Microbacterium plantarum sp. nov. and Microbacterium thalli sp. nov., two endophytic metal-resistant bacteria isolated from Sphaeralcea angustifolia (Cav.) G. Don and Prosopis laevigata (Humb. et Bonpl. ex Willd) M.C. Johnston.

Four Gram-positive, aerobic, catalase- and oxidase-negative, rod-shaped, motile endophytic bacterial strains, designated NM3R9T, NE1TT3, NE2TL11 and NE2HP2T, were isolated from the inner tissues (leaf and stem) of Sphaeralcea angustifolia and roots of Prosopis laevigata. They were characterized using a polyphasic approach, which revealed that they represent two novel Microbacterium species. Phylogenetic analysis based on 16S rRNA gene sequencing showed that the species closest to NE2HP2T was Microbacterium arborescens DSM 20754T (99.6 %) and that closest to NM3R9T, NE2TL11 and NE2TT3 was Microbacterium oleivorans NBRC 103075T (97.4 %). The whole-genome average nucleotide identity value between strain NM3R9T and Microbacterium imperiale DSM 20530T was 90.91 %, and that between strain NE2HP2T and M. arborecens DSM 20754T was 91.03 %. Digital DNA-DNA hybridization showed values of less than 70 % with the type strains of related species. The polar lipids present in both strains included diphosphatidylglycerol, phosphatidylglycerol, glycolipids and unidentified lipids, whereas the major fatty acids included anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and C16 : 0. Whole-cell sugars included mannose, rhamnose and galactose. Strains NM3R9T and NE2HP2T showed physiological characteristics different from those present in closely related Microbacterium species. According to the taxonomic analysis, both strains belong to two novel species. The name Microbacterium plantarum sp. nov. is proposed for strain NE2HP2T (=LMG 30875T=CCBAU 101117T) and Microbacterium thalli sp. nov. for strains NM3R9T (=LMG 30873T=CCBAU 101116T), NE1TT3 (=CCBAU 101114) and NE2TL11 (=CCBAU 101115).

Bacterial lipid diversity.

The glycerophospholipids phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL) are major structural components of bacterial membranes. In some bacteria, phosphatidylcholine or phosphatidylinositol and its derivatives form part of the membrane. PG or CL can be modified with the amino acid residues lysine, alanine, or arginine. Diacylglycerol is the lipid anchor from which syntheses of phosphorus-free glycerolipids, such as glycolipids, sulfolipids, or homoserine-derived lipids initiate. Many membrane lipids are subject to turnover and some of them are recycled. Other lipids associated with the membrane include isoprenoids and their derivatives such as hopanoids. Ornithine-containing lipids are widespread in Bacteria but absent in Archaea and Eukarya. Some lipids are probably associated exclusively with the outer membrane of many bacteria, i.e. lipopolysaccharides, sphingolipids, or sulfonolipids. For certain specialized membrane functions, specific lipid structures might be required. Upon cyst formation in Azotobacter vinelandii, phenolic lipids are accumulated in the membrane. Anammox bacteria contain ladderane lipids in the membrane surrounding the anammoxosome organelle, presumably to impede the passage of highly toxic compounds generated during the anammox reaction. Considering that present knowledge on bacterial lipids was obtained from only a few bacterial species, we are probably only starting to unravel the full scale of lipid diversity in bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

OlsG (Sinac_1600) Is an Ornithine Lipid N-Methyltransferase from the Planctomycete Singulisphaera acidiphila.

Ornithine lipids (OLs) are phosphorus-free membrane lipids widespread in bacteria but absent from archaea and eukaryotes. In addition to the unmodified OLs, a variety of OL derivatives hydroxylated in different structural positions has been reported. Recently, methylated derivatives of OLs were described in several planctomycetes isolated from a peat bog in Northern Russia, although the gene/enzyme responsible for the N-methylation of OL remained obscure. Here we identify and characterize the OL N-methyltransferase OlsG (Sinac_1600) from the planctomycete Singulisphaera acidiphila. When OlsG is co-expressed with the OL synthase OlsF in Escherichia coli, methylated OL derivatives are formed. An in vitro characterization shows that OlsG is responsible for the 3-fold methylation of the terminal δ-nitrogen of OL. Methylation is dependent on the presence of the detergent Triton X-100 and the methyldonor S-adenosylmethionine.

Agrobacteria lacking ornithine lipids induce more rapid tumour formation.

Ornithine lipids (OLs) are phosphorus-free membrane lipids that are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. It has been shown that OLs can be hydroxylated within the amide-linked fatty acyl moiety, the secondary fatty acyl moiety or within the ornithine moiety. These modifications have been related to increased stress tolerance and symbiotic proficiency in different organisms such as Rhizobium tropici or Burkholderia cenocepacia. Analysing the membrane lipid composition of the plant pathogen Agrobacterium tumefaciens we noticed that it forms two different OLs. In the present work we studied if OLs play a role in stress tolerance and pathogenicity in A. tumefaciens. Mutants deficient in the OLs biosynthesis genes olsB or olsE were constructed and characterized. They either completely lack OLs (ΔolsB) or only form the unmodified OL (ΔolsE). Here we present a characterization of both OL mutants under stress conditions and in a plant transformation assay using potato tuber discs. Surprisingly, the lack of agrobacterial OLs promotes earlier tumour formation on the plant host.

Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants.

The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions.

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.