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.

Bioluminescence in Vibrio fischeri is regulated by a quorum-dependent signaling system composed of LuxI and LuxR. LuxI generates N-3-oxohexanoyl homoserine lactone (3OC6-HSL), which triggers LuxR to activate transcription of the luxICDABEG operon responsible for bioluminescence. Surprisingly, a ∆luxI mutant produced more bioluminescence than the wild type in culture. In contrast, a 4 bp duplication within luxI, resulting in a frameshift mutation and null allele, decreased luminescence tenfold. A second signaling system encoded by ainSR affects bioluminescence by increasing levels of LuxR, via the transcriptional activator LitR, and the N-octanoyl homoserine lactone (C8-HSL) signal produced by AinS is considered only a weak activator of LuxR. However, ainS is required for the bright phenotype of the ∆luxI mutant in culture. When 3OC6-HSL was provided either in the medium or by expression of luxI in trans, all cultures were brighter, but the ∆luxI mutant remained significantly brighter than the luxI frameshift mutant. Taken together, these data suggest that the enhanced bioluminescence due to the LuxI product 3OC6-HSL counteracts a negative cis-acting regulatory element within the luxI gene and that when luxI is absent the C8-HSL signal is sufficient to induce luminescence. IMPORTANCE:The regulation of bioluminescence by Vibrio fischeri is a textbook example of bacterial quorum-dependent pheromone signaling. The canonical regulatory model is that an autoinducer pheromone produced by LuxI accumulates as cells achieve a high density, and this LuxI-generated signal stimulates LuxR to activate transcription of the lux operon that underlies bioluminescence. The surprising observation that LuxI is dispensable for inducing bioluminescence forces a re-evaluation of the role of luxI. More broadly, the results underscore the potential deceptiveness of complex regulatory circuits, particularly those in which bacteria produce multiple related signaling molecules.

ABSTRACT The symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes , is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize E. scolopes using D-alanine (D-ala) auxotrophy of the symbiont, controlling the persistence of viable infection by supplying or withholding D-ala. We generated alanine racemase ( alr ) mutants of V. fischeri that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an ∆ alr mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring metC , which encodes cystathionine β-lyase. Likewise, overexpression of metC suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the ∆ alr mutation with deletions of metC and/or bsrF , which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on ∆ alr ∆ bsrF mutant MC13, which has a suppression rate of <10 −9 . When D-ala was removed from a growing culture of MC13, cells rounded and lysed within 40 minutes. Transient colonization of E. scolopes was achieved by inoculating squid in seawater containing MC13 and D-ala, and then transferring the squid into water lacking D-ala, which resulted in loss of viable symbionts within hours. Interestingly, the symbionts within crypt 3 persisted longer than those of crypt 1, suggesting a difference in bacterial growth rate in distinct crypt environments. Our study highlights a new approach for inducing transient colonization and provides insight into the biogeography of the E. scolopes light organ. IMPORTANCE The importance of this study is multi-faceted, providing a valuable methodological tool and insight into the biology of the symbiosis between Vibrio fischeri and Euprymna scolopes . First, the study sheds light on the critical role of D-ala for bacterial growth, and the underpinnings of D-ala synthesis. Our observations that metC obviates the need for D-ala supplementation of an alr mutant in minimal medium and that MetC-dependent growth correlates with D-ala in peptidoglycan, corroborate and extend previous findings in Escherichia coli regarding a role of MetC in D-ala production. Second, our isolation of robust D-ala auxotrophs led us to a novel method for studying the squid- Vibrio symbiosis, allowing for transient colonization without the use of antibiotics, and revealed intriguing differences in symbiont growth parameters in distinct light organ crypts. This work and the methodology developed will contribute to our understanding of the persistence and dynamics of V. fischeri within its host.

ABSTRACT Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in Vibrio fischeri and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic murI racD mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >10 11 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses proB to a gene encoding a putative b road- s pectrum r acemase of V. f ischeri , bsrF . An engineered bsrF allele lacking the putative secretion signal (ΔSS- bsrF ) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS- bsrF allele similarly suppressed the D-alanine auxotrophy of an alr mutant and restored prototrophy to a murI alr double mutant auxotrophic for both D-ala and D-glu. The ΔSS- bsrF allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in V. fischeri that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance. IMPORTANCE D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri , we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing [Tn-Seq, also known as insertion sequencing (INSeq)] to identify genes in P. aeruginosa that contribute to fitness during the colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies on P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance the existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats.IMPORTANCEDrosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from the analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies-and advances in high-throughput sequencing of transposon mutants-support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable the pathogen P. aeruginosa to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

AbstractPseudomonas aeruginosais recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing (Tn-Seq, also known as INSeq) to identify genes inP. aeruginosathat contribute to fitness during colonization ofDrosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies ofP. aeruginosaidentified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen inDrosophila, demonstrate the power of Tn-Seq in the fly model system and advance existing knowledge of intestinal pathogenesis byD. melanogaster,revealing bacterial colonization determinants that contribute to a comprehensive portrait ofP.aeruginosalifestyles across habitats.ImportanceDrosophila melanogasteris a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies – and advances in high-throughput sequencing of transposon mutants – support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable a pathogen,P. aeruginosa, to colonize the fly. Our analysis reveals critical mediators ofP. aeruginosaestablishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

Many bacteria communicate using diffusible pheromone signals known as autoinducers. When the autoinducer concentration reaches a threshold, which requires a minimum population density or ‘quorum’, the bacteria activate specific gene regulatory pathways. Simple diffusion of autoinducer can activate quorum-dependent pathways in cells that are located at substantial distances from the secreting source. However, modeling has predicted that autoinducer diffusion, coupled with positive feedback regulation in autoinducer synthesis, could also allow a quorum-regulated behavior to spread more rapidly through a population by moving as a self-sustaining front at constant speed. Here we show that such propagation can occur in a population of bacteria whose quorum pathway operates under its own natural regulation. We find that in unstirred populations of Vibrio fischeri , introduction of autoinducer at one location triggers a wavelike traveling front of natural bioluminescence. The front moves with a well-defined speed ∼2.5 mm h −1 , eventually outrunning the slower diffusional spreading of the initial stimulus. Consistent with predictions from modeling, the wave travels until late in growth, when population-wide activation occurs due to basal autoinducer production. Subsequent rounds of waves, including waves propagating in the reverse direction, can also be observed late in the growth of V . fischeri under natural regulation. Using an engineered, lac -dependent strain, we show that local stimuli other than autoinducers can also elicit a self-sustaining, propagating response. Our data show that the wavelike dynamics predicted by simple mathematical models of quorum signaling are readily detected in bacterial populations functioning under their own natural regulation, and that other, more complex traveling phenomena are also present. Because a traveling wave can substantially increase the efficiency of intercellular communication over macroscopic distances, our data indicate that very efficient modes of communication over distance are available to unmixed populations of V . fischeri and other microbes.

Many pheromone sensing bacteria produce and detect more than one chemically distinct signal, or autoinducer. The pathways that detect these signals are typically noisy and interlocked through crosstalk and feedback. As a result, the sensing response of individual cells is described by statistical distributions that change under different combinations of signal inputs. Here we examine how signal crosstalk reshapes this response. We measure how combinations of two homoserine lactone (HSL) input signals alter the statistical distributions of individual cell responses in the AinS/R- and LuxI/R-controlled branches of the Vibrio fischeri bioluminescence pathway. We find that, while the distributions of pathway activation in individual cells vary in complex fashion with environmental conditions, these changes have a low-dimensional representation. For both the AinS/R and LuxI/R branches, the distribution of individual cell responses to mixtures of the two HSLs is effectively one-dimensional, so that a single tuning parameter can capture the full range of variability in the distributions. Combinations of crosstalking HSL signals extend the range of responses for each branch of the circuit, so that signals in combination allow population-wide distributions that are not available under a single HSL input. Dimension reduction also simplifies the problem of identifying the HSL conditions to which the pathways and their outputs are most sensitive. A comparison of the maximum sensitivity HSL conditions to actual HSL levels measured during culture growth indicates that the AinS/R and LuxI/R branches lack sensitivity to population density except during the very earliest and latest stages of growth respectively.

N-Acetylmuramoyl-L-alanine amidases are periplasmic hydrolases that cleave the amide bond between N-acetylmuramic acid and alanine in peptidoglycan (PG). Unlike many Gram-negative bacteria that encode redundant periplasmic amidases, Vibrio fischeri appears to encode a single protein that is homologous to AmiB of Vibrio cholerae. We screened a V. fischeri transposon mutant library for strains altered in biofilm production and discovered a biofilm-overproducing strain with an insertion in amiB (VF_2326). Further characterization of biofilm enhancement suggested that this phenotype was due to the overproduction of cellulose, and it was dependent on the bcsA cellulose synthase. Additionally, the amiB mutant was nonmotile, perhaps due to defects in its ability to septate during division. The amidase mutant was unable to compete with the wild type for the colonization of V. fischeri's symbiotic host, the squid Euprymna scolopes. In single-strain inoculations, host squid inoculated with the mutant eventually became colonized but with a much lower efficiency than in squid inoculated with the wild type. This observation was consistent with the pleiotropic effects of the amiB mutation and led us to speculate that motile suppressors of the amiB mutant were responsible for the partially restored colonization. In culture, motile suppressor mutants carried point mutations in a single gene (VF_7477), resulting in a partial restoration of wild-type motility. In addition, these point mutations reversed the effect of the amiB mutation on cellulosic biofilm production. These data are consistent with V. fischeri AmiB possessing amidase activity; they also suggest that AmiB suppresses cellulosic biofilm formation but promotes successful host colonization. IMPORTANCE Peptidoglycan (PG) is a critical microbe-associated molecular pattern (MAMP) that is sloughed by cells of V. fischeri during symbiotic colonization of squid. Specifically, this process induces significant remodeling of a specialized symbiotic light organ within the squid mantle cavity. This phenomenon is reminiscent of the loss of ciliated epithelium in patients with whooping cough due to the production of PG monomers by Bordetella pertussis. Furthermore, PG processing machinery can influence susceptibility to antimicrobials. In this study, we report roles for the V. fischeri PG amidase AmiB, including the beneficial colonization of squid, underscoring the urgency to more deeply understand PG processing machinery and the downstream consequences of their activities.

As our understanding of the human microbiome progresses, so does the need for natural experimental animal models that promote a mechanistic understanding of beneficial microorganism-host interactions. Years of research into the exclusive symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri have permitted a detailed understanding of those bacterial genes underlying signal exchange and rhythmic activities that result in a persistent, beneficial association, as well as glimpses into the evolution of symbiotic competence. Migrating from the ambient seawater to regions deep inside the light-emitting organ of the squid, V. fischeri experiences, recognizes and adjusts to the changing environmental conditions. Here, we review key advances over the past 15 years that are deepening our understanding of these events.

Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an antibiotic-induced response (air) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules. IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbemicrobe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration.

Bacteria communicate by secreting and detecting diffusible small molecule signals or pheromones. Using the local concentrations of these signals to regulate gene expression, individual cells can synchronize changes in phenotype population-wide, a behavior known as quorum sensing (QS). In unstirred media, the interplay between diffusion of signals, bacterial growth, and regulatory feedback can generate complex spatial and temporal patterns of expression of QS-controlled genes. Here we identify the parameters that allow a local signal to trigger a self-sustaining, traveling activation of QS behavior. Using the natural bioluminescence of wild-type Vibrio fischeri as a readout of its lux QS system, we measure the induction of a spreading QS response by a localized triggering stimulus in unstirred media. Our data show that a QS response propagates outward, sustained by positive feedback in synthesis of the diffusible signal, and that this response occurs only if the triggering stimulus exceeds a critical threshold. We also test how the autonomous or untriggered activation of the V. fischeri QS pathway changes at very low initial population densities. At the lowest population densities, clusters of cells do not transition to a self-sensing behavior, but rather remain in communication via signal diffusion until they reach sufficiently large size that their own growth slows. Our data, which are reproduced by simple growth and diffusion simulations, indicate that in part owing to bacterial growth behavior, natural QS systems can be characterized by long distance communication through signal diffusion even in very heterogeneous and spatially dispersed populations.

ABSTRACTPlants expend significant resources to select and maintain rhizosphere communities that benefit their growth and protect them from pathogens. A better understanding of assembly and function of rhizosphere microbial communities will provide new avenues for improving crop production. Secretion of antibiotics is one means by which bacteria interact with neighboring microbes and sometimes change community composition. In our analysis of a taxonomically diverse consortium from the soybean rhizosphere, we found that Pseudomonas koreensis selectively inhibits growth of Flavobacterium johnsoniae and other members of the Bacteroidetes grown in soybean root exudate. A genetic screen in P. koreensis identified a previously uncharacterized biosynthetic gene cluster responsible for the inhibitory activity. The metabolites were isolated based on biological activity and were characterized using tandem-mass spectrometry, multidimensional NMR, and Mosher ester analysis, leading to the discovery of a new family of bacterial piperidine alkaloids, koreenceine A-D (1–4). Three of these metabolites are analogs of the plant alkaloid γ-coniceine. Comparative analysis of the koreenceine cluster with the γ-coniceine pathway revealed distinct polyketide synthase (PKS) routes to the defining piperidine scaffold, suggesting convergent evolution. Koreenceine-type pathways are widely distributed among Pseudomonas species, and koreenceine C was detected in another Pseudomonas sp. from a distantly related cluster. This work suggests that Pseudomonas and plants convergently evolved the ability to produce similar alkaloid metabolites that can mediate inter-bacterial competition in the rhizosphere.IMPORTANCEThe microbiomes of plants are critical to host physiology and development. Microbes are attracted to the rhizosphere due to massive secretion of plant photosynthates from roots. Microorganisms that successfully join the rhizosphere community from bulk soil have access to more abundant and diverse molecules, producing a highly competitive and selective environment. In the rhizosphere, as in other microbiomes, there is little known about the genetic basis for individual species’ behaviors within the community. In this study, we characterized competition between Pseudomonas koreensis and Flavobacterium johnsoniae, two common rhizosphere inhabitants. We identified a widespread gene cluster in several Pseudomonas spp., which is necessary for the production of a novel family of piperidine alkaloids that are structural analogs of plant alkaloids. We expand the known repertoire of antibiotics produced from Pseudomonas in the rhizosphere and demonstrate the role of the metabolites in interactions with other bacteria of the rhizosphere.

The quest to manipulate microbiomes has intensified, but many microbial communities have proven to be recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing an understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa, the Firmicutes, Proteobacteria, and Bacteroidetes We chose Bacillus cereus as a model rhizosphere firmicute and characterized 20 other candidates, including "hitchhikers" that coisolated with B. cereus from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers, Pseudomonas koreensis from the top tier of the competition network and Flavobacterium johnsoniae from the bottom of the network, to represent the Proteobacteria and Bacteroidetes, respectively. The model community has several emergent properties, induction of dendritic expansion of B. cereus colonies by either of the other members, and production of more robust biofilms by the three members together than individually. Moreover, P. koreensis produces a novel family of alkaloid antibiotics that inhibit growth of F. johnsoniae, and production is inhibited by B. cereus We designate this community THOR, because the members are the hitchhikers of the rhizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior.IMPORTANCE The manipulation and engineering of microbiomes could lead to improved human health, environmental sustainability, and agricultural productivity. However, microbiomes have proven difficult to alter in predictable ways, and their emergent properties are poorly understood. The history of biology has demonstrated the power of model systems to understand complex problems such as gene expression or development. Therefore, a defined and genetically tractable model community would be useful to dissect microbiome assembly, maintenance, and processes. We have developed a tractable model rhizosphere microbiome, designated THOR, containing Pseudomonas koreensis, Flavobacterium johnsoniae, and Bacillus cereus, which represent three dominant phyla in the rhizosphere, as well as in soil and the mammalian gut. The model community demonstrates emergent properties, and the members are amenable to genetic dissection. We propose that THOR will be a useful model for investigations of community-level interactions.

Many organisms produce visible light through enzymatic biological reactions, a process broadly termed bioluminescence. This article focuses specifically on the production of bioluminescence by bacteria. Although several bacterial species produce bioluminescence, they are limited to three families, and the process involves a key set of conserved Lux proteins. Centuries of research have led to a rich understanding of the evolution, ecology, biochemistry, and genetics of bacterial bioluminescence. This chapter summarizes these advances and briefly covers some of the applications that have been developed from bacterial bioluminescence.

Mutagenizing bacterial genomes with selectable transposon insertions is an effective approach for identifying the genes underlying important phenotypes. Specific bacteria may require different tools and methods for effective transposon mutagenesis, and here we describe methods to mutagenize Vibrio fischeri using an engineered mini-Tn5 transposon with synthetic "mosaic" transposon ends. The transposon is delivered by conjugation on a plasmid that cannot replicate in V. fischeri and that encodes a hyperactive transposase outside the transposon itself. The chromosomal location of insertions can be readily identified by cloning and/or PCR-based methods described here. Although developed in V. fischeri, these tools and methods have proven effective in some other bacteria as well.

ABSTRACT Pheromone signaling (PS) underlies many important bacterial behaviors, yet its ecological functions remain unresolved. Because pheromone-mediated behaviors require high cell density, the term “quorum sensing” is widely used to describe and make sense of PS. However, while this term has unified and popularized the field, bacterial PS clearly has roles beyond census taking, and the complexities of PS circuits indicate broader functional capacities. Two common features of bacterial PS are its regulation in response to environmental conditions and positive-feedback loops. Combined, these could enable PS to coordinate quorum-dependent group behaviors in response to heterogeneous environmental cues. Particularly in PS systems where positive feedback is strong, cells that are relatively far from a stimulatory environment could be recruited to a group response. Testing this model will benefit from in situ examination of relevant environmental cues and PS outputs in cells across populations, with and without positive feedback, in heterogeneous environments.

Mounting evidence suggests that D-amino acids play previously under-appreciated roles in diverse organisms. In bacteria, even D-amino acids that are absent from canonical peptidoglycan (PG) may act as growth substrates, as signals, or in other functions. Given these proposed roles and the ubiquity of D-amino acids, the paucity of known D-amino-acid-responsive transcriptional control mechanisms in bacteria suggests that such regulation awaits discovery. We found that DarR, a LysR-type transcriptional regulator (LTTR), activates transcription in response to D-Asp. The D-Glu auxotrophy of a Vibrio fischeri murI:: Tn mutant was suppressed, with the wildtype PG structure maintained, by a point mutation in darR. This darR mutation resulted in the overexpression of an adjacent operon encoding a putative aspartate racemase, RacD, which compensated for the loss of the glutamate racemase encoded by murI. Using transcriptional reporters, we found that wild-type DarR activated racD transcription in response to exogenous D-Asp but not upon the addition of L-Asp, L-Glu, or D-Glu. A DNA sequence typical of LTTR-binding sites was identified between darR and the divergently oriented racD operon, and scrambling this sequence eliminated activation of the reporter in response to D-Asp. In several proteobacteria, genes encoding LTTRs similar to DarR are linked to genes with predicted roles in D-and/or L-Asp metabolism. To test the functional similarities in another bacterium, darR and racD mutants were also generated in Acinetobacter baylyi. In V. fischeri and A. baylyi, growth on D-Asp required the presence of both darR and racD. Our results suggest that multiple bacteria have the ability to sense and respond to D-Asp. IMPORTANCE D-Amino acids are prevalent in the environment and are generated by organisms from all domains of life. Although some biological roles for D-amino acids are understood, in other cases, their functions remain uncertain. Given the ubiquity of D-amino acids, it seems likely that bacteria will initiate transcriptional responses to them. Elucidating D-amino acid-responsive regulators along with the genes they control will help uncover bacterial uses of D-amino acids. Here, we report the discovery of DarR, a novel LTTR in V. fischeri that mediates a transcriptional response to environmental D-Asp and underpins the catabolism of D-Asp. DarR represents the founding member of a group of bacterial homologs that we hypothesize control aspects of aspartate metabolism in response to D-Asp and/or to D-Asp-containing peptides.