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


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.
Zi-Chao
ZC
Yu
Dian-Li
DL
Zhao
Li-Yuan
LY
Ran
Zi-Hao
ZH
Mi
Zhao-Yu
ZY
Wu
Xiuhua
X
Pang
Xi-Ying
XY
Zhang
Hai-Nan
HN
Su
Mei
M
Shi
Xiao-Yan
XY
Song
Bin-Bin
BB
Xie
Qi-Long
QL
Qin
Bai-Cheng
BC
Zhou
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. cxl0423@sdu.edu.cn.
Xiu-Lan
XL
Chen
Yu-Zhong
YZ
Zhang
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004917:Erythromycin / Q000494:pharmacology; D055786:Gene Knockout Techniques; D005822:Genetic Vectors / Q000378:metabolism; D016680:Genome, Bacterial; D019015:Geologic Sediments / Q000382:microbiology; D059765:Homologous Recombination; D008826:Microbial Sensitivity Tests; D009792:Oceans and Seas; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D031161:Pseudoalteromonas / Q000187:drug effects / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D004917:Erythromycin