Rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility

Abstract

The lipopolysaccharide (LPS) of the Gram-negative legume symbiont Rhizobium leguminosarum biovar viciae 3841 contains several unique modifications, including the addition of a 27-hydroxyoctacosanoic acid (27OHC28 : 0), also termed the very long chain fatty acid (VLCFA), attached at the 29 position of lipid A. A transposon mutant that lacks expression of two putative 3-oxo-acyl [acyl-carrier protein] synthase II genes, fabF1 and fabF2, from the VLCFA biosynthetic cluster, was isolated and characterized. MS indicated that the lipid A of the mutant lacked the VLCFA modification, and sodium deoxycholate (DOC)-PAGE of the LPS indicated further structural alterations. The mutant was characteristically sensitive to several stresses that would be experienced in the soil environment, such as desiccation and osmotic stresses. An increase in the excretion of neutral surface polysaccharides was observed in the mutant. This mutant was also altered in its attachment to solid surfaces, and was non-motile, with most of the mutant cells lacking flagella. Despite the pleiotropic effects of the mutation, these mutants were still able to nodulate legumes and fix atmospheric nitrogen. This report emphasizes that a structurally intact VLCFAcontaining lipid A is critical to cellular traits that are important for survival in the rhizosphere.

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

“…It is interesting that an inability to grow on TY often correlates with cell envelope deficiencies (Gibson et al, 2008;Vanderlinde et al, 2009Vanderlinde et al, , 2010Vanderlinde et al, , 2011 yet we observe here that suppressors that restore growth on TY remain sensitive to membrane disruptive agents. It is also interesting to note there is an increase in sensitivity when both genes are mutated in the strain cmdB-S/ttpA : : Tn5, when compared with the WT and other mutants.…”

“…Due to the importance of a functional cell envelope in desiccation tolerance (Gilbert et al, 2007;Vanderlinde et al, 2009Vanderlinde et al, , 2010, desiccation tolerance of the mutants was investigated. A filtration method described by Ophir & Gutnick (1994) and modified by Gilbert et al (2007) was used to perform desiccation sensitivity assays.…”

“…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).…”

“…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.…”

The Physiological Mechanisms of Desiccation Tolerance in Rhizobia

“…A mutant of R. leguminosarum bv. viciae 3841 unable to synthesise 27-hydroxyoctacosanoate (one of the components of lipid A) was found to be more sensitive to desiccation stress than the parent strain (Vanderlinde et al, 2009). …”