Identification of functional dnd genes clusters in two Flavobacterium species
We identified functional dnd gene clusters in the genome of two bacterial strains belonging to two different Flavobacterium species. In contrast with all previously described dnd loci, the typical gene order was not conserved in the F. indicum genome, and a hybrid DndEi-encoding gene was identified. DndEi may result from the fusion between a typical DndE and an AAA+ ATPase domain and possess a ‘nicked dsDNA-binding activity’ and a NTPase activity of unknown biological role. F. psychrophilum KU060626-59 contains only the dndCDE gene homologs, suggesting that the minimal functional dnd gene cluster is limited to dndCDE. It has been recently shown that IscS, another cysteine sulfurtransferase, could complement for DndA protein in Escherichia coli (An et al., 2012) to supply this PT modification of DNA, while disruption of dndB does not abolish the Dnd phenotype (Liang et al., 2007; Xu et al., 2009). In genome of F. psychrophilum KU060626-59, we identified a gene encoding an IscS homologous protein. One can speculate that this gene could substitute dndA.
Distribution of dnd clusters in members of the phylum Bacteroidetes
All previously reported dnd gene clusters show a conserved genetic organization with the dndBCDE genes invariably oriented in the same order and direction (Ou et al., 2009). The variety of genomic organization and gene composition of dnd loci within members of the phylum Bacteroidetes is obvious, and these loci seem therefore particularly prone to gene rearrangements in this phylum. Based on their gene composition and gene order, one might conclude that at least two main types of dnd gene clusters coexist within members of the phylum Bacteroidetes. One is found in the genomes of Bacteroides xylanisolvans XB1A, Bacteroides finegoldii CL09T03C10, Paraprevotella xylaniphila YIT 11841, Flavobacteriaceae bacterium 3519-10, and F. psychrophilum KU060626-59, while the other one occurs in the genomes of K. algicida OT-1, R. anatipestifer DSM 15868, Bacteroides sp. 2_1_33B, P. amnii CRIS 21A-A, P. bivia JCVIHMP010, H. hydrossis DSM 1100, P. goldsteinii CLT02T12C30, and B. faecis MAJ27.
The presence of dnd gene clusters seems to occur at low frequency in bacteria. They have never been reported so far among Flavobacterium species or other members of flavobacteria. In this study, dnd gene clusters were only identified into one Flavobacterium genome (i.e. F. indicum) of the 12 publicly available to date. Among the 28 draft genomes of F. psychrophilum strains from many worldwide geographic origins and different host fish (Duchaud et al., 2007 and E. Duchaud, unpublished data), only strain KU060626-59 contained a dnd gene cluster. As such a cluster has been found in only five among the 204 sequenced genomes in the family, the presence of a complete dnd gene cluster in flavobacteria is a rare event. Moreover, the phylogenetic tree of concatenated DndC and DndD protein sequences reported here and the phylogenetic tree constructed on 16S rRNA gene sequences are obviously noncongruent (Fig. S3). Together with the GC% and the dinucleotide distribution bias (Table 1), this confirms that dnd gene clusters are the result of horizontal genetic transfer events.
Although the process of evolution and dissemination of dnd gene clusters across different bacterial species is unknown so far, plasmids have been proposed to play a major role in the dissemination of these clusters. In particular, large plasmids have been suggested to serve as ‘natural depository’ for dnd loci that could be probably sourced from diverse bacterial donors (He et al., 2007). Indeed, dnd gene clusters have been never found on complete phage genomes so far. The only defined dnd island shown to be functionally mobile is the S. lividans SLG island and is known to function as a typical, self-circularizing, site-specific integrative element (He et al., 2007). Using probabilistic model (HMM profiles) (Eddy, 1996) searches across the NCBI plasmid database (that contains 3867 complete plasmid genomes), we detected remote DndC-, DndD- and DndEi-encoding homologs (YP_002967204.1, YP_002967205.1, and YP_002967206.1, respectively) on the Methylobacterium extorquens AM1 megaplasmid (Vuilleumier et al., 2009). The presence of this remote homologous gene cluster on a megaplasmid suggests that large plasmids could indeed serve as ‘natural depository’ and/or vectors for the spreading across bacterial phyla of dnd gene clusters, including the unusual cluster identified in this study.
In addition, the high degree of conservation of gene organization in all previously reported dnd gene clusters may suggest that these elements evolved from a common ancient ancestor (He et al., 2007; Ou et al., 2009). Our study reveals a contrasted situation: the variety of genomic organization and gene composition of dnd loci within members of the phylum Bacteroidetes is obvious and differs from those already described. However, the frequent absence of dnd islands in members of the same species and the presence of two distinct dnd loci within a genome, for instance in M. marina (Table 1 and Fig S1), confirmed that the diverse dnd clusters islands had been acquired independently on many occasions (Ou et al., 2009). As two main types of dnd gene clusters coexist within members of the phylum Bacteroidetes, one might suggest at least two independent ways of acquisition possibly through distinct horizontal genetic transfer events where large plasmids likely play an important role.