Here, the authors present the 7.2-megabase genome of the marine bacterium Hahella …

• Abstract
• Introduction
• Materials and Methods
• Results and Discussion
• Conclusions
• Acknowledgements
• References
• Figures
• Table

Biology Articles » Biotechnology » Blue Biotechnology » Genomic blueprint of Hahella chejuensis, a marine microbe producing an algicidal agent » Results and Discussion

Results and Discussion

- Genomic blueprint of Hahella chejuensis, a marine microbe producing an algicidal agent

RESULTS AND DISCUSSIONS 

General features
The genome of H.chejuensis KCTC 2396^T consists of one circular chromosome of 7 215 267 bp (Figure 2). This makes it the largest among the marine prokaryotic genomes whose genome sequences are available and also among the -proteobacterial genomes sequenced. Among the 6783 predicted genes, 76.3% showed significant database matches and 49.6% were assigned a putative function (Table 1). While about a quarter of the predicted genes are unique, comparison of broadly conserved H.chejuensis genes with those of other completely sequenced prokaryotes indicated that H.chejuensis is distantly affiliated to Pseudomonas spp. (Supplementary Figures S1 and S2).

Basic metabolic capabilities are well equipped to support a free-living, marine heterotrophic lifestyle. The bacterium has a complete repertoire of enzymes for central carbon metabolism including glycolysis, pentose phosphate pathway and TCA cycle, as well as those required for biosynthesis of nucleotides and 20 amino acids. The presence of genes for one putative carbon monoxide dehydrogenases and one hydrogenase complex without other genes for autotrophy implies that, when available organic nutrients are scarce, H.chejuensis might rely on the lithoheterotrophic strategy (25). Genes for inorganic sulfur oxidation, however, were not identified. We also found genes for the respiratory nitrate reductase complex.

Adaptation to the marine environment
The number of genes dedicated to transcriptional regulation or environmental sensing amounts to 362, which corresponds to 5.3% of total predicted genes (Supplementary Table S1). This is in accordance with the tendency that the number of regulatory genes increases as the genome size increases (26). Most common regulator types in H.chejuesnsis include LysR, AraC, TetR and MerR. The bacterium also possesses four major sigma-70 factors, two extracytoplamic function sigma factors and one sigma-54 factor. There are more than 20 proteins that have the sigma-54 interaction module. The number of putative two-component system (47 sensors, 103 response regulators and 23 sensor-response regulator hybrids) is overrepresented compared with other bacterial genomes. In addition, the bacterium has a complex chemosensory system with 35 genes encoding putative chemotactic sensory transducer proteins. However, the typical quorum-sensing system seems absent as no homologs of luxI could be found.

H.chejuensis has a wide range of transporters for sugars, peptides/amino acids, phosphate, manganese, molybdate, nickel and drugs. Sugar transport systems, however, appear highly biased to ABC transport systems as there are 11 ABC-type transporters but phosphotransferase system is incomplete. This phenomenon is rather scarce but often observed in some pathogens (27). A variety of extracellular hydrolytic enzymes represented by the H.chejuensis genome, such as proteases, lipases, nucleases, chitinases and cellulases, could be advantageous once macromolecular nutrients become available. Along with the high portion of regulatory proteins and transporters for a variety of nutrients, these features imply the functional diversity and adaptability of H.chejuensis to changing marine environments.

Like other marine bacteria, H.chejuensis requires 2% NaCl for optimal growth (8). Na⁺ is essential for marine or halophilic bacteria as transmembrane Na⁺ gradient is utilized for uptake of nutrients and flagellar rotation (28). In general, Na⁺/H⁺ antiporter generates the sodium motive force for these cellular processes, but Na⁺-translocating respiratory NADH:ubiquinone oxidoreducatase is widely distributed among Gram-negative marine bacteria in addition to the primary H⁺ pump and Na⁺/H⁺ antiporter. Genome analysis identified the same type of respiratory complex in H.chejuensis and multiple Na⁺/H⁺ antiporters including a multi-subunit Na⁺/H⁺ antiporter system.

Redundant genes and genomic islands
An interesting feature of the H.chejuensis genome is the multiplicity of homologous genes encoding functionally equivalent proteins (Supplementary Table S2). There are dozens of cases where the same function is redundantly encoded by two to four independent genes. As for gene sets, there are two loci each for F₀F₁-type ATP synthesis, flagellar biogenesis and type III protein secretion. When all-against-all similarity searches were performed to identify recent gene duplication within the genome, overall identities among the homologous genes were far below than those of the closest proteins from other sequenced genomes. While in many cases one member best matches to proteins in -Proteobacteria, the other members are similar to those in various other taxa. These observations support that the origin of multiplicity is likely horizontal gene transfer rather than duplication of genes in the H.chejuensis genome.

Like many other bacteria (29), horizontal gene transfer seems to have had essential roles in shaping the H.chejuensis genome. Based on genomic anomalies and phylogenetic context, the bacterium appears to have at least 69 genomic islands (GIs) constituting 23.0% of the chromosome (Figure 2). Genes or gene clusters contained in the islands include those involved in biosynthesis of exopolysacchrides, toxins, polyketides or non-ribosomal peptides, iron utilization, motility, type III protein secretion, or pigmentation. Of them 32 contained homologs of genes associated with mobile elements such as Rhs elements, insertion sequence elements, transposons, bacteriophages and group II introns (Supplementary Table S3). Genes encoding Rhs family proteins are the largest group in the H.chejuensis genome in terms of both abundance and length (1.7% of the chromosome). In most cases, the Rhs proteins are very closely related to those in the archaeon Methanosarcina barkeri or the firmicute Clostridium thermocellum.

Potential virulence-associated genes
H.chejuensis produces a large amount of extracellular polysaccharides (EPSs) (30). EPSs are responsible for development of biofilms, and often act as a virulence factor in pathogenic bacteria. We found five gene clusters that may be involved in the synthesis of exopolysaccharides, all of which overlap GIs partly or entirely (Supplementary Figure S3 and Table S3). Among the gene clusters is one located at the 4.8 Mb region encoding genes for key enzymes such as UDP-glucose dehydrogenase (ugd), Wzy-type polymerase and Wzx flippase. UGD produces UDP-D-glucuronate, which is known to be a building block for production of capsular polysaccharides in several pathogenic bacteria and colanic acid in E.coli (31,32).

Pore-forming hemolysin and RTX toxin play important roles in many pathogenic Gram-negative bacteria with their cytotoxic activities (33). Out of the seven RTX toxin homologs and three hemolysin homologs found in H.chejuensis, five are included in GIs. One of the striking findings from the H.chejuensis genome is the unexpected presence of two type III secretion systems (TTSSs) (34,35) located at positions 3.34–3.37 and 5.25–5.29 Mb that are similar to those present in Yersinia spp., Vibrio spp., Pseudomonas aeruginosa and Aeromonas spp. (Figure 3). While the two TTSSs in H.chejuensis belong to the same subfamily of TTSSs, only one of them is located in a GI. Presence of the homologs of these virulence determinants suggests that H.chejuensis probably is a pathogen of marine eukaryotes.

 
Identification of an algicidal pigment
Experiments with the extract of H.chejuensis indicated that both crude and purified preparations of the red pigment are responsible for the rapid cell lysis of C.polykrikoides (data not shown and Figure 4). Therefore, genes possibly involved in synthesis of secondary metabolites were searched for in the genome sequence of H.chejuensis, and a gene cluster harboring genes similar to the red genes of Streptomyces coelicolor A3(2) (36) was suspected to be responsible for the biosynthesis of the red pigment (Figure 5A).  
When fosmid clones carrying the complete gene cluster were grown on agar plates, colonies located close to those of H.chejuensis or grown in a medium containing the H.chejuensis extract turned red, indicating that this genomic region has the gene set required for pigment biosynthesis (Figure 5A). Further supporting the observation, transposon insertions in the red homologs resulted in loss of colony color when tested with a variant clone that constitutively produces the red pigment.

A maximum absorbance of the purified red pigment at 535 and 470 nm in acidic and basic conditions, respectively, suggested that the pigment is a prodigiosin-like compound. Through LC-ESI-MS/MS analysis, the fragmentation pattern of a base peak [23.73 min; m/z 324.2, (M+H)⁺] from the red pigment was shown to be identical to that of the antibiotic prodigiosin, which was further confirmed by ¹H NMR and ¹³C NMR analyses (Materials and Methods and Figure 5B).