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.
Condensins are essential for global chromosome organization in diverse bacteria. Atypically, Pseudomonas aeruginosa encodes condensins from two superfamilies, SMC-ScpAB and MksBEF. We report that the two proteins play specialized roles in chromosome packing and segregation and are synthetically lethal with ParB. Inactivation of SMC or MksB asymmetrically affected global chromosome layout, its timing of segregation and sometimes triggered a chromosomal inversion. Localization pattern was also unique to each protein. SMC clusters colocalized with oriC throughout cell cycle except shortly after origin duplication, whereas MksB clusters emerged at cell quarters shortly prior to oriC duplication and stayed there even after cell division. Relocation of the proteins was abrupt and coordinated with oriC dynamic. These data reveal that the two condensins asymmetrically play dual roles in chromosome maintenance by organizing it and mediating its segregation. Furthermore, the choreography of condensins and oriC relocations suggest an elegant mechanism for the birth and maturation of chromosomes. ImportanceMechanisms that define the chromosome as a structural entity remain unknown. A key element in this process are condensins, which globally organize chromosomes and contribute to their segregation.This study characterized condensin and chromosome dynamics in Pseudomonas aeruginosa, which harbors condensins from two major protein superfamilies, SMC and MksBEF. The study revealed that both proteins asymmetrically play a dual role in chromosome maintenance by spatially organizing the chromosomes and guiding their segregation but can substitute for each other in some activities. The timing of chromosome, SMC and MksBEF relocation was highly ordered and interdependent revealing causative relationships in the process. Moreover, MksBEF was found to produce clusters at the site of chromosome replication that survived cell division and remained in place until chromosome replication was complete. Overall, these data delineate the functions of condensins from the SMC MksBEF superfamilies, reveal the existence of a chromosome organizing center and suggest a mechanism that might explain the biogenesis of chromosomes.3
Mechanisms that define the chromosome as a structural entity remain unknown. Key elements in this process are condensins, which globally organize chromosomes and contribute to their segregation. This study characterized condensin and chromosome dynamics in Pseudomonas aeruginosa, which harbors condensins from two major protein superfamilies, SMC and MksBEF. The study revealed that both proteins play a dual role in chromosome maintenance by spatially organizing the chromosomes and guiding their segregation but can substitute for each other in some activities. The timing of chromosome, SMC, and MksBEF relocation was highly ordered and interdependent, revealing causative relationships in the process. Moreover, MksBEF produced clusters at the site of chromosome replication that survived cell division and remained in place until replication was complete. Overall, these data delineate the functions of condensins from the SMC and MksBEF superfamilies, reveal the existence of a chromosome organizing center, and suggest a mechanism that might explain the biogenesis of chromosomes.
Salmonella enterica is one of the most common foodborne illnesses in the United States and worldwide, with nearly one-third of the cases attributed to contaminated eggs and poultry products. Vaccination has proven to be an effective strategy to reduce Salmonella load in poultry. The Salmonella Typhimurium Δcrp-cya (MeganVac1) strain is the most commonly used vaccine in the United States; however, the mechanisms of virulence attenuation and host response to this vaccine strain are poorly understood. Here, we profiled the invasion and intracellular survival phenotypes of Δcrp-cya and its derivatives (lacking key genes required for intra-macrophage survival) in HD11 macrophages and the transcriptome response in primary chicken macrophages using RNA-seq. Compared to the parent strain UK1, all the mutant strains were highly defective in metabolizing carbon sources related to the TCA cycle and had greater doubling times in macrophage-simulating conditions. Compared to UK1, the majority of the mutants were attenuated for invasion and intra-macrophage survival. Compared to Δcrp-cya, while derivatives lacking phoPQ, ompR-envZ, feoABC and sifA were highly attenuated for invasion and intracellular survival within macrophages, derivatives lacking ssrAB, SPI13, SPI2, mgtRBC, sitABCD, sopF, sseJ and sspH2 showed increased ability to invade and survive within macrophages. Transcriptome analyses of macrophages infected with UK1, Δcrp-cya and its derivatives lacking phoPQ, sifA and sopF demonstrated that, compared to uninfected macrophages, 138, 148, 153, 155 and 142 genes were differentially expressed in these strains, respectively. Similar changes in gene expression were observed in macrophages infected with these strains; the upregulated genes belonged to innate immune response and host defense and the downregulated genes belonged to various metabolic pathways. Together, these data provide novel insights on the relative phenotypes and early response of macrophages to the vaccine strain and its derivatives. The Δcrp-cya derivatives could facilitate development of next-generation vaccines with improved safety.
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