A siderophore biosynthesis gene cluster from the fish pathogen Photobacterium damselae subsp. piscicida is structurally and functionally related to the Yersinia high-pathogenicity island Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis, produces a siderophore which is distinct from that produced by P. damselae subsp. damselae. Using suppression subtractive hybridization, a subsp. piscicida-specific DNA region of 35 kb was identified in strain DI21, and 11 genes were defined: dahP, araC1, araC2, frpA, irp8, irp2, irp1, irp3, irp4, irp9 and irp5. The sequence of the predicted proteins encoded by these genes showed significant similarity with the proteins responsible for the synthesis and transport of the siderophore yersiniabactin, encoded within the Yersinia high-pathogenicity island (HPI). Southern hybridization demonstrated that this gene cluster is exclusive to some European subsp. piscicida isolates. Database searches revealed that a similar gene cluster is present in Photobacterium profundum SS9 and Vibrio cholerae RC385. An irp1 gene (encoding a putative non-ribosomal peptide synthetase) insertional mutant (CS31) was impaired for growth under iron-limiting conditions and unable to produce siderophores, and showed an approximately 100-fold decrease in degree of virulence for fish. The subsp. piscicida DI21 strain, but not CS31, promoted the growth of a Yersinia enterocolitica irp1 mutant. Furthermore, a yersiniabactin-producing Y. enterocolitica strain as well as purified yersiniabactin were able to cross-feed strains DI21 and CS31, suggesting that the subsp. piscicida siderophore might be functionally and structurally related to yersiniabactin. The differential occurrence among P. damselae strains, and the low sequence similarity to siderophore synthesis genes described in other members of the Vibrionaceae, suggest that this genetic system might have been acquired by horizontal transfer in P. damselae subsp. piscicida, and might have a common evolutionary origin with the Yersinia HPI. INTRODUCTIONIron is an essential element for most bacteria; however, most of the iron in biological systems is chelated by high-affinity iron-binding proteins. Therefore, pathogens have developed efficient mechanisms to obtain iron from the host and to establish an infection (Ratledge & Dover, 2000). The ability to obtain iron from free haem or haem proteins from the host tissues constitutes an important source of iron for many bacterial pathogens (Genco & Dixon, 2001;Osorio & Lemos, 2002). However, one of the main strategies to obtain iron is the synthesis and secretion of Fe(III) chelators, named siderophores, which are low molecular mass iron-chelating molecules that can remove iron from host iron-binding proteins (Crosa, 1989) and then enter the cell through outer-membrane receptor proteins. Many siderophores are small peptides synthesized by nonribosomal peptide synthetases (NRPSs), which are multimodular enzymes that produce peptide products of a particular sequence without an RNA temp...

Photobacterium damselae subsp. piscicida is the causative agent of fish pasteurellosis, a severe disease affecting cultured marine fish worldwide. In this study, suppression subtractive hybridization was used to identify DNA fragments present in the virulent strain PC554.2, but absent in the avirulent strain EPOY 8803-II. Twenty-one genomic regions of this type (that included twenty-six distinct putative ORFs) were analysed by DNA sequencing. Twenty ORFs encoded proteins with homology to proteins in other bacteria, including four homologues involved in siderophore biosynthesis, and four homologues related to mobile elements; three of these were putative transposases and one was a putative conjugative transposon related to the Vibrio cholerae SXT element. This sequence was shown to be integrated into a prfC gene homologue. Six ORFs showed no significant homology to known bacterial proteins. Among the 21 DNA fragments specific to strain PC554.2, 5 DNA fragments (representing 7 ORFs) were also absent in the avirulent strain ATCC 29690. The analysis of these differential regions, as well as the screening of their presence in a collection of strains, demonstrated the high genetic heterogeneity of this pathogen.

The gene encoding the ferric uptake regulator protein (fur gene) of the fish pathogenic bacterium Photobacterium damselae ssp. piscicida Strain DI21 was partially amplified using degenerate oligonucleotides. Complete sequencing of the fur gene and neighbouring DNA was accomplished by primer walking. An open reading frame of 447 bp, coding for a protein of 148 amino acids, and with high homology to previously described Fur proteins, was identified. The fur gene of P. damselae ssp. damselae ATCC 35083 was subsequently amplified by PCR with specific primers and its sequence determined, showing a 99.3% similarity to the P. damselae ssp. piscicida fur gene. The P. damselae fur gene was able to complement the fur mutation of Escherichia coli Strain H1681 in an iron-dependent fashion.KEY WORDS: Photobacterium damselae · Pasteurellosis · fur gene · Fur protein · Iron uptake Resale or republication not permitted without written consent of the publisherDis Aquat Org 58: [151][152][153][154][155][156] 2004 2001). This protein requires ferrous iron as a cofactor for dimerisation and DNA binding. When Fur is associated with ferrous iron, it is able to bind operator sites (called Fur boxes) within the promoter region of iron uptake genes, so that transcription of these genes is shut off in iron-replete cells. In irondepleted conditions, Fur cannot bind operator sites, thus allowing transcription of iron uptake-related genes. In this study, the fur gene has been cloned and sequenced in the 2 P. damselae subspecies, and its ability to function as an iron-dependent corepressor has been tested. MATERIALS AND METHODSStrains used are listed in Table 1. Photobacterium damselae strains were routinely grown at 25°C in Tryptic Soy Agar (Difco) supplemented with 1% NaCl (TSA-1). Escherichia coli strains were routinely grown at 37°C in Luria Bertani (LB) medium. MacConkey Agar (Cultimed) was supplemented either with 200 µM 2, 2'-dipyridil (iron-restricted conditions) or with 0.1 mM FeCl 3 (iron-sufficient conditions). All strains were stored frozen at -80°C in LB broth with 20% glycerol. Ampicillin sodium salt stock solutions (100 mg ml -1 in water) were filter sterilized and stored at -20°C.Standard methods for DNA manipulations were carried out as described by Sambrook & Russell (2001). Total genomic DNA from Photobacterium damselae was prepared with the Easy-DNA kit (Invitrogen). Plasmid DNA purification and extraction of DNA from agarose gels were done with kits from Qiagen. DNAprobe labeling and Southern blot analysis were carried out with the ECL DNA labeling and detection system (Amersham Biosciences), following the manufacturer's instructions. Plasmids used in cloning experiments and those derived from this study are summarised in Table 1 6).Escherichia coli H1681 is a fur-defective strain that harbours a β-galactosidase reporter gene placed under control of the fur-regulated fhuF gene promoter (Heidreich et al. 1996). Thus, this strain is ideal for testing the function of a putative fur gene as an iron-regulated tr...

The marine fish pathogen Photobacterium damselae subsp. piscicida utilizes haem compounds as the sole iron source. In a previous work, we characterized a gene cluster with ten potential haem uptake and utilization genes. Two of these genes, hutC and hutD, which are iron-regulated, conform a putative inner membrane haem ABC transporter. In this study, we constructed an insertional mutant, leading to the inactivation of hutCD genes. Reverse transcriptase-PCR analyses demonstrated that an insertion between the hutB and hutC genes abolished transcription of the downstream hutC and hutD genes. The hutCD mutant was unable to utilize haem as the sole iron source, demonstrating that the putative ABC-transporter proteins HutC and HutD are essential for haem utilization as an iron source in P. damselae subsp. piscicida. In addition, reverse transcriptase-PCR assays conducted with RNA samples isolated from experimentally infected fish revealed the presence of hutCD transcripts. The results demonstrate for the first time that haem uptake genes of a fish pathogen are expressed during the infective process in fish.