Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti.

BACKGROUND: The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell. RESULTS: Comparison of tolC mutant and wild-type strains transcriptomes showed 1177 genes with significantly increased expression while 325 had significantly decreased expression levels. The genes with an increased expression suggest the activation of a cytoplasmic and extracytoplasmic stress responses possibly mediated by the sigma factor RpoH1 and protein homologues of the CpxRA two-component regulatory system of Enterobacteria, respectively. Stress conditions are probably caused by perturbation of the cell envelope. Consistent with gene expression data, biochemical analysis indicates that the tolC mutant suffers from oxidative stress. This is illustrated by the elevated enzyme activity levels detected for catalase, superoxide dismutase and glutathione reductase. The observed increase in the expression of genes encoding products involved in central metabolism and transporters for nutrient uptake suggests a higher metabolic rate of the tolC mutant. We also demonstrated increased swarming motility in the tolC mutant strain. Absence of functional TolC caused decreased expression mainly of genes encoding products involved in nitrogen metabolism and transport. CONCLUSION: This work shows how a mutation in the outer membrane protein TolC, common to many bacterial transport systems, affects expression of a large number of genes that act in concert to restore cell homeostasis. This finding further underlines the fundamental role of this protein in Sinorhizobium meliloti biology.

The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis.

Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

Functional analysis of Burkholderia cepacia genes bceD and bceF, encoding a phosphotyrosine phosphatase and a tyrosine autokinase, respectively: role in exopolysaccharide biosynthesis and biofilm formation.

The biosynthesis of the exopolysaccharide (EPS) cepacian by Burkholderia cepacia complex strains requires the 16.2-kb bce cluster of genes. Two of the clustered genes, bceD and bceF, code for two proteins homologous to phosphotyrosine phosphatases and tyrosine kinases, respectively. We show experimental evidence indicating that BceF is phosphorylated on tyrosine and that the conserved lysine residue present at position 563 in the Walker A ATP-binding motif is required for this autophosphorylation. It was also proved that BceD is capable of dephosphorylating the phosphorylated BceF. Using the artificial substrate p-nitrophenyl phosphate (PNPP), BceD exhibited a V(max) of 8.8 mumol of PNPP min(-1) mg(-1) and a K(m) of 3.7 mM PNPP at 30 degrees C. The disruption of bceF resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental strain's EPS production yield was still registered for the bceD mutant. The exopolysaccharide produced by the bceD mutant led to less viscous solutions and exhibited the same degree of acetylation as the wild-type cepacian, suggesting a lower molecular mass for this mutant biopolymer. The size of the biofilm produced in vitro by bceD and bceF mutant strains is smaller than the size of the biofilm formed by the parental strain, and this phenotype was confirmed by complementation assays, indicating that BceD and BceF play a role in the establishment of biofilms of maximal size.

High-throughput measurement of protein stability in microtiter plates.

The direct determination of protein stability at high throughput has applications in proteomics, directed evolution, and formulation. Each application places different requirements on the accuracy of stability or transition midpoint determination. The measurement of protein stability by chemical denaturation has been previously performed at medium throughput and high accuracy using autotitrating fluorometers, after removal of proteins from the 96-well plate format in which they were expressed and purified. Herein we present a higher-throughput method for measuring and indexing the stability of proteins maintained within the 96-well format using a fluorescence microplate reader. Protein unfolding transitions were monitored by tryptophan fluorescence at 340 nm and assessed using bovine and equine cytochrome c (cyt c), as well as bovine serum albumin (BSA) stabilized with various amounts of palmitic acid. Two different approaches for generating unfolding curves in microtiter plates have been evaluated for their accuracy and applicability. Unfolding curves generated by the serial addition of denaturant into single wells allowed high-throughput stability screens capable of identifying protein variants with unfolding midpoint differences of 0.15 M denaturant concentration or larger. Such a method would be suitable for screening large numbers of proteins, as typically generated for directed evolution. Unfolding curves generated using one well per denaturant concentration allowed for medium-throughput stability screening and generated more accurate and precise stability values (C(1/2) +/- 0.05 M, m(G), and DeltaG(H2O)) for cyt c that are similar to values reported in literature. This method is suitable for screening the smaller numbers of proteins generated in proteomic research programmes. By using BSA stabilized with various palmitate concentrations and simple numerical indexing, it was shown that both experimental methods can successfully rank the order of protein stability.