Increased ParB level affects expression of stress response, adaptation and virulence operons and potentiates repression of promoters adjacent to the high affinity binding sites parS3 and parS4 in Pseudomonas aeruginosa

Abstract

Similarly to its homologs in other bacteria, Pseudomonas aeruginosa partitioning protein ParB facilitates segregation of newly replicated chromosomes. Lack of ParB is not lethal but results in increased frequency of anucleate cells production, longer division time, cell elongation, altered colony morphology and defective swarming and swimming motility. Unlike in other bacteria, inactivation of parB leads to major changes of the transcriptome, suggesting that, directly or indirectly, ParB plays a role in regulation of gene expression in this organism. ParB overproduction affects growth rate, cell division and motility in a similar way as ParB deficiency. To identify primary ParB targets, here we analysed the impact of a slight increase in ParB level on P. aeruginosa transcriptome. ParB excess, which does not cause changes in growth rate and chromosome segregation, significantly alters the expression of 176 loci. Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4 close to oriC is reduced. Conversely, in cells lacking either parB or functional parS sequences the orfs adjacent to parS3 and parS4 are upregulated, indicating that direct ParB- parS3/parS4 interactions repress the transcription in this region. In addition, increased ParB level brings about repression or activation of numerous genes including several transcriptional regulators involved in SOS response, virulence and adaptation. Overall, our data support the role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa.

Pseudomonas aeruginosapartitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of aparS-related motif

“…Since a lack of ParB in P. aeruginosa PAO1161 parB null mutant produced clear-cut effects under conditions of exponential growth in rich medium at 37°C, but not as severe as during growth in minimal medium (24, 76), such milder conditions were used for these experiments. The ChIP procedure was performed with PAO1161 (WT) strain, PAO1161 parB null mutant (negative control), and PAO1161/pKGB9 ( araC-araBAD p- parB ) strain (ParB+++) with 5-fold increased ParB level which does not retard bacterial growth (63). Additionally, three parS mutants were included: parS null with all ten parS sites mutated, parS1 - 4 mutant with the four high affinity ParB sites inactivated, and parS2 + strain with nine parS sequences modified and only parS2 left intact (59).…”

“…It has been demonstrated that ParA, ParB and parS sequences are engaged in genome partitioning in many bacteria (9, 12–23, 25, 31). Additionally, they have been shown to be involved, in a species-specific manner, in the regulation of DNA replication initiation by DnaA (14, 15), in the formation of a loading platform for SMC on newly replicated DNA to assure its proper condensation (46, 47), in positioning of oriC domains at defined cell locations (9, 12, 24, 25, 43, 44, 79), coordination of replication and cell division (18, 39, 80) but also in regulation of gene expression (53, 57, 63, 80).…”

“…A large fraction of the 1166 genes with altered expression seemed to be involved in the stress response due to the disturbances in chromosome segregation (84). A complementary study with a strain showing 5-fold overproduction of ParB (ParB+++), a ParB excess which did not lead to visible changes in growth rate or genome segregation, revealed altered expression of 211 loci (63). The present ChIP-seq data suggested that ParB-DNA binding relies on a presence of specific sequence(s).…”

“…A ParB-overproducing strain (designated ParB+++) was obtained by transformation of WT strain with pKGB9 ( araC-araBAD p- parB ). Chloramphenicol was added to 75 μg ml -1 (liquid cultures) or 150 μg ml -1 (solid media) to maintain the plasmid and 0.02% arabinose was added to induce the expression of parB (63).…”

Genome sequence ofPseudomonas aeruginosaPAO1161, a PAO1 derivative with the ICEFP2 integrative and conjugative element

“…To facilitate the use of PAO1161 in conjugation experiments spontaneous rifampicin resistant clone was obtained 18 . The PAO1161 strain was used in studies on chromosome segregation and gene expression in P. aeruginosa using genome wide approaches [19][20][21] as well as in other physiological and genetic studies [22][23][24][25][26][27][28] .…”