The goal of this research is to understand the mechanism of biosynthesis of peptide siderophores in bacteria. Our paradigms are two peptide siderophores produced by the pathogen Vibrio anguillarum. One is anguibactin, an important component of a plasmid-mediated iron uptake system that is essential for virulence of this bacterium. The other is vanchrobactin, a chromosomally-encoded siderophore that in the absence of the pJM1-encoded anguibactin system becomes an important component of the virulence repertoire of V. anguillarum. The specific mechanisms that govern the biosynthesis of these siderophores in this biologically significant pathogen will be further elucidated. In order to dissect these mechanisms we will use a combination of genetic and biochemical approaches.
The specific aims are: 1. Dissection of the mechanisms of assembly line enzymology in anguibactin biosynthesis. We plan to ascertain by using in vitro approaches the role of the different domains in each of anguibactin production steps. Specifically, we will assess: the role of the ArCP domain from AngB, the condensation (C) and (PPC) with AngM, the cysteine adenylation domain of AngR in activating cysteine;each Cy domain of AngN in cysteine condensation with 2,3-dihydroxybenzoic acid (DHBA) and cyclization to generate dihydroxyphenylthiazolyn (DHPT);the transfer of DHPT to N-hydroxy- histamine by the C domain of AngM. 2. Do NRPSs communicate with each other through specific domains? Our evidence indicates that several anguibactin biosynthetic genes are encoded redundantly on both the pJM1 plasmid and the chromosome of V. anguillarum and that one, angA is only chromosomally mediated. Our recent work demonstrated that the redundant genes and angA are shared by the biosynthetic apparatus for a chromosomal gene cluster that intervenes in the production of vanchrobactin (N-[N'-(2,3- dihydroxybenzoyl)-arginyl]-serine) and uptake of its ferric complexes and that consists of VabE, VabB, VabD and VabF, as well as other tailoring enzymes. The plasmid-mediated AngB and the chromosomal VabB are NRPSs with two domains each: an isochorysmate lyase and an ArCP that work as the first acceptors of an activated molecule of DHBA in the biosynthesis of anguibactin (AngB) and vanchrobactin (VabB). VabB is slightly larger than AngB due to extensions at the amino and carboxy terminus. Although VabB operates in vanchrobactin biosynthesis it cannot replace AngB in anguibactin biosynthesis synthesis. We believe that these results are due to the presence of specific communication-mediating domains in VabB and AngB that allow them to only interact with their specific NRPSs, VabF for VabB and AngM for AngB. We propose experiments to understand the basis of protein-protein communication between these NRPSs that facilitates the selective interaction in the multienzyme complexes. Our results could lead to the exploration of new avenues in the fascinating field of NRPSs communication during siderophore biosynthesis. These novel approaches will likely lead to a better understanding of siderophore biosynthesis and virulence in bacteria.

Public Health Relevance

We intend to dissect the nonribosomal peptide synthetase systems of pathogenic bacteria into sets of well characterized, interchangeable modular domains that can be used as catalytic reagents for the designed biosynthesis of novel pharmacological and chemotherapeutic products that can be used to combat diseases caused by pathogenic bacteria. These approaches will likely lead to the exploration of new avenues in the process of siderophore biosynthesis and virulence in bacteria.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Bacterial Pathogenesis Study Section (BACP)
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Chin, Jean
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Oregon Health and Science University
Schools of Medicine
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Naka, Hiroaki; Liu, Moqing; Actis, Luis A et al. (2013) Plasmid- and chromosome-encoded siderophore anguibactin systems found in marine vibrios: biosynthesis, transport and evolution. Biometals 26:537-47
Naka, Hiroaki; Liu, Moqing; Crosa, Jorge H (2013) Two ABC transporter systems participate in siderophore transport in the marine pathogen Vibrio anguillarum 775 (pJM1). FEMS Microbiol Lett 341:79-86
Naka, Hiroaki; Actis, Luis A; Crosa, Jorge H (2013) The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid-encoded system of Vibrio anguillarum? Microbiologyopen 2:182-94
Dias, Graciela M; Thompson, Cristiane C; Fishman, Brian et al. (2012) Genome sequence of the marine bacterium Vibrio campbellii DS40M4, isolated from open ocean water. J Bacteriol 194:904
Di Lorenzo, Manuela; Stork, Michiel; Crosa, Jorge H (2011) Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrio anguillarum strain 775. Biometals 24:629-43
Naka, Hiroaki; Crosa, Jorge H (2011) Genetic Determinants of Virulence in the Marine Fish Pathogen Vibrio anguillarum. Fish Pathol 46:1-10
Kustusch, Ryan J; Kuehl, Carole J; Crosa, Jorge H (2011) Power plays: iron transport and energy transduction in pathogenic vibrios. Biometals 24:559-66
López, Claudia S; Peacock, R Sean; Crosa, Jorge H et al. (2009) Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum. Biochem J 418:49-59
Di Lorenzo, Manuela; Stork, Michiel; Naka, Hiroaki et al. (2008) Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum. Biometals 21:635-48
Naka, Hiroaki; Lopez, Claudia S; Crosa, Jorge H (2008) Reactivation of the vanchrobactin siderophore system of Vibrio anguillarum by removal of a chromosomal insertion sequence originated in plasmid pJM1 encoding the anguibactin siderophore system. Environ Microbiol 10:265-77

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