Characterization of chromosomal and megaplasmid partitioning loci in Thermus thermophilus HB27

Abstract

BackgroundIn low-copy-number plasmids, the partitioning loci (par) act to ensure proper plasmid segregation and copy number maintenance in the daughter cells. In many bacterial species, par gene homologues are encoded on the chromosome, but their function is much less understood. In the two-replicon, polyploid genome of the hyperthermophilic bacterium Thermus thermophilus, both the chromosome and the megaplasmid encode par gene homologues (parABc and parABm, respectively). The mode of partitioning of the two replicons and the role of the two Par systems in the replication, segregation and maintenance of the genome copies are completely unknown in this organism.ResultsWe generated a series of chromosomal and megaplasmid par mutants and sGFP reporter strains and analyzed them with respect to DNA segregation defects, genome copy number and replication origin localization. We show that the two ParB proteins specifically bind their cognate centromere-like sequences parS, and that both ParB-parS complexes localize at the cell poles. Deletion of the chromosomal parAB genes did not apparently affect the cell growth, the frequency of cells with aberrant nucleoids, or the chromosome and megaplasmid replication. In contrast, deletion of the megaplasmid parAB operon or of the parB gene was not possible, indicating essentiality of the megaplasmid-encoded Par system. A mutant expressing lower amounts of ParABm showed growth defects, a high frequency of cells with irregular nucleoids and a loss of a large portion of the megaplasmid. The truncated megaplasmid could not be partitioned appropriately, as interlinked megaplasmid molecules (catenenes) could be detected, and the ParBm-parSm complexes in this mutant lost their polar localization.ConclusionsWe show that in T. thermophilus the chromosomal par locus is not required for either the chromosomal or megaplasmid bulk DNA replication and segregation. In contrast, the megaplasmid Par system of T. thermophilus is needed for the proper replication and segregation of the megaplasmid, and is essential for its maintenance. The two Par sets in T. thermophilus appear to function in a replicon-specific manner. To our knowledge, this is the first analysis of Par systems in a polyploid bacterium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1523-3) contains supplementary material, which is available to authorized users.

The Divided Bacterial Genome: Structure, Function, and Evolution

“…The chromids and/or megaplasmids whose copy numbers have been examined in the family Rhizobiaceae (48)(49)(50) and the genera Burkholderia (51) have a copy number approximately equal to that of the chromosome. Similarly, the copy numbers of the large plasmids of Thermus thermophilus (52) and Sphingomonas wittichii (53) are similar to those of the chromosomes. However, the copy number of the chromid of the fast-replicating organism V. cholerae can actually be lower than that of the chromosome depending on the growth medium (54).…”

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

“…Pseudomonas aeruginosa 9/4/TGTTCCACGtGGAACa half-parSs GTTCCAC or GTTTCAC [89][90][91] non-essential 2-4% in LB medium, to 7% in an M9 medium (wt < 0.01%) [92] Reduced growth rate, 10-15% increase in cell size and 10% longer generation time, altered colony morphology, affected motility; decreased ParA stability [92] Pseudomonas putida ? */3 TGTTCCACGTGGAACA [63] non-essential 5-10% in minimal medium during the transition from exponential to stationary phase [93] Defects in chromosome partitioning, abnormal cell morphologies during the deceleration phase of growth independent of the medium used [63,93] Vibrio cholerae chrI: 3/3/NGTTNCACGTGAAACN chrII: 10/9/NTTTACANTGTAAAN [94] non-essential chr1 essential chr2 no change in parB1 mutant [95] Increased frequency of replication initiation, disturbed ori positioning in cell poles [95], no segregation defect for V. cholerae chrI [94] Deinococci Deinococcus radiodurans chrI: 3/1/NGTTTcgcGtgaAACN [96] non-essential 8%-13% for ∆parB1, (wt >1%) [96] Reduced growth rate for ∆parB1 [96] Thermus thermophilus 1/1/TGTTTCCCGTGAAACA [97] non-essential 3% for ∆parAB (wt 1.2%) [97] No apparent growth defects for ∆parAB [97,98] Abbreviations: chrI/chrII-primary/secondary chromosome in the multipartite genome; wt-wild-type; ? * -only contig with P. putida oriC was analyzed for presence of parSs in the cited reference.…”

“…ParB nucleation around the parS sequence correlates with the formation of ParB foci in fluorescence microscopy analyses [76,92,97,117,[127][128][129][130][131] or the presence of wide peaks (up to 50 kbp), encompassing parS in chromatin immunoprecipitation analyses (ChIP-seq or ChIP-microarray), indicating the incorporation of parS proximal DNA in the ParB complex [68,71,72,79,80,89,90,129,132].…”

Random Chromosome Partitioning in the Polyploid BacteriumThermus thermophilusHB27

“…The real-time quantitative PCR was essentially performed based on the method described (Breuert et al 2006; Li et al 2015). Specifically, two loci (TTC1610 (near oriC ) and TTC0574 (closer to terC ) on the chromosome were chosen as the investigation targets.…”

“…The mutagenized pJ- pyrFE was then digested with Nde I to get rid of the pyrE gene, and ligated with a bleomycin resistance marker blm (sequence data from Brouns et al 2005). The plasmid pMK- parB-sgfp allowing expression of ParB-sGFP in T. thermophilus was constructed in a former study (Li et al 2015). The plasmids pUC-Δ mreB :: kat and pUC-Δ parA :: blm were gene deletion vectors for generation of double knockout mutant Δ mreB :: kat /Δ parA :: blm in T. thermophilus HB27.…”