Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315

Abstract

Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of the cell cycle.

The Divided Bacterial Genome: Structure, Function, and Evolution

“…Interestingly, all S. meliloti replicons contain genes whose transcription is cell cycle dependent (e.g., groEL2 on pSymA, minCDE on pSymB, and divK on the chromosome), with most cell cycle-regulated genes on the chromosome, an intermediate number on the chromid, and the least on the megaplasmid, consistent with each element being integrated into the cell cycle to various extents (56). The segregation machinery of each replicon in B. cenocepacia is specific to the corresponding replicon (51,66,67). Similarly, the segregation machinery of each of the four secondary replicons in Rhizobium leguminosarum can distinguish between the self replicon and the others (68).…”

“…The segregation of the three large replicons of Burkholderia cenocepacia follows an orderly manner, with the segregation of the origin of the newly replicated chromosome generally preceding that of the origin of the chromid, after which the segregation of the origins of the small chromid/megaplasmid occurs (51). In contrast, chromosomal segregation in S. meliloti is initiated first, followed by the segregation of the megaplasmid and, finally, the chromid (65).…”

“…At the same time, the gain of essential genes from the chromosome results in the formation of a chromid (41,84). This interreplicon gene flow also contributes to the increased stability of the secondary replicon, in terms of reducing both the rate of gene loss and the loss of the entire replicon (11,51,84,152), and contributes to the integration of the replicon into the cell's core metabolism (187). In this way, environmental adaptation can drive the emergence of a multipartite genome.…”

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

“…Caulobacter crescentus 5/5/t/cGTTt/cCACGTGAAAca [80,81] essential Indispensable, severe chromosome segregation defects, ParB depletion results in defective Z-ring formation and cell division, formation of long polyploid cells [82] Hyphomonas neptunium 2/2/TGTTTCACGTGAAACA [83] essential anucleate buds [83] parB mutants could not be obtained, depletion of ParA blocks cell division [83] Betaproteobacteria Burkholderia cenocepacia chrI: 2/2/tGTTNCACGTGAAACa chrII: 6/6/gTTTATGCGCATAAAc [84][85][86][87] non-essential 1-14% (depending on mutated system) [85] Reduced growth rate, reduction in cell size, compromised viability, defects in ori positioning [85] Deltaproteobacteria…”

The Effect of 2-Thiocyanatopyridine Derivative 11026103 on Burkholderia Cenocepacia: Resistance Mechanisms and Systemic Impact

“…Furthermore, on both chromosomes 1 and 2, mutated regions showed roughly symmetrical distribution around the replication terminus ( Figure 1a). These stretches of mutations are likely to have originated from segments of replicating DNA which escaped repair mechanisms; the symmetrical distribution between chromosomes 1 and 2 reflects coordinated replication of both replicons [14]. The only genetic locus where mutations converged among all isolates selected after EMS mutagenesis was the bcal2461-bcal2462 gene pair (Figure 1b).…”