scholarly journals LitR Is a Repressor of syp Genes and Has a Temperature-Sensitive Regulatory Effect on Biofilm Formation and Colony Morphology in Vibrio (Aliivibrio) salmonicida

2014 ◽  
Vol 80 (17) ◽  
pp. 5530-5541 ◽  
Author(s):  
Hilde Hansen ◽  
Ane Mohn Bjelland ◽  
Maria Ronessen ◽  
Espen Robertsen ◽  
Nils Peder Willassen
Keyword(s):  
Biofilm Formation ◽  
Cold Water ◽  
Vibrio Fischeri ◽  
Seawater Temperature ◽  
Colony Morphology ◽  
Effective Vaccine ◽  
Temperature Sensitive ◽  
Regulatory Effect ◽  
Content Type ◽  
Fish Model

ABSTRACTVibrio(Aliivibrio)salmonicidais the etiological agent of cold water vibriosis, a disease in farmed Atlantic salmon (Salmo salar) that is kept under control due to an effective vaccine. A seawater temperature below 12°C is normally required for disease development. Quorum sensing (QS) is a cell density-regulated communication system that bacteria use to coordinate activities involved in colonization and pathogenesis, and we have previously shown that inactivation of the QS master regulator LitR attenuates theV. salmonicidastrain LFI1238 in a fish model. We show here that strain LFI1238 and a panel of naturally occurringV. salmonicidastrains are poor biofilm producers. Inactivation oflitRin the LFI1238 strain enhances medium- and temperature-dependent adhesion, rugose colony morphology, and biofilm formation. Chemical treatment and electron microscopy of the biofilm identified an extracellular matrix consisting mainly of a fibrous network, proteins, and polysaccharides. Further, by microarray analysis of planktonic and biofilm cells, we identified a number of genes regulated by LitR and, among these, were homologues of theVibrio fischerisymbiosis polysaccharide (syp) genes. Thesypgenes were regulated by LitR in both planktonic and biofilm lifestyle analyses. Disruption ofsypgenes in theV. salmonicidaΔlitRmutant alleviated adhesion, rugose colony morphology, and biofilm formation. Hence, LitR is a repressor ofsyptranscription that is necessary for expression of the phenotypes examined. The regulatory effect of LitR on colony morphology and biofilm formation is temperature sensitive and weak or absent at temperatures above the bacterium's upper threshold for pathogenicity.

scholarly journals Tools for Rapid Genetic Engineering of Vibrio fischeri

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Karen L. Visick ◽  
Kelsey M. Hodge-Hanson ◽  
Alice H. Tischler ◽  
Allison K. Bennett ◽  
Vincent Mastrodomenico
Keyword(s):  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Vibrio Fischeri ◽  
Single Step ◽  
Universal Primers ◽  
Resistance Marker ◽  
Content Type ◽  
Flp Recombinase ◽  
Overlap Extension

ABSTRACT Vibrio fischeri is used as a model for a number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multistep process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the V. fischeri chromosome without cloning. We developed a uniform approach for generating deletions using PCR splicing by overlap extension (SOEing) with antibiotic cassettes flanked by standardized linker sequences. PCR SOEing of the cassettes to sequences up- and downstream of the target gene generates a DNA product that can be directly introduced by natural transformation. Selection for the introduced antibiotic resistance marker yields the deletion of interest in a single step. Because these cassettes also contain FRT (FLP recognition target) sequences flanking the resistance marker, Flp recombinase can be used to generate an unmarked, in-frame deletion. We developed a similar methodology and tools for the rapid insertion of specific genes at a benign site in the chromosome for purposes such as complementation. Finally, we generated derivatives of these tools to facilitate different applications, such as inducible gene expression and assessing protein production. We demonstrated the utility of these tools by deleting and inserting genes known or predicted to be involved in motility. While developed for V. fischeri strain ES114, we anticipate that these tools can be adapted for use in other V. fischeri strains and, potentially, other microbes. IMPORTANCE Vibrio fischeri is a model organism for studying a variety of important processes, including symbiosis, biofilm formation, and quorum sensing. To facilitate investigation of these biological mechanisms, we developed approaches for rapidly generating deletions and insertions and demonstrated their utility using two genes of interest. The ease, consistency, and speed of the engineering is facilitated by a set of antibiotic resistance cassettes with common linker sequences that can be amplified by PCR with universal primers and fused to adjacent sequences using splicing by overlap extension and then introduced directly into V. fischeri , eliminating the need for cloning and plasmid conjugation. The antibiotic cassettes are flanked by FRT sequences, permitting their removal using Flp recombinase. We augmented these basic tools with a family of constructs for different applications. We anticipate that these tools will greatly accelerate mechanistic studies of biological processes in V. fischeri and potentially other Vibrio species.

scholarly journals LitR of Vibrio salmonicida Is a Salinity-Sensitive Quorum-Sensing Regulator of Phenotypes Involved in Host Interactions and Virulence

2012 ◽  
Vol 80 (5) ◽  
pp. 1681-1689 ◽  
Author(s):  
Ane Mohn Bjelland ◽  
Henning Sørum ◽  
Daget Ayana Tegegne ◽  
Hanne C. Winther-Larsen ◽  
Nils Peder Willassen ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Cold Water ◽  
Bacterial Species ◽  
Cell Communication ◽  
Cell Aggregation ◽  
Fish Host ◽  
Content Type ◽  
Vibrio Salmonicida

ABSTRACTVibrio(Aliivibrio)salmonicidais the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth ofV. salmonicidain the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome ofV. salmonicidaLFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS inV. fischeri, was deleted. Compared to the parental strain, thelitRmutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, thelitRmutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with thelitRmutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.

scholarly journals Arabinose Induces Pellicle Formation by Vibrio fischeri

2013 ◽  
Vol 79 (6) ◽  
pp. 2069-2080 ◽  
Author(s):  
Karen L. Visick ◽  
Kevin P. Quirke ◽  
Sheila M. McEwen
Keyword(s):  
Biofilm Formation ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Vibrio Fischeri ◽  
Luria Bertani ◽  
Phenol Red ◽  
Liquid Interfaces ◽  
Acid Base ◽  
Pellicle Formation ◽  
Content Type

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

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

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Alba Chavez-Dozal ◽  
William Soto ◽  
Michele K. Nishiguchi
Keyword(s):  
Biofilm Formation ◽  
Marine Bacterium ◽  
Vibrio Fischeri ◽  
Life History Strategy ◽  
Complex Form ◽  
Bacterial Colony ◽  
Content Type ◽  
Naturally Occurring ◽  
Light Organs ◽  
Genetic Mechanisms

ABSTRACT 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.

scholarly journals Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

2014 ◽  
Vol 81 (1) ◽  
pp. 422-431 ◽  
Author(s):  
Chuping Luo ◽  
Xuehui Liu ◽  
Huafei Zhou ◽  
Xiaoyu Wang ◽  
Zhiyi Chen
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Hemolytic Activity ◽  
Biofilm Formation ◽  
Gene Clusters ◽  
Colony Morphology ◽  
Swarming Motility ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Phenotypic Features

ABSTRACTBacilluscyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features ofBacillusstrains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology.Bacillus subtilis916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome ofB. subtilis916 contains four nonribosomal peptide synthase (NRPS) gene clusters,srf,bmy,fen, andloc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studyingB. subtilis916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activityin vitro, the strain mutated insrfAAhad significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other thanfenresulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion,B. subtilis916 coproduces four families of LPs which contribute to the phenotypic features ofB. subtilis916 in an intricate way.

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

mBio ◽  
2021 ◽  
Author(s):  
Alice H. Tischler ◽  
Michael E. Vanek ◽  
Natasha Peterson ◽  
Karen L. Visick
Keyword(s):  
Simple Model ◽  
Biofilm Formation ◽  
Vibrio Fischeri ◽  
Diguanylate Cyclase ◽  
Host Organism ◽  
Signaling Molecule ◽  
Content Type ◽  
Environmental Signal ◽  
Successful Colonization

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.

scholarly journals CysK Plays a Role in Biofilm Formation and Colonization by Vibrio fischeri

2015 ◽  
Vol 81 (15) ◽  
pp. 5223-5234 ◽  
Author(s):  
Priyanka Singh ◽  
John F. Brooks ◽  
Valerie A. Ray ◽  
Mark J. Mandel ◽  
Karen L. Visick
Keyword(s):  
Colony Formation ◽  
Biofilm Formation ◽  
Parent Strain ◽  
Vibrio Fischeri ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Agar Surface ◽  
Control Activation

ABSTRACTA biofilm, or a matrix-embedded community of cells, promotes the ability of the bacteriumVibrio fischerito colonize its symbiotic host, the Hawaiian squidEuprymna scolopes. Biofilm formation and colonization depend onsyp, an 18-gene polysaccharide locus. To identify other genes necessary for biofilm formation, we screened for mutants that failed to form wrinkled colonies, a type of biofilm. We obtained several with defects in genes required for cysteine metabolism, includingcysH,cysJ,cysK, andcysN. ThecysKmutant exhibited the most severe wrinkling defect. It could be complemented with a wild-type copy of thecysKgene, which encodesO-acetylserine sulfhydrolase, or by supplementing the medium with additional cysteine. None of a number of other mutants defective for biosynthetic genes negatively impacted wrinkled colony formation, suggesting a specific role for CysK. CysK did not appear to control activation of Syp regulators or transcription of thesyplocus, but it did influence production of the Syp polysaccharide. Under biofilm-inducing conditions, thecysKmutant retained the same ability as that of the parent strain to adhere to the agar surface. ThecysKmutant also exhibited a defect in pellicle production that could be complemented by thecysKgene but not by cysteine, suggesting that, under these conditions, CysK is important for more than the production of cysteine. Finally, our data reveal a role forcysKin symbiotic colonization byV. fischeri. Although many questions remain, this work provides insights into additional factors required for biofilm formation and colonization byV. fischeri.

scholarly journals Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in Mycobacterium tuberculosis

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Paras Jain ◽  
Tsungda Hsu ◽  
Masayoshi Arai ◽  
Karolin Biermann ◽  
David S. Thaler ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Single Step ◽  
Open Reading Frame ◽  
Model Systems ◽  
Effective Vaccine ◽  
Temperature Sensitive ◽  
Content Type ◽  
Reading Frame ◽  
New Methods

ABSTRACTSpecialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulentMycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library ofM. tuberculosisnull strains, where each strain is deleted for a single defined open reading frame inM. tuberculosis.IMPORTANCEThis work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulentM. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.

scholarly journals The Hybrid Sensor Kinase RscS Integrates Positive and Negative Signals To Modulate Biofilm Formation in Vibrio fischeri

2008 ◽  
Vol 190 (13) ◽  
pp. 4437-4446 ◽  
Author(s):  
Kati Geszvain ◽  
Karen L. Visick
Keyword(s):  
Biofilm Formation ◽  
Vibrio Fischeri ◽  
Transmembrane Helix ◽  
Bioinformatic Analysis ◽  
Colony Morphology ◽  
Sensor Kinase ◽  
Pas Domain ◽  
Transmembrane Helices ◽  
Pellicle Formation ◽  
Carboxy Terminal

ABSTRACT Overexpression of the Vibrio fischeri sensor kinase RscS induces expression of the syp (symbiosis polysaccharide) gene cluster and promotes biofilm phenotypes such as wrinkled colony morphology, pellicle formation, and surface adherence. RscS is predicted to be a hybrid sensor kinase with a histidine kinase/ATPase (HATPase) domain, a receiver (Rec) domain, and a histidine phosphotransferase (Hpt) domain. Bioinformatic analysis also revealed the following three potential signal detection domains within RscS: two transmembrane helices forming a transmembrane region (TMR), a large periplasmic (PP) domain, and a cytoplasmic PAS domain. In this work, we genetically dissected the contributions of these domains to RscS function. Substitutions within the carboxy-terminal domain supported identification of RscS as a hybrid sensor kinase; disruption of both the HATPase and Rec domains eliminated induction of syp transcription, wrinkled colony morphology, pellicle formation, and surface adherence, while disruption of Hpt resulted in decreased activity. The PAS domain was also critical for RscS activity; substitutions in PAS resulted in a loss of activity. Generation of a cytoplasmic, N-terminal deletion derivative of RscS resulted in a partial loss of activity, suggesting a role for localization to the membrane and/or sequences within the TMR and PP domain. Finally, substitutions within the first transmembrane helix of the TMR and deletions within the PP domain both resulted in increased activity. Thus, RscS integrates both inhibitory and stimulatory signals from the environment to regulate biofilm formation by V. fischeri.

scholarly journals Chromosomal Insertions in the Lactobacillus caseiuppGene That Are Useful for Vaccine Expression

2014 ◽  
Vol 80 (11) ◽  
pp. 3321-3326 ◽  
Author(s):  
Bai-fen Song ◽  
Long-zhu Ju ◽  
Yi-jing Li ◽  
Li-jie Tang
Keyword(s):  
Pcr Amplification ◽  
Effective Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Toxin Gene ◽  
Temperature Sensitive ◽  
Alpha Toxin ◽  
Wild Type ◽  
Recombinant Bacteria ◽  
Content Type ◽  
Marker Free

ABSTRACTTo develop a stable and marker-freeLactobacillusstrain useful for the expression of vaccines, we developed a temperature-sensitive suicide plasmid with expression cassettes containing an HCE promoter, a PgsA anchor, the alpha-toxin gene, and anrrnBT1T2 terminator (PPαT) that uses a 5-fluorouracil (5-FU) counterselectable marker forLactobacillus casei. Three strains containing the correct PPαT expression cassettes were produced via the selective pressure of 5-FU screening. We confirmed that theuppgene was deleted and that the PPαT expression cassettes were inserted into theuppsite ofL. caseiATCC 393 by genomic PCR amplification and sequencing. 5-FU resistance in recombinant bacteria could be stably inherited for as long as 40 generations following insertion. However, bacteria containing the integrated DNA grew more slowly than wild-typeL. casei. An indirect enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that the alpha-toxin gene was expressed. Also, we visualized expression of the protein on the surface ofL. caseicells using laser confocal microscopy. These results taken together demonstrate that these recombinant bacteria should provide a safe tool for effective vaccine production.

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