Examination of Prokaryotic Multipartite Genome Evolution through Experimental Genome Reduction

<div><p>Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as <i>Vibrio cholerea</i> and <i>Brucella abortis</i> as well as in many N₂-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria <i>Sinorhizobium meliloti</i>. 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 <i>S. meliloti</i> 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).</p></div

The decreased stationary phase density of strains lacking pSymB in bulk soil is due to carbon limitation and can be traced to two loci.

<p>(<b>A</b> – top panel) The strain with a deletion of B116 (orange) shows a slight, but repeatable, decrease in stationary phase density relative to the wild type (dark blue). (<b>A</b> – bottom panel) The strains with deletions of B123 (dark teal) and B122 (light teal), a sub-region of B123, show a large decrease in stationary phase density relative to the wild type (dark blue). (<b>B</b>) Stationary phase soil populations were supplemented with either 15 mM glucose (solid bars) or 5 mM NH₄Cl, 2 mM KH₂PO₄, and 1 mM MgSO₄ (striped bars). Only the addition of a carbon source (glucose) stimulated further growth for the wild type (dark blue), the ΔpSymAB strain (purple), and strains with deletions of B123 (dark teal), which includes that entire B122 region, and B116 (orange). (<b>A</b> and <b>B</b>) Data points represent the average from duplicate experiments, while error bars represent the range from duplicate samples.</p

Nutrient sources supporting growth of <i>S. meliloti</i>.

<p>*Includes only those sources supporting good growth of the wild type.</p>†<p>In several cases, the presence of either pSymA or pSymB improved growth.</p>‡<p>For both carbon and nitrogen, the usage of one source required both pSymA and pSymB.</p>§<p>In several cases, a no growth was only observed when both pSymA and pSymB were removed.</p><p>Nutrient sources supporting growth of <i>S. meliloti</i>.</p

The effect of the removal of pSymA and/or pSymB on the growth of <i>S. meliloti</i>.

<p>The growth of <i>S. meliloti</i> was examined in M9 minimal medium (<b>A</b> – top panel), TY complex medium (<b>A</b> – bottom panel), or sterile bulk soil mesocosms (<b>C</b>). Data points represent averages from triplicate (<b>A</b>) or duplicate (<b>C</b>) samples. Error bars represent +/− one standard deviation from triplicate samples (<b>A</b>) or the range from duplicate samples (<b>C</b>). (<b>B</b>) Average generation times and standard deviations for each strain grown in M9 or TY media, calculated from a total of six replicates from two independent experiments. Blue – wild type; red – ΔpSymA; yellow –ΔpSymB; purple – ΔpSymAB.</p

Effect of competing species on the growth of the <i>S. meliloti</i> ΔpSymAB strain in bulk soil mesocosms.

<p>The <i>S. meliloti</i> ΔpSymAB strain was grown in bulk soil mesocosms in the presence of either an <i>Aspergillus</i> species, <i>Pseudomonas syringae</i>, or <i>Streptomyces ceolicolor</i> and the growth of the ΔpSymAB strain was examined. The decreased stationary phase density during competition is presumably reflective of competition for nutrients and the reduced availability of nutrients due to usage by the competitor. On the other hand, there is a relative lack of effect on the early exponential growth of the ΔpSymAB strain, and it is able to establish a stable stationary phase population in the presence of the competing species. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004742#s3" target="_blank">materials and methods</a> for details on experimental set-up. Purple – ΔpSymAB alone; teal – ΔpSymAB with a soil-isolated <i>Aspergillus</i> species; brown – ΔpSymAB with <i>P. syringae</i>; orange – ΔpSymAB with <i>S. ceolicolor</i>. Data points represent single values.</p

Environment specific growth inhibition by a pSymA-encoded siderophore.

<p>Growth of the ΔpSymAB strain is inhibited by a siderophore produced by the wild type (left stab) when grown in TY medium (<b>A</b> – left panel), but not if the overlay is supplemented with 150 µM FeCl₃ (<b>A</b> – right panel). This inhibition fails to occur when the siderophore biosynthesis genes are knocked out in the wild type, as is the case in <i>S. meliloti</i> RmFL2950 (58) (right stab). (<b>B</b>) When co-inoculated in the same soil mesocosm, the ΔpSymA strain easily outcompetes the wild type, and the wild type fails to inhibit growth of the ΔpSymA strain. Data points represent averages of duplicate samples, while error bars represent the range from duplicate samples. Solid lines indicate growth pattern during co-inoculation, while dotted lines indicate growth pattern when individually inoculated. Blue – wild type; red –ΔpSymA.</p

Carbon sources supporting growth of <i>S. meliloti</i>.^*

<p>*Substrate requiring pSymA and/or pSymB are indicated in italics.</p>†<p>Growth on this substrate is improved by the presence of pSymA.</p>‡<p>Growth on this substrate is improved by the presence of pSymB.</p><p>Carbon sources supporting growth of <i>S. meliloti</i>.^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004742#nt105" target="_blank">*</a>}</p

Schematic illustrating the described model of multipartite genome evolution and chromid formation.

<p>The acquisition of a megaplasmid (orange) expands the niche range of the cell. Subsequently, this replicon accumulates horizontally acquired genes that provide a fitness advantage in this novel environment (purple). This results in a large metabolic load being associated with the megaplasmid, and its loss is favoured in the cell's original niche. However, gene transfer from the chromosome (black) renders the megaplasmid (now a chromid) indespensible in all environments. See the text ‘model of multipartite genome evolution’ for additional details.</p

Phenotype scoring of <i>35S:GFP–SAP54</i> transgenic lines.

<p>Phenotype scoring of <i>35S:GFP–SAP54</i> transgenic lines.</p

Phytoplasma SAP54 interacts specifically with the Keratin-like (K) domain of selected Type II MADS-box transcription factors (MTFs).

<p>(A) A comprehensive yeast two-hybrid screen of 106 Arabidopsis MTFs reveals that SAP54 interacts with members of the Type II subfamily of MTFs (proteins that interact with SAP54 are indicated in red font). For simplicity, not all MTFs are included in the phylogenetic tree. (B) SAP54 interacts primarily with the K domain of AP1. AD, GAL4-activation domain; BD, GAL4-DNA binding domain; EV, empty vector control. (C) Flowers produced from healthy (left) and AY-WB–infected (right) Arabidopsis lines approximately 4 wk postinoculation. (D) SAP54 (indicated by an arrow) co-immunoprecipitates with SEP3–GFP but not FUL–GFP or AG–GFP. Flowers for immunoprecipitation experiments were harvested from transgenic lines pictured in panel C at an early point of infection (approximately 2 wk postinoculation) to minimize MTF loss due to destabilization. Equal loading of samples was confirmed via Bradford assays to quantify protein concentration.</p