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

Abstract

The molecular mechanism(s) by which deep-sea bacteria grow optimally under high hydrostatic pressure at low temperatures is poorly understood. To gain further insight into the mechanism(s), a previous study screened transposon mutant libraries of the deep-sea bacterium Photobacterium profundum SS9 and identified mutants which exhibited alterations in growth at high pressure relative to that of the parent strain. Two of these mutants, FL23 (PBPRA3229::mini-Tn10) and FL28 (PBPRA1039::mini-Tn10), were found to have highpressure sensitivity and enhanced-growth phenotypes, respectively. The PBPRA3229 and PBPRA1039 genes encode proteins which are highly similar to Escherichia coli DiaA, a positive regulator, and SeqA, a negative regulator, respectively, of the initiation of DNA replication. In this study, we investigated the hypothesis that PBPRA3229 and PBPRA1039 encode DiaA and SeqA homologs, respectively. Consistent with this, we determined that the plasmid-carried PBPRA3229 and PBPRA1039 genes restored synchrony to the initiation of DNA replication in E. coli mutants lacking DiaA and SeqA, respectively. Additionally, PBPRA3229 restored the cold sensitivity phenotype of an E. coli dnaA(Cs) diaA double mutant whereas PBPRA1039 suppressed the cold sensitivity phenotype of an E. coli dnaA(Cs) single mutant. Taken together, these findings show that the genes disrupted in FL23 and FL28 encode DiaA and SeqA homologs, respectively. Consequently, our findings add support to a model whereby high pressure affects the initiation of DNA replication in P. profundum SS9 and either the presence of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) promotes growth under these conditions. Despite the fact that more than 70% of the earth's surface is covered by oceans, which have an average temperature of 3°C and exert an average hydrostatic pressure of 38 MPa (atmospheric pressure is ϳ0.1 MPa), little is understood about the molecular basis of cold-and high-pressure-adapted deepocean life. The discovery and isolation of the pyschrotolerant facultative piezophile (high-pressure-loving organism) Photobacterium profundum SS9 (8) have made it possible to more readily address the mechanisms of piezophilic growth at cold temperatures (for a recent review, see reference 3). P. profundum SS9 is a gammaproteobacterium originally isolated from an amphipod homogenate obtained from the Sulu Sea in the Philippines at a depth of 2.5 km and a temperature of 9°C (8). Although it grows optimally at 28 MPa and 15°C, P. profundum SS9 can also grow over a wide range of pressures (0.1 to 90 MPa) and temperatures (2 to 20°C). The ability to grow at atmospheric pressure has made P. profundum SS9 more amenable to genetic manipulation than obligate piezophiles. The P. profundum SS9 genome has been sequenced and annotated (26) and consists of two chromosomes and an 80-kb plasmid. It was determined that the 80-kb plasmid is nonessential for the piezophilic growth of P. profundum SS9 (26

DiaA/HobA and DnaA: A Pair of Proteins Co-evolved to Cooperate During Bacterial Orisome Assembly

“…It is important to add that, using gene replacement, it has been shown recently that PBPRA3229 of the Gram-negative, deep-sea Photobacterium profundum is a functional homolog of DiaA. 17 However, first, the proteins were highly similar (75% identity and 85% similarity) and second, nothing is known about initiation of P. profundum chromosome replication. Thus, it is difficult to discuss the role of PBPRA3229 in P. profundum orisome assembly and to compare it to the known initiation systems.…”

The First Genomic and Proteomic Characterization of a Deep-Sea Sulfate Reducer: Insights into the Piezophilic Lifestyle of Desulfovibrio piezophilus

“…Within these ecosystems, the hydrostatic pressure can reach up to 110 MPa (Mariana Trench) with an average hydrostatic pressure of approximately 38 MPa, corresponding to depth of 3800 m. Organisms that experience optimal growth at hydrostatic pressure higher than atmospheric pressure (0.1 MPa) are called "piezophiles" [14]. Until now, factors involved in piezophilic lifestyle have mainly been analyzed in two facultative anaerobic heterotrophic genera, Photobacterium and Shewanella [15], [16], [17], [18], [19]. However, these two genera do not accurately represent the complexity and diversity of the deep-sea ecosystems; anaerobes and SRB, as discussed above, are also important members of these biotopes that have to be considered [1], [20], [21], [22].…”

Microbial Ecology of the Dark Ocean above, at, and below the Seafloor

“…75,2011 MICROBIAL ECOLOGY OF THE DARK OCEAN 407 on May 11, 2018 by guest http://mmbr.asm.org/ traits. As mentioned above, Photobacterium profundum strain SS9 is used as a model for studying adaptation to life at high pressure (48,139,576). It is amenable to transposon mutagenesis (307) and generation of mutants through deletions (140).…”