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.
Highlights
The long-held view that bacteria carry the essential part of their genomes on a single chromosome blurred about 25 years ago, when the species Rhodobacter sphaeroides was found to carry certain essential genes on a large replicon distinct from the main chromosome [1]
That the partition system of the main chromosome is a major participant in the choreography of the cell cycle. Bacteria typically carry their genetic information on a single chromosome
In a few bacterial families the genome includes one to three additional chromosome-like DNA molecules. Because these families are rich in pathogenic and environmentally versatile species, it is important to understand how their split genomes evolved and how their maintenance is managed without confusion
Summary
The long-held view that bacteria carry the essential part of their genomes on a single chromosome blurred about 25 years ago, when the species Rhodobacter sphaeroides was found to carry certain essential genes on a large replicon distinct from the main chromosome [1]. All Vibrio species carry one secondary chromosome [2,3] and all Burkholderia species have at least one and typically two [4]. They are thought to have arisen by transfer of essential genes to coresident lowcopy number plasmids, which thereupon grew through further recombination events. Whether the split-genome arrangements resulting from such events persisted by conferring selective advantage is speculative, but it is reasonable to view expansion of secondary chromosomes as a means of incorporating large numbers of beneficial genes without unduly disturbing the regulation and organization of essential genes on the main chromosome. Our aim here is to determine how the maintenance of one principal and two secondary chromosomes is accommodated within the cell cycle of the beta-proteobacterium Burkholderia cenocepacia J2315, an opportunistic pathogen of sufferers from cystic fibrosis. (We use the term "secondary chromosome" for convenience, and deal with the nomenclature of such replicons in the Discussion.)
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