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1.
Applied Biochemistry and Microbiology - The functioning and mechanism of P-glycoprotein (Pgp) regulation under conditions of inhibition of glutathione synthesis in human colon adenocarcinoma...  相似文献   

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The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.  相似文献   

4.
The Survival of Marine Bacteria under Starvation Conditions   总被引:1,自引:1,他引:1  
The survival under starvation conditions of two selected strains of marine bacteria, a yellow Pseudomonas sp. (strain 95A) and an unidentified oxidative peritrichate Gram negative rod (strain 41), was investigated. The 50% survival times of suspensions in phosphate buffer depended on cell density and were often more than 20 d. A capacity to scavenge atmospheric nitrogenous compounds led to a marked increase in the viability of cell suspensions of 104 cells/ml. Intracellular poly-β-hydroxybutyrate (PHB) prolonged the survival of strain 95A. Strain 41 contained more intracellular protein and this was degraded during starvation in ammonia-free air. Prolonged survival was not explicable in terms of low adenylate charge states. The 'maintenance energy'requirements of strains 95A and 41 in chemostat cultures were 0.042 and 0.04 g glucose/g dry wt/h respectively, compared with dilution-rate-dependent values of 0.051 to 0.856 for Escherichia coli. The low maintenance energy requirements would not alone explain the long viability. Thus no peculiar physiological property such as nitrogen-scavenging, ability to survive at the expense of intracellular PHB or protein, abnormally low cellular protein content, low maintenance energy requirements or a low adenylate charge state fully account for the starvation resistance of these marine bacteria.  相似文献   

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The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.The cell envelope of gram-negative bacteria mediates the communication of the cell with the environment, and it is responsible for many vital functions, including nutrient uptake and interaction with other bacteria and host cells. These activities are performed by a large collection of proteins that make the periplasm a cellular compartment with an even higher protein concentration than the cytoplasm (2). Bacteria are frequently exposed to multiple stresses such as heat shock, osmotic stress, and pH changes and are regularly challenged by the host immune system. Thus, the maintenance of periplasmic proteins in a fully functional state is a challenging task undertaken by the protein quality control system (5). It is generally accepted that under stress conditions misfolded proteins, protein fragments, and mislocalized membrane proteins appear, activating a stress response through three different signal transduction pathways (σE, Cpx, and Bae) (21, 22). Activation of this stress response in the periplasm triggers the upregulation of molecular chaperones, peptidases, proteases, and other enzymes with a role in eliminating or refolding damaged periplasmic proteins.The Escherichia coli HtrA protein (also called DegP or protease Do) is a periplasmic protein (4) that is upregulated under stress conditions such as heat shock (14, 15). HtrA functions as a chaperone and a protease in a temperature-dependent fashion (24). Recent studies have also shown that HtrA substrates targeted for degradation or refolding are recognized differently, suggesting that the mechanisms through which HtrA recognizes the substrate may play a role in the protease-chaperone switch (8).HtrA contains an N-terminal protease domain, followed by the PDZ1 and PDZ2 domains. In the absence of substrates, HtrA oligomerizes into a hexameric cage (12) that represents the resting state of the protein (10, 13). Upon binding to protein substrates, HtrA transforms into bigger cages formed by 12 or 24 monomers that encapsulate substrates for degradation or refolding (9, 13).HtrA is a 474-residue protein whose first 26 amino acids are removed at the N terminus most likely by a signal peptidase rendering the mature 48-kDa protein (14, 15). This form of the protein will hereon be referred to as full-length HtrA. However, it has been described (11, 23) that mature HtrA undergoes partial degradation both in vivo and in vitro as a consequence of self-cleavage occurring after Cys69 and Gln82 of the mature protein. These forms of the protein have been named short-HtrA (s-HtrA) (23).A similar phenomenon of autocleavage has been observed in other members of the HtrA family such as the human homologs HtrA1 (7) and HtrA2 proteins (6). The autocleavage process is not specific for proteases of the HtrA family. Several prokaryotic proteins involved in regulation of gene expression, such as the SOS response proteins LexA (16-18) and UmuD (3), are inactivated through a self-cleaving mechanism. Conversely, many mammalian proteases are produced as longer inactive precursors and depend on an intramolecular cleavage event to become active. This is the case for some gastric proteases such as pepsin and chymosin or the lysosome cathepsins D and E (1).Although autocleavage as a mechanism of activation or inactivation of certain proteases is well documented, the physiological role and the events triggering the self-cleavage of HtrA are poorly understood. In this study, we observed that the hexameric cage structure is required to observe autocleavage of HtrA. In addition, we analyzed the conditions that led to self-cleavage of HtrA and we found that the only requirement to observe accumulation of the s-HtrA form in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the degradation of protein substrates were sufficient to induce autocleavage. Therefore, considering the current functional model for HtrA (9, 13), our data suggest that the physiological role of the HtrA autocleavage is to eliminate the excess of HtrA protein expressed under stress conditions when the enzymatic activities of the protein are no longer needed.  相似文献   

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We present a study about AFM imaging of living, moving or self-immobilized bacteria in their genuine physiological liquid medium. No external immobilization protocol, neither chemical nor mechanical, was needed. For the first time, the native gliding movements of Gram-negative Nostoc cyanobacteria upon the surface, at speeds up to 900 µm/h, were studied by AFM. This was possible thanks to an improved combination of a gentle sample preparation process and an AFM procedure based on fast and complete force-distance curves made at every pixel, drastically reducing lateral forces. No limitation in spatial resolution or imaging rate was detected. Gram-positive and non-motile Rhodococcus wratislaviensis bacteria were studied as well. From the approach curves, Young modulus and turgor pressure were measured for both strains at different gliding speeds and are ranging from 20±3 to 105±5 MPa and 40±5 to 310±30 kPa depending on the bacterium and the gliding speed. For Nostoc, spatially limited zones with higher values of stiffness were observed. The related spatial period is much higher than the mean length of Nostoc nodules. This was explained by an inhomogeneous mechanical activation of nodules in the cyanobacterium. We also observed the presence of a soft extra cellular matrix (ECM) around the Nostoc bacterium. Both strains left a track of polymeric slime with variable thicknesses. For Rhodococcus, it is equal to few hundreds of nanometers, likely to promote its adhesion to the sample. While gliding, the Nostoc secretes a slime layer the thickness of which is in the nanometer range and increases with the gliding speed. This result reinforces the hypothesis of a propulsion mechanism based, for Nostoc cyanobacteria, on ejection of slime. These results open a large window on new studies of both dynamical phenomena of practical and fundamental interests such as the formation of biofilms and dynamic properties of bacteria in real physiological conditions.  相似文献   

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A summary of results of investigations by the author and a brief review of some literature data on human bone tissue deprived of mechanical loading (spaceflight, hypokinesia) is given. The direction and markedness of changes in bone mass—the bone mineral density and the bone mineral content—in different skeletal segments depend on their position relative to the gravity vector. A theoretically expected bone mass reduction was revealed in the trabecular structures of the bones of the lower part of the skeleton (local osteopenia). In the upper part of the skeleton, an increase in the bone mineral content is observed, which is considered as a secondary response and is due to redistribution of body fluids cephalad. The main cause of osteopenia is mechanical unloading. Arguments are presented that osteocyte osteolysis, delayed osteoblast histogenesis, and osteoclast resorption provoked by rearrangement in the hierarchy of the systems of volume regulation, ion regulation, and the endocrine regulation of calcium homeostasis are the main mechanisms of osteopenia.  相似文献   

9.
Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes of Mycobacterium smegmatis and other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain a penicillin-binding protein and serine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that an M. smegmatis ΔponA2 mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a ΔponA3 mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 when ponA3 is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied by M. smegmatis and other soil-dwelling mycobacteria.The cell envelope of mycobacteria is a complex carbohydrate- and lipid-rich entity and is a major factor contributing to the success of these organisms as saprophytic and pathogenic bacteria (7, 8, 29, 35). The innermost layer of the cell envelope is a peptidoglycan (PG) composed of N-acylmuramic acid and N-acetylglucosamine with l-alanyl (or glycyl in the case of Mycobacterium leprae)-d-isoglutaminyl-meso-diaminopimelyl-d-alanyl-d-alanine pentapeptides attached to the muramic acid residues (13, 16, 54). While some of the muramyl residues are N acetylated, as they are in most other bacteria, a majority of the muramyl residues are N glycolylated (2, 37, 48, 49), a modification that confers lysozyme resistance (53) and also influences the innate immune response to mycobacterial cell walls (10). The pentapeptide chains of the mycobacterial PG can be modified by amidation, glycylation, or methylation, but the functional significance of these modifications is unknown (28, 31, 32, 38, 54).Approximately 80% of the pentapeptides in mycobacterial PG are cross-linked, and a majority of the links are between the carboxyl group of a penultimate d-Ala residue in a pentapeptide precursor and the amino group of the side chain d center of a meso-diaminopimelic acid (DAP) residue from an adjacent peptide (referred to as a 4-3 cross-link), while approximately one-third of the links are between the carboxyl group of the l center of a DAP residue of one peptide and the amino group of the side chain d center of the DAP residue in an adjacent peptide (referred to as a 3-3 cross-link) (17, 65). The 4-3 linkage is considered the “standard” linkage and is catalyzed by classical, penicillin-sensitive dd-transpeptidases, while the novel 3-3 linkage is thought to be catalyzed by the concerted action of dd-carboxypeptidases and novel ld-transpeptidases (31, 34, 39-41). The reasons why bacteria produce both 4-3 and 3-3 linkages are unknown. Some workers have suggested that the 3-3 linkages might reinforce the wall during times of stress and under nonreplicating conditions or stabilize complex cell envelopes (17, 50, 51, 55). In this regard, the high percentage of 3-3 linkages found in the PG of mycobacteria and their predominance in stationary-phase M. tuberculosis cells (31) suggest that these linkages may have an important role in maintaining cell envelope integrity during periods of growth and under nonreplicating conditions.The enzymes involved in peptidoglycan assembly, the penicillin-binding proteins (PBPs), have a triad sequence motif that forms the transpeptidation active site ([SxxK]——[S/YxN/C]——[K/H][T/S]G), which is the target of the β-lactam class of antibiotics (for a review, see reference 18). The PBPs have been grouped into several classes based on this motif, surrounding sequences, and other structural features (18). Of interest here are the class A PBPs, which are high-molecular-weight (HMW) proteins with both a transglycosylase domain (also called a non-penicillin-binding module [n-PB]) and a transpeptidase domain (also called a penicillin-binding module [PB]) (18). These proteins are tethered to the cytoplasmic membrane by a transmembrane helix with the catalytic domains facing the outside of the cell. Mycobacteria have been reported to have only two genes that encode class A PBPs, ponA1 and ponA2, which are annotated Rv0051 and Rv3682 in the sequence genome of M. tuberculosis H37Rv (9, 17). Previous studies that analyzed collections of transposon mutants to obtain clones with various phenotypes identified strains with insertions in these two genes. The phenotypes of these mutants have clearly shown that these PBPs play a complex role in mycobacterial physiology. One group of workers found a ponA1 mutant of M. smegmatis in a search for mutants with an altered dye-binding phenotype (an indicator of changes in the cell envelope) and showed that this slowly growing mutant was hypersusceptible to β-lactam antibiotics and had altered permeability (6). A ponA2 mutant of M. smegmatis was discovered in a screen for mutants defective for survival during long-term culture (25), while other workers isolated an M. tuberculosis ponA2 mutant in a screen for mutants sensitive to low pH (61). The same group of workers also showed that the M. tuberculosis mutant was more sensitive to heat, H2O2, and NO and was attenuated for persistence in the mouse model of inhalation tuberculosis (62). We previously identified an M. tuberculosis ponA2 mutant in a screen for mutants hypersusceptible to β-lactam antibiotics (14). All of these studies identified transposon mutants in searches for mutants with specific phenotypes, but there have been no direct genetic studies that have specifically examined the function of these PBPs in peptidoglycan metabolism.In this study we demonstrated that M. smegmatis has three class A PBPs. We show here that a newly recognized protein, which we designated PonA3, is a paralog of the PonA2 protein and is found only in certain environmental species of mycobacteria. We analyzed the phenotypes of M. smegmatis mutants with in-frame deletions of ponA2 and ponA3 singly and in combination to increase our understanding of the role that these PBPs play in mycobacterial peptidoglycan biology.  相似文献   

10.
Direct Selection for P1-Sensitive Mutants of Enteric Bacteria   总被引:12,自引:36,他引:12       下载免费PDF全文
A method has been developed to isolate mutants sensitive to coliphage P1 from bacterial genera normally not sensitive to this phage. P1clr100KM was used. This phage is heat inducible and confers kanamycin resistance when present as a prophage (in lysogens). P1-sensitive mutants of Klebsiella, Enterobacter, Citrobacter, and Erwinia have been found. This technique provides a well-known genetic system for the study of many bacterial genera that previously had either no such system or only a marginally useful means of genetic manipulation. It also extends the range of possible intergeneric hybrids that may be constructed and studied.  相似文献   

11.
Escherichia coli contains two PII-like signal trans-duction proteins, PII and GlnK, involved in nitrogen assimilation. We examined the roles of PII and GlnK in controlling expression of glnALG, glnK and nac during the transition from growth on ammonia to nitrogen starvation and vice versa. The PII protein exclusively controlled glnALG expression in cells adapted to growth on ammonia, but was unable to limit nac and glnK expression under conditions of nitrogen starvation. Conversely, GlnK was unable to limit glnALG expression in cells adapted to growth on ammonia, but was required to limit expression of the glnK and nac promoters during nitrogen starvation. In the absence of GlnK, very high expression of the glnK and nac promoters occurred in nitrogen-starved cells, and the cells did not reduce glnK and nac expression when given ammonia. Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation. GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia. After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia. In contrast, wild-type cells that were starved immediately resumed rapid growth when fed ammonia. Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild-type cells. This complex phenotype resulted partly from the requirement for GlnK to regulate nac expression; deletion of nac restored wild-type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation. The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the glnK promoter in cells lacking GlnK restored the wild-type phenotypes.  相似文献   

12.
Bryan JK 《Plant physiology》1990,92(3):785-791
Homoserine dehydrogenase is associated with the multibranched pathway of amino acid biosynthesis originating with aspartic acid. Like most of the related pathway enzymes, this enzyme is localized in chloroplasts. The activity and regulatory properties of the threonine-sensitive isozyme of homoserine dehydrogenase isolated from Zea mays var earliking were examined under variable conditions that could exist within chloroplasts. Catalytic activity is not significantly altered within the range of pHs that occur within these organelles, but inhibition of the enzyme by the pathway product, l-threonine, is markedly diminished at the alkaline pHs characteristic of illuminated chloroplasts. Inhibition by threonine is also subject to modulation by physiological levels of NADPH. Under conditions considered to represent the environment within unilluminated chloroplasts, the enzyme is severely inhibited by micromolar concentrations of threonine, but significant enzyme activity is retained under conditions that are likely to occur during illumination, even in the presence of millimolar levels of threonine. These results indicate that homoserine dehydrogenase may be subject to environmentally mediated regulation in vivo. Other observations support this concept and suggest that the intrinsic catalytic and regulatory properties of key enzymes could facilitate a direct link between light-dependent carbon and nitrogen assimilation and amino acid biosynthesis in chloroplasts of higher plants.  相似文献   

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Rapid Determination of the Presence of Enteric Bacteria in Water   总被引:1,自引:4,他引:1       下载免费PDF全文
A rapid and sensitive method is described for the detection of bacteria in water and various other natural substrates by the isolation of specific bacteriophage. By the addition of large numbers of the organism in question to the sample, the presence of virulent bacteriophage can be demonstrated in as little as 6 to 8 h. Fecal coliform, total coliform, and total coliphage counts were determined for over 150 water samples from several geographical areas over a period of 2 years. Computer analysis of the data shows a high degree of correlation between fecal coliforms and the coliphage present in the samples. With a high correlation coefficient between fecal coliform and coliphage counts, predictions of the fecal coliforms may be made by enumeration of the phage.  相似文献   

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Constant high rates of dislocation-related complications of total hip replacements (THRs) show that contributing factors like implant position and design, soft tissue condition and dynamics of physiological motions have not yet been fully understood. As in vivo measurements of excessive motions are not possible due to ethical objections, a comprehensive approach is proposed which is capable of testing THR stability under dynamic, reproducible and physiological conditions. The approach is based on a hardware-in-the-loop (HiL) simulation where a robotic physical setup interacts with a computational musculoskeletal model based on inverse dynamics. A major objective of this work was the validation of the HiL test system against in vivo data derived from patients with instrumented THRs. Moreover, the impact of certain test conditions, such as joint lubrication, implant position, load level in terms of body mass and removal of muscle structures, was evaluated within several HiL simulations. The outcomes for a normal sitting down and standing up maneuver revealed good agreement in trend and magnitude compared with in vivo measured hip joint forces. For a deep maneuver with femoral adduction, lubrication was shown to cause less friction torques than under dry conditions. Similarly, it could be demonstrated that less cup anteversion and inclination lead to earlier impingement in flexion motion including pelvic tilt for selected combinations of cup and stem positions. Reducing body mass did not influence impingement-free range of motion and dislocation behavior; however, higher resisting torques were observed under higher loads. Muscle removal emulating a posterior surgical approach indicated alterations in THR loading and the instability process in contrast to a reference case with intact musculature. Based on the presented data, it can be concluded that the HiL test system is able to reproduce comparable joint dynamics as present in THR patients.  相似文献   

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Pantoja O  Gelli A  Blumwald E 《Plant physiology》1992,100(3):1137-1141
Patch-clamp techniques were employed to study the electrical properties of vacuoles from sugar beet (Beta vulgaris) cell suspensions at physiological concentrations of cytoplasmic Ca2+. Vacuoles exposed to K+ malate revealed the activation of instantaneous and time-dependent outward currents by positive membrane potentials. Negative potentials induced only instantaneous inward currents. The time-dependent outward currents were 10 times more selective for malate than for K+ and were completely blocked by zinc. Vacuoles exposed to KCl developed instantaneous currents when polarized to positive or negative membrane potentials. The time-dependent outward channels could serve as the route for the movement of malate into the vacuole, whereas K+ could move through the time-independent inward and outward channels.  相似文献   

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Previous studies showed that Salmonella typhimurium apparently senses external nitrogen limitation as a decrease in the concentration of the internal glutamine pool. To determine whether the inverse relationship observed between doubling time and the glutamine pool size in enteric bacteria was also seen in phylogenetically distant organisms, we studied this correlation in Bacillus subtilis, a gram-positive, sporulating bacterium. We measured the sizes of the glutamine and glutamate pools for cells grown in batch culture on different nitrogen sources that yielded a range of doubling times, for cells grown in ammonia-limited continuous culture, and for mutant strains (glnA) in which the catalytic activity of glutamine synthetase was lowered. Although the glutamine pool size of B. subtilis clearly decreased under certain conditions of nitrogen limitation, particularly in continuous culture, the inverse relationship seen between glutamine pool size and doubling time in enteric bacteria was far less obvious in B. subtilis. To rule out the possibility that differences were due to the fact that B. subtilis has only a single pathway for ammonia assimilation, we disrupted the gene (gdh) that encodes the biosynthetic glutamate dehydrogenase in Salmonella. Studies of the S. typhimurium gdh strain in ammonia-limited continuous culture and of gdh glnA double-mutant strains indicated that decreases in the glutamine pool remained profound in strains with a single pathway for ammonia assimilation. Simple working hypotheses to account for the results with B. subtilis are that this organism refills an initially low glutamine pool by diminishing the utilization of glutamine for biosynthetic reactions and/or replenishes the pool by means of macromolecular degradation.  相似文献   

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