Extracellular release of protease III (ptr) by Escherichia coli K12

Escherichia coli strains carrying the protease III structural gene (ptr) on a plasmid secreted the protein into the growth medium. Plasmid-encoded beta-lactamase and chloramphenicol acetyl transferase, which served as periplasmic and cytoplasmic markers during cell fractionation, were not released into the growth medium. There appeared to be some strain dependence on the proficiency of the secretion system. Protease III was not detectably processed upon export through the outer cell membrane.     

Complete nucleotide sequence of the Escherichia coli ptr gene encoding protease III.

The nucleotide sequence of a 3120 bp region of the E. coli chromosome that includes the entire ptr gene has been determined. The proposed coding region for Protease III is 2889 nucleotides long, which would encode a protein consisting of 962 amino acids with a calculated molecular mass of 107,719 daltons. The predicted primary structure of the protein includes a 23-residue signal sequence, cleavage of which would give rise to a mature protein of molecular mass 105,124 daltons. At its 3' end, the ptr gene overlaps the start of the recB coding sequence by 8 bases, suggesting that these genes may form part of an operon.     

Physical characterization of the cloned protease III gene from Escherichia coli K-12.

Analysis of the cloned protease III gene (ptr) from Escherichia coli K-12 has demonstrated that in addition to the previously characterized 110,000-Mr protease III protein, a second 50,000-Mr polypeptide (p50) is derived from the amino-terminal end of the coding sequence. The p50 polypeptide is found predominantly in the periplasmic space along with protease III, but does not proteolytically degrade insulin, a substrate for protease III. p50 does not appear to originate from autolysis of the larger protein. Protease III is not essential for normal cell growth since deletion of the structural gene causes no observed alterations in the phenotypic properties of the bacteria. A 30-fold overproduction of protease III does not affect cell viability. A simple new purification method for protease III is described.     

The regulatory region of the divergent argECBH operon in Escherichia coli K-12

The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes. The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites. A long leader sequence - not involved in attenuation - precedes argCBH. Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations. Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC. Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed.     

Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12.

The narL gene product, NarL, is the nitrate-responsive regulator of anaerobic respiratory gene expression. We used genetic analysis of narL mutants to better understand the mechanism of NarL-mediated gene regulation. We selected and analyzed seven nitrate-independent narL mutants. Each of three independent, strongly constitutive mutants had changes of Val-88 to Ala. The other four mutants were weakly constitutive. The narL505(V88A) allele was largely dominant to narL+, while narX+ had a negative influence on its constitutive phenotype, suggesting that NarX may play a negative role in nitrate regulation. We also constructed two narL mutations that are analogous to previously characterized constitutive degU alleles. The first, narL503(H15L), was a recessive null allele. The second, narL504(D110K), functioned essentially as wild type but was dependent on narX+ for full activity. We changed Asp-59 of NarL, which corresponds to the site of phosphorylation of other response regulators, to Asn. This change, narL502(D59N), was a recessive null allele, which is consistent with the hypothesis that NarL requires phosphorylation for activation. Finally, we tested the requirement for molybdate on regulation in a narL505(V88A) strain. Although narL505(V88A) conferred some nitrate-independent expression of fdnGHI (encoding formate dehydrogenase-N) in limiting molybdate, it required excess molybdate for full induction both in the absence and in the presence of nitrate. This finding suggests that narL505(V88A) did not confer molybdate-independent expression of fdnGHI.     

New outer membrane-associated protease of Escherichia coli K-12.

The gene for a new outer membrane-associated protease, designated OmpP, of Escherichia coli has been cloned and sequenced. The gene encodes a 315-residue precursor protein possessing a 23-residue signal sequence. Including conservative substitutions and omitting the signal peptides, OmpP is 87% identical to the outer membrane protease OmpT. OmpP possessed the same enzymatic activity as OmpT. Immuno-electron microscopy demonstrated the exposure of the protein at the cell surface. Digestion of intact cells with proteinase K removed 155 N-terminal residues of OmpP, while the C-terminal half remained protected. It is possible that much of this N-terminal part is cell surface exposed and carries the enzymatic activity. Synthesis of OmpP was found to be thermoregulated, as is the expression of ompT (i.e., there is a low rate of synthesis at low temperatures) and, in addition, was found to be controlled by the cyclic AMP system.     

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene

The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.     

Point mutations in the regulatory region of the ilvGMEDA operon of Escherichia coli K-12.

The ilvGMEDA operon of Escherichia coli K-12 is preceded by a regulatory region containing a promoter, a leader, and an attenuator. This region has been extensively characterized biochemically. In this note point mutations of the regulatory region are reported. The effect of these mutations on expression from the ilv regulatory region supports the previous biochemical analysis.     

Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12

The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA).     

Hsp31 of Escherichia coli K-12 is glyoxalase III

Hsp31 encoded by hchA is known as a heat‐inducible molecular chaperone. Although structure studies revealed that Hsp31 has a putative catalytic triad consisting of Asp‐214, His‐186 and Cys‐185, its enzymatic function, besides weak amino‐peptidase activity, is still unknown. We found that Hsp31 displays glyoxalase activity that catalyses the conversion of methylglyoxal (MG) to d ‐lactate without an additional cofactor. The glyoxalase activity was completely abolished in the hchA‐deficient strain, confirming the relationship between the hchA gene and its enzymatic activity in vivo. Hsp31 exhibits Michaelis–Menten kinetics for substrates MG with K_m and k_cat of 1.43 ± 0.12 mM and 156.9 ± 5.5 min^?1 respectively. The highest glyoxalase activity was found at 35–40°C and pH of 6.0–8.0, and the activity was significantly inhibited by Cu²⁺, Fe³⁺ and Zn²⁺. Mutagenesis studies based on our evaluation of conserved catalytic residues revealed that the Cys‐185 and Glu‐77 were essential for catalysis, whereas His‐186 was less crucial for enzymatic function, although it participates in the catalytic process. The stationary‐phase Escherichia coli cells became more susceptible to MG when hchA was deleted, which was complemented by an expression of plasmid‐encoded hchA. Furthermore, an accumulation of intracellular MG was observed in hchA‐deficient strains.     

Inversion in the lactose region of Escherichia coli K-12.

A spontaneous mutant of Escherichia coli K-12, strain SY99, with an inversion in the lactose region was isolated and partially characterized. The inversion was detected due to inverse chromosomal conjugational transfer after introduction of an F42 (F'lac) episome. The termini of the inversion are between proAB and lac on one side and lac and proC on the other. The inverse conjugational transfer in SY99 did not appear to be absolute but was always accompanied by a residual "normal" counterclockwise mobilization. This residual transfer was further shown to be caused by the intrinsic instability of this region (at least in the line W3110). The possible involvement of IS3 elements flanking the lactose operon is discussed.     

Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12.

beta-Ketoacyl-acyl carrier protein (ACP) synthase III catalyzes the condensation of acetyl-CoA with malonyl-ACP in dissociated (Type II) fatty acid synthase systems. A synthase III mutant was used to localize the structural gene to the 24.5-min region of the Escherichia coli chromosome, and the defective synthase III allele was designated fabH1. The fabH gene was identified on a 1.3-kilobase NruI-HindIII chromosomal DNA fragment (plasmid pWO114) that complemented the enzymatic defect in fabH1 strains. The NruI-HindIII fragment was sequenced and contained a single open reading frame predicted to encode a 33,517-dalton protein with an isoelectric point of 4.85. The fabH sequence contained an Ala-Cys-Ala tripeptide characteristic of condensing enzyme active sites. A T7 expression system showed that the NruI-HindIII fragment directed the synthesis of a single 34,800-dalton protein. This protein was purified and the order of the amino-terminal 30 residues of the protein corresponded exactly to the amino acid structure predicted from the DNA sequence. The purified protein possessed both acetoacetyl-ACP synthase and acetyl-CoA:ACP transacylase activities, and cells harboring plasmid pWO114 overproduced the two activities, supporting the conclusion that a single protein carries out both reactions. Overproduction of synthase III resulted in a significant increase in shorter-chain fatty acids in the membrane phospholipids. These catalytic properties are consistent with the proposed role of synthase III in the initiation of fatty acid synthesis.     

Cloning of the Escherichia coli K-12 hemB gene.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.     

Genetic analysis of the Escherichia coli K-12 srl region.

Specialized transducing lambda derivatives, deletion mapping, and Plkc transductional crosses have been used to analyze the genetic organization and regulation of the srl genes. Transducing phages obtained from a secondary site lambda insertion in srlA are of two types: lambdapsrlC1 and lambdaprecA are substituted in the b2 region of the lambda chromosome (galtype) and carry the srlC gene but not srlD; lambdapsrlD is substituted in the early region of the phage deoxyribonucleic acid (biotype) and carries the srlD gene but not srlC. The lambdapsrlC1 phage, which lysogenizes at attlambda, complements srlC mutants in trans, indicating that this gene codes for a diffusable positive regulatory element. The srl genes have been ordered relative to the cysC, recA, and alaS genes by two- and three-factor P1kc crosses. The order, cysC...srlD-srlA-srlC-recA-alaS, has been obtained. The srlA and srlD genes comprise an operon with srlD operator distal. From the secondary site lysogen, it has been possible to obtain deletion mutants of this region that are sensitive to ultraviolet light and are recombination deficient. Genetic evidence suggests that these deletions extend from srl into the recA gene.     

Mapping of the gene for cytidine deaminase (cdd) in Escherichia coli K-12

The structural gene encoding cytidine deaminase (cdd) has been mapped in Escherichia coli K-12. It is located counterclockwise to ptsF between 46 and 47 min. The gene order in this region of the E. coli chromosome was found to be his-udk-gat-dld-cdd-ptsF.