Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity

Abstract

The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of Rhizobium leguminosarum is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of R. leguminosarum strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative moxR-like AAA ؉ ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membranedisruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a chvG mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in Rhizobium leguminosarum. Given the broad conservation of these genes among the Alphaproteobacteria, the results of this study may also provide insight into the physiological role of these genes in other Alphaproteobacteria, including the animal pathogen Brucella.

A previously uncharacterized tetratricopeptide-repeat-containing protein is involved in cell envelope function in Rhizobium leguminosarum

“…DNase treatment was conducted using Turbo DNase (Life Technologies). Reverse transcription reactions were carried out according to a previously described protocol (Manzon et al, 2007), with modifications described by Vanderlinde et al (2009Vanderlinde et al ( , 2011. Quantitative PCR was conducted on an Applied Biosystems StepOne Real-Time PCR System using SYBR Green PCR Master Mix (Life Technologies) as per manufacturer's instructions and using primers RL0936B and RL0936C (Table 1).…”

“…The shared mutant phenotype of an inability to grow on complex peptide rich media suggests a possible network between these cell envelope related genes, and further experimentation is needed to identify potential interaction networks. Vanderlinde et al (2011) identified a link between the uncharacterized, broadly conserved MoxR AAA+ operon, and proper cell envelope function in R. leguminosarum. The genes in this operon are subsequently referred to as complex media growth deficient (cmdA-cmdD), based on the mutant phenotype of an inability to grow on complex peptide rich growth media.…”

“…The genes in this operon are subsequently referred to as complex media growth deficient (cmdA-cmdD), based on the mutant phenotype of an inability to grow on complex peptide rich growth media. Mutations in the operon resulted in a number of phenotypes related to envelope disfunction and cell morphology (Vanderlinde et al, 2011). The large MoxR AAA+ family contains a diverse group of ATPases that are found throughout bacteria and are suggested to have chaperone-like activities (Snider & Houry, 2006;Wong & Houry, 2012).…”

“…Vanderlinde et al (2011) noted that culturing cmdA-cmdD mutants on TY agar at high cell density (10 8 c.f.u. ml 21 ) resulted in the appearance of suppressor mutants capable of growth on the TY agar.…”

“…Our earlier work and that of others has identified a link between Rhizobium genes involved in cell envelope development and their requirement for growth on complex peptide rich media such as tryptone-yeast extract medium (TY). Specific examples include genes involved in the synthesis of the very long chain fatty acid attached to the lipid A of the outer membrane lipopolysaccharide (Vanderlinde et al, 2009), a gene with homology to the cell-envelope-associated carboxyl-terminal protease (ctpA) (Gilbert et al, 2007), a two-component sensor histidine kinase (ctpA) (Gibson et al, 2008), a two-component signal transduction system (chvG/chvI) (Bélanger et al, 2009;Foreman et al, 2010;Vanderlinde & Yost, 2012) and a broadly conserved four gene operon with a MoxR AAA+ and von Wilderbrand A domain containing gene (RL3499-RL3502) (Vanderlinde et al, 2011). The shared mutant phenotype of an inability to grow on complex peptide rich media suggests a possible network between these cell envelope related genes, and further experimentation is needed to identify potential interaction networks.…”