Large-scale transposon mutagenesis of Photobacterium profundum SS9 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.

FIG. 1.

Microtiter plate growth assay used in the first screening of the transposon mutants. Growth was detected by addition of phenol red to the growth medium, which changed color following the production of acid by the bacteria growing fermentatively. Yellow, growth; red, no growth.

FIG. 2.

Genomic localization of the transposon insertions in the two chromosomes of P. profundum SS9 (not to scale). (Left panel) Chromosome 1. (Right panel) Chromosome 2. From the outside in, the first two circles show the predicted protein coding on the two strands, with the colors indicating the COG functional classes. The third circle shows the locations of the pressure-sensitive (green) and pressure-enhanced (red) genes. The fourth circle shows the locations of the cold-sensitive (blue) genes. The fifth circle shows the syntheny with the draft genome of P. profundum 3TCK (www.venterinstitute.org). The sixth circle shows the mean fluorescence intensities obtained in the microarray experiments at 28 MPa, and the sixth circle shows the codon adaptation index, with scores of >0.5 indicated by red (87).

FIG. 3.

Cold sensitivity (CS) ratios for the mutants isolated in this study. The values were computed as described in Materials and Methods. The green bars indicate the ratios for the mutants that displayed a reproducible phenotype only on plates. C, control strain. For some strains, the lag phase was 40 to 59 h (indicated by one asterisk), 60 to 79 h (two asterisks), or >79 h (three asterisks) longer than the lag phase for the control strain. The error bars indicate one standard deviation.

FIG. 4.

Pressure sensitivity (PS) ratios for the mutants isolated in this study. The values were computed as described in Materials and Methods. C, control strain. The error bars indicate one standard deviation.

FIG. 5.

Cold sensitivity (CS) and pressure sensitivity (PS) ratios for a selected subset of mutants after reintroduction of the wild-type copy of the allele on the plasmid vector pFL122. (Top panel) Complementation of cold-sensitive mutants. (Bottom panel) Complementation of pressure-sensitive mutants. The error bars indicate one standard deviation. Complementation of the mutation eliminated the increase in the lag phase observed in FL12 for the cold sensitivity ratio. CTRL, control.