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

Summary Efficient 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 M_r 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.     

Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids

The hom-1-thrB operon encodes homoserine dehydrogenase resistant to feedback inhibition by L-threonine and homoserine kinase. Stable expression of this operon has not yet been attained in different Corynebacterium glutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the hom-1-thrB operon to enable an analysis of the physiological consequences of its expression in C. glutamicum. Strains carrying one, two, or three copies of hom-1-thrB were obtained. They showed proportionally increased enzyme activity of feedback-resistant homoserine dehydrogenase and of homoserine kinase. This phenotype was stably maintained in all recombinants for more than 70 generations. In a lysine-producing C. glutamicum strain which does not produce any threonine, expression of one copy of hom-1-thrB resulted in the secretion of 39 mM threonine. Additional copies resulted in a higher, although not proportional, accumulation of threonine (up to 69 mM). This indicates further limitations of threonine production. As the copy number of hom-1-thrB increased, increasing amounts of homoserine (up to 23 mM) and isoleucine (up to 34 mM) were secreted. Determination of the cytosolic concentration of the respective amino acids revealed an increase of intracellular threonine from 9 to 100 mM and of intracellular homoserine from 4 to 74 mM as the copy number of hom-1-thrB increased. These results suggest that threonine production with C. glutamicum is limited by the efflux system for this amino acid. Furthermore, the results show the successful use of moderate and stable hom-1-thrB expression for directing the carbon flux from aspartate to threonine.     

In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the ?10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.     

Identification of cfxR, an activator gene of autotrophic CO2 fixation in Alcaligenes eutrophus

A regulatory gene, cfxR, involved in the carbon dioxide assimilation of Alcaligenes eutrophus H16 has been characterized through the analysis of mutants. The function of cfxR is required for the expression of two cfx operons that comprise structural genes encoding Calvin cycle enzymes. CfxR (34.8 kDa) corresponds with an open reading frame of 954 bp, with a translational initiation codon 167 bp upstream of the chromosomal cfx operon. The cfx operon and cfxR are transcribed divergently. The N-terminal sequence of CfxR is very similar to those of bacterial regulatory proteins belonging to the LysR family. Heterologous expression of cfxR in Escherichia coli was achieved using the pT7-7 system. Mobility shift experiments demonstrated that CfxR is a DNA-binding protein with a target site upstream of both the chromosomal and the plasmid-encoded cfx operons.     

An ABC transporter is essential for resistance to the antitumor agent mithramycin in the producerStreptomyces argillaceus

The organization of the phosphate-specific transport (pst) operon inPseudomonas aeruginosa has been determined. The gene order of thepst operon ispstC, pstA, pstB, phoU, and a well-conserved Pho box sequence (16/18 bases identical) exists in the promoter region. The most striking difference from the knownEscherichia coli pst operon is the lack of thepstS gene encoding a periplasmic phosphate (P_i)-binding protein. Even though the threepst genes were absolutely required for P_i-specific transport, expression of thepst operon at high levels did not increase P_i uptake inP. aeruginosa. DNA sequences for thepstB andphoU genes have been determined previously. The newly identifiedpstC andpstA genes encode possible integral membrane proteins of 677 amino acids (M _r 73 844) and 513 amino acids (M _r 56 394), respectively. The amino acid sequences of PstC and PstA predict that these proteins contain a long hydrophilic domain not seen in theirE. coli counterparts. A chromosomal deletion of the entirepst operon renderedP. aeruginosa unable to repress P_i taxis under conditions of P_i excess. ThephoU andpstB genes are essential for repressing P_i taxis. However, mutants lacking either PstC or PstA alone were able to repress P_i taxis under conditions of P_i excess.     

In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the –10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.     

Identification and characterization of genes (xapA, xapB, and xapR) involved in xanthosine catabolism in Escherichia coli.

We have characterized four genes from the 52-min region on the Escherichia coli linkage map. Three of these genes are directly involved in the metabolism of xanthosine, whereas the function of the fourth gene is unknown. One of the genes (xapA) encodes xanthosine phosphorylase. The second gene, named xapB, encodes a polypeptide that shows strong similarity to the nucleoside transport protein NupG. The genes xapA and xapB are located clockwise of a gene identified as xapR, which encodes a positive regulator belonging to the LysR family and is required for the expression of xapA and xapB. The genes xapA and xapB form an operon, and their expression was strictly dependent on the presence of both the XapR protein and the inducer xanthosine. Expression of the xapR gene is constitutive and not autoregulated, unlike the case for many other LysR family proteins. In minicells, the XapB polypeptide was found primarily in the membrane fraction, indicating that XapB is a transport protein like NupG and is involved in the transport of xanthosine.     

Regulatory Structure of the Biosynthetic Pathway for the Aspartate Family of Amino Acids in Lemna paucicostata Hegelm. 6746, with Special Reference to the Role of Aspartokinase

Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [¹⁴C]threonine and [¹⁴C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants.     

DNA sequence conservation at the gene level in a conserved chromosomal segment in twoPseudomonas species

A comparative analysis has been made of the DNA sequences of the isofunctional genes encodingN-acetylglutamate synthase of the arginine biosynthetic pathway of the bacterial speciesPseudomonas aeruginosa andPseudomonas putida. Overall homologies of 81% and 84% at the nucleotide and deduced amino acid sequence levels, respectively, were observed. This high homology was also reflected in the strikingly similar hydropathy profiles of the encoded proteins; patterns of codon usage, including rare codon usage; and amino acid composition of the proteins. This high level of homology at the DNA sequence level is consistent with the location of these genes in the genetically conserved chromosomal region (called auxotrophic-rich region) of the respectivePseudomonas species. Despite chromosomal rearrangements identified in this region the conservation observed at the chromosomal level between thesePseudomonas species is also maintained at the level of the DNA sequence, and in the deduced amino acid sequence, of the genes reported here and of six other pairs of genes of the tryptophan biosynthetic pathway, reported by others, which are also located within this chromosomal region.     

Identification of a Gene Encoding a Transporter Essential for Utilization of C4 Dicarboxylates in Corynebacterium glutamicum

The Corynebacterium glutamicum R genome contains a total of eight genes encoding proteins with sequence similarity to C₄-dicarboxylate transporters identified from other bacteria. Three of the genes encode proteins within the dicarboxylate/amino acid:cation symporter (DAACS) family, another three encode proteins within the tripartite ATP-independent periplasmic transporter family, and two encode proteins within the divalent anion:Na⁺ symporter (DASS) family. We observed that a mutant strain deficient in one of these genes, designated dcsT, of the DASS family did not aerobically grow on the C₄ dicarboxylates succinate, fumarate, and malate as the sole carbon sources. Mutant strains deficient in each of the other seven genes grew as well as the wild-type strain under the same conditions, although one of these genes is a homologue of dctA of the DAACS family, involved in aerobic growth on C₄ dicarboxylates in various bacteria. The utilization of C₄ dicarboxylates was markedly enhanced by overexpression of the dcsT gene. We confirmed that the uptake of [¹³C]labeled succinate observed for the wild-type cells was hardly detected in the dcsT-deficient mutant but was markedly enhanced in a dcsT-overexpressing strain. These results suggested that in C. glutamicum, the uptake of C₄ dicarboxylates for aerobic growth was mainly mediated by the DASS transporter encoded by dcsT. The expression level of the dcsT gene transiently increased in the early exponential phase during growth on nutrient-rich medium. This expression was enhanced by the addition of succinate in the mid-exponential phase and was repressed by the addition of glucose in the early exponential phase.     

Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I4

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I₄ has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I₄. Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI₄ DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI₄ when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.     

Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli

The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.