Abstract
Violacein, a purple pigment produced by some Gram-negative bacteria, has various physiological properties, such as antitrypanosomal and antitumoral activities. A gene cluster that encodes five enzymes, VioA–VioE, is responsible for synthesizing violacein. The expression of these enzymes is known to be regulated by a quorum sensing mechanism in Chromobacterium violaceum and Pseudoalteromonas sp. 520P1. To clarify the molecular mechanism of regulation of violacein synthesis, we cloned and characterized the gene cluster from Pseudoalteromonas sp. 520P1. A fosmid library of strain 520P1 was constructed and clones containing the gene cluster were isolated. The gene cluster was 7383 bp in length and encoded five enzyme genes, vioA–vioE. A putative promoter sequence was predicted in the upstream region of the cluster. In the promoter region, two contiguous palindromic sequences, a possible quorum sensing regulatory site, were found. However, the isolated Escherichia coli clones harboring the gene cluster and its upstream region were unable to produce violacein probably due to the lack of quorum sensing machinery for expression. To further examine the ability of vioA–vioE genes to synthesize violacein in vivo, the upstream promoter region was removed from the cluster and heterologous expression of the treated cluster was performed in E. coli using a recombinant pET vector with T7 promoter. Purple pigment was expressed, and the pigment was identified to be violacein using ultraviolet and visible light and HPLC analysis. These results will contribute to further studies regarding violacein biosynthesis and its mass production.
Similar content being viewed by others
References
Ahmetagic A, Pemberton JM (2010) Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12. Plasmid 63:79–85
Andrighetti-Fröhner CR, Antonio RV, Creczynski-Pasa TB, Barardi CR, Simões CM (2003) Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum. Mem Inst Oswaldo Cruz 98:843–848
August PR, Grossman TH, Minor C, Draper MP, MacNeil IA, Pemberton JM, Call KM, Holt D, Osburne MS (2000) Sequence analysis and functional characterization of the violacein biosynthetic pathway from Chromobacterium violaceum. J Mol Microbiol Biotechnol 2:513–519
Balibar CJ, Walsh CT (2006) In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum. Biochemistry 45:15444–15457
Becker MH, Brucker RM, Schwantes CR, Harris RN, Minbiole KPC (2009) The bacterially produced metabolite violacein is associated with survival of amphibians infected with a lethal fungus. Appl Environ Microbiol 75:6635–6638
Brady SF, Chao CJ, Handelsman J, Clardy J (2001) Cloning and heterologous expression of a natural product biosynthetic gene cluster from eDNA. Org Lett 3:1981–1984
Devine JH, Countryman C, Baldwin TO (1988) Nucleotide sequence of the luxR and luxI genes and the structure of the primary regulatory region of the lux regulon of Vibrio fischeri ATCC 7744. Biochemistry 27:837–842
Durán N, Menck CFM (2001) Chromobacterium violaceum: a review of pharmacological and industiral perspectives. Crit Rev Microbiol 27:201–222
Gauthier MJ (1982) Validation of the name Alteromonas luteoviolacea. Int J Syst Bacteriol 32:82–86
Hakvåg S, Fjaervik E, Klinkenberg G, Borgos SE, Josefsen KD, Ellingsen TE, Zotchev SB (2009) Violacein-producing Collimonas sp. from the sea surface microlayer of coastal waters in Trøndelag, Norway. Mar Drugs 7:576–588
Hirano S, Asamizu S, Onaka H, Shiro Y, Nagano S (2008) Crystal structure of VioE, a key player in the construction of the molecular skeleton of violacein. J Biol Chem 283:6459–6466
Hrvatin S, Piel J (2007) Rapid isolation of rare clones from highly complex DNA libraries by PCR analysis of liquid gel pools. J Microbiol Methods 68:434–436
Jiang PX, Wang HS, Zhang C, Lou K, Xing XH (2010) Reconstruction of the violacein biosynthetic pathway from Duganella sp. B2 in different heterologous hosts. Appl Microbiol Biotechnol 86:1077–1088
Leon LL, Miranda CC, De Souza AO, Durán N (2001) Antileishmanial activity of the violacein extracted from Chromobacterium violaceum. J Antimicrob Chemother 48:449–450
Lopes SCP, Blanco YC, Justo GZ, Nogueira PA, Rodrigues FLS, Goelnitz U, Wunderlich G, Facchini G, Brocchi M, Duran N, Costa FTM (2009) Violacein extracted from Chromobacterium violaceum inhibits Plasmodium growth in vitro and in vivo. Antimicrob Agents Chemother 53:2149–2152
McCarthy SA, Sakata T, Kakimoto D, Johnson RM (1985) Production and isolation of purple pigment by Alteromonas luteoviolacea. Bull Jpn Soc Sci Fish 51:479–484
McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR, Camara M, Daykin M, Lamb JH, Swift S, Bycroft BW, Stewart GSAB, Williams P (1997) Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703–3711
Melo PS, Maria SS, Vidal BC, Haun M, Durán N (2000) Violacein cytotoxicity and induction of apoptosis in V79 cells. In Vitro Cell Dev Biol Anim 36:539–543
Momen AZ, Hoshino T (2000) Biosynthesis of violacein: intact incorporation of the tryptophan molecule on the oxindole side, with intramolecular rearrangement of the indole ring on the 5-hydroxyindole side. Biosci Biotechnol Biochem 64:539–549
Morohoshi T, Fukamachi K, Kato M, Kato N, Ikeda T (2010) Regulation of the violacein biosynthetic gene cluster by acylhomoserine lactone-mediated quorum sensing in Chromobacterium violaceum ATCC 12472. Biosci Biotechnol Biochem 74:2116–2119
Pantanella F, Berlutti F, Passariello C, Sarli S, Morea C, Schippa S (2007) Violacein and biofilm production in Janthinobacterium lividum. J Appl Microbiol 102:992–999
Pemberton JM, Vincent KM, Penfold RJ (1991) Cloning and heterologous expression of the violacein biosynthesis gene cluster from Chromobacterium violaceum. Curr Microbiol 22:355–358
Ryan KS, Balibar CJ, Turo KE, Walsh CT, Drennan CL (2008) The violacein biosynthetic enzyme VioE shares a fold with lipoprotein transporter proteins. J Biol Chem 283:6467–6475
Sánchez C, Braña AF, Méndez C, Salas JA (2006) Reevaluation of the violacein biosynthetic pathway and its relationship to indolocarbazole biosynthesis. Chembiochem 7:1231–1240
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Schloss PD, Allen HK, Klimowicz AK, Mlot C, Gross JA, Savengsuksa S, McEllin J, Clardy J, Ruess RW, Handelsman J (2010) Psychrotrophic strain of Janthinobacterium lividum from a cold Alaskan soil produces prodigiosin. DNA Cell Biol 29:1–9
Shirata A, Tsukamoto T, Yasui H, Hata H, Hayasaka S, Kojima A, Kato H (2000) Isolation of bacteria producing bluish-purple pigment and use for dyeing. J Agrc Res Q 34:131–140
Stevens AM, Dolan KM, Greenberg EP (1994) Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region. Proc Natl Acad Sci USA 91:12619–12623
Studier FM (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234
Taga N (1968) Some ecological aspects of marine bacteria in the Kuroshio current. Bull Misaki Mar Biol Inst Kyoto Univ 12:65–76
Thomas T, Evans FF, Schleheck D, Mai-Prochnow A, Burke C, Penesyan A, Dalisay DS, Stelzer-Braid S, Saunders N, Johnson J, Ferriera S, Kjelleberg S, Egan S (2008) Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment. PLoS ONE 3:e3252
Wang Y, Ikawa A, Okaue S, Taniguchi S, Osaka I, Yoshimoto A, Kishida Y, Arakawa R, Enomoto K (2008) Quorum sensing signaling molecules involved in the production of violacein by Pseudoalteromonas. Biosci Biotechnol Biochem 72:1958–1961
Wang HS, Jiang PX, Lu Y, Ruan ZY, Jiang RB, Xing XH, Lou K, Wei D (2009) Optimization of culture conditions for violacein production by a new strain of Duganella sp. B2. Biochem Eng J 44:119–124
Yada S, Wang Y, Zou Y, Nagasaki K, Hosokawa K, Osaka I, Arakawa R, Enomoto K (2008) Isolation and characterization of two groups of novel marine bacteria producing violacein. Mar Biotechnol 10:128–132
Acknowledgments
This work was partly supported by a Grant-in-Aid (07F011) for the promotion of academic frontier from the Ministry of Education, Culture, Science, Sports and Technology, Japan.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhang, X., Enomoto, K. Characterization of a gene cluster and its putative promoter region for violacein biosynthesis in Pseudoalteromonas sp. 520P1. Appl Microbiol Biotechnol 90, 1963–1971 (2011). https://doi.org/10.1007/s00253-011-3203-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-011-3203-9