Computational protocol: Draft Genome Sequence of a Pseudomonas aeruginosa NA04 Bacterium Isolated from an Entomopathogenic Nematode

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Protocol publication

[…] The gammaproteobacterium Pseudomonas aeruginosa is Gram negative, aerobic, and highly versatile. It is distributed in different ecological niches, including water and soil, and in association with host organisms (). The genus Pseudomonas is particularly interesting because of its importance as an opportunistic pathogen in humans and its biotechnological potential (, ).We isolated a bacterium from the entomopathogenic nematode Heterorhabditis indica MOR03, recovered from soil samples cultivated with sugarcane in Yautepec, Morelos, Mexico (18°55′16.9″N and 99°02′24.0″W’). A previous analysis of 16S rRNA gene sequences showed that the bacterium has a 100% sequence identity with Pseudomonas aeruginosa. In addition, this strain shows potential for the biological control of pests and is interesting as a model for studying the interactions between bacteria and host nematodes.We obtained the genome sequence of Pseudomonas aeruginosa NA04. The strain was grown in Luria broth (LB) and incubated for 12 h at 30°C with shaking at 250 rpm. Genomic DNA was extracted with the ZR fungal/bacterial miniprep kit (Zymo Research), and 5 µg of genomic DNA was sequenced using the Illumina HiSeq platform (2 × 300 bp paired-end) approach. We obtained a total of 9,447,636 reads that were quality trimmed and error corrected using DynamicTrim (SolexaQA++) Perl script (). Reads were assembled using SPAdes version 3.5.0 (). The draft genome consists of 54 contigs with a total length of 6,375,895 bp. The largest contig was 891,545 bp, the N50 value was 757,654 bp, and L50 was reached in 4 contigs. The genome sequence of P. aeruginosa PAO1 was used as a reference. The total percentage of aligned bases in the reference is 96.08%, which was used to order the contigs in Mauve (). Automated annotation was made with Rapid Annotations using Subsystems Technology (RAST) (). P. aeruginosa NA04 has a G+C content of 66.49% and includes 5,823 coding sequences. RNAmmer and ARAGORN obtained 7 rRNAs (5S, 16S, 23S) and 67 tRNAs (, ). The analysis of the concatenated sequences (gyrB,16S, 23S of rRNA genes) of P. aeruginosa NA04 shows that it is closely related to P. aeruginosa M18 (NC_017548), isolated from the rhizosphere soil of sweet melon (). The genome of P. aeruginosa NA04 includes genes that code for chitinase and a chitin binding protein. Because the exoskeletons of the insects are constituted with chitin, these could influence the insecticide activity. Also, the genome contains the gene coding for a potent toxin (exotoxin A) that affects the protein synthesis of the host cells and genes encoding the RTX-like toxins found in the entomopathogenic bacterium Photorhabdus luminescens (). These genes are necessary for the production of siderophores involved in the efficient uptake of iron, an important factor for the colonization of the host (). […]

Pipeline specifications

Software tools SolexaQA, SPAdes, Mauve, RAST, RNAmmer, ARAGORN
Applications Genome annotation, Nucleotide sequence alignment
Organisms Pseudomonas aeruginosa, Caenorhabditis elegans