Genetic Analysis of Regulation of the RecF Pathway of Recombination in Escherichia coli K-12

Genetic evidence is provided supporting the hypothesis that one or more genes of the RecF pathway of recombination other than recA are controlled by the lexA repressor. Using lexA, recA, and recA operator mutations, we also analyze the role of recA and sbcB in regulating the RecF pathway.     

Pathway Choice in Glutamate Synthesis in Escherichia coli

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog α-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.     

Regulation of the methionine regulon in Escherichia coli

The genes involved in methionine biosynthesis are scattered throughout the Escherichia coli chromosome and are controlled in a similar but not coordinated manner. The product of the metJ gene and S-adenosylmethionine are involved in the repression of this ‘regulon’.     

MAL1基因启动子区的克隆及其在大肠杆菌中的启动功能分析

目的：MAL1基因启动子区在原核生物大肠杆菌中是否有双向启动外源基因的功能。方法：利用PCR扩增MAL1基因启动子区,将不同方向的MAL1基因启动子区后接上Zeocin基因,构建成重组报告质粒,转化大肠杆菌DH5α,验证该启动子区是否双向都具有启动外源基因的功能。结果：将含不同方向启动子区的重组质粒PUCMZ1和PUCMZ2,转化大肠杆菌,转化子均有Zeoein抗性。结论：证明了MAL1结构基因上游启动子区在原核生物大肠杆菌中具有双向启动的功能,且3′-5′方向的启动能力强于5′-3′方向。     

Promoter recognition and promoter strength in the Escherichia coli system.

The strength of Escherichia coli promoters in vivo as well as the rates of association between RNA polymerase and promoter sequences differ by more than an order of magnitude. Since efficient promoter recognition and rapid binding of the enzyme might be a prerequisite for exceptional promoter strength we have determined the forward rate constants kon (as well as koff) for nine promoters including PL, PA1, and PN25 from phages lambda, T7, and T5, respectively as well as Pbla and PlacUV5 from E. coli. The second order forward rate constants span a 30-fold range from 1 X 10(7) M-1 s-1 for Pbla and PL up to 2.9 X 10(8) M-1 S-1 for PN25. Little correlation between 'promoter recognition' as defined by the rate of complex formation of a promoter sequence with RNA polymerase and its strength in vivo as defined by the rate of RNA synthesis has been found. This adds to the evidence that the complex functional pathway encoded in a promoter sequence can be limited at various levels and that promoter strength in vivo is the result of an optimization process involving more than just one functional parameter.     

Regulation of Escherichia coli phosphofructokinase in situ

The activity of E. coli phosphofructokinase in situ has been studied in cells permeabilized to its substrates, products and effectors by a toluene-freezing treatment. The in situ enzyme exhibits moderate cooperativity in respect to F6P (n_H up to 2.0), rather low affinity for ATP (with Km up to 1 mM when saturated with F6P), activation by ADP, and inhibition, within the physiological range of concentrations, by high ATP and phosphoenolpyruvate. This behaviour of the enzyme in situ at concentrations of the effector metabolites as those reported in intact cells in glycolytic and gluconeogenic conditions could account for the changes of phosphofructokinase activity needed for metabolic regulation in vivo.     

Regulation of Cell Division in Escherichia coli

The rate of cell division was measured in cultures of Escherichia coli B/r strain after periods of partial or complete inhibition of deoxyribonucleic acid (DNA) synthesis. The rate of DNA synthesis was temporarily decreased by removing thymidine from the growth medium or replacing it with 5-bromouracil. After restoration of DNA synthesis, a temporary period of accelerated cell division was observed. The results were consistent with the idea that chromosome replication begins when an initiator complement of fixed size accumulated in the cell. The increase in the potential for the initiation of new replication points during inhibition of DNA synthesis results in an increase in the rate of cell division after an interval which encompasses the time for the arrival of these replication points to the termini of the chromosomes and the time from this event to division.     

Regulation of Intracellular Proteolysis in Escherichia coli

Individual nitrogenous metabolites have been examined as regulating agents for the breakdown of intracellular proteins in Escherichia coli. Generally, NH(4) (+) is the most effective regulator. Its depletion progressively increases the basal proteolytic rate to maximum in most strains when the doubling time is increased to 2 h. In E. coli 9723, the rate is further increased at longer doubling times. Amino acids have individual effects on intracellular proteolysis. The basal rate in amino acid-requiring auxotrophs of E. coli 9723 is stimulated weakly by starvation for histidine, tryptophan, or tyrosine, moderately by four other amino acid depletions, and more strongly by eight others. The degree of stimulation roughly correlates with the frequency of the amino acid in the cell proteins. Amino acid analogues that incorporate extensively into protein generally slightly inhibit intracellular proteolysis, except for selenomethionine, which is slightly stimulatory. Metabolic inhibitors were studied at graded concentrations. Chloramphenicol inhibits the basal level of intracellular proteolysis when protein synthesis is slightly or moderately inhibited, and stimulates proteolysis slightly at higher levels. Graded inhibition of ribonucleic acid synthesis with rifampin progressively stimulates intracellular proteolysis. Uracil depletion is also stimulatory. Inhibition of deoxyribonucleic acid synthesis with mitomycin C or by thymine starvation slightly inhibits intracellular proteolysis. Intracellular proteolysis is postulated to be regulated primarily by active ribosomal function. At 43 to 45 C, intracellular proteolysis becomes maximally induced and unresponsive to normal regulatory control by metabolites. Most regulation is directed towards the breakdown of the more stable cell proteins. Total proteolysis in all cell proteins is no more than doubled by the most effective conditions of starvation.     

Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.