Construction and application of an expression vector from the new plasmid pLAtc1 of Acidithiobacillus caldus.
In this study, a recently sequenced 9.8-kb plasmid, pLAtc1, from Acidithiobacillus caldus strain SM-1 was characterized and developed into an expression vector. The pLAtc1 backbone carried an oriV, three rep genes, five mob genes, a Nic site, and an addiction system. Multilocus sequence analysis indicated that pLAtc1 was phylogenetically more related to the IncQ-like broad host range plasmids than to other IncQ plasmids. pLAtc1 was able to replicate and reside in Gram-negative Escherichia coli, Comamonas testosteroni, but not in Gram-positive Corynebacterium glutamicum. pLAtc1 was mobilized via conjugation into E. coli BL21 and A. caldus SM-1 from E. coli S17-1. Quantitative PCR revealed seven and four copies of plasmid in A. caldus and E. coli cells, respectively. The expression vector pLAtcE was constructed from pLAtc1 by introducing a regulatable promoter (P tetH ), a transcriptional terminator, a multiple cloning site, a kanamycin resistance gene, and a streptomycin resistance gene. The functionality of pLAtcE was demonstrated by expressing a gene encoding enhanced green fluorescence protein in E. coli and in A. caldus. pLAtcE was used to express α-ketoglutarate dehydrogenase (sucAB) and succinate dehydrogenase (sdhA) genes in A. caldus. The newly engineered strain that harbored sucAB and sdhA on a plasmid pLAtcE-sucA-sucB-sdhA grew better than the parent strain SM-1/pLAtcE in tetrathionate and glucose-supplemented medium and produced more acidity and resulted in a more oxidative environment. This study created a useful molecular tool for genetic manipulation of the thermoacidophilic and autotrophic A. caldus.
State Key Laboratory of Microbial Resources, Chinese Academy of Sciences, Beichen West Road No.1, Chaoyang District, Beijing, 100101, People's Republic of China.
Ming-Jiang
MJ
Zhang
Cheng-Ying
CY
Jiang
Xiao-Yan
XY
You
Shuang-Jiang
SJ
Liu
D042763:Acidithiobacillus / Q000235:genetics*; D003001:Cloning, Molecular; D020604:Comamonas testosteroni / Q000235:genetics; D003227:Conjugation, Genetic; D048230:Corynebacterium glutamicum / Q000235:genetics; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000145:classification / Q000302:isolation & purification*; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D058885:Multilocus Sequence Typing; D010957:Plasmids / Q000145:classification / Q000302:isolation & purification*; D011401:Promoter Regions, Genetic; D018741:Replication Origin; D012641:Selection, Genetic; D061806:Transcription Termination, Genetic
D004269:DNA, Bacterial; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins