The cysteine transport system of Saccharomyces cerevisiae.

Although Saccharomyces cerevisiae strains had different cysteine uptake activities, they revealed monophasic uptake kinetics and had the same KT (83.3 microM). The optimal pH of cysteine uptake was between 4.5 and 5.0, but the activity was quickly lost if cells were kept in buffer. When the activity was measured in the growth medium, it increased in the presence of EDTA and greatly decreased in the presence of mercuric chloride. Thioglycol as well as metabolic inhibitors such as dinitrophenol and azide were inhibitory. Homocysteine and methionine were competitive and non-competitive inhibitors, respectively. Cysteamine and cysteic acid were not inhibitory. From these observations, we conclude that the system mediating uptake of cysteine is specific (we thus name it the cysteine transport system) and that the cysteine transport system recognizes not only the SH-group but also amino- and carboxyl-groups. In wild-type strains the cysteine transport system was derepressed only when the cells were incubated without any sulfur source. On the other hand, in cysteine-dependent mutants, cysteine uptake activity increased with increase of exogenous supply of cysteine, glutathione or methionine. From this result, we suspect that the cellular cysteine level is the limiting factor for biosynthesis of the cysteine transport system in cysteine-dependent strains.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats VI. Nitrite monitoring during storage of pasteurized pork slurries

Residual nitrite levels were monitored during storage for up to 6 months, in a model pork slurry system used to study the relative effects of curing ingredients and additives used in pasteurized cured meats to control the growth of Clostridium botulinum.
In 'low' pH slurries the rate of loss of nitrite fell with reducing storage temperature. Less residual nitrite remained after HIGH heat treatment but the rate of loss of that residual nitrite was slower during storage than nitrite remainly after LOW heat treatment. Inclusion of nitrate resulted in higher residual nitrite levels, particularly after HIGH heat and if stored below 20°C. If isoascorbate was added nitrite became undetectable within circa 30 days, even when nitrate had been added. The rate of loss of nitrite was slower in 'high' pH slurries (pH 6.3–6.8).
Monitoring levels of nitrite in the product soon after production would detect its accidental overuse but monitoring nitrite in the product during distribution or at retail, without knowledge of the composition and prior history of the product, gives little indication of the amount used at manufacture. The level of residual nitrite was not directly related to the ability of the curing mixture to control the growth of CI. botulinum types A and B. Some slurries in which C1. botulinum grew least during 6 months' storage contained no residual nitrite because isoascorbate was also present.     

发酵红皮萝卜中硝酸还原菌的分离筛选

研究了发酵红皮萝卜中细菌和大肠杆菌数量与亚硝酸盐含量变化的规律,从中分离得到10株菌株,通过硝酸盐还原能力的测定,筛选出9株具有硝酸盐还原能力的菌株,其中E5和E6两个菌株硝酸盐还原能力最强,并测定了2个菌株硝酸还原酶(NR)的酶活及pH对酶活的影响。进而对筛选的E5、E6两个菌株进行了分子生物学鉴定。结果表明,E5为阴沟肠杆菌(Enterobacter cloacae),E6为克雷伯氏菌(Klebsiella oxytoca)。     

The KlTrk1 gene encodes a low affinity transporter of the K+ uptake system in the budding yeast Kluyveromyces lactis

Potassium uptake in Saccharomyces cerevisiae is mediated by at least two proteins, known as Trk1p and Trk2p. Direct involvement in cation movements has been demonstrated for Trk1p, which is the high affinity transporter. S. cerevisiae cells also show low affinity potassium uptake, perhaps mediated by Trk2p. Mutants lacking Trk1p, lose high affinity system, but when grown with moderate potassium concentrations, Trk2p seems to replace it. Mutants lacking both proteins are viable but require at least 10 mM K(+) in the medium to sustain growth. Here we report the cloning and characterization of a gene from Kluyveromyces lactis encoding a homologue of these two proteins. KlTrkp is a 1070 amino acid peptide that shows, overall, higher homology with Trk2p than with Trk1p, and its disruption gives rise to cells with deficient potassium transport and with an increased K(+) requirement for normal growth. Determination of kinetic parameters in the K. lactis wild-type and Kltrk1Delta strains, as well as in Sctrk1Delta Sctrk2Delta S. cerevisiae cells expressing KlTrk1, indicated that this is a low affinity component of a major potassium uptake system in K. lactis.     

High-affinity glucose uptake in Saccharomyces cerevisiae is not dependent on the presence of glucose-phosphorylating enzymes

Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.     

Relationship between nitrate/nitrite reductase activities in meat associated staphylococci and nitrosylmyoglobin formation in a cured meat model system

Quantitative determination of catalase, nitrate reductase, nitrite reductase and nitric oxide synthase activities (NOS) was performed on 11 different bacterial strains, mainly staphylococci, isolated from fermented sausages, bacon brine or cured meat products. All except one strain possessed catalase activity in the range from 1.0 to 6.1 μmol min^− 1 ml^− 1. Ten out of 11 bacteria strains showed nitrate reductase activity in the range between 50 and 796 nmol min^− 1 ml^− 1 and nine showed nitrite reductase activity in the range between 6 and 42 nmol min^− 1 ml^− 1. No evidence of NOS activity of the selected strains was detected. In a colour formation assay containing myoglobin all strains affected nitrosylmyoglobin (MbFe^IINO) formation in assays containing nitrite, whereas only strains having nitrate reductase activity generated MbFe^IINO in assays containing nitrate as the sole nitrosylating agent. The quantitative nitrate and nitrite reductase activity did not fully explain or correlate well with the observed rate of formation of MbFe^IINO, which seemed to be more affected by the growth rate of the different strains. The mechanism of the reduction of nitrite into NO of strains not having nitrite reductase activity remains to be fully elucidated, but could be due to a dual-mode action of nitrate reductase capable of acting on nitrate.     

白肋烟晾制期间烟叶中细菌的分离和鉴定

对晾制烟叶中分离到的33株优势菌株进行16S rDNA鉴定,鉴定结果为Pseudomonas、Bacillus、Enterobacter、Rhizobium、Corynebacterium、Pantoea、Acinetobacter、Arthrobacter、Xanthomonas、Paracoccus、Achromobacter、Rhodococcus。经硝酸盐和亚硝酸盐还原能力测定,结果表明:6株不能还原硝酸盐和亚硝酸盐,占总菌株数的18.18%;20株能还原硝酸盐而不能还原亚硝酸盐,占总菌株数的60.61%;7株既可以还原硝酸盐又可以还原亚硝酸盐(2株Rhizobium、1株Paracoccus、4株Pseudomonas),占总菌株数的21.21%,可能是抑制白肋烟叶中TSNA形成的菌株。      

Effects of nitrate or nitro supplementation, with or without added chlorate, on Salmonella enterica serovar Typhimurium and Escherichia coli in swine feces

The effects of coincubating the active agent of an experimental chlorate product with nitrate or select nitro compounds, possible inducers and competing substrates for the targeted respiratory nitrate reductase, on concentrations of experimentally inoculated Salmonella enterica serovar Typhimurium and indigenous Escherichia coli were determined. Studies were completed in swine fecal suspensions as a prelude to the administration of these inhibitors to pigs. Results confirmed the bactericidal effect of chlorate (5 to 10 mM) against these fecal enterobacteria, reducing (P < 0.05) concentrations by > 2 log CFU ml(-1) after 3 to 6 h of incubation. An effect (P < 0.05) of pH was observed, with considerable regrowth of Salmonella and E. coli occurring after 24 h of incubation in suspensions buffered to pH 7.1 but not in suspensions buffered to pH 6.5 or 5.6. A 24-h coincubation of fecal suspensions with 5 to 10 mM chlorate and as little as 2.5 mM nitrate or 10 to 20 mM 2-nitro-1-propanol, 2-nitroethanol, and, sometimes, nitroethane decreased (P < 0.05) Salmonella but not necessarily E. coli concentrations. 2-Nitro-1-propanol and 2-nitroethanol exhibited inhibitory activity against Salmonella and E. coli by an undetermined mechanism, even in the absence of added chlorate.     

Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella typhimurium DT104 in rumen contents in vitro

Escherichia coli O157:H7 and Salmonella Typhimurium DT104 are important foodborne pathogens affecting the beef and dairy industries and strategies are sought to rid these organisms from cattle at slaughter. Both pathogens possess respiratory nitrate reductase that also reduces chlorate to the lethal chlorite ion. Because most anaerobes lack respiratory nitrate reductase, we hypothesized that chlorate may selectively kill E. coli O157:H7 and Salmonella Typhimurium DT104 but not potentially beneficial anaerobes. In support of this hypothesis, we found that concentrations of E. coli O157:H7 and Salmonella Typhimurium DT104 were reduced from approximately 1,000,000 colony forming units (CFU) to below our level of detection (< or = 10 CFU) following in vitro incubation (24 h) in buffered ruminal contents (pH 6.8) containing 5 mM added chlorate. In contrast, chlorate had little effect on the most probable number (mean +/- SD) of total culturable anaerobes (ranging from 9.9 +/- 0.72 to 10.7 +/- 0.01 log10 cells/ml). Thus, chlorate was bactericidal to E. coli O157:H7 and Salmonella Typhimurium DT104 but not to potentially beneficial bacteria. The bactericidal effect of chlorate was concentration dependent (less at 1.25 mM) and markedly affected by pH (more bactericidal at pH 6.8 than pH 5.6).     

Dried sausages fermented with Staphylococcus xylosus at different temperatures and with different ingredient levels - Part I. Chemical and bacteriological data

Sausages with added Staphylococcus xylosus were fermented at different temperatures and with different added levels of salt, glucose, nitrite, nitrate and Pediococcus pentosaceus in accordance with a six factor fractional design. The numbers of surviving Staphylococcus xylosus, lactic acid bacteria, pH, free fatty acids and residual amounts of nitrite and nitrate were measured. The effects of temperature and different ingredients on the chemical and bacterial data were tested using multiple linear regression and analysis of variance. The study showed that numbers of surviving Staphylococcus xylosus were reduced by high fermentation temperature, especially when salt concentration was low and glucose concentration high. High levels of nitrite and addition of Pediococcus pentosaceus had adverse effects as well. pH was lowered by high fermentation temperature, low salt concentration and addition of glucose and Pediococcus pentosaceus. On the other hand p H was increased by addition of nitrate. The pH-lowering effect of glucose was small when temperature was low. The residual levels of nitrite and nitrate were increased by addition of nitrate, but then increased and decreased, respectively, by increasing temperature. Addition of glucose and Pedio coccus pentosaceus lowered the residual amount of nitrite. The level of free fatty acids was reduced by salt and increased by higher temperature. Of the individual acids, the amount of linoleic acid was also increased by addition of Pediococcus pentosaceus and decreased by nitrate.     

A comparative study on the transport of L(-)malic acid and other short-chain carboxylic acids in the yeast Candida utilis: Evidence for a general organic acid permease

Cells of the yeast Candida utilis grown in medium with short-chain mono-, di- or tricarboxylic acids transported L(-)malic acid by two transport systems at pH 3·0. Results indicate that probably a proton symport for the ionized form of the acid and a facilitated diffusion for the undissociated form were present. Dicarboxylic acids such as succinic, fumaric, oxaloacetic and α-ketoglutaric acids were competitive inhibitors of the malic acid for the high-affinity system, suggesting that these acids used the same transport system. In turn, competitive inhibition uptake studies of labelled carboxylic acid in the low-affinity range indicated that this system was non-specific and able to accept not only carboxylic (mono-, di- or tri-) acids but also some amino acids. Additionally, under the same growth conditions, C. utilis produced two mediated transport systems for lactic acid: a proton symport for the anionic form which appeared to be a common monocarboxylate carrier and a facilitated diffusion system for the undissociated acid displaying a substrate specificity similar to that observed for the low-affinity dicarboxylic acid transport. The mediated carboxylic acid transport systems were inducible and subjected to repression by glucose. In glucose-grown cells the undissociated dicarboxylic acids entered the cells slowly by simple diffusion. Repressed glucose-grown cells were only able to produce both transport systems if an inducer, at low concentration (0·5%, w/v), was present during starvation in buffer. This process was inhibited by the presence of cycloheximide indicating that induction requires de novo protein synthesis. If a higher acid concentration was used, only the low-affinity transport system was detectable, showing that the high-affinity system was also repressed by high concentrations of the inducer.     

Effect of sodium chlorate on Salmonella typhimurium concentrations in the weaned pig gut

Salmonella cause economic losses to the swine industry due to disease and compromised food safety. Since the gut is a major reservoir for Salmonella, strategies are sought to reduce their concentration in pigs immediately before processing. Respiratory nitrate reductase activity possessed by Salmonella also catalyzes the intracellular reduction of chlorate (an analog of nitrate) to chlorite, which is lethal to the microbe. Since most gastrointestinal anaerobes lack respiratory nitrate reductase, we conducted a study to determine if chlorate may selectively kill Salmonella within the pig gut. Weaned pigs orally infected with 8 x 10(7) CFU of a novobiocin- and nalidixic acid-resistant strain of Salmonella Typhimurium were treated 8 and 16 h later via oral gavage (10 ml) with 0 or 100 mM sodium chlorate. Pigs were euthanized at 8-h intervals after receiving the last treatment. Samples collected by necropsy were cultured qualitatively and quantitatively for Salmonella and for most probable numbers of total culturable anaerobes. A significant (P < 0.05) chlorate treatment effect was observed on cecal concentrations of Salmonella, with the largest reductions occurring 16 h after receiving the last chlorate treatment. An observed treatment by time after treatment interaction suggests the chlorate effect was concentration dependent. Chlorate treatment may provide a means to reduce foodborne pathogens immediately before harvest.     

Transport of L-leucine in the fission yeast Schizosaccharomyces pombe

Transport of L -leucine into Schizosaccharomyces pombe cells from the stationary phase of growth (after preincubation for 60 min with 1% glucose) proceeds uphill, practically unidirectionally, and is mediated by at least two systems: a high-affinity system with a K_T of 0·045 mmol 1^?1 and J_max of 3·3 nmol min^?1 (mg dry weight)^?1 and a low-affinity system with a K_T of 1·25 mmol 1^?1 and J_max of 16·0 nmol min^?1 (mg dry weight)^?1. The high-affinity system has a pH optimum at 3.2, the accumulation ratio is highest at a cell density of 2–4 mg dry weight per ml and decreases with increasing leucine concentration. Transport of leucine by the high-affinity system is strongly inhibited by proton conductors, ammonium ions and by most amino acids, but only L -phenylalanine, L -isoleucine, L -valine and L -cysteine behave as fully competitive inhibitors. Systems of L -leucine transport in S. pombe are not constitutive. Transport activity appears only after preincubation of cells with a suitable source of energy. If cycloheximide is added during preincubation with glucose, no transport systems for leucine are synthesized. After removal of glucose, the activity of transport systems decays with a half-time of about 20 min. The presence of cyclic AMP increases the initial rate of leucine uptake only in cells preincubated with glucose and in the absence of cycloheximide.     

Monitoring nitrite and nitrate residues in frankfurters during processing and storage

Frankfurter-type sausages were prepared in a pilot plant with different concentrations of NaNO(2) (75, 125 or 250 ppm) combined or not with 200 ppm KNO(3). A meat system, free of curing agents, was also used as control. Nitrite and nitrate levels were tested in various processing steps and over 120 days storage at 3 °C of the vacuum-packaged frankfurters. Little influence of the originally added nitrite level on the amount of nitrate formed was observed. Important losses of nitrite and nitrate were due to cooking. Thereafter about 50% of the nitrite added initially remained in this form in all samples (39, 59 and 146 ppm, respectively) and between 10 and 15% as nitrate. When only nitrate was initially added, formation of nitrite after cooking was observed (maximum level 43 ppm NaNO(2)). Formulations prepared with both nitrate and nitrite showed no significant differences (p < 0.01) respect to their nitrite or nitrate counterparts. A good correlation among nitrite and nitrate levels and storage time was showed by multiple linear regression analysis. It is concluded that the use of nitrate in combination with nitrite in cooked meat products seems to have little technological significance and adds to the total body burden of nitrite.     

Metabolic behavior of denitrifying phosphate-accumulating organisms under nitrate and nitrite electron acceptor conditions

The effects of various types of electron acceptors on anoxic phosphorus uptake were investigated in detail to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms. Batch experimental tests under three different electron acceptor conditions, i.e., nitrate, nitrite and mixtures of nitrate and nitrite, were carried out using activated sludge cultivated in a sequencing batch reactor. The experimental results confirmed no inhibition of the utilization of nitrate or nitrite as an electron acceptor for anoxic phosphorus uptake. Anoxic phosphorus uptake occurred provided there was an electron acceptor present regardless of whether it was nitrate or nitrite. However, for nitrite a relatively small amount of anoxic phosphorus was taken up per nitrogen denitrified compared to nitrate. On the other hand, the amount of anoxic phosphorus taken up per nitrogen denitrified increased with an increase in the initial loading amount of electron acceptor in the case of nitrate, whereas it slightly decreased nitrite. Moreover, the amount of phosphorus taken up per nitrogen denitrified decreased with increasing mixed liquor suspended solid (MLSS) concentration in the case of nitrate, while it slightly increased for nitrite. From these results, it was confirmed that the activity of anoxic phosphorus uptake is strongly associated with the type and the initial loading amount of electron acceptor and the MLSS concentration under anoxic conditions.     

Kinetics of nitrate, nitrite, and Cr(VI) reduction by iron metal

The kinetics of nitrate, nitrite, and Cr(VI) reduction by three types of iron metal (Fe0) were studied in batch reactors for a range of Fe0 surface area concentrations and solution pH values (5.5-9.0). At pH 7.0, there was only a modest difference (2-4x) in first-order rate coefficients (k(obs)) for each contaminant among the three Fe0 types investigated (Fisher, Peerless, and Connelly). The k(obs) values at pH 7.0 for both nitrite and Cr(VI) reduction were first-order with respect to Fe0 surface area concentration, and average surface area normalized rate coefficients (kSA) of 9.0 x 10(-3) and 2.2 x 10(-1) L m(-2) h(-1) were determined for nitrite and Cr(VI), respectively. Unlike nitrite and Cr(VI), Fe0 surface area concentration had little effect on rates of nitrate reduction (with the exception of Connelly Fe0, which reduced nitrate at slower rates at higher Fe0 surface areas). The rates of nitrate, nitrite, and Cr(VI) reduction by Fisher Fe0 decreased with increasing pH with apparent reaction orders of 0.49 +/- 0.04 for nitrate, 0.61 +/- 0.02 for nitrite, and 0.72 +/- 0.07 for Cr(VI). Buffer type had minimal effects on reduction rates, indicating that pH was primarily responsible for the differences in rate. At high pH values, Cr(VI) reduction ceased after a short time period, and negligible nitrite reduction was observed over 48 h.     

大白菜发酵过程中亚硝酸盐消长规律的研究

对大白菜在发酵过程中亚硝酸盐的形成规律进行了研究。结果发现 ,亚硝酸盐的出现期主要集中在发酵前期 ,随发酵液酸度的增加亚硝酸盐逐渐消失。亚硝峰的形成主要是由杂菌将硝酸盐还原为亚硝酸盐的作用 ,与大白菜体内硝酸还原酶无直接关系。亚硝峰消失的原因一是在酸性条件下的化学降解 ,二是乳酸菌对亚硝酸盐的还原作用。纯接种发酵可降低亚硝峰 ,无论是接入单一菌株还是接入混合菌株都能明显地降低亚硝峰 ,尤以混合接种效果最佳     

Transport of lactic acid in Kluyveromyces marxianus: evidence for a monocarboxylate uniport.

Lactic acid-grown cells of a strain of Kluyveromyces marxianus transported D- and L-lactic acid by a saturable mechanism that was partially inducible and subject to glucose repression, with the following kinetic parameters at pH 5.4: Vmax = 1.00 (+/- 0.13) mmol h-1 per g dry weight and Ks = 0.42 (+/- 0.08) mM. Lactic acid transport was competitively inhibited by pyruvic, glycolic, acetic and bromoacetic acids. The latter, a non-metabolizable analogue, was transiently accumulated, the extent depending on the extracellular pH. The pH dependence of the Ks values for undissociated lactic acid and for the lactate anion indicated that the latter was the transported species. Lactate uptake was not accompanied by the simultaneous uptake of protons, potassium ions or sodium ions excluding symport mechanisms. Initial lactic acid uptake led to transient membrane hyperpolarization as measured with a fluorescent dye excluding also an electroneutral anion antiport mechanism. It was concluded that lactate anions use a monocarboxylate uniport and that the counter anion, possibly bicarbonate, uses a separate channel, the coupling being electrical and loose.