Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam.

cysB and cysE strains were obtained as spontaneous mecillinam-resistant mutants of Salmonella typhimurium. The resistance to mecillinam was caused by the cys mutations which also conferred tolerance to lethal cell shape mutations. Most, but not all, cysB and cysE mutations from other origins displayed the same behavior. Resistance was abolished by O- and N-acetylserine in cysE mutants; by thiosulfate, sulfite, and sulfide in cysB mutants; and by cysteine in both types of mutants. It is concluded that an event involved in mecillinam action requires the inducer and the activator protein of the cysteine regulon.     

Cysteine auxotrophs of Salmonella typhimurium which grow without cysteine in a hydrogen/carbon dioxide atmosphere.

Cysteine auxotrophs of Salmonella typhimurium mutated in cysB, cysI or cysJ grew with sulphate as a sulphur source when incubated under a hydrogen/carbon dioxide atmosphere. Yields obtained under these conditions were equivalent to those characteristic of wild-type S. typhimurium. The same mutants failed to grow with sulphate as a sulphur source when incubated aerobically. Auxotrophs mutated in cysA, cysC, cysD, cysE, cysG and cysH required cysteine for growth under both incubation conditions. The results suggest that mutations in cysB (regulation of the several cys operons) and also cysI and cysJ (sulphite reductase activity) can be circumvented during anaerobic growth under hydrogen.     

Positive control of sulphate reduction in Escherichia coli. The nature of the pleiotropic cysteineless mutants of E. coli K 12

1. The function of the wild-type alleles of the pleiotropic mutants cysB and cysE of Escherichia coli was investigated. 2. The wild-type allele cysB(+) is dominant to the mutant allele cysB in stable and transient heterozygotes. 3. The wild-type allele cysE(+) is dominant to the mutant allele cysE, as predicted. 4. Sulphur-starved cultures of cysB or cysE strains contain less than 0.2nmole of free cysteine/mg. dry wt. 5. Complementation in vitro is not observed between extracts of cysB mutants and mutants lacking sulphite reductase only. 6. A scheme, involving positive control of the enzymes of sulphate activation and reduction, is suggested to account for the control of cysteine biosynthesis.     

Regulation of L-cystine transport in Salmonella typhimurium.

A kinetic analysis of L-cystine uptake in wild-type Salmonella typhimurium indicates the presence of at least two, and possibly three, separate transport systems. CTS-1 accounts for the majority of uptake at 20 muM L-cystine, with a Vmax of 9.5 nmol/min per mg and a Km of 2.0 muM; CTS-2 is a low-capacity, higher-affinity system with a Vmax of 0.22 nmol/min per mg and a Km of 0.05 muM; a third, nonsaturable process has been designated CTS-3. We find that wild-type CTS-1 levels are at least 11 times higher in sulfur-limited cells than in L-cystine-grown cells. Pleiotropic cysteine auxotrophs of the types cysE (lacking serine transacetylase) and cysB- (lacking a regulatory element of positive control) have very low levels of CTS-1 even when grown under conditions of sulfur limitation, which response is analogous to that previously observed for cysteine biosynthetic enzymes (N . M. Kredich, J. Biol. Chem. 246:3474-3484, 1971). CTS-1 is induced in cysE mutants by growth in the presence of O-acetyl-L-serine (the product of serine transacetylase), again paralleling the behavior of the cysteine biosynthetic pathway. Strain DW25, a prototrophic cysBc mutant, which is constitutive for cysteine biosynthesis, is also derepressed for CTS-1 when grown on L-cystine. Since CTS-1 is regulated by sulfur limitation, O-acetyl-L-serine, and the cysB gene product, the same three conditions controlling cysteine biosynthesis, we propose that this transport system is a part of the cysteine regulon.     

Yeast cytochrome c messenger RNA. In vitro translation and specific immunoprecipitation of the CYC1 gene product.

An assay based upon indirect immunoprecipitation has been developed for yeast cytochrome c and apocytochrome c. The specificity of this assay was demonstrated by its ability to selectively precipitate cytochrome c from an autolysate of yeast cell proteins. Translation of the polypeptide chain of cytochrome c in a wheat germ extract programmed with yeast poly(A) RNA was demonstrated using this immunoprecipitation assay. Translation of poly(A) RNA from yeast strains carrying nonsense mutations in the cyc1 gene yielded in vitro cytochrome c polypeptides which were shorter than the wild type protein by the amount expected for polypeptide chains which had terminated at the nonsense codon. The in vivo rate of cytochrome c synthesis was shown to be 6-fold greater in derepressed cells than in glucose-repressed cells. The 6-fold difference is sufficient to account for the 6-fold higher level of cytochrome c in derepressed than in repressed cells. The level of translatable cytochrome c mRNA is at least 4 times as high in derepressed as in glucose-repressed cells, suggesting that regulation occurs at some step in the synthesis of this messenger.     

Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1.

Phage N-1 grown in Anabaena strain 7120 [N-1 . 7120] forms plaques on A. variabilis about 10(-7) to 10(-6) as efficiently as on Anabaena 7120. By manipulating different characteristics of the interaction between phage and host, it was possible to increase the relative efficiency of plaque formation to 0.38. Growth of A. variabilis at 40 degrees C for at least three generations resulted in an increase in the rate of phage adsorption and a 10-fold increase in the efficiency of plaque formation. The efficiency of plaque formation was further increased about 42-fold, with little or no further increase in rate of adsorption, in a variant strain. A. variabilis strain FD, isolated from a culture of A. variabilis which had grown for more than 30 generations at 40 degrees C. The low relative efficiency of plaque formation by N-1 . 7120 on A. variabilis could be partially accounted for if A. variabilis contains a deoxyribonucleic acid restriction endonuclease which is absent from Anabaena 7120. Indirect evidence for such an endonuclease included the following: (i) phage N-1 grown in A. variabilis (N-1 . Av) had approximately a 7 X 10(3)-fold higher relative efficiency of plaque formation on A. variabilis than had N-1 . 7120; and (ii) the efficiency of plaque formation by N-1 . 7120 on A. variabilis strain FD was increased by up to 146-fold after heating the latter organism at 51 degrees C.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Restoration of immunity in lysogens carrying prophage P2, derepressed at high temperature

Summary The possibility that a strain lysogenic for phage P2 could be brought into the so-called antiimmune state in which the synthesis of phage repressor is permanently turned off, was tested in the following way. Two lysogenic strains that could be derepressed at 42°C were prepared. In one, the prophage had, in addition to a temperature-sensitive repressor mutation (c5), amber mutations in the two early genes A and B. In the other, the prophage had an unknown defect that blocked expression of the A and B genes. Both strains could multiply at 42° C as well as or almost as well as a non-lysogen. After the strains had grown for several generations at 42° C, they were returned to 30° and the resynthesis of repressor was followed by measuring the restoration of immunity to super-infection. In both cases, the immunity returned slowly over a period of 2 to 3 h. In a strain made doubly lysogenic for two amA amB c5 prophages, immunity was restored at a more rapid rate, suggesting that the rate of restoration depended mainly on the number of copies of repressor gene present. Attempts to demonstrate channeling towards the lytic pathway in the derepressed lysogens was also negative. The temperature treatment tended instead to increase the frequency of lysogenization of superinfecting P2. Thus, the presence of a system, similar to the cro system described for phage lambda, to regulate repressor synthesis in phage P2 could not be demonstrated.     

Suppression of Amber and Ochre Mutants in Salmonella typhimurium by a Mutant F′-1-gal Factor Carrying an Ochre Suppressor Gene

A Salmonella typhimurium strain was given the amber mutation hisC527 by transduction, made galactose-negative by mutation, then infected with the F'-1-gal factor. Of 107 spontaneous and mutagen-induced histidine-independent mutants tested, 3 proved to result from suppressor mutations within the F' factor. The mutant F' factors, when transferred to S. typhimurium and E. coli auxotrophs, suppressed amber and ochre but not UGA or missense mutants, and are inferred to carry ochre suppressor genes. Attempts to isolate an F' amber suppressor mutant were unsuccessful. A suppressor F' factor was transferred to 14 rough mutants which had been isolated from LT2 hisC527 (amber) by selection for resistance to phage P22.c2. One rough mutant was partly suppressed, as shown by its acquisition of O agglutinability and by alterations in its phage resistance pattern. Phage P22h grown on the suppressed mutant contransduced its rf. gene with cysE(+) and with pyrE(+), and the affected locus is inferred to be rfaL. Both the original and the mutant F' factors conferred resistance to the rough-specific phage Br60, which is therefore "female-specific."     

Meal cysteine improves postprandial glucose control in rats fed a high-sucrose meal

Whey protein, particularly the alpha-lactalbumin fraction, are rich in cysteine (cys) and could therefore favor postprandial glucose homeostasis by a glutathione-mediated effect. This work investigates the effects of the ingestion of an alpha-lactalbumin-rich whey concentrate (alpha-LAC) during a high-sucrose (HS) meal on postprandial glucose homeostasis in healthy rats. In the first experiment, rats received an HS meal containing 14% protein, in which the protein source was either alpha-LAC (HS(a)) or total milk proteins, alone (HS(0)) or supplemented with 17 mg (HS(1)) or 59 mg (HS(2)) of N-acetylcysteine (NAC). This resulted in a total cys content 3.6-fold higher in the HS(1) and HS(a) meals and 12-fold higher in the HS(2) meal, when compared to the HS(0) meal. Postprandial parameters were monitored for 3 h after ingestion of the meal. The same measurements were performed on rats injected with 4 mmol/kg of buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis. Increasing the meal's cys content dose-dependently reduced both postprandial glucose and insulin (P<.05). The inhibition of glutathione synthesis with BSO injection abrogated the beneficial effects of NAC supplementation on postprandial glucose response but did not affect those of alpha-LAC. These results show that (1) the substitution of alpha-LAC for total milk protein reduces glucose response, as does the addition of a cys donor to the meal, (2) but contrary to those of a simple cys donor, the beneficial effects of alpha-LAC are not entirely mediated by glutathione synthesis, suggesting additional mechanisms.     

Rat glutathione S-transferase. Cloning of double-stranded cDNA and induction of its mRNA

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.     

The effect of urea on the expression of the cysteine regulon

Urea inhibits the expression of those cysteine genes which are regulated by the cysB gene products. It has no effect, however, on the constitutively expressed cysE gene.     

Demonstration of anaerobic catalase synthesis in the cz1 mutant of Saccharomyces cerevisiae.

Anaerobic synthesis of catalase T (a typical oxygen-inducible haem containing enzyme) in the cz1 mutant of yeast was demonstrated. The synthesis of catalase T in anaerobically grown mutant cells is stimulated by haemin under carbon-derepressed conditions of growth (galactose as carbon source) but not in glucose repressed cultures. Haem is practically undetectable in the anaerobically grown glucose repressed wild type strain and its level in derepressed cells amounts to 3% of the fully derepressed aerobically grown cells. In the cz1 mutant cultures grown in anoxia both on galactose and glucose the haem level usually exceeds 10% of that in the aerobic control.     

Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF

The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to alpha-thrombin (from 15- to 33-fold), but not to PDGF. As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF- A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75% the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation. Thus, the mechanism of strain-induced growth appears to involve the intermediary action of secreted PDGF.     

High-level resistance to mecillinam produced by inactivation of soluble lytic transglycosylase in Salmonella enterica serovar Typhimurium

By screening for high-level mecillinam resistant derivatives of a low-level resistant strain (cysB403 galE1922 relA21::Tn10) of Salmonella enterica serovar Typhimurium, a MudJ insertion in the gene for soluble lytic transglycosylase (slt) was isolated. This insertion (slt-1::MudJ) increased the resistance to mecillinam of cysB and cysE strains (MIC: about 20-40 microg mL(-1)) to a strikingly high level (MIC: 160 microg mL(-1)). As in Escherichia coli K-12, the slt mutation slightly increased the sensitivity of the wild type and of several strains that carried mutations that did not increase mecillinam resistance. All the strains acquired a spherical cell shape when treated with mecillinam. The effect of slt-1::MudJ was limited to mecillinam, the response to several other antibiotics remaining unaltered by the insertion. The results presented in this paper demonstrate that soluble lytic transglycosylase performs an important role in the response to mecillinam, which only becomes evident when failure of CysB/CysE function causes medium-level resistance. The results also suggest that soluble lytic transglycosylase interacts with, and is partially inhibited by normal lipopolysaccharide.     

Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4.

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.     

Improving the nutritive value of tubers: elevation of cysteine and glutathione contents in the potato cultivar White Lady by marker-free transformation

The amino acids that limit the nutritive value of potato are the sulfur containing amino acids methionine and cysteine. Manipulation of the targeted amino acid biosynthesis is a way to circumvent this problem. Cysteine is synthesised from O-acetyl-l-serine formed by serine acetyltransferase (SAT). To increase the cysteine content of the commercial potato cultivar White Lady the chimeric SAT-coding cysE gene from Escherichia coli under the control of the constitutive CaMV 35S promoter and fused to the chloroplast targeting rbcS 5'-transit peptide sequence was introduced into the White Lady genome. Novelty of the approach was the application of marker-free transformation. Two transgenic lines were obtained that accumulated the cysE mRNA in high amounts. Crude leaf extracts of these plants exhibited up to 80- and 20-fold higher SAT activity in leaves and tubers, respectively, than those prepared from non-transformed plants. Levels of cysteine and glutathione both in leaves and tubers were 1.5-fold higher in average than in control plants. The alterations observed had no effect on tuber yield and sprouting behaviour. Gas chromatography coupled to mass spectrometry showed that all other amino acids than cysteine were unaffected. Here we demonstrate for the first time that the cysteine content of tubers can be enhanced by metabolic engineering.