*For correspondence. E-mail: santuzat@icb.ufmg.br; Tel.: +55-31-3409- 2665 § Supplemental material for this article may be found at http://www.springerlink.com/content/120956. Thiago Castro-Gomes 1 , Mariana S. Cardoso 1 , Wanderson D. DaRocha 3 , Letícia A. Laibida 3 , Andréa M. A. Nascimento 2 , Luciana W. Zuccherato 2 , Maria Fátima Horta 1 , Marcelo P. Bemquerer 4 , and Santuza M. R. Teixeira 1 * 1 Departamento de Bioquímica e Imunologia and 2 Departamento de Biologia Geral, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil 3 Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brazil 4 EMBRAPA Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil (Received Apr 5, 2013 / Revised Aug 1, 2013 / Accepted Aug 8, 2013) Journal of Microbiology (2014) Vol. 52, No. 4, pp. 350–353 Copyright 2014, The Microbiological Society of Korea DOI 10.1007/s12275-014-3202-5 NOTE Identification of Secreted Virulence Factors of Chromobacterium violaceum § Chromobacterium violaceum, a component of tropical soil microbiota, is an opportunistic pathogenic bacterium that can infect humans and other animals. In addition to identi- fying a large number of genes that demonstrate the vast bio- technological potential of this bacterium, genome sequenc- ing revealed several virulence factors, including different cy- tolysins, which can be related to its pathogenicity. Here we confirmed these predictions from genomic analyses by iden- tifying, through mass spectrometry, proteins present in the culture supernatant of C. violaceum that may constitute se- creted virulence factors. Among them, we identified a secreted collagenase and the product of a gene with sequence sim- ilarity to previously characterized bacterial porins. Keywords : Chromobacterium violaceum, genome, hemolysin, virulence factor Chromobacterium violaceum is a Gram-negative β-proteo- bacterium that inhabits a variety of ecosystems such as wa- ter and soil of tropical and subtropical regions (Durán and Menck, 2001). The most striking feature of C. violaceum is its, purple-colored pigment derived from tryptophan, known as violacein, a bacterial secondary metabolite that has anti- oxidant and antimicrobial activities (Hoshino, 2011). C. vio- laceum is also recognized as an opportunistic pathogen that can infect humans and other animals. Although rare, infec- tions with this bacterium are characterized by rapid dissemi- nation and high mortality rates (Chattopadhyay et al., 2002; Brito et al., 2004). The sequence of the C. violaceum genome revealed nume- rous genes related to the biotechnological and pharmaceutical potential of this extremely versatile bacterium (Vasconcelos et al., 2003). Moreover, the identification of pathogenicity islands harboring genes encoding virulence factors shows that C. violaceum has a genetic background also compatible with a potentially pathogenic bacterium (Vasconcelos et al., 2003; Brito et al., 2004). In addition to the twelve open rea- ding frames (ORFs) previously described in the original ge- nome analysis of C. violaceum as sequences encoding hemo- lysin-like proteins, we have identified another seven genes, five of them encoding proteins with sequence homology to porins from Gram-negative bacteria (Supplementary data Table S1). Among a total of nineteen C. violaceum genes that can be considered potential virulence factors, there are members of the hlyCABD operon found in uropathogenic Escherichia coli and in various Gram-negative bacteria, which includes a gene encoding a calcium binding hemolysin, hlyA, containing the RTX (repeat-in-toxin) motif (CV0516) as well as components of the type I transport apparatus present in the inner membrane of various pathogenic bacteria (CV0513 and CV0514). Two other genes encoding hemolysins were also found associated with activator/transporter proteins: CV1918, whose product has been characterized as a Serratia- like hemolysin (Brumbach et al., 2007) and is linked to a gene (CV1917) encoding an outer membrane protein that activates and secretes the cytolysin. Similarly, CV2872 en- codes a large protein homologous to hemolysin-type calcium- binding proteins and is adjacent to a gene encoding a hemo- lysin activator protein (CV2873). Two genes encoding pro- teins with high similarity to hemolysins that have lipolytic activity (CV0360 and CV0362) were also found next to each other. Interestingly, a gene encoding a protein similar to sphingomyelinases from several bacteria, including Pseu- domonas aeruginosa and the β-hemolysin of Staphylococcus aureus (CV3275) was found in the operon that encodes com- ponents of the violacein biosynthetic pathway. Lastly, five genes (CV1646, CV1989, CV3104, CV3424, and CV3829) showing sequence homology to porins from Gram-negative bacteria, as well as genes encoding a hemolysin III family channel protein (CV3342), a hemolysin A (CV4301) and two other putative hemolysin genes (CV0231 and CV0656), are sequences encoding potentially membrane-damaging pro-