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 '.     

Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase.

The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D. N. Crowell, M. S. Anderson, and C. R. H. Raetz, J. Bacteriol. 168:152-159, 1986). We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene. This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB. There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA. The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled. A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction. These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E. coli chromosome. The accompanying paper by Tomasiewicz and McHenry (J. Bacteriol. 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon.     

Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid. A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A. R. Harker, R. H. Olsen, and R. J. Seidler, J. Bacteriol. 171:314-320, 1989; B. Kaphammer, J. J. Kukor, and R. H. Olsen, J. Bacteriol. 172:2280-2286, 1990). When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently. The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F. The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4. An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII. The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family. The complete ORF represents the regulatory gene tfdR. The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively. This ORF is identified as part of a putative isofunctional gene, tfdDII.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.     

Determination of the precise location and orientation of the Escherichia coli dnaE gene.

The minimal region required for expression of the dnaE gene of Escherichia coli has been determined relative to a detailed restriction endonuclease map. This has been accomplished by analysis of Bal 31 exonuclease-generated deletions from the termini of the E. coli DNA contained in plasmid pMWE303 , a plasmid that we have previously demonstrated to contain the dnaE gene (M. M. Welch and C. S. McHenry , J. Bacteriol . 152:351-356, 1982). The competence of these deletion-containing plasmids in expressing the alpha subunit of DNA polymerase III holoenzyme has been determined by their ability both to complement a dnaE mutant and to direct the synthesis of a complete alpha subunit. The carboxyl-terminal coding region of dnaE has been identified through the detection of partial alpha polypeptides encoded by plasmids containing deletions from one end of the gene. This approach has permitted the precise determination of both termini of the dnaE gene and the determination of the orientation of the gene within the E. coli chromosome.     

Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon

A second nitrogen fixation (nif) operon in the cyanobacterium (blue-green alga) Anabaena (Nostoc) sp. strain PCC 7120 has been identified and sequenced. It is located just upstream of the nifHDK operon and consists of four genes in the order nifB, fdxN, nifS, and nifU. The three nif genes were identified on the basis of their similarity with the corresponding genes from other diazotrophs. The fourth gene, fdxN, codes for a bacterial type ferredoxin (Mulligan, M. E., Buikema, W. J., and Haselkorn, R. (1988) J. Bacteriol. 167, 4406-4410). The four genes are probably transcribed as a single operon, but are expressed at a lower level than the nifHDK operon, and only after a developmentally induced DNA rearrangement occurs that excises a 55-kilobase pair element from within the fdxN gene (Golden, J. W., Mulligan, M. E., and Haselkorn, R. (1987) Nature 327, 526-529; Golden, J. W., Carrasco, C. D., Mulligan, M. E., Schneider, G. J., and Haselkorn, R. (1988) J. Bacteriol. 170, 5034-5041). The promoter for the nifB operon was located by primer extension. Comparison of the nifB 5'-flanking sequence with the nifH 5'-flanking sequence did not reveal any consensus base pairs that would define a nif promoter for Anabaena. The operon contains two instances of 7-base pair directly repeated sequences: seven copies of the repeated sequence are found between the nifB and fdxN genes and six copies are found between the nifS and nifU genes. The function of these repeats is unknown.     

Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum.

Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J. W. Hastings, M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7491-7500, 1991). When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene. The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters. The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A. The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli.

The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene.     

Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.     

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.