lactone appeared to increase UV resistance, but this was independent of photolyase or bioluminescence. Moreover, although bioluminescence confers an advantage for V. fischeri during colonization of its natural host, Euprymna scolopes, the phr mutant colonized this host to the same level as the wild type. Taken together, our results indicate that at least in V. fischeri strain ES114, the benefits of bioluminescence during symbiotic colonization are not mediated by photolyase, and although some UV resistance mechanism may be coregulated with bioluminescence, we found no evidence that light production benefits cells by stimulating photolyase in this strain.

Aliivibrio fischeri↗
PubMed2006

Breaching the great wall: peptidoglycan and microbial interactions.

Once thought to be a process that occurred only in a few human pathogens, release of biologically active peptidoglycan fragments during growth by Gram-negative bacteria controls many types of bacterial interaction, including symbioses and interactions between microorganisms. This Perspective explores the role of peptidoglycan fragments in mediating a range of microbial-host interactions, and discusses the many systems in which peptidoglycan fragments released during bacterial growth might be active.

Animals↗
PubMed2006

New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ.

Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by approximately 99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.

Acyltransferases↗
PubMed2005

Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri.

Most Vibrio fischeri strains isolated from the Euprymna scolopes light organ carry plasmids, often including both a large (>40kb) plasmid, and one or more small (<12kb) plasmids. The large plasmids share homology with pES100, which is the lone plasmid in V. fischeri type strain ES114. pES100 appears to encode a conjugative system similar to that on plasmid R721. The small plasmids lack extensive similarity to pES100, but they almost always occur in cells that also harbor a large plasmid resembling pES100. We found that many or all of these small plasmids share homology with pES213, a plasmid in strain ES213. We determined the 5501-bp pES213 sequence and generated selectable antibiotic resistance encoding pES213 derivatives, which enabled us to examine replication, retention, and transfer in V. fischeri. An 863-bp fragment of pES213 with features characteristic of theta-type replicons conferred replication without requiring any pES213 open reading frame (ORF). We estimated that pES213 derivatives were maintained at 9.4 copies per genome, which corresponds well with a model of random plasmid segregation to daughter cells and the approximately 10(-4) per generation frequency of plasmid loss. pES213 derivatives mobilized between V. fischeri strains at frequencies up to approximately 10(-4) in culture and in the host, apparently by employing the pES100 conjugative apparatus. pES213 carries two homologs of the putative pES100 origin of transfer (oriT), and V. fischeri strains lacking the pES100 conjugative relaxase, including a relaxase mutant, failed to serve as donors for transmission of pES213 derivatives. In other systems, genes directing conjugative transfer can function in trans to oriT, so it was noteworthy that ORFs adjacent to oriT, VFB51 in pES100 and traYZ in pES213, enhanced transfer 100- to 1000-fold when provided in cis. We also identified and disrupted the V. fischeri recA gene. RecA was not required for stable pES213 replication but surprisingly was required in donors for efficient transfer of pES213 derivatives. These studies provide an explanation for the prevalence and co-occurrence of pES100- and pES213-type plasmids, illuminate novel elements of pES213 mobilization, and provide the foundation for new genetic tools in V. fischeri.

Aliivibrio fischeri↗
PubMed2005

Localization and bacteriostasis of Vibrio introduced into the Pacific white shrimp, Litopenaeus vannamei.

Although numerous mechanisms of immune defense have been described in crustaceans, the tissue distribution and fate of live bacteria introduced into the host remain unclear. In the present study, Litopenaeus vannamei were injected with a sub-lethal dose of kanamycin-resistant Vibrio campbellii expressing green fluorescent protein. Accumulation of intact bacteria was quantified by real-time PCR, while bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. Over the 240 min examined, the lymphoid organ contained the greatest number of intact V. campbellii per gram tissue as well as the lowest percentage of culturable V. campbellii compared to other tissues, including the hemolymph. In contrast, the gills and hepatopancreas accumulated intact bacteria, but contained a significantly greater percentage of culturable bacteria than the hemolymph after 240 min. These data suggest that the lymphoid organ plays a major role in bacterial uptake and bacteriostasis in penaeid shrimp.

Animals↗
PubMed2005

Culture-independent characterization of the microbiota of the ant lion Myrmeleon mobilis (Neuroptera: Myrmeleontidae).

Ant lions are insect larvae that feed on the liquefied internal components of insect prey. Prey capture is assisted by the injection of toxins that are reportedly derived from both the insect and bacterial symbionts. These larvae display interesting gut physiology where the midgut is not connected to the hindgut, preventing elimination of solid waste until adulthood. The presence of a discontinuous gut and the potential involvement of bacteria in prey paralyzation suggest an interesting microbial role in ant lion biology; however, the ant lion microbiota has not been described in detail. We therefore performed culture-independent 16S rRNA gene sequence analysis of the bacteria associated with tissues of an ant lion, Myrmeleon mobilis. All 222 sequences were identified as Proteobacteria and could be subdivided into two main groups, the alpha-Proteobacteria with similarity to Wolbachia spp. (75 clones) and the gamma-Proteobacteria with similarity to the family Enterobacteriaceae (144 clones). The Enterobacteriaceae-like 16S rRNA gene sequences were most commonly isolated from gut tissue, and Wolbachia-like sequences were predominant in the head and body tissue. Fluorescence in situ hybridization analyses supported the localization of enterics to gut tissue and Wolbachia to nongut tissue. The diversity of sequences isolated from freshly caught, laboratory-fed, and laboratory-starved ant lions were qualitatively similar, although the libraries from each treatment were significantly different (P = 0.05). These results represent the first culture-independent analysis of the microbiota associated with a discontinuous insect gut and suggest that the ant lion microbial community is relatively simple, which may be a reflection of the diet and gut physiology of these insects.

Alphaproteobacteria↗
PubMed2004

Microbial factor-mediated development in a host-bacterial mutualism.

Tracheal cytotoxin (TCT), a fragment of the bacterial surface molecule peptidoglycan (PGN), is the factor responsible for the extensive tissue damage characteristic of whooping cough and gonorrhea infections. Here, we report that Vibrio fischeri also releases TCT, which acts in synergy with lipopolysaccharide (LPS) to trigger tissue development in its mutualistic symbiosis with the squid Euprymna scolopes. As components of PGN and LPS have commonly been linked with pathogenesis in animals, these findings demonstrate that host interpretation of these bacterial signal molecules is context dependent. Therefore, such differences in interpretation can lead to either inflammation and disease or to the establishment of a mutually beneficial animal-microbe association.

Aliivibrio fischeri↗
PubMed2004

Correlation between osmolarity and luminescence of symbiotic Vibrio fischeri strain ES114.

Vibrio fischeri isolates from Euprymna scolopes are dim in culture but bright in the host. We found the luminescence of V. fischeri to be correlated with external osmolarity both in culture and in this symbiosis. Luminescence enhancement by osmolarity was independent of the lux promoter and unaffected by autoinducers or the level of lux expression, but the addition of an aldehyde substrate for luciferase raised the luminescence of cells grown at high and low osmolarities to the same high level. V. fischeri culture media have lower osmolarities than are typical in seawater or in cephalopods, partially accounting for the bacterium's low light output in culture.

Culture Media↗
PubMed2003

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.

Animal Structures↗
PubMed2003

Contribution of pilA to competitive colonization of the squid Euprymna scolopes by Vibrio fischeri.

Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.

Amino Acid Sequence↗