Cell Growth Inhibition upon Deletion of Four Toxin-Antitoxin Loci from the Megaplasmids of Sinorhizobium meliloti

Abstract

Toxin and antitoxin (TA) gene pairs are addiction systems that are present in many microbial genomes. Sinorhizobium meliloti is an N 2 -fixing bacterial symbiont of alfalfa and other leguminous plants, and its genome consists of three large replicons, a circular chromosome (3.7 Mb) and the megaplasmids pSymA (1.4 Mb) and pSymB (1.7 Mb). S. meliloti carries 211 predicted type II TA genes, each encoding a toxin or an antitoxin. We constructed defined deletion strains that collectively removed the entire pSymA and pSymB megaplasmids except for their oriV regions. Of approximately 100 TA genes on pSymA and pSymB, we identified four whose loss was associated with cell death or stasis unless copies of the genes were supplied in trans. [orf2230/sma2231]), and this report contains the first experimental proof that RES/Xre (smb21127/smb21128) loci can function as a TA system. Transcriptome sequencing (RNA-seq) analysis did not reveal transcriptional differences between the TA systems to account for why deletion of the four "active" systems resulted in cell toxicity. These data suggest that severe cell growth phenotypes result from the loss of a few TA systems and that loss of most TA systems may result in more subtle phenotypes. These four TA systems do not appear to play a direct role in the S. meliloti-alfalfa symbiosis, as strains lacking these TA systems had a symbiotic N 2 fixation phenotype that was indistinguishable from the wild type.

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

“…General DNA manipulations and recombinant techniques, bacterial matings, isolation of genomic S. meliloti DNA, and ⌽M12 transductions were performed as previously described (42,44,45). Genomic DNA was sequenced and the presence of nucleotide variations examined as described elsewhere (46).…”

“…LB, LBmc, and tryptone-yeast extract (TY) media were prepared as previously described (42 (43). For MM9 agar, agar was added to reach 15 g/liter.…”

Counter-transcribed RNAs of Rhizobium leguminosarum repABC plasmids exert incompatibility effects only when highly expressed

“…The temperate, fast-growing rhizobia (genera Rhizobium and Sinorhizobium/Ensifer) as well as their close relatives, Agrobacterium spp., are characterized by having complex partitioned genomes consisting of one of more chromosomes, and multiple plasmids (Cevallos et al, 2008;Harrison et al, 2010;Hynes and McGregor, 1990;Young et al, 2006), which can constitute up to 45% of the total genome of the bacteria (Milunovic et al, 2014;Young et al, 2006). Most of the genes required for nodulation and N fixation are located on a plasmid or megaplasmid known as a Symbiosis plasmid or pSym (Hynes and McGregor, 1990;López-Guerrero et al, 2012;Mazur and Koper, 2012).…”

l-Hydroxyproline andd-Proline Catabolism in Sinorhizobium meliloti

“…The proline auxotroph RmP3155 was constructed by transducing ⌬B161 (an ϳ53-kb deletion of the pSymB plasmid that removes the smb20003 gene; Nm r Gm r ) into a proC::Tn5-B20 strain (19,20). The putA mutant SmFL5502 was identified in our previously constructed pTH1522 reporter gene fusion library of RmP110 (21).…”

“…When we tested for growth on nitrogen sources, NH 4 Cl was omitted from the M9 growth medium. 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).…”