Genomics/Technical resourcesWhole genome sequences of a free-living Pseudomonas sp. strain ML96 isolated from a freshwater Maar Lake
Introduction
The gammaproteobacterial genus Pseudomonas represents a metabolically versatile and genetically plastic group of bacteria. Members of Pseudomonas are frequently found in various environments, e.g. soils, sediments, lakes and oceans (Moore et al., 2006). They have received extensive research attention largely due to that some members are pathogenic to plants, animals or humans. Consequently, most of Pseudomonas species with genomes sequenced are either pathogens or mutualistic partners to higher organisms, e.g. Pseudomonas aeruginosa PAO1 which is one of the top three causes of opportunistic human infections (Stover et al., 2000). We surveyed the source environments of Pseudomonas spp. with genome sequences available in public databases (Fig. 1A) and found that most of them are associated with either plants or humans. The only two genomes that came from freshwater Pseudomonas species are Pseudomonas pseudoalcaligenes CECT5344 isolated from the Guadalquir River (Córdoba, Spain) (Luque-Almagro et al., 2013) and Pseudomonas alcaligenes NBRC 14159 isolated from swimming pool water with an origin of tap water (Hugh and Ikari, 1964). Such sequencing bias may limit our capability of using comparative genomics tools to reach a deep understanding of this both environmentally and medically important group of bacteria.
Here we isolated a free living Pseudomonas strain ML96 in Dec. 2011 from the Huguangyan Maar Lake (21.143 N 110.279 E) in southern China. The 3 μm-filter-prefiltered water (at depth ~ 13 m) was directly plated onto R2A agar. After a three-day incubation, the round, smooth colonies of strain ML96 displayed a light pink color and was identified as Pseudomonas through 16S rRNA gene sequencing. Strain ML96's genome was sequenced with the aim to provide further genomic insight into freshwater Pseudomonas spp. The strain has been deposited to the Marine Culture Collection of China under the accession number MCCC 1K00453.
Pseudomonas sp. ML96 was grown in liquid R2A media and DNA was extracted using a commercial genomic DNA extraction kit. A 300-bp DNA library for Illumina sequencing technology was constructed and sequenced at Shanghai Majorbio Pharm Technology Co. Ltd., China. Approximately 834.2 M bases from 101 bp long pair-end reads were obtained. Reads were subjected to quality control and trimming on the Galaxy server (Goecks et al., 2010) and then were de novo assembled into contigs using the Velvet program (ver. 1.2.08) (Zerbino and Birney, 2008). Various hash lengths between 31 and 71 were tested and an optimal assembly was achieved with a kmer size of 55. Less than 1 kb long contigs were removed from the final assembly. Annotation was performed with the RAST server (Aziz et al., 2008).
The draft genome consisted of 47 contigs with a N50 value of 165,116 bp and a total length of 4.7 Mb, containing 4457 ORFs and 45 tRNAs (Table 1). The G + C content was 64.8%. The genome size of ML96 was similar to those of two other freshwater Pseudomonas species and it appears that free-living freshwater Pseudomonas tend to have a smaller size of genome (Fig. 1B), suggesting that a genomic streamlining is occurring in aquatic Pseudomonas that perform a free-living planktonic life strategy. We calculated orthologous genes in ML96 using the web server EDGAR (https://edgar.computational.bio.uni-giessen.de) (Blom et al., 2009). The five genomes analyzed shared 2403 CDS in the core genome, accounting for 40.4–54.4% of all CDS in investigated genomes (Fig. 2). Approximately 13.1% (583/4449) of CDS in ML96 were unique. Comparing the number of shared CDS between Pseudomonas sp. ML96 and reference genomes, P. alcaligenes NBRC 14159 shared the most with ML 96 (3256 CDS), among which 281 CDS are unique in strains ML96 and NBRC 14159, in consistent with their close relationship on the phylogenetic tree (Fig. 1A). The genome data from a freshwater Pseudomonas strain would deepen our understanding of genomic diversity and evolution of this important group of bacteria.
Section snippets
Nucleotide sequence accession number
This whole genome shotgun project has been deposited at GenBank under the BioProject PRJNA255572 with the accession no. JPMY00000000. Raw Illumina reads were deposited at the NCBI Sequence Read Archive under the BioSample SAMN02927160.
Acknowledgments
This work was supported by the Open Research Fund of Shandong Provincial Key Laboratory of Water and Soil Conservation and Environmental Protection (Linyi University, China) and the Natural Science Foundation of Shandong Province Grant No. ZR2013CM002.
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2022, Science of the Total EnvironmentCitation Excerpt :The 500 mL water sample were filtered by 0.22 μm membrane for prokaryotic enrichment, followed by total DNA extraction using a DNeasy Power Soil kit (QIAGEN, Germany). Bacterial 16S rRNA gene sequences were amplified with the 341F / 806R PCR primer sets and sequenced on the Illumina MiSeq platform at Majorbio (Shanghai, China) (Li et al., 2015). Sequencing data were analyzed on the Majorbio Cloud Platform (www.majorbio.com).