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 » Materials and Methods

Materials and Methods

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

Sequencing and annotation
Genome sequence was determined by standard whole-genome shotgun method. Paired end reads of 8.1-fold genomic coverage were produced from 2, 5 and 40 kb clones, all prepared from randomly sheared chromosomal DNA. Chromatograms were processed by the phred/phrap/consed software package (http://www.phrap.org). Gap closure and additional sequencing of low-coverage regions were done by primer walking of gap-spanning clones or PCR products. Physical structure of the final genome sequence was further confirmed by comparing the hypothetical restriction patterns of the sequence for PmeI, SmaI and SwaI and the patterns produced by pulsed-field gel electrophoresis after restriction digestion of the genomic DNA with the three enzymes. The putative origin of replication was determined by GC skew analysis and by identification of genes known to cluster near the prokaryotic oriC site (14,15). Putative CDSs of 100 bp were predicted by amalgamating the results from CRITICA (16) and GLIMMER (17). Intergenic sequences were reanalyzed for short CDSs by running BLASTX. Functional assignment of genes was performed by searching translated CDSs against public protein databases. Manual validation of the annotation results and final refinement including sequence and feature editing were done using ARTEMIS (18). Metabolic pathways were examined using the KEGG database (19) and Pathway Tools (20).

Phylogenetic analysis
The 16s rDNA sequences or 34 concatenated protein sequences that are conserved as the genetic core of the universal ancestor (21) were retrieved from GenBank and used as common tracers of genome evolution. To identify counterparts of 34 COGs in each species, the retrieved genomes were searched with series of 34 hidden Markov models made up of each COG protein cluster. The 16s rDNA sequences or concatenated universal 34 protein sequences under E-value cutoff (1.0 x 10^–06) were analyzed with neighbor-joining and maximum parsimony methods in CLUSTAL W (22) and PHYLIP (23). To correct multiple substitutions in protein residues, the Kimura 2-parameter model was used, and to evaluate the reliability of the branching patterns, 1000 random bootstrap re-samplings were executed.

Identification of genomic islands
Horizontally transferred genes (HTGs) were inferred from genomic anomalies or phylogenetic context. A gene was considered anomalous if both G+C content and codon usage are aberrant (G+C content 1.5 and Mahalanobis distance as a degree of the codon usage deviation 80.23) (24). In addition, genes having orthologs in other prokaryotes but not in -Proteobacteria were added to the list of HTGs. BLASTP searches were performed to find pairs of reciprocal best hits between in the H.chejuensis CDSs and those in each of the 208 completely sequenced prokaryotes. If the CDS in the searched genomes can be aligned with that of H.chejuensis (80% of its length and 30% identity), the pair was considered orthologs. Then, a genome scan of a 10-gene window was run and the regions containing four or more HTGs were identified. Neighboring regions were merged into larger regions and each was manually examined to exclude fragments consisting of housekeeping genes or only small-sized hypothetical genes.

Algicidal activity assay
The medium for production of red pigment was M-RP10356 medium (5% glucose, 0.1% peptone, 0.42 g KH₂PO₄, 0.34 g K₂HPO₄, 0.5 g MgSO₄, 2.0 g CaCl₂, 0.001 g CoCl₂·6H₂O, 0.001 g MnCl₃, 0.001 g ZnSO₄ and 0.001 g NaMoO₄, pH 7.0, where 250 ml distilled water and aged seawater were added to the final volume of one liter). Cultivation was carried out in a 5 liters jar-fermentor for 72 h at 25°C with aeration of 1.5 volume of air added to liquid volume per minute after inoculation (2.0%). From the culture broth, crude RP10356 was extracted by chloroform, which was further concentrated. Crude preparation of the pigment was purified with silica gel 60 (0.063 mm, Merck, Germany) using chloroform (100, v), and re-purified with YMC-Pack ODS-A (250 x 10 mm, YMC Co., Japan) using methanol:water:acetic acid (81:14:5, v/v/v). All algal strains were maintained in f/2 culture medium at 22.5°C, and under a light intensity of 55 µmol/m²/s using a 16 h light/8 h dark illumination cycle. Purified pigment was dissolved in ethanol, and aliquots of 20 µl solution were added to 980 µl of microalgal suspension in test tubes. Cell number was counted with a microscope after 1.0% Lugol's solution staining. Following formula was used to calculate the algicidal effect:

Heterologous expression and mutagenesis
Five fosmid clones of Escherichia coli EPI300, HC81010E03, HC81008E02, HC81002H12, HC81006F09 and HC81004F05 containing either part or the whole of the pigment gene cluster were plated and incubated overnight at 37°C on Luria–Bertani agar containing 20 µg/ml chloramphenicol, CopyControl Induction Solution (Epicentre, USA) and, sometimes, 20% crude extract of H.chejuensis prepared by filtration (pore size 0.22 µm). Transposon mutagenesis on a variant of HC81006F09 that constitutively expresses the red pigment was carried out using EZ::TN Insertion Kit (Epicentre).

Structural determination of the pigment
Red pigment was extracted with a mixture of methanol/1N HCl (24:1, v/v) from the supernatant of H.chejuensis culture which was grown on Marine Broth (Difco) for 24–48 h at 30°C with vigorous shaking. Red-colored fraction was purified through high-performance liquid chromatography. Following LC using acetonitrile and water (with 0.1% formic acid) as the mobile phase at a flow rate of 0.2 ml/min, ESI-MS was carried out with a Finnigan LCQ Advantage MAX ion trap mass spectrometer equipped with a Finnigan electrospray source. To determine the molecular structure, ¹H NMR (CD₃OD, 300 MHz) and ¹³C NMR (CD₃OD, 75 MHz) analyses were performed, resulting in the raw data of 6.94 (m, 1H), 6.71 (m, 1H), 6.66 (s, 1H), 6.39 (s, 1H), 6.21 (m, 1H), 6.01 (s, 1H), 3.89 (s, 3H), 2.37 (t, 2H), 2.27 (s, 3H), 1.53 (m, 2H), 1.33 (m, 4H), 0.90 (t, 3H) (¹H NMR) and 169.6, 160.2, 141.0, 135.8, 129.9, 129.2, 125.1, 123.0, 120.6, 115.9, 113.1, 110.9, 95.9, 58.9, 32.7, 31.8, 26.6, 23.6, 14.4, 11.5 (¹³C NMR).