Kandelia candel is being established as a model xylophyte for ecoadaptation due to its salt tolerance. To adapt to high salinity, the photosynthesis apparatus must function efficiently under these conditions. Proteomic analysis of chloroplasts isolated from plants under different degrees of salt stress was performed to quantify the changes of individual proteins and to gain a global view of the total chloroplast protein dynamics. Among the 1030 proteins quantified (unique peptide ≥ 1), 76 showed a more than 1.5-fold change in abundance, of which 36 are involved in the light-dependent reactions and 12 in the Calvin cycle. The dynamic change of these proteins indicates that light-dependent reactions are maintained by up-regulating the levels of component proteins at both moderate and high salinity, and the Calvin cycle remained functional at moderate salinity but showed a decline at high salinity. In addition to proteins related to photosynthesis, some known abiotic-stress proteins and plastoglobuli were up-regulated in salt-stressed plants. Plastoglobuli might contribute to maintaining membrane integrity and fluidity. In conclusion, this extensive proteomic investigation on intact chloroplasts of the salt-tolerant xylophyte under salt stress provides some important novel information on adaptative mechanisms involving photosynthesis in responses to salt stress in K. candel.

The bacteriophage P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA). These studies indicate that both P and DnaC contain a cryptic ssDNAbinding activity that is mobilized when each forms a complex with the DnaB helicase. Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P⅐DnaB or a DnaB⅐DnaC complex. This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication. Further, we find that the O replication initiator enhances interaction of the P⅐DnaB complex with ssDNA. Partial disassembly of a ssDNA:O⅐P⅐DnaB complex by the DnaK͞DnaJ͞GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template. On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.

Salt stress is a major abiotic stress that limits crop productivity in many regions of the world. A comparative proteomic approach to identify salt stress-responsive proteins and to understand the molecular mechanisms was carried out in the woody halophyte Kandelia candel. Four-leaf-old K. candel seedlings were exposed to 150 (control), 300, 450, and 600 mM NaCl for 3 days. Proteins extracted from the leaves of K. candel seedlings were separated by two-dimensional gel electrophoresis (2-DE). More than 900 protein spots were detected on each gel, and 53 differentially expressed protein spots were located with at least two-fold differences in abundance on 2-DE maps, of which 48 were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF/MS). The results showed that K. candel could withstand up to 450 mM NaCl stress by up-regulating proteins that are mainly involved in photosynthesis, respiration and energy metabolism, Na+ compartmentalization, protein folding and assembly, and signal transduction. Physiological data, including superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities, hydrogen peroxide (H2O2) and superoxide anion radicals (O2 −) contents, as well as Na+ content and K+/Na+ ratios all correlated well with our proteomic results. This study provides new global insights into woody halophyte salt stress responses. Identification of differentially expressed proteins promotes better understanding of the molecular basis for salt stress reduction in K. candel.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [I4C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (ApH) probes and [I4C]triphenylmethylphosphonium as a membrane potential (A*) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The ApH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, A4 was approximately-90 mV, whereas no negative A* was detectable at pH 4.5. The proton motive force was calculated to be-106 mV at pH 6.5 and-102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.

Adhesion is a critical step in the initial stage of Vibrio alginolyticus infection; therefore, it is important to understand the underlying mechanisms governing the adhesion of V. alginolyticus and determine if environmental factors have any effect. A greater understanding of this process may assist in developing preventive measures for reducing infection. In our previous research, we presented the first RNA-seq data from V. alginolyticus cultured under stress conditions that resulted in reduced adhesion. Based on the RNA-seq data, we found that the Tricarboxylic acid cycle (TCA pathway) might be closely related to adhesion. Environmental interactions with the TCA pathway might alter adhesion. To validate this, bioinformatics analysis, quantitative Real-Time PCR (qPCR), RNAi, and in vitro adhesion assays were performed, while V. alginolyticus was treated with various stresses including temperature, pH, salinity, and starvation. The expression of genes involved in the TCA pathway was confirmed by qPCR, which reinforced the reliability of the sequencing data. Silencing of these genes was capable of reducing the adhesion ability of V. alginolyticus. Adhesion of V. alginolyticus is influenced substantially by environmental factors and the TCA pathway is sensitive to some environmental stresses, especially changes in pH and starvation. Our results indicated that (1) the TCA pathway plays a key role in V. alginolyticus adhesion: (2) the TCA pathway is sensitive to environmental stresses.

A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gramnegative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39°C and pH 6.5, the activity of the enzyme with p-nitrophenyli--D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.o mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45°C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl -cellulose.Of the cellulolytic rumen bacteria, Bacteroides succinogenes has been described as a predominant cellulose degrader (8, 24). Its cellulase system has been the subject of extensive research in the past few years (16, 19-21, 23, 32, 37, 40). Both endoglucanase and cellobiase activities have been demonstrated in this bacterium for some time (19)(20)(21)37). Recently, two endoglucanases were purified from the extracellular-culture fluid of B. succinogenes (32). These two enzymes hydrolyzed cellooligosaccharides and amorphous cellulose but produced different end products. Examination of the exo-type cellulase activities in B. succinogenes has also been carried out. In a previous study, we identified an exo-type cellodextrinase in the periplasm of B. succinogenes cells grown on cellulose (23). This enzyme was capable of the successive removal of terminal cellobiose units from water-soluble cellodextrins, but not from longer cellulose chains, and therefore appeared to be different from the well-characterized fungal exoglucanases. We therefore decided to undertake further studies on exo-type cellulase activity.Here we report on the purification and characterization of a unique cellobiosidase from B. succinogenes, the activity of which was significantly enhanced by chloride and several other anions. This cellobiosidase may be involved in the initial steps of cellulose hydrolysis. MATERIALS AND METHODSOrganism and growth conditions. B. succinogenes S85 (previously obtained from M. P. Bryant, University of Illinois, Urbana) was grown continuously in a Multigen bench top fermentor (Multigen; New Brunswick Scientific Co., Inc., Edison, N.J.) equipped with a 2-liter chemstat vessel (C-30) and an automatic pH control, as described previously (23). The chemostat medium use...

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Clostridium acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at pH 4.5 had intracellular concentrations of acetate, butyrate, and butanol which were 13-, 7-, and 1.3-fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at pH 6.5 had intracellular concentrations of acetate and butyrate which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at ca. pH 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at pH 4.5, exogenous acetate and butyrate caused a concentration-dependent decrease in the intracellular pH, while butanol had relatively little * Corresponding author.