Nucleotide sequence of traQ and adjacent loci in the Escherichia coli K-12 F-plasmid transfer operon.

The F tra operon region that includes genes trbA, traQ, and trbB was analyzed. Determination of the DNA sequence showed that on the tra operon strand, the trbA gene begins 19 nucleotides (nt) distal to traF and encodes a 115-amino-acid, Mr-12,946 protein. The traQ gene begins 399 nt distal to trbA and encodes a 94-amino-acid, Mr-10,867 protein. The trbB gene, which encodes a 179-amino-acid, Mr-19,507 protein, was found to overlap slightly with traQ; its start codon begins 11 nt before the traQ stop codon. Protein analysis and subcellular fractionation of the products expressed by these genes indicated that the trbB product was processed and that the mature form of this protein accumulated in the periplasm. In contrast, the protein products of trbA and traQ appeared to be unprocessed, membrane-associated proteins. The DNA sequence also revealed the presence of a previously unsuspected locus, artA, in the region between trbA and traQ. The artA open reading frame was found to lie on the DNA strand complementary to that of the F tra operon and could encode a 104-amino-acid, 12,132-dalton polypeptide. Since this sequence would not be expressed as part of the tra operon, the activity of a potential artA promoter region was assessed in a galK fusion vector system. In vivo utilization of the artA promoter and translational start sites was also examined by testing expression of an artA-beta-galactosidase fusion protein. These results indicated that the artA gene is expressed from its own promoter.     

Regulation of arginine biosynthesis in the psychropiezophilic bacterium Moritella profunda: in vivo repressibility and in vitro repressor-operator contact probing

We report the cloning of the arginine repressor gene from the psychropiezophilic Gram-negative bacterium Moritella profunda, the purification of its product (ArgR(Mp)), the identification of the operator in the bipolar argECBFGH(A) operon, in vivo repressibility studies, and an in vitro analysis of the repressor-operator interaction, including binding to mutant and heterologous arginine operators. The ArgR(Mp) subunit shows about 70% amino acid sequence identity with Escherichia coli ArgR (ArgR(Ec)). Binding of purified hexameric ArgR(Mp) to the control region of the divergent operon proved to be arginine-dependent, sequence-specific, and significantly more sensitive to heat than complex formation with ArgR(Ec). ArgR(Mp) binds E.coli arginine operators very efficiently, but hardly recognizes the operator from Bacillus stearothermophilus or Thermotoga maritima. ArgR(Mp) binds to a single site overlapping the -35 element of argC(P), but not argE(P). Therefore, the arrangement of promoter and operator sites in the bipolar argECBFGH(A) operon of M.profunda is very different from the organization of control elements in the bipolar argECBH operon of E.coli, where both promoters overlap the common operator and are equally repressible. We demonstrate that M.profunda argC(P) is about 44-fold repressible, whereas argE(P) is fully constitutive. A high-resolution contact map of the ArgR(Mp)-operator interaction was established by enzymatic and chemical footprinting, missing contact and base-specific premodification binding interference studies. The results indicate that the argC operator consists of two ARG box-like sequences (18bp imperfect palindromes) separated by 3bp. ArgR(Mp) binds to one face of the DNA helix and establishes contacts with two major groove segments and the intervening minor groove of each ARG box, whereas the minor groove segment facing the repressor at the center of the operator remains largely uncontacted. This pattern is reminiscent of complex formation with the repressors of E.coli and B.stearothermophilus, and suggests that each ARG box is contacted by two ArgR subunits belonging to opposite trimers. Moreover, the premodification interference patterns and mutant studies clearly indicate that the inner, center proximal halves of each ARG box in the M.profunda argC operator are more important for complex formation and repression than the outermost halves. A close inspection of sequence conservation and of single base-pair O(c)-type mutations indicate that the same conclusion can be generalized to E.coli operators.     

Bacteriophage P22 Cro protein: sequence, purification, and properties

The DNA sequence of part of the bacteriophage P22 early regulatory region, including genes cro and c1, was determined. The protein product of the cro gene consists of 61 amino acid residues, and that of c1, 92 amino acid residues. Both genes were placed separately in plasmids from which they are expressed from a controllable promoter in vivo. Induced cells bearing the cro-expressing plasmid were used as a source for purifying and characterizing the Cro protein. The amino-terminal sequence of this protein was found to be as predicted by the DNA sequence; close agreement was also observed between its predicted and experimentally determined amino acid composition and molar extinction coefficient at 280 nm. In gel filtration experiments, Cro protein at concentrations around 10(-5) M appears to have a molecular weight of 8600, which is more consistent with monomers (6800) than with dimers (13 600). Cro protein binds specifically to the three repressor binding sites in the P22 right operator; in order of decreasing affinity, these are OR3, OR1, and OR2.     

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.     

Integration host factor is required for positive regulation of the tdc operon of Escherichia coli.

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.     

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.     

Purification of the integration host factor homolog of Rhodobacter capsulatus: cloning and sequencing of the hip gene, which encodes the beta subunit.

We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an alpha beta heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the beta subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the beta subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup- mutant IR4, which is mutated in the himA gene (coding for the alpha subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does.