The Regulatory Network of Natural Competence and Transformation of
The human pathogen Vibrio cholerae is an aquatic bacterium frequently encountered in rivers, lakes, estuaries, and coastal regions. Within these environmental reservoirs, the bacterium is often found associated with zooplankton and more specifically with their chitinous exoskeleton. Upon growth on such chitinous surfaces, V. cholerae initiates a developmental program termed “natural competence for genetic transformation.” Natural competence for transformation is a mode of horizontal gene transfer in bacteria and contributes to the maintenance and evolution of bacterial genomes. In this study, we investigated competence gene expression within this organism at the single cell level. We provide evidence that under homogeneous inducing conditions the majority of the cells express competence genes. A more heterogeneous expression pattern was observable on chitin surfaces. We hypothesize that this was the case due to the heterogeneity around the chitin surface, which might vary extensively with respect to chitin degradation products and autoinducers; these molecules contribute to competence induction based on carbon catabolite repression and quorum-sensing pathways, respectively. Therefore, we investigated the contribution of these two signaling pathways to natural competence in detail using natural transformation assays, transcriptional reporter fusions, quantitative RT–PCR, and immunological detection of protein levels using Western blot analysis. The results illustrate that all tested competence genes are dependent on the transformation regulator TfoX. Furthermore, intracellular cAMP levels play a major role in natural transformation. Finally, we demonstrate that only a minority of genes involved in natural transformation are regulated in a quorum-sensing-dependent manner and that these genes determine the fate of the surrounding DNA. We conclude with a model of the regulatory circuit of chitin-induced natural competence in V. cholerae.
Vyšlo v časopise:
The Regulatory Network of Natural Competence and Transformation of. PLoS Genet 8(6): e32767. doi:10.1371/journal.pgen.1002778
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002778
Souhrn
The human pathogen Vibrio cholerae is an aquatic bacterium frequently encountered in rivers, lakes, estuaries, and coastal regions. Within these environmental reservoirs, the bacterium is often found associated with zooplankton and more specifically with their chitinous exoskeleton. Upon growth on such chitinous surfaces, V. cholerae initiates a developmental program termed “natural competence for genetic transformation.” Natural competence for transformation is a mode of horizontal gene transfer in bacteria and contributes to the maintenance and evolution of bacterial genomes. In this study, we investigated competence gene expression within this organism at the single cell level. We provide evidence that under homogeneous inducing conditions the majority of the cells express competence genes. A more heterogeneous expression pattern was observable on chitin surfaces. We hypothesize that this was the case due to the heterogeneity around the chitin surface, which might vary extensively with respect to chitin degradation products and autoinducers; these molecules contribute to competence induction based on carbon catabolite repression and quorum-sensing pathways, respectively. Therefore, we investigated the contribution of these two signaling pathways to natural competence in detail using natural transformation assays, transcriptional reporter fusions, quantitative RT–PCR, and immunological detection of protein levels using Western blot analysis. The results illustrate that all tested competence genes are dependent on the transformation regulator TfoX. Furthermore, intracellular cAMP levels play a major role in natural transformation. Finally, we demonstrate that only a minority of genes involved in natural transformation are regulated in a quorum-sensing-dependent manner and that these genes determine the fate of the surrounding DNA. We conclude with a model of the regulatory circuit of chitin-induced natural competence in V. cholerae.
Zdroje
1. MorensDMFolkersGKFauciAS 2004 The challenge of emerging and re-emerging infectious diseases. Nature 430 242 249
2. BertuzzoEMariLRighettoLGattoMCasagrandiR 2011 Prediction of the spatial evolution and effects of control measures for the unfolding Haiti cholera outbreak. Geophys Res Lett 38doi:10.1029/2011GL046823
3. AndrewsJRBasuS 2011 Transmission dynamics and control of cholera in Haiti: an epidemic model. Lancet 377 1248 1255
4. TuiteARTienJEisenbergMEarnDJMaJ 2011 Cholera Epidemic in Haiti, 2010: Using a Transmission Model to Explain Spatial Spread of Disease and Identify Optimal Control Interventions. Ann Intern Med 154 593 601
5. RinaldoABlokeschMBertuzzoEMariLRighettoL 2011 A transmission model of the 2010 cholera epidemic in haiti. Ann Intern Med 155 403 404
6. ChinCSSorensonJHarrisJBRobinsWPCharlesRC 2010 The origin of the Haitian cholera outbreak strain. N Engl J Med 364 33 42
7. ChunJGrimCJHasanNALeeJHChoiSY 2009 Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae. Proc Natl Acad Sci USA 106 15442 15447
8. LippEKHuqAColwellRR 2002 Effects of global climate on infectious disease: the cholera model. Clin Microbiol Rev 15 757 770
9. ColwellRR 1996 Global climate and infectious disease: the cholera paradigm. Science 274 2025 2031
10. PruzzoCVezzulliLColwellRR 2008 Global impact of Vibrio cholerae interactions with chitin. Environ Microbiol 10 1400 1410
11. MeibomKLBlokeschMDolganovNAWuC-Y Schoolnik GK 2005 Chitin induces natural competence in Vibrio cholerae. Science 310 1824 1827
12. ChenYDaiJMorrisJGJrJohnsonJA 2010 Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic Vibrio parahaemolyticus O3:K6. BMC Microbiol 10 274
13. GuligPATuckerMSThiavillePCJosephJLBrownRN 2009 USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus. Appl Environ Microbiol 75 4936 4949
14. Pollack-BertiAWollenbergMSRubyEG 2010 Natural transformation of Vibrio fischeri requires tfoX and tfoY. Environ Microbiol 12 2302 2311
15. BlokeschM Schoolnik GK 2007 Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs. PLoS Pathog 3 e81 doi:10.1371/journal.ppat.0030081
16. FaruqueSMMekalanosJJ 2003 Pathogenicity islands and phages in Vibrio cholerae evolution. Trends Microbiol 11 505 510
17. BlokeschM Schoolnik GK 2008 The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae. J Bacteriol 190 7232 7240
18. YamamotoSMoritaMIzumiyaHWatanabeH 2010 Chitin disaccharide (GlcNAc)2 induces natural competence in Vibrio cholerae through transcriptional and translational activation of a positive regulatory gene tfoXVC. Gene 457 42 49
19. MarvigRLBlokeschM 2010 Natural transformation of Vibrio cholerae as a tool-optimizing the procedure. BMC Microbiol 10 155
20. YamamotoSIzumiyaHMitobeJMoritaMArakawaE 2011 Identification of a Chitin-Induced Small RNA That Regulates Translation of the tfoX Gene, Encoding a Positive Regulator of Natural Competence in Vibrio cholerae. J Bacteriol 193 1953 1965
21. AntonovaESHammerBK 2011 Quorum-sensing autoinducer molecules produced by members of a multispecies biofilm promote horizontal gene transfer to Vibrio cholerae. FEMS Microbiol Lett 322 68 76
22. SuckowGSeitzPBlokeschM 2011 Quorum Sensing Contributes to Natural Transformation of Vibrio cholerae in a Species-Specific Manner. J Bacteriol 193 4914 4924
23. BlokeschM 2012 Chitin colonization, chitin degradation and chitin-induced natural competence of Vibrio cholerae are subject to catabolite repression. Environ Microbiol doi:10.1111/j.1462-2920.2011.02689.x (article first published online: 6 JAN 2012)
24. NesterEWStockerBA 1963 Biosynthetic Latency in Early Stages of Deoxyribonucleic Acidtransformation in Bacillus Subtilis. J Bacteriol 86 785 796
25. MaamarHDubnauD 2005 Bistability in the Bacillus subtilis K-state (competence) system requires a positive feedback loop. Mol Microbiol 56 615 624
26. MaamarHRajADubnauD 2007 Noise in gene expression determines cell fate in Bacillus subtilis. Science 317 526 529
27. CormackBPValdiviaRHFalkowS 1996 FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173 33 38
28. BevisBJGlickBS 2002 Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20 83 87
29. DunnAKMillikanDSAdinDMBoseJLStabbEV 2006 New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl Environ Microbiol 72 802 810
30. FullnerKJMekalanosJJ 1999 Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect Immun 67 1393 1404
31. MeibomKLLiXBNielsenATWuCYRosemanS 2004 The Vibrio cholerae chitin utilization program. Proc Natl Acad Sci USA 101 2524 2529
32. ProvvediRDubnauD 1999 ComEA is a DNA receptor for transformation of competent Bacillus subtilis. Mol Microbiol 31 271 280
33. DohertyAJSerpellLCPontingCP 1996 The helix-hairpin-helix DNA-binding motif: a structural basis for non-sequence-specific recognition of DNA. Nucleic Acids Res 24 2488 2497
34. MenzelRGellertM 1983 Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell 34 105 113
35. LaubMTMcAdamsHHFeldblyumTFraserCMShapiroL 2000 Global analysis of the genetic network controlling a bacterial cell cycle. Science 290 2144 2148
36. FongJCYildizFH 2008 Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio cholerae biofilm formation. J Bacteriol 190 6646 6659
37. NielsenATDolganovNARasmussenTOttoGMillerMC 2010 A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine. PLoS Pathog 6 doi:10.1371/journal.ppat.1001102 e1001102
38. SrivastavaPChattorajDK 2007 Selective chromosome amplification in Vibrio cholerae. Mol Microbiol 66 1016 1028
39. SilanderOKNikolicNZaslaverABrenAKikoinI 2012 A genome-wide analysis of promoter-mediated phenotypic noise in Escherichia coli. PLoS Genet 8 doi:10.1371/journal.pgen.1002443 e1002443
40. RedfieldRJCameronADQianQHindsJAliTR 2005 A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae. J Mol Biol 347 735 747
41. CameronADRedfieldRJ 2008 CRP binding and transcription activation at CRP-S sites. J Mol Biol 383 313 323
42. SchleifR 2010 AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev 34 779 796
43. LambertsenLSternbergCMolinS 2004 Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environ Microbiol 6 726 732
44. DeutscherJFranckeCPostmaPW 2006 How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev 70 939 1031
45. KimHSKimSMLeeHJParkSJLeeKH 2009 Expression of the cpdA gene, encoding a 3′,5′-cyclic AMP (cAMP) phosphodiesterase, is positively regulated by the cAMP-cAMP receptor protein complex. J Bacteriol 191 922 930
46. HigginsDAPomianekMEKramlCMTaylorRKSemmelhackMF 2007 The major Vibrio cholerae autoinducer and its role in virulence factor production. Nature 450 883 886
47. NgWLBasslerBL 2009 Bacterial quorum-sensing network architectures. Annu Rev Genet 43 197 222
48. XavierKBBasslerBL 2005 Interference with AI-2-mediated bacterial cell-cell communication. Nature 437 750 753
49. MillerMBSkorupskiKLenzDHTaylorRKBasslerBL 2002 Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. Cell 110 303 314
50. JoblingMGHolmesRK 1997 Characterization of hapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol Microbiol 26 1023 1034
51. WatersCMBasslerBL 2006 The Vibrio harveyi quorum-sensing system uses shared regulatory components to discriminate between multiple autoinducers. Genes Dev 20 2754 2767
52. Mortier-BarriereIVeltenMDupaignePMirouzeNPietrementO 2007 A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell 130 824 836
53. LiXRosemanS 2004 The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase. Proc Natl Acad Sci USA 101 627 631
54. MacfadyenLP 2000 Regulation of competence development in Haemophilus influenzae. J Theor Biol 207 349 359
55. HeidelbergJFEisenJANelsonWCClaytonRAGwinnML 2000 DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406 477 483
56. ZhuJMillerMBVanceREDziejmanMBasslerBL 2002 Quorum-sensing regulators control virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA 99 3129 3134
57. SeperAFenglerVHRoierSWolinskiHKohlweinSD 2011 Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation. Mol Microbiol 82 1015 1037
58. Yanisch-PerronCVieiraJMessingJ 1985 Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33 103 119
59. SimonRPrieferUPühlerA 1983 A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria. Nat Biotechnol 1 784 791
60. BaoYLiesDPFuHRobertsGP 1991 An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of gram-negative bacteria. Gene 109 167 168
61. BolivarFRodriguezRLGreenePJBetlachMCHeynekerHL 1977 Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2 95 113
62. SliusarenkoOHeinritzJEmonetTJacobs-WagnerC 2011 High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics. Mol Microbiol 80 612 627
63. MilesAAMisraSSIrwinJO 1938 The estimation of the bactericidal power of the blood. J Hyg 38 732 749
64. KeeneON 1995 The log transformation is special. Stat Med 14 811 819
65. LiuXBeyhanSLimBLiningtonRGYildizFH 2010 Identification and characterization of a phosphodiesterase that inversely regulates motility and biofilm formation in Vibrio cholerae. J Bacteriol 192 4541 4552
66. LaemmliUK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680 685
67. SambrookJFritschEFManiatisT 2001 Molecular Cloning: A Laboratory Manual (Third Edition). FordNNolanCFergusonM New York Cold Spring Harbor Laboratory Press editors
68. YildizFH Schoolnik GK 1998 Role of rpoS in stress survival and virulence of Vibrio cholerae. J Bacteriol 180 773 784
69. De Souza SilvaOBlokeschM 2010 Genetic manipulation of Vibrio cholerae by combining natural transformation with FLP recombination. Plasmid 64 186 195
70. NielsenATDolganovNAOttoGMillerMCWuCY 2006 RpoS controls the Vibrio cholerae mucosal escape response. PLoS Pathog 2 doi:10.1371/journal.ppat.0020109 e109
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
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