Examination of Prokaryotic Multipartite Genome Evolution through Experimental Genome Reduction

Abstract

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N2-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism's niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).

Neo-formation of chromosomes in bacteria

“…The additional, secondary, ERs (SERs) are proposed to derive from accessory replicons (plasmids 5,6 ) that were acquired by a mono-chromosome progenitor bacterium and stabilized through the transfer from the chromosome of genes essential to the cell viability 7,8 . The existence of plasmid-like replication and partition systems in SERs 7,9-14 as well as experimental results 15 support this view. Yet, the duplication and maintenance processes of SERs are in stark contrast to the common behaviour of plasmids for which both the timing of replication initiation and the centromere movement are random 16,17 .…”

“…1) and/or oriented 36 (presence of plasmid-type systems for genome maintenance and replication initiation) criteria to characterize the SER(s). While clarifying the functional and evolutionary contributions of each type of replicon to a multipartite genome in a given bacterial lineages 7,10,32,36 , these studies did not produce an incontrovertible definition of SERs 4,36 or a universal model for their emergence 4,7,15,32,36 . We thus attempted to investigate the nature(s) and origin(s) of these replicons using as few assumptions as possible.…”

“…Beside the exploration of the replication/segregation mechanistic, studies of multipartite genomes, targeting a single bacterial species or genus 3,7,8,10,15 or using a more extensive set of taxa 4,36 , relied on debatable 4,36,37 (replicon size, nucleotide composition, coding of core essential genes for growth and survival; Extended data Fig. 1) and/or oriented 36 (presence of plasmid-type systems for genome maintenance and replication initiation) criteria to characterize the SER(s).…”

“…The presence in SERs of plasmid-type replication initiation systems signs the involvement of plasmids in the coming into being of these replicons. Yet, the existence of several types of SERs characterized by specific features, and the extreme similitude between chromosomes and SERs within a few genomes provides evidence that processes other than the capture and amelioration of megaplasmids 7,8,10,13,15,36 are at play in the stabilization of additional essential replicons and the formation of multipartite genomes. Indeed, the presence on Asticaccaulis, Paracoccus and Prevotella SERs of high numbers of IS genes found on chromosomes in closely related species, and their complementarity between the SER and chromosome (Extended Data facilitate the exchange of genes and the translocation of replicon fragments and, through several cell division and possibly several rounds of integration-disintegration, provide time for the stabilisation process to take place, finally resulting in the separation of the shuffled cointegrate into multiple viable essential replicons forming a divided genome.…”

l-Hydroxyproline andd-Proline Catabolism in Sinorhizobium meliloti

“…For growth curves, inoculum cultures grown overnight in LBmc (19) were washed with 0.85% NaCl and inoculated into each test medium to an optical density at 600 nm (OD 600 ) of 0.05. The growth profiles of 0.15-ml cultures were measured for 48 h with shaking in 96-well microtiter plates at 30°C, and the data were analyzed as previously described (22). The OD 600 values presented are not corrected for path length, and unless stated otherwise, generation times were calculated between uncorrected OD 600 values of 0.1 and 0.3.…”