A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12

While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min. These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.     

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

Immunological cross reactivity of four enzymes involved in the biosynthetic pathway of lysine, methionine and threonine in Escherichia coli K12.

In Escherichia coli K12 the biosynthetic pathway of lysine, methionine and threonine is characterized by three isofunctional aspartokinases and two homoserine dehydrogenases. A single polypeptide chain carries the threonine-sensitive aspartokinase and homoserine dehydrogenase (AK I-HDH I), and a different polypeptide chain carries the methionine-repressible aspartokinase and homoserine dehydrogenase (AK II-HDH II). Immuno-adsorbants prepared with rabbit antibodies against AK I-HDH I bind the lysine-sensitive aspartokinase (AK III), the AK II-HDH II, and the homoserine kinase (HSK), an enzyme of the threonine biosynthetic pathway. Saturation of the immunoadsorbant with AK I-HDH I results in a decreased binding capacity for the other enzymes. Displacement of bound AK III or HSK can be obtained with pure AK I-HDH I, showing that the affinity of the antibodies to homologous antigens is higher than to heterologous ones. Immunoadsorbants prepared with anti-HSK antibodies show the same type of recognition: binding of the three aspartkinases and a capacity to displace the heterologous antigens bound. Accordingly, the same antibodies, implicated in the binding of the homologous antigen, bind the other enzymes. None of the other enzymes of the pathway, or the other kinases tested are recognized by the two immunoadsorbants. It can be postulated that in E. coli K12, duplication of a common ancestor gene gave rise to the three aspartokinases and to the homoserine kinase; two of the genes coding for the aspartokinases fused with those coding for the homoserine dehydrogenases. Indicating that only few epitopes are shared by these enzymes, by conventional immuno-diffusion techniques no precipitation lines appeared with antibodies against AK I-HDH I and the other proteins.     

Limited homology between trg and the other transducer proteins of Escherichia coli.

Transducers are transmembrane proteins that are central to the chemotactic system of Escherichia coli. The proteins transduce ligand recognition into an excitatory signal and function in adaptation as methyl-accepting proteins. The transducer genes tsr, tar, and tap have extensive homology with each other. However, previous studies revealed little indication of homology between those three transducer genes and a fourth gene, trg. We investigated the relationship between trg and the other genes by blot-hybridization experiments and the relationship between Trg and the other transducer proteins by immune precipitation and experiments with an antiserum raised to purified Trg protein. In experiments in which 35% mismatch would be tolerated, weak hybridization of trg was detected to a DNA fragment containing tar and tap but not to a fragment containing tsr. In experiments in which only 30% mismatch would be tolerated, no trg hybridization was apparent either to total chromosomal DNA or to DNA from hybrid plasmids carrying the other transducer genes. An anti-Trg serum formed immune precipitates with the Tsr and Tar proteins as well as with the Trg protein to which it was raised. We conclude that there is homology between Trg and the other transducer, but the homology is more limited than that shared among the other transducers. Furthermore, we found no indication of additional transducer genes closely related to trg. Thus, the trg gene is a somewhat distant cousin within a single transducer gene family of E. coli.     

Reduction of methionine sulfoxide to methionine by Escherichia coli.

L-Methionine-dl-sulfoxide can support the growth of an Escherichia coli methionine auxotroph, suggesting the presence of an enzyme(s) capable of reducing the sulfoxide to methionine. This was verified by showing that a cell-free extract of E. coli catalyzes the conversion of methionine sulfoxide to methionine. This reaction required reduced nicotinamide adenine dinucleotide phosphate and a generating system for this compound. The specific activity of the enzyme increased during logarithmic growth and was maximal when the culture attained a density of about 10(9) cells per ml.     

Derivation of glycine from threonine in Escherichia coli K-12 mutants.

Escherichia coli AT2046 has been shown previously to lack the enzyme serine transhydroxymethylase and to require exogenous glycine for growth as a consequence. Strains JEV73 and JEV73R, mutants derived from strain AT2046, are shown here to be serine transhydroxymethylase deficient, but able to derive their glycine from endogenously synthesized threonine. Leucine is shown to be closely involved in the regulation of biosynthesis of glycine, to spare glycine in strain AT2046T, to replace glycine in strain JEV73, and to increase threonine conversion to glycine in a representative prototroph of E. coli. An interpretation of strains JEV73 and JEV73R as regulatory mutants of strain AT2046 is given. A hypothesis as to the role of leucine as a signal for nitrogen scavenging is suggested.     

Isolation and purification of Brucella antigens synthesized by Escherichia coli K12 cells]

The homogeneous preparations of the brucella protein antigens were isolated from the hybrid producer strains Escherichia coli 6SE579 and 6SE800 by the cold osmotic shock technique and further purification on immunosorbents. The 18 + 38 and 38 kDa antigens were obtained. The antiserum specific to brucella 38 kDa antigen was obtained and used for isolation of the 18 kDa antigen from the producer strain 6SE579 synthesizing two brucella antigens. The immunosorbent developed on the basis of BrCn-agarose conjugated with antibodies from the serum has permitted isolation of 18 kDa protein antigen preparation. Thus, the combined technique of cold osmotic shock and affinity chromatography on immunosorbents permits one to isolate highly purified individual antigens of brucella from Escherichia coli K12 producer cells.     

Immunological cross-reactivity between outer membrane pore proteins of Campylobacter jejuni and Escherichia coli

Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin.     

Properties of the penicillin-binding proteins of Escherichia coli K12,.

Benzyl[14C]penicillin binds to six proteins with molecular weights between 40000 and 91000 in the inner membrane of Escherichia coli. Two additional binding proteins with molecular weights of 29000 and 32000 were sometimes detected. All proteins were accessible to benzyl[14C]penicillin in whole cells. Proteins 5 and 6 released bound benzyl[14C]penicillin with half times of 5 and 19 min at 30 degrees C but the other binding proteins showed less than 50% release during a 60-min period at 30 degrees C. The rate of release of bound penicillin from some of the proteins was greatly stimulated by 2-mercaptoethanol and neutral hydroxylamine. Release of benzyl[14C]penicillin did not occur if the binding proteins were denatured in anionic detergent and so was probably enzymic. No additional binding proteins were detected with two [14C]cephalosporins. These beta-lactams bound to either all or some of those proteins to which benzyl[14C]penicillin bound. No binding proteins have been detected in the outer membrane of E coli with any beta-[14C]lactam. The binding of a range of unlabelled penicillins and cephalosporins were studied by measuring their competition for the binding of benzyl[14C]penicillin to the six penicillin-binding proteins. These results, together with those obtained by direct binding experiments with beta-[14C]lactams, showed that penicillins bind to all six proteins but that at least some cephalosporins fail to bind, or bind very slowly, to proteins 2, 5 and 6, although they bind to the other proteins. Since these cephalosporins inhibited cell division and caused cell lysis at concentrations where we could detect no binding to proteins 2, 5 and 6, we believe that these latter proteins are not the target at which beta-lactams bind to elicit the above physiological responses. The binding properties of proteins 1, 3, and 4 correlate reasonably well with those expected for the above killing targets.     

Operator-constitutive mutants in the threonine operon of Escherichia coli K-12.

Three Escherichia coli K-12 mutant strains resistant to DL-alpha-amino-beta-hydroxyvaleric acid were isolated in which the expression of the thr operon is constitutive. The localization and dominance properties of the mutations involved, called thrO, are those of operator mutations. The gene sequence is OABC as suggested by earlier studies.     

Effect of methionine and vitamin B-12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12

The effects of supplementation of growth medium with high concentrations of methionine (5 mm) and/or vitamin B₁₂ (10 nm) on the activities of five enzymes of the methionine regulon were measured in wild-type Escherichia coli K12, a metJ prototrophic and three metJ methionine auxotrophic derivatives. Growth on vitamin B₁₂ causes lowering of the activities of the non-B₁₂ methyltransferase while growth on methionine causes elevation of its activity in all four metJ mutants. The previous observation that this enzyme is not repressed by vitamin B₁₂ addition in metH mutants together with our observation that vitamin B₁₂ causes repression in mutants (metF) unable to synthesize the donor for homocysteine methylation supports the model of Kung et al. (10) that the holo-B₁₂-methyltransferase functions as a repressor of synthesis of the non-B₁₂-methyltransferase. Growth on methionine causes lowering of cystathionase activity, and growth on vitamin B₁₂ results in elevation of cystathionase activity in a metJ prototroph and one metJ auxotroph. The metJmetA strain (RG326) has a higher than normal level of cystathionase while the metJmetF strain (RG191) has lower than normal cystathionase activity. These results indicate the existence of a metJ independent system that modulates the activity of cystathionase possibly in response to changes in concentration of unidentified metabolite(s).     

Myosin and actin from Escherichia coli K12 C600.

Myosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2 M KCl, a large number of "thick filaments" of uniform size (about 0.6-0.7 micron long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2 M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing "arrowheads". The arrowhead structure disappeared in the presence of ATP. A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the additon of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein. These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.     

Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12.

Summary To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Mel and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b^-c⁺), meo A (phenotype b⁺c^-) and ompB (phenotypes b^-c^-, b^- c⁺, b⁺⁺ c^- and b⁺⁺ c^±). It has recently been described that also a b⁺ c^- phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781–786 (1977)]. Among ompB (b^- c⁺)/meoA (b⁺ c^-) double mutants strains were found with the b⁺ c^- phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095–1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins.     

Purification and properties of nitrite reductase from Escherichia coli K12.

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.