First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene.

The min 4 region of the Escherichia coli genome contains genes (lpxA and lpxB) that encode proteins involved in lipid A biosynthesis. We have determined the sequence of 1,350 base pairs of DNA upstream of the lpxB gene. This fragment of DNA contains the complete coding sequence for the 28.0-kilodalton lpxA gene product and an upstream open reading frame capable of encoding a 17-kilodalton protein (ORF17). In addition there appears to be an additional open reading frame (ORF?) immediately upstream of ORF17. The initiation codon for lpxA is a GUG codon, and the start codon for ORF17 is apparently a UUG codon. The start and stop codons overlap between ORF? and ORF17, ORF17 and lpxA, and lpxA and lpxB. This overlap is suggestive of translational coupling and argues that the genes are cotranscribed. Crowell et al. (D.N. Crowell, W.S. Reznikoff, and C.R.H. Raetz, J. Bacteriol. 169:5727-5734, 1987) and Tomasiewicz and McHenry (H.G. Tomasiewicz and C.S. McHenry, J. Bacteriol. 169:5735-5744, 1987) have demonstrated that there are three similarly overlapping coding regions downstream of lpxB including dnaE, suggesting the existence of a complex operon of at least seven genes: 5'-ORF?-ORF17-lpxA-lpxB-ORF23-dnaE-ORF37-3 '.     

Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli.

Several enzymes have been discovered recently in crude extracts of Escherichia coli that appear to be involved in the biosynthesis of the lipid A component of lipopolysaccharide. Two of these are lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase. Lipid A disaccharide synthase activity is barely detectable in cells harboring a lesion in the lpxB (pgsB) gene. We subcloned the lpxB gene from plasmid pLC26-43 of the Clarke and Carbon collection (L. Clarke and J. Carbon, Cell 9:91-99, 1976) and localized it to a 1.7-kilobase-pair fragment of DNA counterclockwise of dnaE on the E. coli chromosome. Furthermore, we discovered a new gene (lpxA) located adjacent to and counterclockwise of lpxB that encodes or controls UDP-N-acetylglucosamine acyltransferase. Our data prove that lpxB and lpxA are transcribed in the clockwise direction and suggest that they may be cotranscribed.     

Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA.

In a previous paper, we reported identification of the 5' part of hprA of Methylobacterium extorquens AM1, which encodes the serine cycle enzyme hydroxypyruvate reductase (L. V. Chistoserdova and M. E. Lidstrom, J. Bacteriol. 174:71-77, 1992). Here we present the complete sequence of hprA and partial sequence of genes adjacent to hprA. Upstream of hprA, the 3' part of an open reading frame was discovered, separated from hprA by 263 bp. This open reading frame was identified as the gene encoding another serine cycle enzyme, serine glyoxylate aminotransferase (sgaA). Cells containing an insertion mutation into sgaA were unable to grow on C1 compounds, demonstrating that the gene is required for C1 metabolism. Sequencing downstream of hprA has revealed the presence of another open reading frame (mtdA), which is probably cotranscribed with hprA. This open reading frame was identified as the gene required for the synthesis of 5,10-methylenetetrahydrofolate dehydrogenase. Our data suggest that this enzyme plays an integral role in methylotrophic metabolism in M. extorquens AM1, either in formaldehyde oxidation or as part of the serine cycle.     

Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanobacterium Synechococcus sp. PCC7942

The genome of the cyanobacterium Synechococcus sp. PCC7942 contains two genes encoding the D2 polypeptide of photosystem II (PSII), which are designated here as psbDI and psbDII. The psbDI gene, like the psbD gene of plant chloroplasts, is cotranscribed with and overlaps the open reading frame of the psbC gene, encoding the PSII protein CP43. The psbDII gene is not linked to psbC, and appears to be transcribed as a monocistronic message. The two psbD genes encode identical polypeptides of 352 amino acids, which are 86% conserved with the D2 polypeptide of spinach. In plants, the translational start codon of the psbC gene has been reported to be an ATG codon 50 bp upstream from the end of the psbD gene. This triplet is not present in the psbDI sequence of Synechococcus sp., but is replaced by ACG, a codon which is very unlikely to initiate translation. Translation of the psbC gene may begin at a GTG codon which overlap the psbDI open reading frame by 14 bp and is preceded by a block of homology to the 3' end of the 16S ribosomal RNA, a potential ribosome-binding site. There are only two bp differences between the sequences of the two psbD genes; one of these results in substitution in psbDII of GCG for the presumed GTG start codon in psbDI.     

Nucleotide sequence of the gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex.

The nucleotide sequence was determined for the first part of the Bacillus subtilis sdh operon. An open reading frame corresponding to the structural gene, sdhA, for cytochrome b558 was identified. The predicted molecular weight of the cytochrome (excluding the N-terminal methionine) is 22,770. It is a very hydrophobic protein with five probable membrane-spanning segments. There is little homology between the B. subtilis cytochrome b558 and cytochrome b of mitochondrial complex III from different organisms or between cytochrome b558 and the hydrophobic sdhC and sdhD peptides of the Escherichia coli sdh operon. About 30 bases downstream of the sdhA stop codon, a new open reading frame starts. The nucleotide sequence predicts the presence of a typical flavin-binding peptide which identifies this reading frame as part of the sdhB gene. Seven bases upstream of the sdhA initiation codon ATG there is a typical B. subtilis ribosome binding site (free energy of interaction, -63 kJ), and further upstream, tentative sigma 55 and sigma 32 promoter sequences were found. The upstream region also contains two 12-base-long direct repeats; their significance is unknown.     

Nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes.

2,3-Dihydroxybiphenyl dioxygenase, which catalyzes ring metacleavage of 2,3-dihydroxybiphenyl, is encoded by the bphC gene of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and T. Miyazaki, J. Bacteriol. 166:392-398, 1986). We determined the nucleotide sequence of a DNA fragment of 2,040 base pairs which included the bphC gene. The fragment included one open reading frame of 912 base pairs to accommodate the enzyme. The predicted processed amino acid sequence of the enzyme subunit consisted of 302 residues, and its 12 NH2-terminal residues were in perfect agreement with those determined for the enzyme. Approximately 10 base pairs upstream from the initiation codon for 2,3-dihydroxybiphenyl dioxygenase, there was a base sequence complementary to the 3' end of the 16S rRNA from Pseudomonas aeruginosa. There was no promoterlike sequence in the region upstream of the bphC gene, but another long open reading frame was present. A putative bphD gene encoding a metacleavage compound-hydrolyzing enzyme was suggested in the region downstream of the bphC gene.     

The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.