Histidine decarboxylase of Lactobacillus 30a: function and reactivity of sulfhydryl groups.

Two classes of sulfhydryl groups in histidine decarboxylase from Lactobacillus 30 a can be differentiated by their reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Five cysteinyl residues (class I) of the native enzyme are titrated by DTNB as the pH of the reaction medium is increased from 6.5 to 7.5; the pH-rate profile for their reaction is described by a pKa of 9.2. An additional five thiol groups (class II) are titrated only when denaturing agents are added above neutral pH. Histidine decarboxylase is completely inactivated by DTNB in a kinetically second-order process (Kapp = 660 +/- 20 M-1 min-1 at pH 7.6 and 25 degrees C) which occurs coincident with and at the same rate as modification of the five class-I SH groups of the enzyme, i.e., one thiol group per pyruvoyl prosthetic group. The competitive inhibitors, histamine and imidazole, markedly enhanced the reactivity of these cysteinyl residues toward DTNB; this enhancement is accompanied by a concomitant increase in the rate of inactivation. A single SH group in each of the five catalytic units of histidine decarboxylase is thus implicated as being critical for the expression of enzymatic activity.     

Isolation and Characterization of Aminopeptidase-Deficient Lactobacillus bulgaricus Mutants

Lactobacillus bulgaricus CNRZ 397 is able to hydrolyze many amino-acyl- and dipeptidyl-β-naphthylamides. Analysis of heat inactivation kinetics, protease inhibitor effects, and the subcellular location of aminopeptidase (AP) activities from the parental strain and mutant derivatives dificient in alanyl- or leucyl-β-naphthylamide hydrolysis pointed out the existence of four APs. All mutants isolated were totally deficient in AP II, a cell wall metallo-enzyme with a broad substrate specificity but that is specifically responsible for lysyl-AP activity and is characterized by a molecular mass of 95,000 daltons. AP I and AP III are cytoplasmic enzymes that exhibit arginyl-AP activity; both enzymes are inducible during growth in rich peptide MRS medium (Difco Laboratories, Detroit, Mich.). The existence of a fourth AP (AP IV) that is involved in leucyl-AP activity was suggested. Moreover, we showed that X-prolyl-dipeptidyl-AP activity, which was not catalyzed by an AP, involved an enzyme(s) that is controlled by a regulatory mechanism that is common to that of AP II.     

Histidine Production by Auxotrophic Histidine Analog-resistant Mutants of Corynebacterinm glutamicum

Corynebacterium glutamicum mutants carrying both auxotrophy and histidine analog-resistance were derived by a mutagenic treatment, and their histidine productivity was compared with that of a triazolealanine (TRA)-resistant histidine producer, C. glutamicum KY-10260. As a result, a leucine auxotrophic TRA-resistant mutant, Rα-88 was selected out of 164 auxotrophic derivatives of KY-10260. It produced histidine at a distinctly higher concentration than the parent strain under every condition tested. The concentration reached 11 mg/ml or 5.8% (w/w) of the initial sugar. Addition of an excessive amount of leucine to the medium inhibited the histidine production together with the by-production of valine by this mutant. Thiazolealanine-resistant mutants derived from a tyrosine auxotroph, a phenylalanine auxotroph and a tryptophan auxotroph gave the same or lower production in comparison with KY-10260.     

Isolation and Properties of Selenomethionine-Resistant Mutants of NEUROSPORA CRASSA

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.     

Isolation and Properties of Escherichia coli Mutants Which Are Nonpermissive for the Growth of Phage Lambda

By selecting survivors of λ phage infection, mutants of Escherichia coli K12 that block reproduction cycle of the phage have been isolated. Fourteen of these phage-tolerant mutants (lam mutants) were chosen and characterized biochemically and genetically. It was shown that these mutants were tolerant to infection by all the lambdoid phages, except for few cases, but they were susceptible to infection by a non-lambdoid temperate phage (φ299), P1 or T phages. The mutants can be divided into at least three groups: (1) A mutant (lam 16) strain that seems to block normal penetration of phage DNA: (2) Three mutant (lam 64, lam 67 and lam 71) strains that block an “early” step(s) of phage growth, including phage DNA synthesis: (3) Six mutant (lam 24, lam 25, lam 26, lam 27, lam 646 and lam 6) strains that block normal functioning of the gene E products and produce unusual head structures. Some lambdoid phages and λ mutants that overcome the interference by the lam mutations have been obtained, and were used as tools for characterizing the host mutations. Two (lam 12 and lam 13) mutant strains and one (lam 1) mutant were inferred as affecting the expression of “late” genes, and early gene, respectively, by this test.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the cyanogen bromide peptides from the alpha chain

The alpha chain of histidine decarboxylase contains eight internal methionine residues. After reductive amination to convert the NH2-terminal pyruvoyl residue to an alanyl residue and cyanogen bromide treatment, 13 pure peptides were isolated. Four of these are incomplete cleavage products. The sequence of each of the remaining nine peptides was established by automated and manual degradation of the intact peptides and of smaller peptides obtained from tryptic, chymotryptic, and staphylococcal protease digests of the cyanogen bromide peptides. These results, together with the data on overlapping peptides reported in the accompanying paper (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), establish the complete amino acid sequence of the alpha chain.     

Salmonella typhimurium Mutants Lacking Ribonuclease I: Effect on the Polarity of Histidine Mutants

Mutants of Salmonella typhimurium containing 1 to 2% of wild-type ribonuclease I activity were isolated. The rns mutation had no effect on the polarity of mutations in the S. typhimurium histidine operon. Even in the presence of an rns mutation, it was not possible to obtain strong suppressors of the polarity of two polar mutations in the his operon.     

Histidine decarboxylase: Isolation and molecular characteristics

High levels of histidine decarboxylase activity were measured in rat basophilic leukemia cells grown in ascitic form in 4 week old WKY/N rats. The potent inhibition of this enzyme by brocresine and -methylhistidine but not by -methyl DOPA identified it as a specific histidine decarboxylase. Gel filtration and polyacrylamide gel electrophoresis revealed a molecular weight of 125,000 for the native enzyme, similar to that of fetal rat liver histidine decarboxylase. Using rat basophilic leukemia cells as starting material, histidine decarboxylase was purified extensively in a seven step procedure. Electrophoresis under denaturing conditions revealed that histidine decarboxylase is a dimeric protein consisting of two identical subunits with a molecular weight of 62,000. The results indicate that rat basophilic leukemia cells provide a new and rich source for the purification of histidine decarboxylase.     

Histidine decarboxylase of Lactobacillus 30a. Comparative sequences of the beta chain from wild type and mutant enzymes

Manual and automated sequencing of peptides isolated from tryptic, chymotryptic, and Staphylococcus aureus V8 protease digests of the beta chain of histidine decarboxylase from Lactobacillus 30a have established the following sequence for the wild type protein: NH2-Ser-Gly-Leu-Asp-Ala-Lys-Leu-Asn-Lys-Leu-Gly-Val-Asp-Arg-Ile-Ala-Ile-Ser-Pro -Tyr-Lys-Gln-Trp-Thr-Arg-Gly-Tyr-Met-Glu-Pro-Gly-Asn-Ile-Gly-Asn-Gly-Tyr-Val-Thr-Gly-Leu-Lys-Val-Asp-Ala-Gly-Val-Arg-Asp-Lys-Ser-Asp-Asp-Asp-Val-Leu-Asp-Gly-I le-Val-Ser-Tyr-Asp-Arg-Ala-Glu-Thr-Lys-Asn-Ala-Tyr-Ile-Gly-Gln-Ile-Asn-Met-Thr- Thr-Ala-Ser-COOH The beta chain from mutant 3 of this organism shows two amino acid replacements: Ser-51 is replaced by Ala and Gly-58 by Asp. These amino acid replacements result in a significant increase in the predicted alpha-helical content and a significant decrease in the isoelectric point of the mutant beta chain and are consistent with changes in physical and catalytic properties of the mutant histidine decarboxylase. In addition, about 15% of the mutant chains contain Met-Ser at the NH2 terminus rather than Ser. Asn-35 is partially deamidated in both proteins. Structural comparisons show that the histidine decarboxylase from Lactobacillus 30a and a similar pyruvoyl enzyme from Micrococcus sp. n. have evolved from a common ancestral protein.     

Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.     

Histidine Mutants Requiring Adenine: Selection of Mutants with Reduced hisG Expression in SALMONELLA TYPHIMURIUM

A method is described for the selection of Salmonella typhimurium mutants with reduced levels of hisG enzyme activity. This method is based on the fact that the hisG enzyme catalyzes the consumption of ATP in the first step of histidine biosynthesis. Normally, this reaction is closely regulated, both by feedback inhibition and by repression of the operon. However, conditions can be set up that result in the uncontrolled use of adenine in histidine biosynthesis. Cells grown under these conditions become phenotypic adenine auxotrophs. Some revertant clones that no longer require adenine contain mutations in hisG, hisE, or the his-control region. The hisG mutations are of all types (nonsense, frameshift, missense, deletion and leaky types), and they map throughout the hisG gene.     

pH-Conditional, Ammonia Assimilation-Deficient Mutants of Hydrogenomonas eutropha: Isolation and Growth Characteristics

Two mutants of the facultative autotroph Hydrogenomonas eutropha were isolated by using a modified penicillin selection method. The mutation involved was unusual in that its effect on cellular growth was conditional with regard to extracellular pH and the type of substrate employed. Growth of both mutants was abnormal under autotrophic conditions and during heterotrophic cultivation in the presence of organic substrates which lacked an amino group. Abnormal growth was characterized by linear growth rates which were low at pH 6.0 and moderate at pH 7.2. In contrast, growth of the mutants was normal on most amino acids. Those substrates yielding abnormal growth were oxidized at normal rates by the mutants, indicating the mutation did not impair their uptake or metabolism. The data suggest that the mutants are defective in their ability to assimilate inorganic nitrogen into organic forms, and this defect is strongly influenced by pH.     

Cold-Sensitive Cell-Division-Cycle Mutants of Yeast: Isolation, Properties, and Pseudoreversion Studies

We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.—Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37°. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.