GENOME ANNOUNCEMENT
Draft Genome Sequence of High-Melanin-Yielding Aeromonas media Strain WS Baozhong Chai, He Wang, and Xiangdong Chen State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China
We sequenced the genome of the high-melanin-yielding Aeromonas media strain WS and then analyzed genes potentially involved in melanin formation. The 4.2-Mb draft genome carries multiple genes responsible for pyomelanin synthesis and other candidate genes identified in our separate study, which have no homolog in other strains of Aeromonas species.
M
elanin is an enigmatic pigment existing widely spread in a variety of life forms ranging from bacteria to humans. It can enhance survival and competitive abilities of organisms via providing protection from environmental stress conditions such as UV radiation damage (21) and toxic free radicals (3). Aeromonas media strain WS was isolated from water samples collected from East Lake, Wuhan, China, and exhibits a significantly high yield of melanin (22). Our previous studies have suggested that this kind of pigment has the potential to be used as a general photoprotective agent for bioinsecticides and an active sunscreen for patients with skin photosensitivity to sun exposure (9, 22). Production of melanin has been described in many Aeromonas species (1, 6, 10), but the molecular determinant involved in this metabolic pathway has not been characterized. We once reported the isolation of the distinct tyrosinase TyrA from A. media strain WS (23), but subsequent research indicated that this protein is not a key factor responsible for melanin formation in the strain, since the deletion of tyrA from the bacterium does not bring any obvious change regarding the pigment production. Thus, whole-genome analysis was carried out in order to gain insight into the melanin formation mechanism in Aeromonas species. Genomic DNA was extracted using a genomic DNA purification kit (Promega) and sequenced with Solexa paired-end sequencing technology (4). A total of 860 Mbp of sequence reads were generated from a 70-bp paired-end library (204-fold genome coverage). Reads were assembled into 258 contigs with more than 500 bp using Velvet 1.2.03 (24). Annotation was performed using KEGG, UniProt, and COG (Clusters of Orthologous Group) databases and the RAST server (2). All tRNAs and rRNAs were predicted by tRNAscan-SE 1.21 and RNAmmer1.2 (14, 15), respectively. The draft genome of strain WS comprised 4,317,138 bp with 61.39% mean GC content. There are 4,020 predicted open reading frames, three rRNAs, and 54 tRNAs in its genome. Comparison with genome sequences available in GenBank (Aeromonas salmonicida A449, Aeromonas hydrophila ATCC 7966, and Aeromonas veronii B565) showed 659 unique genes in A. media WS, most of which are transposases (insertion elements) and hypothetical proteins with unknown functions. This notable feature implies potential gene exchange between the strain WS and related environments (19). In addition, we have obtained several melanindeficient mutants in a separate study and identified some candidate genes involved in the melanin formation process (data not shown). There are several known pathways in bacteria by which ty-
December 2012 Volume 194 Number 23
rosine can be converted into melanin (8, 12, 13, 16, 18); production of L-3,4-dihydroxyphenylalanine (L-DOPA) by tyrosinase is considered a major one among them. That L-DOPA could be found in the bacterial culture suggested that tyrosinase may also be involved in melanin formation in strain WS (22). However, the predicted protein coincident with typical bacterial tyrosinase has not been found by searching the whole genome against HMM PF00264 yet (7). In contrast, the genome of strain WS carries multiple other genes potentially involved in melanin formation, such as genes encoding phenylalanine 4-monooxygenase and pterin-4-alpha-carbinolamine dehydratase. Genes responsible for pyomelanin synthesis were also predicted, including those encoding 4-hydroxyphenylpyruvate dioxygenase, homogentisate 1,2-dioxygenase, outer membrane lipoprotein, 4-hydroxy-3methylbut-2-enyl diphosphate reductase, and ABC transporter ATP-binding protein (11). On the other hand, melanin has also been associated with pathogenic virulence in pathogenic microorganisms (5, 17, 20). Therefore, the publication of the genome will also be helpful to expand our knowledge on the biological significance of the pigment in this kind of opportunistic pathogen. Nucleotide sequence accession numbers. This Whole-Genome Shotgun project has been deposited at DDBJ/EMBL/ GenBank under the accession no. ALJZ00000000. The version described in this paper is the first version, ALJZ02000000. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (no. 31070077). We are grateful to the members of the Tianjin Biochip Corporation sequencing team for technical help.
REFERENCES 1. Abbott SL, Cheung WK, Janda JM. 2003. The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. J. Clin. Microbiol. 41:2348 –2357. 2. Aziz RK, et al. 2008. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75. doi:10.1186/1471-2164-9-75. 3. Bell AA, Wheeler MH. 1986. Biosynthesis and functions of fungal melanins. Annu. Rev. Phytopathol. 24:411– 451.
Received 23 September 2012 Accepted 1 October 2012 Address correspondence to Xiangdong Chen,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.01807-12
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4. Bentley DR, et al. 2008. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53–59. 5. Dixon DM, Polak A, Conner GW. 1989. Mel- mutants of Wangiella dermatitidis in mice: evaluation of multiple mouse and fungal strains. J. Med. Vet. Mycol. 27:335–341. 6. Donlon J, McGettigan S, O’Brien P, O’Carra P. 1983. Re-appraisal of the nature of the pigment produced by Aeromonas salmonicida. FEMS Microbiol. Lett. 19:285–290. 7. Finn RD, et al. 2006. Pfam: clans, web tools and services. Nucleic Acids Res. 34:D247–D251. 8. Funa N, et al. 1999. A new pathway for polyketide synthesis in microorganisms. Nature 400:897– 899. 9. Geng J, et al. 2008. Photoprotection of bacterial-derived melanin against ultraviolet A-induced cell death and its potential application as an active sunscreen. J. Eur. Acad. Dermatol. Venereol. 22:852– 858. 10. Gibson LF, George AM. 1998. Melanin and novel melanin precursors from Aeromonas media. FEMS Microbiol. Lett. 169:261–268. 11. Hunter RC, Newman DK. 2010. A putative ABC transporter, hatABCDE, is among molecular determinants of pyomelanin production in Pseudomonas aeruginosa. J. Bacteriol. 192:5962–5971. 12. Kobayashi T, et al. 1995. Modulation of melanogenic protein expression during the switch from eu- to pheomelanogenesis. J. Cell Sci. 108:2301–2309. 13. Kotob SI, Coon SL, Quintero EJ, Weiner RM. 1995. Homogentisic acid is the primary precursor of melanin synthesis in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. Appl. Environ. Microbiol. 61:1620 –1622. 14. Lagesen K, et al. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 35:3100 –3108.
6694
jb.asm.org
15. Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25: 955–964. 16. Mason HS. 1948. The chemistry of melanin. J. Biol. Chem. 172:83–99. 17. Nosanchuk JD, Casadevall A. 2003. The contribution of melanin to microbial pathogenesis. Cell. Microbiol. 5:203–223. 18. Raper HS. 1927. The tyrosinase-tyrosine reaction: production from tyrosine of 5: 6-dihydroxyindole and 5: 6-dihydroxyindole-2-carboxylic acid—the precursors of melanin. Biochem. J. 21:89 –96. 19. Reith ME, et al. 2008. The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen. BMC Genomics 9:427. doi:10.1186/1471-2164-9-427. 20. Salas SD, et al. 1996. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J. Exp. Med. 184:377–386. 21. Seagle BL, Gasyna EM, Mieler WF, Norris JR, Jr. 2006. Photoprotection of human retinal pigment epithelium cells against blue light-induced apoptosis by melanin free radicals from Sepia officinalis. Proc. Natl. Acad. Sci. U. S. A. 103:16644 –16648. 22. Wan X, et al. 2007. Isolation of a novel strain of Aeromonas media producing high levels of DOPA-melanin and assessment of the photoprotective role of the melanin in bioinsecticide applications. J. Appl. Microbiol. 103:2533–2541. 23. Wan X, et al. 2009. Molecular and biochemical characterization of a distinct tyrosinase involved in melanin production from Aeromonas media. Appl. Microbiol. Biotechnol. 82:261–269. 24. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18:821– 829.
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