The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein–protein interactions are discussed.

Channel-mediated lactic acid transport: a novel function for aquaglyceroporins in bacteria

MIPs (major intrinsic proteins), also known as aquaporins, are membrane proteins that channel water and/or uncharged solutes across membranes in all kingdoms of life. Considering the enormous number of different bacteria on earth, functional information on bacterial MIPs is scarce. In the present study, six MIPs [glpF1 (glycerol facilitator 1)–glpF6] were identified in the genome of the Gram-positive lactic acid bacterium Lactobacillus plantarum. Heterologous expression in Xenopus laevis oocytes revealed that GlpF2, GlpF3 and GlpF4 each facilitated the transmembrane diffusion of water, dihydroxyacetone and glycerol. As several lactic acid bacteria have GlpFs in their lactate racemization operon (GlpF1/F4 phylogenetic group), their ability to transport this organic acid was tested. Both GlpF1 and GlpF4 facilitated the diffusion of D/L-lactic acid. Deletion of glpF1 and/or glpF4 in Lb. plantarum showed that both genes were involved in the racemization of lactic acid and, in addition, the double glpF1 glpF4 mutant showed a growth delay under conditions of mild lactic acid stress. This provides further evidence that GlpFs contribute to lactic acid metabolism in this species. This lactic acid transport capacity was shown to be conserved in the GlpF1/F4 group of Lactobacillales. In conclusion, we have functionally analysed the largest set of bacterial MIPs and demonstrated that the lactic acid membrane permeability of bacteria can be regulated by aquaglyceroporins.

Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR

ABSTRACTThe proteobacteriumPantoea stewartiisubsp.stewartiicauses Stewart's wilt disease in maize when it colonizes the xylem and secretes large amounts of stewartan, an exopolysaccharide. The success of disease pathogenesis lies in the timing of bacterial virulence factor expression through the different stages of infection. Regulation is achieved through a quorum-sensing (QS) system consisting of the acyl-homoserine lactone (AHL) synthase, EsaI, and the transcription regulator EsaR. At low cell densities, EsaR represses transcription of itself and ofrcsA, an activator of the stewartan biosynthesis operon; it also activatesesaS, which encodes a small RNA (sRNA). Repression or activation ceases at high cell densities when EsaI synthesizes sufficient levels of the AHL ligandN-3-oxo-hexanoyl-l-homoserine lactone to bind and inactivate EsaR. This study aims to identify other genes activated or repressed by EsaR during the QS response. Proteomic analysis identified a QS regulon of more than 30 proteins. Electrophoretic mobility shift assays of promoters of genes encoding differentially expressed proteins distinguished direct targets of EsaR from indirect targets. Additional quantitative reverse transcription-PCR (qRT-PCR) and DNA footprinting analysis established that EsaR directly regulates the promoters ofdkgA,glpF, andlrhA. The proteins encoded bydkgA,glpF, andlrhAare a 2,5-diketogluconate reductase, glycerol facilitator, and transcriptional regulator of chemotaxis and motility, respectively, indicating a more global QS response inP. stewartiithan previously recognized.

Implication of Glycerol and Phospholipid Transporters in Mycoplasma pneumoniae Growth and Virulence

ABSTRACTMycoplasma pneumoniae, the causative agent of atypical pneumonia, is one of the bacteria with the smallest genomes that are nonetheless capable of independent life. Because of their longstanding close association with their human host, the bacteria have undergone reductive evolution and lost most biosynthetic abilities. Therefore, they depend on nutrients provided by the host that have to be taken up by the cell. Indeed,M. pneumoniaehas a large set of hitherto unexplored transporters and lipoproteins that may be implicated in transport processes. Together, these proteins account for about 17% of the protein complement ofM. pneumoniae. In the natural habitat ofM. pneumoniae, human lung epithelial surfaces, phospholipids are the major available carbon source. Thus, the uptake and utilization of glycerol and glycerophosphodiesters that are generated by the activity of lipases are important for the nutrition ofM. pneumoniaein its common habitat. In this study, we have investigated the roles of several potential transport proteins and lipoproteins in the utilization of glycerol and glycerophosphodiesters. On the basis of experiments with the corresponding mutant strains, our results demonstrate that the newly identified GlpU transport protein (MPN421) is responsible for the uptake of the glycerophosphodiester glycerophosphocholine, which is then intracellularly cleaved to glycerol-3-phosphate and choline. In addition, the proteins MPN076 and MPN077 are accessory factors in glycerophosphocholine uptake. Moreover, the lipoproteins MPN133 and MPN284 are essential for the uptake of glycerol. Our data suggest that they may act as binding proteins for glycerol and deliver glycerol molecules to the glycerol facilitator GlpF.