Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene

Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein. The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues. The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein. When the cloned gene was expressed in E. coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800). The product was labeled with [3H]glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase. We thus designed the gene product as lipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane. A possible orientation of lipoprotein-28 in the E. coli envelope is discussed.     

Evolution of the lipoprotein gene in the enterobacteriaceae. Cloning and DNA sequence of the lpp gene from Proteus mirabilis

We cloned the lipoprotein gene from Proteus mirabilis and determined its DNA sequence. Comparison with the lpp genes from Escherichia coli, Serratia marcescens, Erwinia amylovora and Morganella morganii revealed several unique features of the evolution of the lpp gene in the Enterobacteriaceae and enabled us to establish phylogenetic relationships between these bacteria.     

Recognition of Morganella subspecies, with proposal of Morganella morganii subsp. morganii subsp. nov. and Morganella morganii subsp. sibonii subsp. nov.

The genus name Morganella was established within the family Enterobacteriaceae in 1978. Morganella morganii is the only species described thus far within this genus, and the name M. morganii has been accepted by usage in the scientific community for strains previously known as Proteus morganii. M. morganii isolates differ in their abilities to ferment trehalose and exhibit variable lysine and ornithine decarboxylase patterns, emphasizing the phenotypic heterogeneity within this species. Previous genetic studies failed to reveal separate entities within the genus Morganella. We observed some trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns. Two strains were lysine and ornithine positive, 3 were lysine positive and ornithine negative, and 29 were lysine negative and ornithine positive. These strains and 25 non-trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns were investigated. DNA-DNA hybridization studies and phenotypic characterizations revealed that M. morganii can be separated into three DNA relatedness groups and seven biogroups. Strains from DNA relatedness group 1 were trehalose negative, and strains from DNA relatedness groups 2 and 3 were trehalose positive. One biogroup from DNA relatedness group 2 was phenotypically indistinguishable from DNA relatedness group 3. On the basis of these studies, we propose that M. morganii be subdivided into M. morganii subsp. morganii (type strain ATCC 25830) containing biogroups A, B, C, and D (DNA relatedness group 1) and M. morganii subsp. sibonii (type strain 8103-85; = ATCC 49948) containing biogroups E, F, and G (DNA relatedness groups 2 and 3).     

Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli.

Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. Like porin protein F (OprF), it is a vaccine candidate because it antigenically cross-reacts with all serotype strains of the International Antigenic Typing Scheme. Since lipoprotein I was expressed in Escherichia coli under the control of its own promoter, we were able to isolate the gene by screening a lambda EMBL3 phage library with a mouse monoclonal antibody directed against lipoprotein I. The monocistronic OprI mRNA encodes a precursor protein of 83 amino acid residues including a signal peptide of 19 residues. The mature protein has a molecular weight of 6,950, not including bound glycerol and lipid. Although the amino acid sequences of protein I of P. aeruginosa and Braun's lipoprotein of E. coli differ considerably (only 30.1% identical amino acid residues), peptidoglycan in E. coli, are identical. Using lipoprotein I expressed in E. coli, it can now be tested whether this protein alone, without P. aeruginosa lipopolysaccharide contaminations, has a protective effect against P. aeruginosa infections.     

Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene.

Pseudomonas aeruginosa PAO-E64 is a mutant which produces parental levels of elastase antigen but has no elastolytic activity at 37 degrees C. The lesion (lasA1) in PAO-E64 is not a mutation in the structural gene for P. aeruginosa elastase (P.A. Schad, R.A. Bever, T.I. Nicas, F. Leduce, L.F. Hanne, and B.H. Iglewski, J. Bacteriol. 169: 2691-2696, 1987). A 1.7-kilobase segment of DNA that complements the lasA1 lesion was sequenced. Computer analysis of the DNA sequence showed that it contained an open reading frame which encoded a 41,111-dalton protein. The lasA gene was expressed under an inducible PT-7 promoter, and a 40,000-dalton protein was detected in Escherichia coli lysates. The lasA protein was localized in the outer membrane fraction of E. coli. This lasA protein produced in E. coli activated the extracellular elastase produced by the P. aeruginosa mutant, PAO-E64.     

Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli

Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics. The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 [García et al., Mol. Gen. Genet. 201 (1985) 225-230] and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter. The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one. The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined. A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified. The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration [H?ltje and Tomasz, J. Biol. Chem. 251 (1976) 4199-4207; Briese and Hakenbeck, Eur. J. Biochem. 146 (1985) 417-427]. Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin. The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.     

Escherichia coli glutaminyl-tRNA synthetase. I. Isolation and DNA sequence of the glnS gene

We have isolated a lambda-transducing phage carrying the gene (glnS) for Escherichia coli glutaminyl-tRNA synthetase. The location of the glnS gene within the 13.5-kilobase E. coli DNA transducing fragment was determined by genetic means. The glnS gene was recloned into plasmid pBR322 and its nucleotide sequence was established. The DNA sequence translates to a protein of 550 amino acids.     

DNA sequence of the Escherichia coli K-12 gamma-glutamyltranspeptidase gene, ggt.

The DNA sequence of ggt, the gene that codes for gamma-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12, has been determined. The sequence contains a single open reading frame encoding the signal peptide and large and small subunits, in that order. This result suggests that E. coli gamma-glutamyltranspeptidase is processed posttranslationally.     

DNA sequence of the gene coding for Escherichia coli ribonuclease H

The gene for Escherichia coli ribonuclease H has been studied by use of a plasmid which contains a segment of the E. coli chromosome. The genomic DNA was subcloned from pLC28-22 to pBR322 by use of various restriction enzymes. Such subcloning limited the RNase H gene to a piece of DNA no longer than 760 base pairs. Cells bearing plasmids containing the RNase H gene produce as much as 10-15 times the normal amount of RNase H without any drastic effect on maintenance of the plasmid or cell growth. DNA sequence analysis has permitted the prediction of a protein whose molecular weight is 17,559 (155 amino acid residues). The predicted sequence was confirmed by amino acid analysis, NH2-terminal amino acid sequence, and size determination of highly purified RNase H.     

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.     

Molecular cloning of Proteus morganii phenylalanine deaminase gene in Escherichia coli

The gene for phenylalanine deaminase (PAD) of Proteus morganii strain 2815 has been isolated on a 6.3-kb HindIII restriction fragment and cloned within RP4-prime plasmids, pYB2321 and pYB2322, in both orientations. Expression of the cloned gene in Escherichia coli strains was comparable to that in P. morganii 2815. The hybrid plasmids mobilized the 2815 chromosome with trajectories in reverse directions from an origin between ser-2 and ade-1, suggesting the map location of the PAD gene.     

Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli

Cytochrome P-450cam catalyzes the stereospecific methylene hydroxylation of camphor to form 5-exohydroxycamphor and is encoded by the camC gene on the CAM plasmid of Pseudomonas putida, ATCC 17453. The cytochrome P-450cam structural gene has been cloned by mutant complementation in P. putida (Koga, H., Rauchfuss, B., and Gunsalus, I. C. (1985) Biochem. Biophys. Res. Commun. 130, 412-417). We report the complete nucleotide sequence of the camC gene along with 155 base pairs of 5' and 175 base pairs of 3' flanking sequence. Upon comparison of the amino acid sequence derived from the gene sequence to the one obtained from the purified protein (Haniu, M., Armes, L. G., Yasunobu, K. T., Shastry, B. A., and Gunsalus, I. C. (1982) J. Biol. Chem. 257, 12664-12671), five differences were found. The most significant was the addition of a Trp and a Thr residue between Val-54 and Arg-55, thereby increasing the amino acid numbering scheme by 2 after Val-54, bringing the total number of amino acids to 414. Other differences were: Gln-274----Glu-276, Ser-359----His-361, and Asn-405----Asp-407. N-terminal amino acid sequence analysis of the cloned cytochrome P-450cam enzyme expressed in Escherichia coli under the lac promoter showed a faithful translation of the hemo-protein, with the N-terminal Met removed by processing as found in P. putida. Purification to homogeneity of the cloned protein was accomplished by the method used for the CAM plasmid-encoded enzyme of P. putida. The G + C content of the camC gene was found to be 59.0%, caused by a preferred usage of G and C terminated codons. The gene encoding putidaredoxin reductase, camA, was located 22 nucleotides downstream from the cytochrome P-450cam gene. The camA gene initiated with a novel GUG codon, the first such initiator documented in Pseudomonas.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.