Nucleotide sequence of the sfaA gene coding for the S-fimbrial protein subunit of Escherichia coli

The sfaA gene of the uropathogenic Escherichia coli O6 strain 536, which is responsible for the determination of the S fimbrial protein subunit, was sequenced. The structural gene codes for a polypeptide of 180 amino acids including a 24-residue N-terminal signal sequence. A size of 15.95 kDa was calculated for the processed SfaA protein. The nucleotide and deduced amino acid sequences show significant homology to those of the F1C fimbria and, to a lesser extent, of the mannose-sensitive hemagglutinating fimbria (FimA, PilA). Only week homology to P fimbriae subunits (F7₂, Pap) was found.     

Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12.

Melibiose uptake and hydrolysis in E.coli is performed by the MelB and MelA proteins, respectively. We report the cloning and sequencing of the melA gene. The nucleotide sequence data showed that melA codes for a 450 amino acid long protein with a molecular weight of 50.6 kd. The sequence data also supported the assumption that the mel locus forms an operon with melA in proximal position. A comparison of MelA with alpha-galactosidase proteins from yeast and human origin showed that these proteins have only limited homology, the yeast and human proteins being more related. However, regions common to all three proteins were found indicating sequences that might comprise the active site of alpha-galactosidase.     

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

Nucleotide sequence of the Escherichia coli entE gene

The Escherichia coli entE gene encodes a polypeptide necessary in the latter stages of biosynthesis of the siderophore enterobactin. The entE gene and adjacent DNA were sequenced. The predicted EntE polypeptide consists of 536 amino acids and has a Mr of 58,299 and a net charge of -7.33. Genetic evidence combined with this and previous sequencing data indicate that the genes entCEB(G)A are transcribed as unit from a promoter upstream of entC.     

Nucleotide sequence and expression in Escherichia coli of the recA gene of Neisseria gonorrhoeae

The nucleotide sequence of the recA gene of Neisseria gonorrhoeae MS11 has been determined. The product of this gene can act as a recombinase in Escherichia coli, but does so with a decreased efficiency, probably because of the formation of mixed multimers with the equivalent E. coli protein.     

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 of the phosphofructokinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene

The gene (Bs-pfk) for phosphofructokinase (PFK) from Bacillus stearothermophilus has been cloned and sequenced. The deduced amino acid sequence is nearly identical to the sequence which was previously determined by peptide analysis. The elevated G + C content of Bs-pfk relative to the homologous Ec-pfkA from Escherichia coli is consistent with previous observations concerning genes from thermophilic prokaryotes. A significant degree of homology exists when the deduced amino acid sequence of B. stearothermophilus PFK is compared with the corrected sequences of rabbit muscle PFK or E. coli PFK-1. The cloning and sequencing of Bs-pfk completes the first step toward using site-specific mutagenesis to investigate the structure-function relationships for this allosteric enzyme.     

Nucleotide sequence of the murE gene of Escherichia coli

The nucleotide sequence of the murE gene encoding the diaminopimelic acid adding enzyme of Escherichia coli is reported. The coding region consisted of 1413 base pairs and was separated from the ftsI (penicillin-binding protein 3) gene by 61 base pairs. The deduced primary structure of MurE comprised 471 amino acid residues with a molecular mass of 50.6 kilodaltons.     

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).     

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.