Interacting bactofilins impact cell shape of the MreB-less multicellular Rhodomicrobium vannielii.

Most non-spherical bacteria rely on the actin-like MreB cytoskeleton to control synthesis of a cell-shaping and primarily rod-like cell wall. Diverging from simple rod shape generally requires accessory cytoskeletal elements, which locally interfere with the MreB-guided cell wall synthesis. Conserved and widespread representatives of this accessory cytoskeleton are bactofilins that polymerize into static, non-polar bundles of filaments. Intriguingly, many species of the Actinobacteria and Rhizobiales manage to grow rod-like without MreB by tip extension, yet some of them still possess bactofilin genes, whose function in cell morphogenesis is unknown. An intricate representative of these tip-growing bacteria is Rhodomicrobium vannielii; a member of the hitherto genetically not tractable and poorly studied Hyphomicrobiaceae within the MreB-less Rhizobiales order. R. vannielii displays complex asymmetric cell shapes and differentiation patterns including filamentous hyphae to produce offspring and to build dendritic multicellular arrays. Here, we introduce techniques to genetically access R. vannielii, and we elucidate the role of bactofilins in its sophisticated morphogenesis. By targeted mutagenesis and fluorescence microscopy, protein interaction studies and peptidoglycan incorporation analysis we show that the R. vannielii bactofilins are associated with the hyphal growth zones and that one of them is essential to form proper hyphae. Another paralog is suggested to represent a novel hybrid and co-polymerizing bactofilin. Notably, we present R. vannielii as a powerful new model to understand prokaryotic cell development and control of multipolar cell growth in the absence of the conserved cytoskeletal element, MreB.

The Lipid A from the Lipopolysaccharide of the Phototrophic Bacterium Rhodomicrobium vannielii ATCC 17100 Revisited.

The structure of lipid A from lipopolysaccharide (LPS) of Rhodomicrobium vannielii ATCC 17100 (Rv) a phototrophic, budding bacterium was re-investigated using high-resolution mass spectrometry, NMR, and chemical degradation protocols. It was found that the (GlcpN)-disaccharide lipid A backbone was substituted by a GalpA residue that was connected to C-1 of proximal GlcpN. Some of this GalpA residue was ß-eliminated by alkaline de-acylation, which indicated the possibility of the presence of another so far unidentified substituent at C-4 in non-stoichiometric amounts. One Manp residue substituted C-4' of distal GlcpN. The lipid A backbone was acylated by 16:0(3-OH) at C-2 of proximal GlcpN, and by 16:0(3-OH), i17:0(3-OH), or 18:0(3-OH) at C-2' of distal GlcpN. Two acyloxy-acyl moieties that were mainly formed by 14:0(3-O-14:0) and 16:0(3-O-22:1) occupied the distal GlcpN of lipid A. Genes that were possibly involved in the modification of Rv lipid A were compared with bacterial genes of known function. The biological activity was tested at the model of human mononuclear cells (MNC), showing that Rv lipid A alone does not significantly stimulate MNC. At low concentrations of toxic Escherichia coli O111:B4 LPS, pre-incubation with Rv lipid A resulted in a substantial reduction of activity, but, when higher concentrations of E. coli LPS were used, the stimulatory effect was increased.

Draft genome sequence of Rhodomicrobium udaipurense JA643T with special reference to hopanoid biosynthesis.

Hopanoids are present in vast amounts as integral components of bacteria and plants with their primary function to strengthen rigidity of the plasma membrane. To establish their roles more precisely, we conducted sequencing of the whole genome of Rhodomicrobium udaipurense JA643(T) isolated from a fresh water stream of Udaipur in Himachal Pradesh, India, by using the Illumina HiSeq pair end chemistry of 2 × 100 bp platform. Determined genome showed a high degree of similarity to the genome of R. vannielii ATCC17100(T) and the 13.7 million reads generated a sequence of 3,649,277 bp possessing 3,611 putative genes. The genomic data were subsequently investigated with respect to genes involved in various features. The machinery required for the degradation of aromatic compounds and resistance to solvents as well as all that required for photosynthesis are present in this organism. Also, through extensive functional annotation, 18 genes involved in the biosynthesis of hopanoids are predicted, namely those responsible for the synthesis of diploptene, diplopterol, adenosylhopane, ribosylhopane, aminobacteriohopanetriol, glycosyl group containing hopanoids and unsaturated hopanoids. The hopanoid biosynthetic pathway was then inferred based on the genes identified and through experimental validation of individual hopanoid molecules. The genome data of R. udaipurense JA643(T) will be useful in understanding the functional features of hopanoids in this bacterium.