Functional genomics provides insights into the genetic network(s) associated with the function of the cmd operon in Rhizobium leguminosarum bv. viciae

Abstract

The bacterium Rhizobium leguminosarum forms a symbiotic association with leguminous plants such as peas and lentils, which are important food crops. Rhizobium’s cell envelope is important for establishing a symbiotic association with host plants and for helping the cell adapt to stressful conditions in the rhizosphere. A previously uncharacterized four gene operon, cmdA-D (RL3499-RL3502) in Rhizobium leguminosarum viciae 3841 has been shown to be essential for proper cell envelope function. The broadly conserved cmd operon codes for ATPases which mediate protein-protein interaction and are involved in various cellular activities. cmd mutants cannot grow on complex peptide rich media, therefore the operon was named for the complex media deficient (cmd) phenotype of the mutants. However, cmd mutants can grow on minimal media. Apart from the inability to grow on peptide rich media (TY), these mutants exhibit some additional phenotypes such as distorted enlarged cell shape, sensitivity to cell envelope stressors like detergents, hydrophobic antibiotics, alkaline pH, and production of twice as many nodules in pea plants compared to the wild type. At low frequencies, strains that are able to grow on peptide rich TY media, can be isolated from the cmd operon mutants. These isolates represent secondary site mutations that are supressing the original cmd mutant phenotype. One such isolate 38cmdB_S, able to grow on TY, isolated from a cmd mutant in non-polar RL3500 (38cmdB) mutant background was selected for further characterization. This study aims to increase our understanding of the biological function of the cmd operon by studying the 38cmdB mutant and the suppressor mutant, 38cmdB_S through phenotypic characterisation, genome wide mutagenesis using insertion sequencing (INSeq), and transcriptomic analysis through RNA-Seq of the wild type, 38cmdB and 38cmdB_S mutant. ii INSeq, a high throughput genetic screening technique was used to simultaneously identify the suppressor locus and to identify conditional essential genes in the suppressor mutant background. Using this approach and selection on TY media, 40 conditional essential genes were found in the suppressor mutant. The Insertion sequencing data not only reveals the putative suppressor locus but also other genes which are connected to the function of the cmd operon. Metal chelatase, cell envelope biosynthesis proteins, genes involved in ABC transporter, and in glycolysis and gluconeogenesis were identified as necessary to compensate for the loss of cmd operon’s function in the suppressor strain. In this study, it was discovered that the 38cmdB mutant could regain growth on TY if the culture medium was supplemented with metals (magnesium, iron, calcium) and vitamins (biotin, thiamine, pantothenic acid), indicating the necessity of high concentration of metals and vitamins in the absence of functional cmd operon. Moreover, the comparative transcriptomic data (for the same growth condition) between wild type, and both the cmdB mutant and the suppressor mutant added new information about the genetic network associated with the cmd operon. Genes involved in siderophore synthesis, cell wall biosynthesis, and different transporters such as ABC, MFS, and RND were observed to be upregulated in both mutant strains, suggesting changes in gene regulation in both mutants associated with the loss of cmdB (RL3500) gene and cmd operon’s function. The exact molecular function of the cmd operon is still unknown, however the involvement of cmd operon in protein maturation, in insertion of divalent metals into other proteins, and in maintaining the cell envelope integrity during polar growth in Rhizobium leguminosarum can be suggested from this study.