Role of DiaA and SeqA homologues in the deep-sea adapted growth of photobacterium profundum SS9
Abstract
The mechanism of high pressure-adapted growth in the deep-sea bacterium
Photobacterium profundum SS9 is poorly understood. To gain further insights, two
P. profundum SS9R mutants were investigated. FL23 (pbpra3229::m-Tn10) and
FL28 (pbpra1039::m-Tn10) had been previously characterised as high pressuresensitive
and pressure-enhanced, respectively. FL23 had a growth defect at
atmospheric pressure but failed to show high pressure-adapted growth on solid agar.
Pbpra3229 is 75 % identical to E. coli DiaA (stimulator of DNA replication and
critical for the timely initiation of replication) and 45% identical to E. coli GmhA
(essential for lipopolysaccharide core biosynthesis), which led to an investigation
into whether either process was affected in FL23. However, the lipopolysaccharide
of FL23 and its parent strain were identical, which suggests that Pbpra3229 is not a
GmhA homologue. In contrast, the pbpra3229 and E. coli diaA genes were
functionally interchangeable and both restored the timing of DNA replication in an
E. coli diaA mutant. FL28 had growth and morphological defects at high pressure,
but both phenotypes were exacerbated at atmospheric pressure. Pbpra1039 is 55%
identical to E. coli SeqA, which is a negative regulator of DNA replication and also
essential for timely initiation. Pbpra1039 was shown to be a functional homologue of
E. coli SeqA, as pbpra1039 partially complemented the DNA replication defect of an
E. coli seqA mutant. Combined, these findings provide evidence that Pbpra3229 is a
DiaA homologue, whereas Pbpra1039 is a cold adapted SeqA homologue, and that
both positive and negative regulation of initiation of DNA replication are essential
for the ability of P. profundum SS9 to adapt to deep-sea conditions. A marine
metagenomic library was also screened for clones that produced novel cell envelope
polysaccharides and tools were developed to identify cell envelope polysaccharides
in P. profundum SS9.