Prasanta Datta

TdcA, a transcriptional activator of the tdcABC operon of Escherichia coli, is a member of the LysR family of proteins

The tdcB and tdcC genes of the tdcABC operon of Escherichia coli encode threonine dehydratase and a threonine-serine permease, respectively. These proteins are involved in transport and metabolism of threonine and serine during anaerobic growth. In this study, we functionally characterized tdcA, which encodes a 35 kDa polypeptide consisting of 312 amino acid residues. Non-polar and partially polar mutations introduced into tdcA drastically reduced the expression of the genes down-stream from tdcA. Complementation studies using single-copy chromosomal integrants of a tdcB-lacZ fusion harboring an in-frame deletion of tdcA with chromosomal or plasmid-borne tdcA+ in trans showed complete restoration of tdc operon expression in vivo. The amino acid sequene at the amino-terminal end of TdcA revealed a significant homology to the helix-turn-helix motifs of typical DNA binding proteins. Sequence alignment of TdcA with LysR also showed considerable sequence similarity throughout their entire lengths. Our results suggest that TdcA is related to the LysR family of proteins by common ancestry and, based on its functional role in tdc expression, belongs to the LysR family of transcriptional activators.

Fibroblast growth regulatory factor inhibits dna synthesis in balb/c 3t3 cells by arresting in g1.

SUMMARY A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated tibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late Gl or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early Gl showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the Gl. Further, the regulatory factor delayed the progression of GO/Gl-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early Gl, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.

Threonine Deaminase: A Novel Activity Stain on Polyacrylamide Gels

With phenazine methosulfate and nitro blue tetrazolium, an activity stain for threonine deaminase has been developed. Because the deamination reaction does not involve any overall oxidation or reduction, it is proposed that one of the intermediates of the reaction, α-aminocrotonate or α-iminobutyrate, is the reducing agent. Studies with ferricyanide as the artificial electron acceptor indicate that a decarboxylation of the intermediate occurs during the dye reduction.

Molecular cloning and expression of the biodegradative threonine dehydratase gene (tdc) of Escherichia coli K12

SummaryThe biodegradative threonine dehydratase gene (tdc) of Escherichia coli was cloned by isolating a dehydratase-negative mutant after Tn5 mutagenesis, cloning the tdc::Tn5 DNA into pBR322 and then replacing the Tn5 element on the plasmid in vivo. Subcloning and nucleotide sequence data revealed two distinct procaryotic promoterlike elements each containing a potential CAP-binding site and AT-rich regions, and a Shine-Dalgarno sequence. One of these putative promoters, P2, was located immediately upstream from the tdc coding region, and a second, P1, was approximately 1 kilobase upstream from P2. Deletion of the potential CAP-binding site from P1 prevented tdc gene expression. However, removal of P2 and a large segment of the upstream DNA had no discernible effect on dehydratase synthesis. A 936-base pair open reading frame was found between P1 and the tdc coding region, which produced a polypeptide of about 32 kilodaltons. The data suggest that P1, and not P2, is necessary for tdc gene expression, and that the DNA sequences coding for the 32 KD polypeptide and threonine dehydratase are part of a single transcriptional unit.

Influence of DNA topology on expression of the tdc operon in Escherichia coli K-12

TdcB activity expressed from the chromosomal gene and LacZ expression from single-copy tdc-lacZ transcriptional and translational fusions were measured in Escherichia coli strains harboring mutations in the genes encoding DNA gyrase, topoisomerase I and the HU protein. The pattern of tdc operon expression in these mutants suggests that relaxation of supercoiled DNA enhances tdc transcription in vivo.

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli.

SummaryEfficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of Mr 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.

CHEMICAL CHARACTERIZATION OF BIODEGRADATIVE THREONINE DEHYDRATASES FROM TWO ENTERIC BACTERIA

Abstract Some chemical properties of the purified biodegredative threonine dehydratases ( l -threonine hydro-lyase (deaminating), EC 4.2.1.16) from Escherichia coli and Salmonella typhimurium are described. The overall amino acid compositions of the two enzymes appear similar with some variations in several amino acid residues. Tryptic peptide maps show that in S. typhimurium four peptides of E. coli origin are missing, whereas six peptides unique to Salmonella protein are present. Carboxymethylation reaction with iodo[14C]acetate to detect half-cystine residues indicates that peptides 21 and S5 in S. typhimurium, but not in E. coli enzyme, are labeled, and the reverse is true for peptide 22; four other peptides of S. typhimurium have more half-cystine residues than their counterparts in E. coli. In addition, the Salmonella enzyme appears to have several disulfide bonds. Despite these differences, the amino acid sequence of the amino termini of the two proteins reveals a highly conserved structure, with only three out of 25 residues being different. Reduction with tritium-labeled borohydride followed by tryptic fingerprinting of the two proteins shows that one peptide contains active-site pyridoxal phosphate. Modifier binding studies with the S. typhimurium enzyme indicate that pyruvate and glyoxylate occupy separate sites on the enzyme molecules. Further, there are two distinct sites for glyoxylate binding: in the monoglyoxylated form of the enzyme, only peptide 22 becomes labeled, whereas both peptides 22 and 21 of the tetraglyoxylated form of the dehydratase contain bound glyoxylate. These results support the earlier findings that these two metabolites regulate enzyme activity by two separate, mutually exclusive, mechanisms.

EFFECTS OF K + ON THE CATALYTIC AND REGULATORY PROPERTIES OF HOMOSERINE DEHYDROGENASE OF PSEUDOMONAS FLUORESCENS

Abstract Partially purified homoserine dehydrogenase ( l -homoserine; NADP+ oxidoreductase, EC 1.1.1.3) isolated from Pseudomonas fluorescens requires K+ for its stability. At 1 mM K+, the apparent first-order rate constant for enzyme inactivation at 25° was 25-fold higher than that observed at 20 mM K+. A dissociation constant, KD, of 3 mM of the ion-enzyme complex was calculated. Following inactivation of the enzyme by depletion of K+, the activity could be regenerated to a large extent by incubation with KCl and NADP+; no other cation was nearly as effective as K+. Binding of K+ on the enzyme molecule also plays an important part in the regulation of enzyme activity by the allosteric modifier l -threonine. At 1 mM K+, the catalytic activity was strongly inhibited by 10 mM l -threonine, whereas, at 10 mM K+ concentration in the assay mixture, l -threonine inhibition was almost completely abolished; threonine and K+ were kinetically competitive. It is proposed that K+ can induce specific conformational changes in the protein molecule that are either sensitive or insensitive to feedback inhibition control; in addition, the cation is necessary for the maintenance of the protein structure to ensure catalytic activity.

Physical linkage and transcriptional orientation of the tdc operon on the Escherichia coli chromosome

SummaryThe physical and genetic structure of 37 kilobases of DNA encopassing the tdc region at 68.3 min of the Escherichia coli chromosome was determined by DNA sequence analysis and restriction mapping. Reexamination of new data concerning the direction of transcription of the tdc operon revealed that in strain W3110 the tdc region is located on a transposable segment of DNA.