The Power of Biocatalysis: A One-Pot Total Synthesis of Rhamnolipids from Butane as the Sole Carbon and Energy Source.

Microbially derived surfactants, so-called biosurfactants, have drawn much attention in recent years and are expected to replace current petrochemical surfactants, owing to their environmental and toxicological benefits. One strategy to support that goal is to reduce production costs by replacing relatively expensive sugars with cheaper raw materials, such as short-chain alkanes. Herein, we report the successful one-pot total synthesis of rhamnolipids, a class of biosurfactants with 12 stereocenters, from butane as sole carbon and energy source through the design of a tailored whole-cell biocatalyst.

Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for L-methionine and branched-chain amino acids.

Corynebacterium glutamicum possesses export systems for various amino acids including BrnFE, a two-component export system for L-methionine and the branched-chain amino acids L-valine, L-isoleucine and L-leucine. A gene for a putative transcriptional regulator of the Lrp family is transcribed divergently to the brnFE operon and is required for L-isoleucine export. By comparing global gene expression changes due to L-isoleucine addition we revealed increased brnFE expression in response to L-isoleucine in C. glutamicum wild type but not in an lrp deletion mutant. ChIP-to-chip analysis, band shift experiments and DNAse footprint analysis demonstrated that Lrp binds to the intergenic region between lrp and brnF. Expression analysis of transcriptional fusions with the lrp and brnFE promoters indicated that branched-chain amino acids and L-methionine when added to the growth medium stimulated brnFE expression in the order L-leucine > L-methionine > L-isoleucine > L-valine and that Lrp was required for activation of brnFE expression. Thus, regulation of brnFE by Lrp ensures that BrnFE is synthesized only if its substrate amino acids accumulate in cells which is commensurate with its role to counteract such situations of metabolic imbalance.

Phosphatidylcholine biosynthesis and its significance in bacteria interacting with eukaryotic cells.

Phosphatidylcholine (PC), a typical eukaryotic membrane phospholipid, is present in only about 10% of all bacterial species, in particular in bacteria interacting with eukaryotes. A number of studies revealed that PC plays a fundamental role in symbiotic and pathogenic microbe-host interactions. Agrobacterium tumefaciens mutants lacking PC are unable to elicit plant tumors. The human pathogens Brucella abortus and Legionella pneumophila require PC for full virulence. The plant symbionts Bradyrhizobium japonicum and Sinorhizobium meliloti depend on wild-type levels of PC to establish an efficient root nodule symbiosis. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (Pcs) pathway. The methylation pathway involves a three-step methylation of phosphatidylethanolamine by at least one phospholipid N-methyltransferase to yield phosphatidylcholine. In the Pcs pathway, choline is condensed directly with CDP-diacylglycerol to form PC. This review focuses on the biosynthetic pathways and the significance of PC in bacteria with an emphasis on plant-microbe interactions.

Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes.

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.

Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane.

Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant-transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N-methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of host-microbe interactions.

Identification of a gene cluster in Klebsiella pneumoniae which includes citX, a gene required for biosynthesis of the citrate lyase prosthetic group.

The biosynthesis of the 2'-(5"-phosphoribosyl)-3'-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG. In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing. A search for K. pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes. The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species. The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed >or=40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K. pneumoniae. A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.