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Background   Pseudoalteromonas  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.    Results  The 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 Δ epsT  mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that  epsT  is an important gene involved in the EPS production of SM9913.    Conclusions  A 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.

Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913