Large-Scale Transposon Mutagenesis ofPhotobacterium profundumSS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure

Abstract

Microorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacterium Photobacterium profundum strain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism.

Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9

“…One of the putative high-pressure-sensitive mutants (FL23) isolated from these screens had a mini-Tn10 insertion in the gene PBPRA3229, which encodes a protein with 75% identity (85% similarity) to Escherichia coli DiaA (DnaA initiator-associating factor) (14). Although FL23 shows growth defects at 0.1 MPa (15°C) relative to the parent strain, the ratio of growth at 45 MPa to growth at 0.1 MPa and 15°C is substantially reduced compared to that of the parent strain, confirming that disruption of PBPRA3229 results in a high-pressure sensitivity growth phenotype (19).…”

“…The conjugation of plasmids from E. coli DH5␣ into P. profundum SS9R strains was performed by following a previously described method with modifications (19). Briefly, the P. profundum SS9 acceptor and E. coli donor and helper (MM294A/pRK2013) strains were grown in the appropriate media to early stationary phase and then washed in marine broth.…”

“…However, the effect of DiaA on the timing of DNA replication initiation appears to be SeqA independent (14). Interestingly, a putative P. profundum SS9R seqA transposon insertion mutant (PBPRA1039::Tn10) was identified as having high-pressure-enhanced growth at 45 MPa and 15°C relative to its growth at atmospheric pressure (19). Therefore, this preliminary finding suggests that the removal of a negative regulator of the initiation of DNA replication could promote the growth of P. profundum SS9R at high pressure.…”

“…Previously, it was demonstrated that the FL23 mutant exhibits a high-pressure sensitivity phenotype at 45 MPa relative to its growth at atmospheric pressure (19). To further characterize the effect of pressure on FL23, we investigated the growth of FL23 with pFL190 compared to that of parent strain P. profundum SS9R with pFL190 on marine agar overlay plates at 0.1 and 28 MPa and 15°C (Fig.…”

“…To gain insights into the genetic basis of high-pressureadapted growth, transposon mutant libraries of P. profundum SS9R (a rifampin [rifampicin]-resistant derivative of SS9) were screened in liquid culture for mutants with defects in the ability to grow at high pressure (45 MPa, 15°C) (19). One of the putative high-pressure-sensitive mutants (FL23) isolated from these screens had a mini-Tn10 insertion in the gene PBPRA3229, which encodes a protein with 75% identity (85% similarity) to Escherichia coli DiaA (DnaA initiator-associating factor) (14).…”