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Research Article | Molecular Biology and Physiology

csrB Gene Duplication Drives the Evolution of Redundant Regulatory Pathways Controlling Expression of the Major Toxic Secreted Metalloproteases in Vibrio tasmaniensis LGP32

An Ngoc Nguyen, Elena Disconzi, Guillaume M. Charrière, Delphine Destoumieux-Garzón, Philippe Bouloc, Frédérique Le Roux, Annick Jacq
Craig D. Ellermeier, Editor
An Ngoc Nguyen
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette, FranceBiotechnology Department, Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Viet Nam
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Elena Disconzi
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette, France
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Guillaume M. Charrière
Interactions Hôtes-Pathogènes-Environnements, Université de Montpellier, CNRS, Ifremer, Université de Perpignan Via Domitia, Montpellier, France
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Delphine Destoumieux-Garzón
Interactions Hôtes-Pathogènes-Environnements, Université de Montpellier, CNRS, Ifremer, Université de Perpignan Via Domitia, Montpellier, France
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Philippe Bouloc
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette, France
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  • ORCID record for Philippe Bouloc
Frédérique Le Roux
Ifremer, Unité Physiologie Fonctionnelle des Organismes Marins, Plouzané, FranceSorbonne Universités, UPMC Paris 06, CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff, France
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Annick Jacq
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette, France
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Craig D. Ellermeier
University of Iowa
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DOI: 10.1128/mSphere.00582-18
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  • FIG 1
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    FIG 1

    Phylogenetic tree of Vibrio and Aliivibrio csrB genes. (A) Different copies of Vibrio and Aliivibrio csrB genes were collected, and their phylogenetic tree has been established by a maximum likelihood method as described in Materials and Methods. Alisal, Aliivibrio salmonicida; Phobac, Photobacterium profundum; Vibalg, Vibrio alginolyticus; Vibang, V. anguillarum; Vibchol, V. cholerae; Vibcor, V. coralliilyticus; Vibfis, V. fischeri; Vibfur, V. furnissii; Vibhar, V. harveyi; Vibnig, V. nigripulchritudo; Vibpara, V. parahaemolyticus; Vibtas, V. tasmaniensis; Vibtub, V. tubiashii; Vibvul, V. vulnificus. Each csrB gene name (indicated species_CsrB1 to species_CsrB3) is labeled by one or two small letters (from a to m) characterizing its genomic environment as shown in Tables S1 to S4. csrB genes belonging to the same branch are indicated by matching background colors. Vibtas_csrB3 and Vibtas_csrB4 share no synteny with any other csrB genes in the list. Branch support values represent the results of an approximate likelihood-ratio test (aLRT) and are indicated only for nodes corresponding to complete or partial changes of synteny. Branches with a support value of less than 0.6 were collapsed. (B) Phylogenetic tree of the Vibrio and Aliivibrio strains used in the experiments represented by panel A. Sequences of recA, gyrB, and rpoA for each species were collected, concatenated, and used to construct the tree as described for panel A.

  • FIG 2
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    FIG 2

    VibtasCsrB4 can complement the absence of csrB genes in V. cholerae. Luminescence was measured during growth (see Materials and Methods) in strains carrying a pLux plasmid as follows: V. cholerae wild-type strain (blue diamonds), V. cholerae strain ΔcsrBCD (red squares), and V. cholerae strain ΔcsrBCD/pCsrB4 (green triangles). Relative light unit (RLU) represent counts per minute per milliliter per OD600 value.

  • FIG 3
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    FIG 3

    Regulation of csrB genes in V. tasmaniensis. (A) The level of each CsrB product was determined by dot blot experiments performed with the V. tasmaniensis wild-type strain and ΔvarA and ΔvarS single mutant and ΔvarA ΔvarS double mutant strains as described in Materials and Methods. The values are expressed as fold differences from the tmRNA level. The error bars correspond to standard deviations of the means (SEM). (B) The level of CsrB1 was determined in duplicate by dot blot experiments performed at an OD600 of 0.4 (exponential phase [Exp]) and at an OD600 of 1.4 (stationary phase [St]). Results are expressed as fold changes from the average value corresponding to the WT level in exponential phase, after normalization to the tmRNA level. *, P > 0.05; **, P > 0.01; ***, P > 0.001.

  • FIG 4
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    FIG 4

    Production of Vsm and PrtV is HapR dependent whereas only Vsm production is σS dependent. (A) (Left panel) The protein profiles of vesicle-free supernatants of WT LGP32 culture supernatants at different time points during growth were analyzed by SDS-PAGE (12% acrylamide) after TCA precipitation. The sizes of the molecular weight markers are indicated at the left of the gel. Bands corresponding to proteins analyzed by mass spectroscopy are indicated by an asterisk (*). (Right panel) Supernatants of an O/N culture of WT LGP32 and a ΔhapR mutant were subjected to TCA precipitation and analyzed by SDS-PAGE (4% to 12% gradient gel). Identities of the bands are indicated between the two panels. (B) (Left panel) Vesicle-free supernatants of an LGP32 ΔrpoS mutant collected at different time points during growth were subjected to TCA precipitation and analyzed by SDS-PAGE (12% acrylamide). Sizes of the molecular weight markers are indicated at the left of the gels. (Right panel) Supernatants of an O/N culture of WT LGP32 and the ΔrpoS mutant were subjected to TCA precipitation and analyzed by SDS-PAGE (4% to 12% gradient gel). Identities of the bands are indicated between the two panels. (C) Secreted proteolytic activity was assayed for different strains on milk agar nutrient plates as described in Materials and Methods and detected as a clearing zone around the colonies.

  • FIG 5
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    FIG 5

    A redundant VarS/A-CsrB pathway controls protease production through LuxO. (A) O/N culture supernatants of the WT strain and different mutants were subjected to trichloroacetic acid (TCA) precipitation as described above and analyzed by SDS-PAGE (4% to 12% acrylamide gradient gels). MW, molecular weight. (B and C) Secreted proteolytic activity was assayed on milk marine agar plates without (B) or with (C) supplementation with 2 µg/ml of Cm for the different strains, and the size of the clearing zones was measured after 48 h of incubation at 20°C. Values represent averages of data from a minimum of three independent determinations, and error bars correspond to the SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by pairwise t tests. Values considered to be significantly different (P < 0.05) are indicated by different letters.

  • FIG 6
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    FIG 6

    CsrBs are redundant in V. tasmaniensis. (A) Secreted proteolytic activity was assayed in triplicate for different strains on milk agar nutrient plates as described in Materials and Methods and were detected as a clearing zone around the colonies. The size of the clearing zones was measured after 48 h. Means of results from a minimum of three replicates are presented, and error bars correspond to the SEM. (B) The level of each CsrB was determined by dot blot experiments in V. tasmaniensis strains in which the three other csrB genes had been deleted. Relative density values are expressed as fold change from the tmRNA level and correspond to averages of results from three independent measurements. The error bars correspond to standard deviations of the means (SEM).

  • FIG 7
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    FIG 7

    The VarS/VarA/CsrB system controls production of σS via quorum sensing in a redundant manner. Identical amounts of whole-cell extract from various strains as indicated were analyzed by Western blotting using polyclonal antibodies directed against S. enterica serovar Typhimurium σS. (Top panel) A representative Western blot is shown. (Lower panel) σS levels were quantified using ImageJ. Results (normalized to the WT signal) are expressed as fold change from the WT level and represent averages of four independent determinations (four different samples from four independent cultures). Error bars correspond to SEM. Data were analyzed by one-way ANOVA as described for Fig. 5.

  • FIG 8
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    FIG 8

    Diagrammatic representation of the network controlling production of Vsm and PrtV in V. tasmaniensis. At least 5 overlapping but distinct pathways control production of PrtV and Vsm in V. tasmaniensis LGP32. The QS pathway is depicted in blue. The VarS/VarA-independent–CsrB1/CsrA-dependent pathway is depicted in gray. The VarS/VarA/CsrB2,3,4/CsrA-dependent pathway is depicted in golden yellow. A putative VarS/VarA-dependent pathway controlling hapR and rpoS expression through the activity of a putative positive X factor is depicted in red. Other signals contributing to rpoS expression are depicted in green. The combination of colors in symbols representing LuxO, HapR, σS, PrtV, and Vsm conceptually indicates the combined contributions of all of the pathways/signals to expression. Since we did not quantitatively determine their respective contributions in this study, this is not meant to be a quantitative representation. Positive actions are represented by arrowheads, whereas negative actions are depicted by solid ellipsoids. Solid lines indicate direct actions, whereas dashed lines indicate indirect actions and/or unknown mechanisms. Putative pathways are further indicated by question marks.

Supplemental Material

  • Figures
  • TABLE S1

    List of genes surrounding the csrB1 copies in the Vibrio and Aliivibrio species used in Fig. 1 (in red). Gene homology is indicated by matching background colors. Each csrB is characterized by one or two synteny groups, each labeled by a small letter (a to f). Download Table S1, PDF file, 0.05 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S2

    List of genes surrounding the csrB2 copies in the Vibrio species used in Fig. 1 (in red). Gene homology is indicated by matching background colors, Each csrB is characterized by one or two synteny groups, each labeled by a small letter (g or h). Download Table S2, PDF file, 0.05 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S3

    List of genes surrounding the csrB3 copies in the different species used in Fig. 1 (in red). Gene homology is indicated by matching background colors. Each csrB is characterized by one or two synteny groups, each labeled by a small letter (i to l). Note that VibtascsrB3 does not share synteny with any other csrB3. Download Table S3, PDF file, 0.05 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S4

    List of genes surrounding the csrB2-3 copies in the Aliivibrio species used in Fig. 1 (in red). Gene homology is indicated by matching background colors. The synteny group for these copies is labeled by the small letter m. Download Table S4, PDF file, 0.03 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S5

    Bacterial strains used in this study. Download Table S5, PDF file, 0.05 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S6

    Plasmids used in this study. Download Table S6, PDF file, 0.03 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S1

    VarA binding sites upstream of csrB genes. Upstream regulatory regions of the V. tasmaniensis csrB genes were aligned using Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) followed by some manual curation. The +1 transcription start locations, as determined by transcriptomic data (26, 29), are indicated in bold. Putative sigma 70 promoter −10 and −35 locations are underlined. The conserved VarA binding sites are indicated by red bold letters. Green letters indicate a CsrA biding site. Download FIG S1, PDF file, 0.03 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Oyster mortality in response to experimental infection by various varS, varA, and csrB mutants. Oyster mortality was determined as previously described (54), using two different doses of CFU, as indicated. Download FIG S2, TIFF file, 13.4 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S7

    Oligonucleotides used in this study. Download Table S7, PDF file, 0.03 MB.

    Copyright © 2018 Nguyen et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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csrB Gene Duplication Drives the Evolution of Redundant Regulatory Pathways Controlling Expression of the Major Toxic Secreted Metalloproteases in Vibrio tasmaniensis LGP32
An Ngoc Nguyen, Elena Disconzi, Guillaume M. Charrière, Delphine Destoumieux-Garzón, Philippe Bouloc, Frédérique Le Roux, Annick Jacq
mSphere Nov 2018, 3 (6) e00582-18; DOI: 10.1128/mSphere.00582-18

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csrB Gene Duplication Drives the Evolution of Redundant Regulatory Pathways Controlling Expression of the Major Toxic Secreted Metalloproteases in Vibrio tasmaniensis LGP32
An Ngoc Nguyen, Elena Disconzi, Guillaume M. Charrière, Delphine Destoumieux-Garzón, Philippe Bouloc, Frédérique Le Roux, Annick Jacq
mSphere Nov 2018, 3 (6) e00582-18; DOI: 10.1128/mSphere.00582-18
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KEYWORDS

bacterial gene regulation
bacterial sRNAs
transcriptomics
Vibrio pathogenic to oysters
host-pathogen interactions

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