Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity.

In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy-producing hydrogenase, HupSL, is regulated by the substrate H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65-->Val and Phe113-->Leu in HupV) rendered HupUV sensitive to O2 inactivation. Also, in contrast with the wild-type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild-type). The H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT/HupR, regulates HupSL synthesis in response to H2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhibited the same hydrogenase activity as the wild-type enzyme and was equally able to repress HupSL synthesis in the absence of H2.

Interaction between the H2 sensor HupUV and the histidine kinase HupT controls HupSL hydrogenase synthesis in Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by H(2) through a signal transduction cascade comprising three proteins: the H(2)-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His(6))-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a His(6)-HupUHupV complex present in dimeric and tetrameric forms, both of which had H(2) uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, H(2) affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H(2) regulation.

Role of ctc from Listeria monocytogenes in osmotolerance.

Listeria monocytogenes is a food-borne pathogen with the ability to grow under conditions of high osmolarity. In a previous study, we reported the identification of 12 proteins showing high induction after salt stress. One of these proteins is highly similar to the general stress protein Ctc of Bacillus subtilis. In this study, induction of Ctc after salt stress was confirmed at the transcriptional level by using RNA slot blot experiments. To explore the role of the ctc gene product in resistance to stresses, we constructed a ctc insertional mutant. No difference in growth was observed between the wild-type strain LO28 and the ctc mutant either in rich medium after osmotic or heat stress or in minimal medium after heat stress. However, in minimal medium after osmotic stress, the growth rate of the mutant was increased by a factor of 2. Moreover, electron microscopy analysis showed impaired morphology of the mutant grown under osmotic stress conditions in minimal medium. Addition of the osmoprotectant glycine betaine to the medium completely abolished the osmotic sensitivity phenotype of the ctc mutant. Altogether, these results suggest that the Ctc protein of L. monocytogenes is involved in osmotic stress tolerance in the absence of any osmoprotectant in the medium.

A proteomic analysis of the salt stress response of Listeria monocytogenes.

Protein variations in Listeria monocytogenes were analyzed by 2-D electrophoresis. Bacteria were grown either in a rich medium or in a chemically defined medium. Three proteins, which are more expressed in the chemically defined medium than in the rich medium, were identified by mass spectrometry. They are closely related to AppA, Ctc and YvyD. After an osmotic shock, according to the medium and the NaCl concentration, the synthesis rate (P<0.05) of 59 proteins is altered by salinity. Half of them were more expressed, some of these proteins were closely related to Ctc, GbuA and the 30S ribosomal protein S6. Among the proteins which were down-expressed in the presence of salt, two were similar to AckA and PdhD.

A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode.

Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (D-ribose-binding periplasmic protein, D-galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode.

Mutational analysis of polynucleotide phosphorylase from Escherichia coli.

Polynucleotide phosphorylase (PNPase), a homotrimeric exoribonuclease present in bacteria, is involved in mRNA degradation. In Escherichia coli, expression of this enzyme is autocontrolled at the translational level. We introduced about 30 mutations in the pnp gene by site-directed mutagenesis, most of them in phylogenetically conserved residues, and determined their effects on the three catalytic activities of PNPase, phosphorolysis, polymerisation and phosphate exchange, as well as on the efficiency of translational repression. The data are presented and discussed in the light of the crystallographic structure of PNPase from Streptomyces antibioticus. The results show that both PNPase activity and the presence of the KH and S1 RNA-binding domains are required for autocontrol. Deletions of these RNA-binding domains do not abolish any of the three catalytic activities, indicating that they are contained in a domain independent of the catalytic centre. Moreover, the catalytic centre was located around the tungsten-binding site identified by crystallography. Some mutations affect the three catalytic activities differently, an observation consistent with the presence of different subsites.

Salt stress proteins induced in Listeria monocytogenes.

The ability of Listeria monocytogenes to tolerate salt stress is of particular importance, as this pathogen is often exposed to such environments during both food processing and food preservation. In order to understand the survival mechanisms of L. monocytogenes, an initial approach using two-dimensional polyacrylamide gel electrophoresis was performed to analyze the pattern of protein synthesis in response to salt stress. Of 400 to 500 visible proteins, the synthesis of 40 proteins (P < 0.05) was repressed or induced at a higher rate during salt stress. Some of the proteins were identified on the basis of mass spectrometry or N-terminal sequence analysis and database searching. Twelve proteins showing high induction after salt stress were similar to general stress proteins (Ctc and DnaK), transporters (GbuA and mannose-specific phosphotransferase system enzyme IIAB), and general metabolism proteins (alanine dehydrogenase, CcpA, CysK, EF-Tu, Gap, GuaB, PdhA, and PdhD).