Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

Abstract

ABSTRACT Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae , a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes ( napA , phaA , and the Na + /P i antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction ( napA , nasA ) and corresponding cofactor biosynthesis (genomic moaB , moeB , and dsbC and plasmid-carried dsbD and ccmH ) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps ( nirS , nirJ , nosD ), dimethyl sulfoxide reduction ( dmsA1 ), and fermentation ( pdhB1 , arcA , aceE , pta , acs ) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA ) of the anaerobic electron transfer chain and central metabolism ( pdhB ) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring ( lysM , mipA , fadK ), C4-dicarboxylate transport ( dctM1 , dctM3 ), and protease functions to anaerobic growth require further investigation to unravel the novel underlying adaptation strategies.