Membrane-bound phosphatases in Escherichia coli: sequence of the pgpA gene.

One of the phosphatidyl glycerophosphate phosphatase genes of Escherichia coli, pgpA, was cloned, and its DNA sequence was determined. Its 507-base-pair open reading frame was consistent with the 18,000-molecular-weight product identified by a maxicell experiment. Between its possible promoter and methionine initiation codon, a repetitive extragenic palindromic sequence was found.     

DNA sequence analysis, gene product identification, and localization of flagellar motor components of Escherichia coli.

The Escherichia coli operon designated flaA contains seven flagellar genes; among them are two switch protein genes whose products are believed to interface with the motility and chemotaxis machinery of the cell. Complementation analysis using several plasmids carrying different portions of the flaA operon and analysis of expression of these plasmids in minicells allowed the identification of two flagellar gene products. The MotD (now called FliN) protein, a flagellar switch protein, was determined to have an apparent molecular weight of 16,000, and the FlaAI (FliL) protein, encoded by a previously unidentified gene, had an apparent molecular weight of 17,000. DNA sequence analysis of the motD gene revealed an open reading frame of 414 base pairs. There were two possible initiation codons (ATG) for motD translation, the first of which overlapped with the termination codon of the upstream gene, flaAII (fliN). The wild-type flaAI gene on the chromosome was replaced with a flaAI gene mutated in vitro. Loss of the flaAI gene product resulted in a nonmotile and nonflagellated phenotype. The subcellular location for both the MotD and FlaAI proteins was determined; the FlaAI protein partitioned exclusively in the insoluble fraction of a whole minicell sonic extract, whereas the MotD protein remained in both the soluble and insoluble fractions. In addition, we subcloned a 2.2-kilobase-pair DNA fragment capable of complementing the remaining four genes of the flaA operon (flbD [fliO], flaR [fliP], flaQ [fliQ], and flaP [fliR]).     

Topology and subcellular localization of FtsH protein in Escherichia coli.

FtsH protein in Escherichia coli is an essential protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding sequence. Western blots (immunoblots) of proteins from fractionated cell extracts and immunoelectron microscopy of the FtsH-overproducing strain showed exclusive localization of the FtsH protein in the cytoplasmic membrane. Most of the FtsH-specific labeling with gold particles was observed in the cytoplasmic membrane and the adjacent cytoplasm; much less was observed in the outer membrane and in the bulk cytoplasm. Genetic analysis by TnphoA insertions into ftsH revealed that the 25- to 95-amino-acid region, which is flanked by two hydrophobic stretchs, protrudes into the periplasmic space. From these results, we concluded that FtsH protein is an integral cytoplasmic membrane protein spanning the membrane twice and that it has a large cytoplasmic carboxy-terminal part with a putative ATP-binding domain. The average number of FtsH molecules per cell was estimated to be approximately 400.     

Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product

The dnaJ and dnaK genes are essential for replication of Escherichia coli DNA, and they constitute an operon, dnaJ being downstream from dnaK. The amount of the dnaJ protein in E. coli is substantially less than that of the dnaK protein, which is produced abundantly. In order to construct a system that over-produces the dnaJ protein, we started our study by determining the DNA sequence of the entire dnaJ gene, and an operon fusion was constructed by inserting the gene downstream of the lambda PL promoter of an expression vector plasmid, pPL-lambda. Cells containing the recombinant plasmid produced dnaJ protein amounting to 2% of the total cellular protein when cells were induced. The overproduced protein was purified, and Edman degradation of the protein indicated that the NH2-terminal methionine was found to be processed. From the DNA sequence of the dnaJ gene, the processed gene product is composed of 375 amino acid residues, and its molecular weight is calculated to be 40,975.     

Nucleotide sequence of the Escherichia coli mutH gene.

The complete nucleotide sequence of mutH gene from E. coli has been determined. Based on the deduced amino acid sequence, the MutH protein has a molecular weight of 25.4 kdaltons in agreement with the previous estimates based on SDS-polyacrylamide gel electrophoresis of the purified protein. Deletion analysis of the DNA sequences upstream of mutH has identified the promoter region for this gene. Two independently isolated temperature sensitive alleles of the mutH gene have also been sequenced. One mutation results in an amino acid change at position 27 (thr to leu) while the other occurs at position 156 (asp to asn).     

Membrane-bound beta-lactamase forms in Escherichia coli

Frameshift pseudo-revertants of Escherichia coli RTEM beta-lactamase were obtained by a selection procedure, starting from frameshift mutants at the signal-processing site. These pseudo-revertant proteins, which have a totally altered COOH-terminal part of the signal sequence, are attached to the outer face of the inner membrane. The mutant proteins are enzymatically active in vitro and in vivo, and the membrane localization has no deleterious effect on cell growth. We conclude that initiation of transport across the membrane does not require the COOH-terminal part of the signal, but this part of the sequence determines whether the protein is released to the periplasm either with or without cleavage of the signal, or whether the protein remains anchored to the membrane. Mutants with two signals in series were used to show that a truncated signal is not refractory to transport per se. If neither signal contains a functional cleavage site, the protein is at least partially found on the outer face of the inner membrane. If both signals contain functional cleavage sites, both are removed and the protein is released to the periplasm. If only the first signal contains a cleavage site, a longer fusion protein is transported and released. The results presented here show that a pre-beta-lactamase-like protein can fold properly even as a membrane-bound species.     

Identification of the purC gene product of Escherichia coli.

The purC region of the Escherichia coli chromosome was isolated from in vivo-derived lambda transducing bacteriophages and cloned in high-copy-number plasmids. The product of the purC gene, phosphoribosylaminoimidazolesuccinocarboxamide synthetase, was identified as a protein with an Mr of ca. 27,000. The level of the protein is increased by more than 60-fold in strains carrying the gene on a high-copy-number plasmid. Purine addition represses the enzyme level in both plasmid- and non-plasmid-containing strains.     

Analysis of the relA gene product of Escherichia coli.

The relA gene product, ATP: GTP 3'-pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 degrees C of 120 mumol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 degrees C was found to be 4 mumol guanosine pentaphosphate formed min-1 mg protein-1. These values are about 100 times higher than reported by others. Our further studies of this enzyme led to the following results. Antibodies raised against this enzyme inhibit the ribosome-activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes. The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes. The amount of antibody-binding material in CP79 (relA) is at least 5 times lower.     

Formation of Membrane-bound CDP-diglyceride from Membrane-bound Phosphatidic Acid in Escherichia coli

The title compounds were synthesized from starting materials of microbial origin by employing a Wittig reaction as the key step.     

Cloning the spoT gene of Escherichia coli: identification of the spoT gene product.

We have isolated five specialized transducing lambda bacteriophages (lambda dpyrE spoT) carrying the pyrE and spoT genes of Escherichia coli. A fragment from one of these phages was used as the source of DNA to clone the spoT and pyrE genes on a multicopy plasmid, pBR322. Insertions and deletions in this plasmid were obtained. These plasmids were used to transform a minicell-producing strain, and the gene products synthesized were determined. Our experiments demonstrate that the spoT and pyrE genes are separated by about 4 magadaltons and suggest that the spoT gene product is a protein whose molecular weight is 80,000. The strain in which the spoT+ allele is carried on a plasmid produced nine times more spoT gene activity than a normal spoT+ strain when assayed in crude extracts. This strain was used to prepare partially purified gene product, guanosine 5'-diphosphate, 3'-diphosphate pyrophosphatase. The enzyme has the following characteristics. (i) It hydrolyzes pyrophosphate from the 5'-pyrophosphate of guanosine 5'-diphosphate, 3'-diphosphate, yielding GDP and pyrophosphate. (ii) Its activity is strongly stimulated by Mn2+ and slightly stimulated by salt. (iii) Its activity is inhibited by uncharged tRNA. There are also two additional activities in the cell extract which degrade guanosine in 5'-diphosphate, 3'-diphosphate in vitro but which are not specified by the spoT gene.     

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.     

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning beta-strands, probably forming a beta-barrel. Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCl and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.