Abstract
BackgroundPseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination.ResultsThe sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT-4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDP-glucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration–segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913.ConclusionsA conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment.
Highlights
Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem
Photobacterium profundum SS9, which was isolated from the Sulu Sea in Philippines at a depth of 2.5 km [5], was found to have many special genetic features in DNA replication, fidelity and structure [6,7], as well as membrane integrity and fluidity [8,9,10] to adapt to the high-pressure environment by Tn5 gene replacement mutagenesis
Many genes of this strain were found to be regulated by cold shock [13], and the regulation of fatty acid biosynthesis and NAP-α system in response to different temperatures and pressures were studied by gene knockout [14,15]
Summary
Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. The extreme characteristics of the deep-sea sediments have compelled various bacteria to evolve special features to adapt to the deep-sea environment. Photobacterium profundum SS9, which was isolated from the Sulu Sea in Philippines at a depth of 2.5 km [5], was found to have many special genetic features in DNA replication, fidelity and structure [6,7], as well as membrane integrity and fluidity [8,9,10] to adapt to the high-pressure environment by Tn5 gene replacement mutagenesis. The environmental adaptation mechanisms of Shewanella piezotolerans WP3, a bacterium isolated from a western Pacific Ocean sediment sample at a depth of 1914 m [11], was studied in the genomic level [12]. Many genes of this strain were found to be regulated by cold shock [13], and the regulation of fatty acid biosynthesis and NAP-α system in response to different temperatures and pressures were studied by gene knockout [14,15]
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