Condensins are essential for Pseudomonas aeruginosa corneal virulence through their control of lifestyle and virulence programs.

Pseudomonas aeruginosa is a significant opportunistic pathogen responsible for numerous human infections. Its high pathogenicity resides in a diverse array of virulence factors and an ability to adapt to hostile environments. We report that these factors are tied to the activity of condensins, SMC and MksBEF, which primarily function in structural chromosome maintenance. This study revealed that both proteins are required for P. aeruginosa virulence during corneal infection. The reduction in virulence was traced to broad changes in gene expression. Transcriptional signatures of smc and mksB mutants were largely dissimilar and non-additive, with the double mutant displaying a distinct gene expression profile. Affected regulons included those responsible for lifestyle control, primary metabolism, surface adhesion and biofilm growth, iron and sulfur assimilation, and numerous virulence factors, including type 3 and type 6 secretion systems. The in vitro phenotypes of condensin mutants mirrored their transcriptional profiles and included impaired production and secretion of multiple virulence factors, growth deficiencies under nutrient limiting conditions, and altered c-di-GMP signaling. Notably, c-di-GMP mediated some but not all transcriptional responses of the mutants. Thus, condensins are integrated into the control of multiple genetic programs related to epigenetic and virulent behavior of P. aeruginosa.

Low-temperature effects on docosahexaenoic acid biosynthesis in Schizochytrium sp. TIO01 and its proposed underlying mechanism.

BACKGROUND: Schizochytrium species are known for their abundant production of docosahexaenoic acid (DHA). Low temperatures can promote the biosynthesis of polyunsaturated fatty acids (PUFAs) in many species. This study investigates low-temperature effects on DHA biosynthesis in Schizochytrium sp. TIO01 and its underlying mechanism. RESULTS: The Schizochytrium fatty acid biosynthesis pathway was evaluated based on de novo genome assembly (contig N50 = 2.86 Mb) and iTRAQ-based protein identification. Our findings revealed that desaturases, involved in DHA synthesis via the fatty acid synthase (FAS) pathway, were completely absent. The polyketide synthase (PKS) pathway and the FAS pathway are, respectively, responsible for DHA and saturated fatty acid synthesis in Schizochytrium. Analysis of fatty acid composition profiles indicates that low temperature has a significant impact on the production of DHA in Schizochytrium, increasing the DHA content from 43 to 65% of total fatty acids. However, the expression levels of PKS pathway genes were not significantly regulated as the DHA content increased. Further, gene expression analysis showed that pathways related to the production of substrates (acetyl-CoA and NADPH) for fatty acid synthesis (the branched-chain amino acid degradation pathway and the pentose phosphate pathway) and genes related to saturated fatty acid biosynthesis (the FAS pathway genes and malic enzyme) were, respectively, upregulated and downregulated. These results indicate that low temperatures increase the DHA content by likely promoting the entry of relatively large amounts of substrates into the PKS pathway. CONCLUSIONS: In this study, we provide genomic, proteomic, and transcriptomic evidence for the fatty acid synthesis pathway in Schizochytrium and propose a mechanism by which low temperatures promote the accumulation of DHA in Schizochytrium. The high-quality and nearly complete genome sequence of Schizochytrium provides a valuable reference for investigating the regulation of polyunsaturated fatty acid biosynthesis and the evolutionary characteristics of Thraustochytriidae species.

Segregation but Not Replication of the Pseudomonas aeruginosa Chromosome Terminates at Dif.

Coordination between chromosome replication and segregation is essential for equal partitioning of genetic material between daughter cells. In bacteria, this is achieved through the proximity of the origin of replication, oriC, and the chromosome partitioning site, parS We report here that in Pseudomonas aeruginosa, segregation but not replication is also controlled at the terminus region of the chromosome. Using the fluorescent repressor operator system (FROS), we investigated chromosome segregation in P. aeruginosa strain PAO1-UW, wherein the chromosome dimer resolution site, dif, is asymmetrically positioned relative to oriC In these cells, segregation proceeded sequentially along the two chromosomal arms and terminated at dif In contrast, chromosome replication terminated elsewhere, opposite from oriC We further found two large domains on the longer arm of the chromosome, wherein DNA segregated simultaneously. Notably, GC-skew, which reflects a bias in nucleotide usage between the leading and lagging strands of the chromosome, switches polarity at the dif locus but not necessarily at the terminus of replication. These data demonstrate that termination of chromosome replication and segregation can be physically separated without adverse effects on bacterial fitness. They also reveal the critical role of the dif region in defining the global layout of the chromosome and the progression of chromosome segregation and suggest that chromosome packing adapts to its subcellular layout.IMPORTANCE Segregation of genetic information is a central event in cellular life. In bacteria, chromosome segregation occurs concurrently with replication, sequentially along the two arms from oriC to dif How the two processes are coordinated is unknown. We explored here chromosome segregation in an opportunistic human pathogen, Pseudomonas aeruginosa, using its strain with markedly unequal chromosomal arms. We found that replication and segregation diverge in this strain and terminate at very different locations, whereas the longer chromosomal arm folds into large domains to align itself with the shorter arm. The significance of this research is in establishing that segregation and replication of bacterial chromosomes are largely uncoupled from each other and that the large-scale structure of the chromosome adapts to its subcellular layout.

Pseudomonas aeruginosa Condensins Support Opposite Differentiation States.

Condensins play a key role in global chromosome packing. Pseudomonas aeruginosa encodes two condensins, SMC-ScpAB and MksBEF. We report here that the two proteins are involved in the differentiation of the bacterium and impose opposite physiological states. The inactivation of SMC induced a state characterized by increased adhesion to surfaces as well as defects in competitive growth and colony formation. In contrast, MksB-deficient cells were impaired in biofilm formation with no obvious defects during planktonic growth. The phenotype of the double mutant was dominated by the absence of MksB, indicating that the observed growth defects are regulatory in their nature rather than structural. ATPase mutations recapitulated many of the phenotypes of the condensins, indicating their requirement for a functional protein. Additionally, inactivation of condensins dramatically reduced the virulence of the bacterium in a murine model of lung infection. These data demonstrate that condensins are involved in the differentiation of P. aeruginosa and reveal their importance for pathogenicity. IMPORTANCE: Adaptation and differentiation play key roles in bacterial pathogenicity. In Pseudomonas aeruginosa, an opportunistic human pathogen, these processes are mediated by the activity of an intricate regulatory network. We describe here novel members of this network, condensins. We show that the two P. aeruginosa condensins specialize in the establishment of the sessile and planktonic states of the bacterium. Whereas condensins have well-established roles in global chromosome organization, their roles in regulating bacterial physiology have remained unknown. Our data indicate that the two programs may be linked. We further show that condensins are essential for the pathogenicity of P. aeruginosa.