Bacterial growth dynamics in a rhythmic symbiosis.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for ∼6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage.

A prototrophic suppressor of a Vibrio fischeri D-glutamate auxotroph reveals a member of the periplasmic broad-spectrum racemase family (BsrF).

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 >1011 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 broad-spectrum racemase of V. fischeri, 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.

Vibrio fischeri: a model for host-associated biofilm formation.

Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.

Toxicity evaluation of the contaminated area of Crotone from biological indicators: a multispecies approach.

Contamination of terrestrial and aquatic ecosystems by toxic industrial waste has become a major issue in many countries. Of particular concern is the reuse of toxic hazardous waste in construction materials. This paper examined for the first time the chemical and radiation ecotoxicity of site-specific Technological Enhanced Naturally Occurring Radioactive Materials (TENORM) residues from phosphate processing industry in soil environmental matrices through bioindicators. The area under investigation was the former industrial district of Crotone (Calabria, Italy), recently included within the Sites of National Interest (SIN), comprising the 42 Italian national priority contaminated sites. Major biological exposure pathways considered were absorption and bioaccumulation. The marine bacterium Vibrio fischeri and the freshwater crustacean Daphnia magna were employed as aquatic bioindicators, while for the soil ecosystem, the seeds of Sorghum saccharatum and Lepidium sativum were used. Selection of test species aimed at assessing the toxicity of wastes in soil as well as in freshwater or marine systems. Results indicated V. fischeri as the most sensitive of all the species tested (5.56 g/L), while D. magna was found to be affected at 94.27 g/L. An overall inhibition was observed in seedling growth as compared to control at the highest concentration of the pollutants (100 g/L), while seed germination was not adversely affected by the pollutant. At this preliminary level, data indicated a potential risk for biodiversity of the area. In fact, the measured toxicity thresholds, even if above 100 mg/L, are comparable to concentrations of the toxicants spread all over the territory of Crotone.

Investigating chirality in quorum sensing by analysis of Burkholderia cepacia and Vibrio fischeri with comprehensive chiral LC-MS/MS and GC-MS/MS methods.

N-acyl homoserine lactones (N-HLs) are signaling molecules used by Gram-negative bacteria in a phenomenon called quorum sensing. Bacteria will detect N-HLs as a way of monitoring their population which, upon reaching a critical level, will express a specific phenotype. An example is the expression of bioluminescence by Vibrio fischeri. Most studies have not considered the chirality of these molecules nor have they used highly sensitive detection methods. Here, the production of d,l-N-HLs are monitored for V. fischeri, Burkholderia cepacia, Pseudomonas fluorescens, and P. putida, using highly sensitive tandem mass spectrometry analysis. Novel N-HLs are reported for both V. fischeri and B. cepacia, including a plethora of previously unknown d-N-HLs, including the first d-N-HLs containing oxo and hydroxy functionalities. Anomalously, N-HLs were not detected in any cultures of P. fluorescens and P. putida, which are species that previously were reported to produce N-HLs. However, it is apparent that differences in the reported occurrence and levels of N-HLs can result from (a) different strains of bacteria, (b) different growth media and environmental conditions, and (c) sometimes false-positive results from detection methodologies. Time studies of V. fischeri suggest the possibility that separate synthetic and elimination pathways exist between d- and l-N-HLs. Possible biological processes that could be the source of d-N-HL production are considered.

Calcium Promotes T6SS-Mediated Killing and Aggregation between Competing Symbionts.

Bacteria use a variety of strategies to exclude competitors from accessing resources, including space within a host niche. Because these mechanisms are typically costly to deploy, they are often tightly regulated for use in environments where the benefits outweigh the energetic cost. The type VI secretion system (T6SS) is a competitive mechanism that allows inhibitors to kill competing microbes by physically puncturing and translocating cytotoxic effectors directly into neighboring competitor cells. Although T6SSs are encoded in both symbiotic and free-living taxa where they may be actively secreting into the extracellular milieu during growth in liquid culture, there is little evidence for bacteria engaging in T6SS-mediated, contact-dependent killing under low-viscosity liquid conditions. Here, we determined that calcium acts as a pH-dependent cue to activate the assembly of an antibacterial T6SS in a Vibrio fischeri light organ symbiont in a low-viscosity liquid medium. Moreover, competing V. fischeri isolates formed mixed-strain aggregates that promoted the contact necessary for T6SS-dependent elimination of a target population. Our findings expand our knowledge of V. fischeri T6SS ecology and identify a low-viscosity liquid condition where cells engage in contact-dependent killing. IMPORTANCE Microbes deploy competitive mechanisms to gain access to resources such as nutrients or space within an ecological niche. Identifying when and where these strategies are employed can be challenging given the complexity and variability of most natural systems; therefore, studies evaluating specific cues that conditionally regulate interbacterial competition can inform the ecological context for such competition. In this work, we identified a pH-dependent chemical cue in seawater, calcium, which promotes activation of a contact-dependent interbacterial weapon in the marine symbiont Vibrio fischeri. This finding underscores the importance of using ecologically relevant salts in growth media and the ability of bacterial cells to sense and integrate multiple environmental cues to assess the need for a weapon. Identification of these cues provides insight into the types of environments where employing a weapon is advantageous to the survival and propagation of a bacterial population.

Mutational Analysis of Vibrio fischeri c-di-GMP-Modulating Genes Reveals Complex Regulation of Motility.

The symbiont Vibrio fischeri uses motility to colonize its host. In numerous bacterial species, motility is negatively controlled by cyclic-di-GMP (c-di-GMP), which is produced by diguanylate cyclases (DGCs) with GGDEF domains and degraded by phosphodiesterases with either EAL or HD-GYP domains. To begin to decode the functions of the 50 Vibrio fischeri genes with GGDEF, EAL, and/or HD-GYP domains, we deleted each gene and assessed each mutant's migration through tryptone broth salt (TBS) soft agar medium containing or lacking magnesium (Mg) and calcium (Ca), which are known to influence V. fischeri motility. We identified 6, 13, and 16 mutants with altered migration in TBS-Mg, TBS, and TBS-Ca soft agar, respectively, a result that underscores the importance of medium conditions in assessing gene function. A biosensor-based assay revealed that Mg and Ca affected c-di-GMP levels negatively and positively, respectively; the severe decrease in c-di-GMP caused by Mg addition correlates with its strong positive impact on bacterial migration. A mutant defective for VF_0494, a homolog of V. cholerae rocS, exhibited a severe defect in migration across all conditions. Motility of a VF_1603 VF_2480 double mutant was also severely defective and could be restored by expression of "c-di-GMP-blind" alleles of master flagellar regulator flrA. Together, this work sheds light on the genes and conditions that influence c-di-GMP-mediated control over motility in V. fischeri and provides a foundation for (i) assessing roles of putative c-di-GMP-binding proteins, (ii) evaluating other c-di-GMP-dependent phenotypes in V. fischeri, (iii) uncovering potential redundancy, and (iv) deciphering signal transduction mechanisms. IMPORTANCE Critical bacterial processes, including motility, are influenced by c-di-GMP, which is controlled by environment-responsive synthetic and degradative enzymes. Because bacteria such as Vibrio fischeri use motility to colonize their hosts, understanding the roles of c-di-GMP-modulating enzymes in controlling motility has the potential to inform on microbe-host interactions. We leveraged recent advances in genetic manipulation to generate 50 mutants defective for putative c-di-GMP synthetic and degradative enzymes. We then assessed the consequences on motility, manipulating levels of magnesium and calcium, which inversely influenced motility and levels of c-di-GMP. Distinct subsets of the 50 genes were required under the different conditions. Our data thus provide needed insight into the functions of these enzymes and environmental factors that influence them.

A Putative Lipoprotein Mediates Cell-Cell Contact for Type VI Secretion System-Dependent Killing of Specific Competitors.

Interbacterial competition is prevalent in host-associated microbiota, where it can shape community structure and function, impacting host health in both positive and negative ways. However, the factors that permit bacteria to discriminate among their various neighbors for targeted elimination of competitors remain elusive. We identified a putative lipoprotein (TasL) in Vibrio species that mediates cell-cell attachment with a subset of target strains, allowing inhibitors to target specific competitors for elimination. Here, we describe this putative lipoprotein, which is associated with the broadly distributed type VI secretion system (T6SS), by studying symbiotic Vibrio fischeri, which uses the T6SS to compete for colonization sites in their squid host. We demonstrate that TasL allows V. fischeri cells to restrict T6SS-dependent killing to certain genotypes by selectively integrating competitor cells into aggregates while excluding other cell types. TasL is also required for T6SS-dependent competition within juvenile squid, indicating that the adhesion factor is active in the host. Because TasL homologs are found in other host-associated bacterial species, this newly described cell-cell attachment mechanism has the potential to impact microbiome structure within diverse hosts. IMPORTANCE T6SSs are broadly distributed interbacterial weapons that share an evolutionary history with bacteriophage. Because the T6SS can be used to kill neighboring cells, it can impact the spatial distribution and biological function of both free-living and host-associated microbial communities. Like their phage relatives, T6SS+ cells must sufficiently bind competitor cells to deliver their toxic effector proteins through the syringe-like apparatus. Although phage use receptor-binding proteins (RBPs) and tail fibers to selectively bind prey cells, the biophysical properties that mediate this cell-cell contact for T6SS-mediated killing remain unknown. Here, we identified a large, predicted lipoprotein that is coordinately expressed with T6SS proteins and facilitates the contact that is necessary for the T6SS-dependent elimination of competitors in a natural host. Similar to phage RBPs and tail fibers, this lipoprotein is required for T6SS+ cells to discriminate between prey and nonprey cell types, revealing new insight into prey selection during T6SS-mediated competition.

Three Microbial Musketeers of the Seas: Shewanella baltica, Aliivibrio fischeri and Vibrio harveyi, and Their Adaptation to Different Salinity Probed by a Proteomic Approach.

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria. In this work, we aimed to elucidate the basis of adjustment to prolonged salinity challenges at the proteome level in marine bacteria. The objects of our studies were three representatives of bacteria inhabiting various marine environments, Shewanella baltica, Vibrio harveyi and Aliivibrio fischeri. The proteomic studies were performed with bacteria cultivated in increased and decreased salinity, followed by proteolytic digestion of samples which were then subjected to liquid chromatography with tandem mass spectrometry analysis. We show that bacteria adjust at all levels of their biological processes, from DNA topology through gene expression regulation and proteasome assembly, to transport and cellular metabolism. The finding that many similar adaptation strategies were observed for both low- and high-salinity conditions is particularly striking. The results show that adaptation to salinity challenge involves the accumulation of DNA-binding proteins and increased polyamine uptake. We hypothesize that their function is to coat and protect the nucleoid to counteract adverse changes in DNA topology due to ionic shifts.

Production of both l- and d- N-acyl-homoserine lactones by Burkholderia cepacia and Vibrio fischeri.

Quorum sensing (QS) is a complex process in which molecules, such as l-N-acyl-homoserine lactones (l-AHLs), are produced as essential signaling molecules allowing bacteria to detect and respond to cell population density by gene regulation. Few studies have considered the natural production and role of the opposite enantiomers, d-AHLs. In this work, production of d,l-AHLs by Burkholderia cepacia and Vibrio fischeri was monitored over time, with significant amounts of d-AHLs detected. Bioluminescence of V. fischeri was observed with maximum bioluminescence correlating with the maximum concentrations of both l- and d- octanoyl-homoserine lactones (l- and d-OHL). l-Methionine, a precursor to l-AHLs, was examined via supplementation studies conducted by growing three parallel cultures of B. cepacia in M9 minimal media with added l-, d-, or d,l-methionine and observing their effect on the production of d,l-AHL by B. cepacia. The results show that addition of any methionine (l-, d-, or d,l-) does not affect the overall ratio of l- to d-AHLs, that is d-AHL production was not selectively enhanced by d-methionine addition. However, the overall AHL (l- and d-) concentration does increase with the addition of any methionine supplement. These findings indicate the possibility of a distinct biosynthetic pathway for d-AHL production, possibly exposing a new dimension within bacterial communication.

Calcium-Responsive Diguanylate Cyclase CasA Drives Cellulose-Dependent Biofilm Formation and Inhibits Motility in Vibrio fischeri.

The marine bacterium Vibrio fischeri colonizes its host, the Hawaiian bobtail squid, in a manner requiring both bacterial biofilm formation and motility. The decision to switch between sessile and motile states is often triggered by environmental signals and regulated by the widespread signaling molecule c-di-GMP. Calcium is an environmental signal previously shown to affect both biofilm formation and motility by V. fischeri. In this study, we investigated the link between calcium and c-di-GMP, determining that calcium increases intracellular c-di-GMP dependent on a specific diguanylate cyclase, calcium-sensing protein A (CasA). CasA is activated by calcium, dependent on residues in an N-terminal sensory domain, and synthesizes c-di-GMP through an enzymatic C-terminal domain. CasA is responsible for calcium-dependent inhibition of motility and activation of cellulose-dependent biofilm formation. Calcium regulates cellulose biofilms at the level of transcription, which also requires the transcription factor VpsR. Finally, the Vibrio cholerae CasA homolog, CdgK, is unable to complement CasA and may be inhibited by calcium. Collectively, these results identify CasA as a calcium-responsive regulator, linking an external signal to internal decisions governing behavior, and shed light on divergence between Vibrio spp. IMPORTANCE Biofilm formation and motility are often critical behaviors for bacteria to colonize a host organism. Vibrio fischeri is the exclusive colonizer of its host's symbiotic organ and requires both biofilm formation and motility to initiate successful colonization, providing a relatively simple model to explore complex behaviors. In this study, we determined how the environmental signal calcium alters bacterial behavior through production of the signaling molecule c-di-GMP. Calcium activates the diguanylate cyclase CasA to synthesize c-di-GMP, resulting in inhibition of motility and activation of cellulose production. These activities depend on residues in CasA's N-terminal sensory domain and C-terminal enzymatic domain. These findings thus identify calcium as a signal recognized by a specific diguanylate cyclase to control key bacterial phenotypes. Of note, CasA activity is seemingly inverse to that of the homologous V. cholerae protein, CdgK, providing insight into evolutionary divergence between closely related species.

Para-Aminobenzoic Acid, Calcium, and c-di-GMP Induce Formation of Cohesive, Syp-Polysaccharide-Dependent Biofilms in Vibrio fischeri.

The marine bacterium Vibrio fischeri efficiently colonizes its symbiotic squid host, Euprymna scolopes, by producing a transient biofilm dependent on the symbiosis polysaccharide (SYP). In vitro, however, wild-type strain ES114 fails to form SYP-dependent biofilms. Instead, genetically engineered strains, such as those lacking the negative regulator BinK, have been developed to study this phenomenon. Historically, V. fischeri has been grown using LBS, a complex medium containing tryptone and yeast extract; supplementation with calcium is required to induce biofilm formation by a binK mutant. Here, through our discovery that yeast extract inhibits biofilm formation, we uncover signals and underlying mechanisms that control V. fischeri biofilm formation. In contrast to its inability to form a biofilm on unsupplemented LBS, a binK mutant formed cohesive, SYP-dependent colony biofilms on tTBS, modified LBS that lacks yeast extract. Moreover, wild-type strain ES114 became proficient to form cohesive, SYP-dependent biofilms when grown in tTBS supplemented with both calcium and the vitamin para-aminobenzoic acid (pABA); neither molecule alone was sufficient, indicating that this phenotype relies on coordinating two cues. pABA/calcium supplementation also inhibited bacterial motility. Consistent with these phenotypes, cells grown in tTBS with pABA/calcium were enriched in transcripts for biofilm-related genes and predicted diguanylate cyclases, which produce the second messenger cyclic-di-GMP (c-di-GMP). They also exhibited elevated levels of c-di-GMP, which was required for the observed phenotypes, as phosphodiesterase overproduction abrogated biofilm formation and partially rescued motility. This work thus provides insight into conditions, signals, and processes that promote biofilm formation by V. fischeri. IMPORTANCE Bacteria integrate environmental signals to regulate gene expression and protein production to adapt to their surroundings. One such behavioral adaptation is the formation of a biofilm, which can promote adherence and colonization and provide protection against antimicrobials. Identifying signals that trigger biofilm formation and the underlying mechanism(s) of action remain important and challenging areas of investigation. Here, we determined that yeast extract, commonly used for growth of bacteria in laboratory culture, inhibits biofilm formation by Vibrio fischeri, a model bacterium used for investigating host-relevant biofilm formation. Omitting yeast extract from the growth medium led to the identification of an unusual signal, the vitamin para-aminobenzoic acid (pABA), that when added together with calcium could induce biofilm formation. pABA increased the concentrations of the second messenger, c-di-GMP, which was necessary but not sufficient to induce biofilm formation. This work thus advances our understanding of signals and signal integration controlling bacterial biofilm formation.

Activation of the Type VI Secretion System in the Squid Symbiont Vibrio fischeri Requires the Transcriptional Regulator TasR and the Structural Proteins TssM and TssA.

Bacteria have evolved diverse strategies to compete for a niche, including the type VI secretion system (T6SS), a contact-dependent killing mechanism. T6SSs are common in bacterial pathogens, commensals, and beneficial symbionts, where they affect the diversity and spatial structure of host-associated microbial communities. Although T6SS gene clusters are often located on genomic islands (GIs), which may be transferred as a unit, the regulatory strategies that promote gene expression once the T6SS genes are transferred into a new cell are not known. We used the squid symbiont Vibrio fischeri to identify essential regulatory factors that control expression of a strain-specific T6SS encoded on a GI. We found that a transcriptional reporter for this T6SS is active only in strains that contain the T6SS-encoding GI, suggesting the GI encodes at least one essential regulator. A transposon screen identified seven mutants that could not activate the reporter. These mutations mapped exclusively to three genes on the T6SS-containing GI that encode two essential structural proteins (a TssA-like protein and TssM) and a transcriptional regulator (TasR). Using T6SS reporters, reverse transcription-PCR (RT-PCR), competition assays, and differential proteomics, we found that all three genes are required for expression of many T6SS components, except for the TssA-like protein and TssM, which are constitutively expressed. Based on these findings, we propose a model whereby T6SS expression requires conserved structural proteins, in addition to the essential regulator TasR, and this ability to self-regulate may be a strategy to activate T6SS expression upon transfer of T6SS-encoding elements into a new bacterial host. IMPORTANCE Interbacterial weapons like the T6SS are often located on mobile genetic elements, and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in Vibrio fischeri. These structural proteins also contain predicted GTPase and GTP binding domains, suggesting their role in promoting T6SS expression may involve sensing the energetic state of the cell. Such a mechanism would provide a direct link between T6SS activation and cellular energy levels, providing a "checkpoint" to ensure the cell has sufficient energy to build such a costly weapon. Because these regulatory factors are encoded within the T6SS gene cluster, they are predicted to move with the genetic element to activate T6SS expression in a new host cell.

Crystal structure details of Vibrio fischeri DarR and mutant DarR-M202I from LTTR family reveals their activation mechanism.

DarR, a novel member of the LTTR family derived from Vibrio fischeri, activates transcription in response to d-Asp and regulates the overexpression of the racD genes encoding a putative aspartate racemase, RacD. Here, the crystal structure of full-length DarR and its mutant DarR-M202I were obtained by X-ray crystallography. According to the electron density map analysis of full-length DarR, the effector binding site of DarR is occupied by 2-Morpholinoethanesulfonic acid monohydrate (MES), which could interact with amino acids in the effector binding site and stabilize the effector binding site. Furthermore, we elaborated the structure of DarR-M202I, where methionine is replaced by isoleucine resulting in overexpression of the downstream operon. By comparing DarR-MES and DarR-M202I, we found similar behavior of DarR-MES in terms of the stability of the RD active pocket and the deflection angle of the DBD. The Isothermal titration calorimetry and Gel-filtration chromatography experiments showed that only when the target DNA sequence of a particular quasi-palindromic sequence exceeds 19 bp, DarR can effectively bind to racD promoter. This study will help enhance our understanding of the mechanism in the transcriptional regulation of LTTR family transcription factors.

A Small Molecule Coordinates Symbiotic Behaviors in a Host Organ.

The lifelong relationship between the Hawaiian bobtail squid Euprymna scolopes and its microbial symbiont Vibrio fischeri represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the host in a process termed counterillumination. The squid host hatches without its symbionts, which must be acquired from the ocean amidst a diversity of nonbeneficial bacteria, such that precise molecular communication is required for initiation of the specific relationship. Therefore it is likely there are specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in V. fischeri with altered biofilm production, which correlates directly to colonization capability in its host. "Biofilm-up" and "biofilm-down" mutants were compared to a wild-type strain, and ions that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structural elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(d-histidyl-l-proline). This diketopiperazine modulated luminescence in V. fischeri and, using imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors.IMPORTANCE The complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between Vibrio fischeri and Euprymna scolopes, we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming V. fischeri strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.

Toxicity of Zn-Fe Layered Double Hydroxide to Different Organisms in the Aquatic Environment.

The application of layered double hydroxide (LDH) nanomaterials as catalysts has attracted great interest due to their unique structural features. It also triggered the need to study their fate and behavior in the aquatic environment. In the present study, Zn-Fe nanolayered double hydroxides (Zn-Fe LDHs) were synthesized using a co-precipitation method and characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and nitrogen adsorption-desorption analyses. The toxicity of the home-made Zn-Fe LDHs catalyst was examined by employing a variety of aquatic organisms from different trophic levels, namely the marine photobacterium Vibrio fischeri, the freshwater microalga Pseudokirchneriella subcapitata, the freshwater crustacean Daphnia magna, and the duckweed Spirodela polyrhiza. From the experimental results, it was evident that the acute toxicity of the catalyst depended on the exposure time and type of selected test organism. Zn-Fe LDHs toxicity was also affected by its physical state in suspension, chemical composition, as well as interaction with the bioassay test medium.

Control of Competence in Vibrio fischeri.

Vibrio species, including the squid symbiont Vibrio fischeri, become competent to take up DNA under specific conditions. For example, V. fischeri becomes competent when grown in the presence of chitin oligosaccharides or upon overproduction of the competence regulatory factor TfoX. While little is known about the regulatory pathway(s) that controls V. fischeri competence, this microbe encodes homologs of factors that control competence in the well-studied V. cholerae To further develop V. fischeri as a genetically tractable organism, we evaluated the roles of some of these competence homologs. Using TfoX-overproducing cells, we found that competence depends upon LitR, the homolog of V. cholerae master quorum-sensing and competence regulator HapR, and upon homologs of putative pilus genes that in V. cholerae facilitate DNA uptake. Disruption of genes for negative regulators upstream of LitR, namely, the LuxO protein and the small RNA (sRNA) Qrr1, resulted in increased transformation frequencies. Unlike LitR-controlled light production, however, competence did not vary with cell density under tfoX overexpression conditions. Analogous to the case with V. cholerae, the requirement for LitR could be suppressed by loss of the Dns nuclease. We also found a role for the putative competence regulator CytR. Finally, we determined that transformation frequencies varied depending on the TfoX-encoding plasmid, and we developed a new dual tfoX and litR overexpression construct that substantially increased the transformation frequency of a less genetically tractable strain. By advancing the ease of genetic manipulation of V. fischeri, these findings will facilitate the rapid discovery of genes involved in physiologically relevant processes, such as biofilm formation and host colonization.IMPORTANCE The ability of bacteria to take up DNA (competence) and incorporate foreign DNA into their genomes (transformation) permits them to rapidly evolve and gain new traits and/or acquire antibiotic resistances. It also facilitates laboratory-based investigations into mechanisms of specific phenotypes, such as those involved in host colonization. Vibrio fischeri has long been a model for symbiotic bacterium-host interactions as well as for other aspects of its physiology, such as bioluminescence and biofilm formation. Competence of V. fischeri can be readily induced upon overexpression of the competence factor TfoX. Relatively little is known about the V. fischeri competence pathway, although homologs of factors known to be important in V. cholerae competence exist. By probing the importance of putative competence factors that control transformation of V. fischeri, this work deepens our understanding of the competence process and advances our ability to genetically manipulate this important model organism.

Ecotoxicity profile of heavily contaminated surface water of two rivers in Tunisia.

Persistent organic and inorganic contaminants generated by industrial effluent wastes poses a threat to the maintenance of aquatic ecosystems and public health. The Khniss and Hamdoun rivers, located in the central-east of Tunisia, receive regularly domestic and textile wastewater load. The present study aimed to survey the water quality of these rivers using physicochemical, analytical and toxicological approaches. In the physicochemical analysis, the recorded levels of COD and TSS in both samples exceed the Tunisian standards. Using the analytical approach, several metals and some textile dyes were detected. Indeed, 17 metals were detected in both samples in varying concentrations, which do not exceed the Tunisian standards. The sources of metals pollution can be of natural and anthropogenic origin. Three textile disperse dyes were detected with high levels compared to other studies: the disperse orange 37 was detected in the Khniss river with a concentration of 6.438 µg/L and the disperse red 1 and the disperse yellow 3 were detected in the Hamdoun river with concentrations of 3.873 µg/L and 1895 µg/L, respectively. Textile activities were the major sources of disperse dyes. For both samples, acute and chronic ecotoxicity was observed in all the studied organisms, however, no genotoxic activity was detected. The presence of metals and textile disperse dyes could be associated with the ecotoxicological effects observed in the river waters, in particular due to the industrial activity, a fact that could deteriorate the ecosystem and therefore threaten the human health of the population living in the study areas. Combining chemical and biological approaches, allowed the detection of water ecotoxicity in testing organisms and the identification of possible contributors to the toxicity observed in these multi-stressed water reservoirs.

Identification of a Transcriptomic Network Underlying the Wrinkly and Smooth Phenotypes of Vibrio fischeri.

Vibrio fischeri is a cosmopolitan marine bacterium that oftentimes displays different colony morphologies, switching from a smooth to a wrinkly phenotype in order to adapt to changes in the environment. This wrinkly phenotype has also been associated with increased biofilm formation, an essential characteristic for V. fischeri to adhere to substrates, to suspended debris, and within the light organs of sepiolid squids. Elevated levels of biofilm formation are correlated with increased microbial survival of exposure to environmental stressors and the ability to expand niche breadth. Since V. fischeri has a biphasic life history strategy between its free-living and symbiotic states, we were interested in whether the wrinkly morphotype demonstrated differences in its expression profile in comparison to the naturally occurring and more common smooth variant. We show that genes involved in major biochemical cascades, including those involved in protein sorting, oxidative stress, and membrane transport, play a role in the wrinkly phenotype. Interestingly, only a few unique genes are specifically involved in macromolecule biosynthesis in the wrinkly phenotype, which underlies the importance of other pathways utilized for adaptation under the conditions in which Vibrio bacteria are producing this change in phenotype. These results provide the first comprehensive analysis of the complex form of genetic activation that underlies the diversity in morphologies of V. fischeri when switching between two different colony morphotypes, each representing a unique biofilm ecotype.IMPORTANCE The wrinkly bacterial colony phenotype has been associated with increased squid host colonization in V. fischeri The significance of our research is in identifying the genetic mechanisms that are responsible for heightened biofilm formation in V. fischeri This report also advances our understanding of gene regulation in V. fischeri and brings to the forefront a number of previously overlooked genetic networks. Several loci that were identified in this study were not previously known to be associated with biofilm formation in V. fischeri.

The cytokine MIF controls daily rhythms of symbiont nutrition in an animal-bacterial association.

The recent recognition that many symbioses exhibit daily rhythms has encouraged research into the partner dialogue that drives these biological oscillations. Here we characterized the pivotal role of the versatile cytokine macrophage migration inhibitory factor (MIF) in regulating a metabolic rhythm in the model light-organ symbiosis between Euprymna scolopes and Vibrio fischeri As the juvenile host matures, it develops complex daily rhythms characterized by profound changes in the association, from gene expression to behavior. One such rhythm is a diurnal shift in symbiont metabolism triggered by the periodic provision of a specific nutrient by the mature host: each night the symbionts catabolize chitin released from hemocytes (phagocytic immune cells) that traffic into the light-organ crypts, where the population of V. fischeri cells resides. Nocturnal migration of these macrophage-like cells, together with identification of an E. scolopes MIF (EsMIF) in the light-organ transcriptome, led us to ask whether EsMIF might be the gatekeeper controlling the periodic movement of the hemocytes. Western blots, ELISAs, and confocal immunocytochemistry showed EsMIF was at highest abundance in the light organ. Its concentration there was lowest at night, when hemocytes entered the crypts. EsMIF inhibited migration of isolated hemocytes, whereas exported bacterial products, including peptidoglycan derivatives and secreted chitin catabolites, induced migration. These results provide evidence that the nocturnal decrease in EsMIF concentration permits the hemocytes to be drawn into the crypts, delivering chitin. This nutritional function for a cytokine offers the basis for the diurnal rhythms underlying a dynamic symbiotic conversation.