Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (>10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.Nitrate can be used by many bacteria as a source of assimilable nitrogen or as a terminal electron acceptor under anoxic conditions (nitrate respiration). In the assimilatory process, which may occur aerobically or anaerobically, nitrate is ultimately reduced to ammonia (NH 3 ) and subsequently incorporated into biomass. This pathway is performed by many bacteria, fungi, and plants. In respiration, nitrate is used as an alternative electron acceptor when oxygen is not available. The enzymes for this pathway are found only in bacteria. Two main forms have been described so far, and in both, nitrate reduction is coupled to the generation of a proton motive force (3, 17), which is directly utilized as a source of energy or transformed into ATP by a membrane-associated ATPase. In one form, reported for Escherichia coli and other members of the family Enterobacteriaceae, the organisms reduce nitrate to nitrite, which is then excreted or further reduced to NH 3 by a dissimilatory (e.g., in E. coli [25]) or assimilatory nitrite reductase. The other form of nitrate respiration, denitrification, is defined as the reduction of nitrate to the gaseous oxides nitric oxide and nitrous oxide, which then may be further reduced to nitrogen (18,27). This form, generally found in obligately respiring bacteria such as pseudomonads, is crucial to nitrogen cycling in nature (10).Certain bacteria, such as strict aerobes, are able to perform assimilatory nitrate reduction but cannot use nitrate as the terminal electron acceptor. Other bacteria, such as E. coli and Salmonella typhimurium, do not assimilate nitrogen through nitrate reduction during aer...
Transposon mutagenesis of Staphylococcus carnosus led to the identification of three genes, modABC, which encode an ABC transporter that is involved in molybdate transport. It was shown by [14C]palmitate labeling that ModA represents a lipoprotein that in gram-positive bacteria is the counterpart of the periplasmic binding proteins of gram-negative organisms. The sequence characteristics identify ModB as the integral-membrane, channel-forming protein and ModC as the ATP-binding energizer for the transport system. Mutants defective in modABC had only 0.4% of the wild-type nitrate reductase activity. Molybdate at a non-physiologically high concentration (100 microM) fully restored nitrate reductase activity, suggesting that at least one other system is able to transport molybdate, but with lower affinity. The expression of modA (and most likely of modBC) was independent of oxygen and nitrate. To date, there are no indications for molybdate-specific regulation of modABC expression since in a modB mutant, modA expression was unchanged in comparison to the wild-type.
Lactobaci&ls sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells ofL. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethylp-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog at-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system. In fermentative bacteria, an electrochemical proton gradient (proton motive force) can be generated by proton extrusion via F0F,-ATPase or by electrogenic secondary transport processes (16,18,19). Most frequently, secondary transport systems catalyze symport of solutes with protons or sodium ions (18,19). The proton or sodium motive force is then used to drive solute transport. However, some secondary transport proteins catalyze exclusively an antiport. Examples of this class of transport are the systems which couple the uptake of precursor molecules to the excretion of product (precursor/product antiport) (16, 18). The driving forces for these processes are supplied by the (electro)chemical gradients of both precursor and product. An example of such an antiport system is the arginine/ornithine antiporter which has been detected in several bacteria (6,17 (9,16,20).Lactobacillus sanfrancisco strains are the main organisms in sourdough starter preparations (1,11,23,27). The prevailing sugar in sourdough is maltose, which is formed during degradation of starch by oa-amylase. The metabolism of maltose in cells of L. sanfrancisco is initiated by cleavage of maltose to glucose and glucose-i-phosphate via a maltose phosphorylase (24). Glucose-i-phosphate and part of the free glucose are metabolized, whereas approximately half of the generated glucose is released into the external ...
The gene encoding the dextransucrase DsrD from the industrial strain Leuconostoc mesenteroides Lcc4 was isolated by PCR using degenerate primers recognizing conserved regions present in other dextransucrase-encoding genes from Leuconostoc spp. and Southern blot analyses on total genomic DNA. N-terminal sequence analysis of the active protein recovered in the culture showed that the secreted protein of 165 kDa is devoid of a 42 aa prepeptide which is removed post-translationally, most likely by signal peptidase cleavage. Primer extension and Northern blot analysis identified a monocistronic dsrD mRNA with two transcription initiation sites. Expression of the dextransucrase DsrD was investigated in pH-controlled fed-batch cultures via Northern blot analysis and enzyme activity measurement during the experiments. Sucrose levels of 20 g l 21 wereshown to induce the DsrD biosynthesis around 10-fold. The combination of pH-controlled fed-batch fermentation and Northern analysis clearly showed that dsrD expression was related to the growth of the bacteria. dsrD was transferred to and expressed in Lactococcus lactis MG1363. Controlled fed-batch cultures revealed that active dextransucrase was produced and secreted by the recombinant L. lactis strain. The expression was independent of sucrose levels. These results show that dextransucrase can be efficiently expressed and secreted in a non-Leuconostoc, heterologous host and is able to drive dextran synthesis.
Physiological and genetic characterization of Staphylococcus carnosus nitrate reductase-negative mutants led to the identification of the nitrate reductase operon, narGHJI. Transcription from the nar promoter was stimulated by anaerobiosis, nitrate, and nitrite. This is in accordance with the nitrate reductase activities determined with benzyl viologen as electron donor. However, in the presence of oxygen and nitrate, high transcriptional initiation but low nitrate reductase activity was observed. Since the alphabeta complex of the nitrate reductase formed during anaerobic growth was insensitive to oxygen, other oxygen-sensitive steps (e.g., post-transcriptional mechanisms, molybdenum cofactor biosynthesis) must be involved. The nitrate-reducing system in S. carnosus displays similarities to the dissimilatory nitrate reductases of Escherichia coli. However, in the S. carnosus nar promoter, no obvious Fnr and integration host factor recognition sites are present; only one site that is related to the E. coli NarL consensus sequence was found. Studies to determine whether the E. coli proteins NarL and Fnr are functional at the S. carnosus narGHJI promoter indicated that the promoter is not functional in E. coli.
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