Complete genome sequence of Ilumatobacter coccineum YM16-304^T

Genome project history

Growth conditions and DNA isolation

I. coccineum YM16-304^T cells were grown in a 20 L volume at 27°C in Difco^TM Marine broth 2216 (Beckton Dickinson). DNA was isolated from 0.5 g of wet cells by manual extraction after lysis with lysozyme and SDS.

Genome sequencing and assembly

The genome of I. coccineum YM16-304^T was sequenced using the conventional whole-genome shotgun sequencing method. Plasmid libraries with average insert sizes of 1.5 kb and 6.0 kb were generated in pTS1 (Nippon Gene) and pUC118 (TaKaRa) vectors, respectively, while a fosmid library with average insert size of 38 kb was constructed in pCC1FOS (EPICENTRE) as described previously [16]. A total of 26,592 clones (18,432, 5,376 and 2,784 clones from libraries with 1.5 kb, 6.0 kb and 38 kb inserts, respectively) were subjected to sequencing from both ends of the inserts on a ABI 3730xl DNA Analyzer (Applied Biosystems). Sequence reads were trimmed at a threshold of 20 in Phred score and assembled by using Phrap and CONSED assembly tools [12,13]. Gaps between contigs were closed by sequencing PCR products which bridge two neighboring contigs. Finally, each base of the genome was ensured to be sequenced from multiple clones either from both directions with Phrap quality score ≥ 70 or from one direction with Phrap quality score ≥40.

Genome annotation

The complete sequence of the chromosome was analyzed using Glimmer3 [14] for predicting protein-coding genes, tRNAscan-SE [17] and ARAGORN [18] for tRNA genes, and RNAmmer [19] for rRNA genes. The functions of predicted protein-coding genes were assigned manually, using the in-house genome annotation system OCSS (unpublished), in comparison with Uniprot [20], Interpro [21], HAMAP [22] and KEGG [23] databases.

Primary metabolism

Strain YM16-304^T lacks the dapE gene for succinyl-diaminopimelate desuccinylase (EC:3.5.1.18) in the biosynthesis pathway of lysine and diaminopimelic acids (DAPs). Instead, two candidate genes (YM304_26990 and YM304_19190) for LL-DAP aminotransferase (EC:2.6.1.83, dapL), that constitutes an alternative DAP-lysine biosynthesis pathway (DAP aminotransferase pathway [24,25]), were identified. The dapL gene is found in discrete lineages of Bacteria and Archaea, and is known to complement Escherichia coli dapD and dapE mutants, although purified proteins favor the reverse reaction rather than the synthesis of LL-DAP [25].

Among the genes of serine biosynthesis pathway, the serB gene for phosphoserine phosphatase (EC:3.1.3.3) was not identified by similarity searches. On the other hand, the thrH gene for phosphoserine / homoserine phosphotransferase [26] (EC:3.1.3.3, 2.7.1.39) was identified (YM304_28950). The possibility of using thrH gene product for serine biosynthesis instead of serB gene product was suggested.

Strain YM16-304^Tseems to possess an alternative form of histidine biosynthesis pathway in which hisB gene for the synthesis of L-histidinol was replaced with the hisN gene (YM304_12240) as typically found in Corynebacterium glutamicum ATCC13032 [27] and other actinomycetes. However, the hisE gene for phosphoribosyl-ATP pyrophosphohydrolase (EC:3.6.1.31), which is responsible for the second step in histidine biosynthesis pathway, was not identified by similarity searches.

Metabolic reconstruction based on the annotation suggested that strain YM16-304^T possesses the enzymes required for the biosynthesis of saturated fatty acids, unsaturated fatty acids, branched-chain fatty acids and carotenoids. The putative carotenoid biosynthesis pathway comprises crtE (YM304_37400), crtB (YM304_37420), crtI (YM304_37410) and crtLm (YM304_23780) gene homologs, which most probably synthesizes γ-carotene from isopentenyl pyrophosphate derived from non-mevalonate pathway [28–30]. Strain YM16-304^T also possesses genes homologous to crtO (YM304_25370) and crtZ (YM304_38780), which were suggested to be involved in the synthesis of ketolated carotenoid such as canthaxanthin and astaxanthin [30]. Actual products of this pathway need to be experimentally verified.

The annotation also suggests that strain YM16-304^T possesses the enzymes required for the biosynthesis of menaquinone (vitamin K), vitamin B₆, nicotinate and nicotinamide, pantothenate and CoA, lipoic acid, protoheme, mycothiol and coenzyme F₄₂₀, while biosynthetic pathways for folate, thiamine, riboflavin, biotin and adenosylcobalamin (coenzyme B₁₂) are either missing or incomplete.

Secondary metabolism

The phylogenetic analysis based on 16S rRNA gene sequences showed that three species in the genus Ilumatobacter were closely related to some uncultured actinobacteria including marine sponge symbionts [31]. Marine sponges are noted as a rich source of biologically active secondary metabolites, true producers of such compound being suspected to be symbiotic bacteria [32–34]. However, only a small percentage of these symbiotic microorganisms are culturable [35,36], and genes involved in the synthesis of bioactive compounds such as polyketide synthases have often been isolated by metagenomic approaches [37,38].

The strain YM16-304^T genome seemed to encode only a limited number of secondary metabolic enzymes, i.e., two type I polyketide synthases (PKS). The genome does not contain genes for type II and type III PKS nor a gene for nonribosomal peptide synthetase.

The type I PKS genes of the strain YM16-304^T (YM304_13420, YM304_13410), together with the adjacent pfaD homolog (YM304_13430), most probably encode omega-3 polyunsaturated fatty acid (PUFA) synthase gene cluster. In some Gammaproteobacteria from marine sources such as Photobacterium profundum strain SS9, omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) are known to be synthesized by a PKS system consisting of pfaA, pfaB, pfaC and pfaD genes [39–41]. The domain organization of YM304_13420 was identical to that of the pfaA gene of P. profundum SS9. The N-terminal ketosynthase domain and the C-terminal dehydratase domains of YM304_13410 were similar to those of the pfaC gene of P. profundum, while the internal acyltransferase domain of YM304_13410 was moderately similar to that of the pfaB gene of P. profundum, representing a presumed chimeric form of PKS. As PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis [42], the actual product of this PKS system may need to be clarified experimentally. Some PUFA-producing bacteria such as Moritella marina MP-1 [39,43] were reported to require an additional gene, pfaE, encoding a phosphopantheteinyl transferase. However, the pfaE gene was not identified in strain YM16-304^T. Other classes of phosphopantheteinyl transferase (e.g. YM304_08850) may substitute the function of PfaE, similar to the case suggested in P. profundum SS9 [44].

Cell surface

Strain YM16-304 seemed to possess 13 ORFs containing LPXTG motif (InterPro ID: IPR001899), the presumed sorting signal of cell surface proteins in Gram-positive bacteria [45]. It was reported that several cell surface proteins containing LPXTG motif act as an adhesion factor known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) [46]. The genome of strain YM16-304 contained extracellular polysaccharide gene cluster (YM304_29910-YM304_30490), including gene cluster for the synthesis of sialic acids (YM304_30300-YM304_30320), which are also crucial for cell adhesion [47]. These extracellular components might serve for the bacterium to adhere to host tissues such as marine sponges.

Many marine bacteria use the Na⁺ cycle and require Na⁺ for their growth [48]. In these bacteria, Na⁺ is often used in the respiratory chain, ATP synthase, flagellar rotation and solute uptake instead of H⁺ [49]. Some bacteria can use both Na⁺ and H⁺ to expand the range of environments in which the bacteria can grow [50]. Strain YM16-304 was isolated from a sand sample collected at a beach and grows optimally in marine broth media, suggesting its marine origin. However, the gene products for the respiratory chain and ATP synthase were predicted to be of the H⁺-dependent type by similarity search. The Na⁺-dependent amino acid symporters were also not identified, nor was the H⁺-dependent symporters.