The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

Summary The meta-cleavage operon of TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are The xyIXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xyIE, xyIG, xylF, xylJ, xylK, xylI and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxalocrotonate tautomerase, respectively. The functions of xyIT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xyIX and xyIH consists of coding sequences.     

Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWWO

Summary Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWWO are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointergrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.     

Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida

Summary Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida. A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon. In parallel, a series of N-methyl-N-nitro-N-nitrosoguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon. Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined. Four cistrons for toluate 1,2-dioxygenase were thus identified. DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate. This analysis also identified four cistrons. Examination of the products of these cistrons, by means of E. coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xy/Y, encodes a 20 kilodalton protein.     

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

Summary A new procedure is described to recombine plasmid-bornelacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flankinglacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncatedlacZ andaadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carryinglacZ andaadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy inPseudomonas putida the regulatory elements controlling expression of the Xy1S-activatedPm promoter of the TOL catabolic plasmid pWWO. We show here that thePm promoter undergoes a Xy1S-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.     

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli

Summary Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M_r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

The Azotobacter vinelandii nifL-like gene: nucleotide sequence analysis and regulation of expression

Summary The nucleotide sequence of the Azotobacter vinelandii nifL-like gene (Av-nifL) was determined. The 1.9 kb sequence shows an open reading frame (ORF) of 1557 by which encodes a polypeptide of 519 amino acids, with a calculated molecular weight of 57 793. Av-nifL has about 50 % homology with the Klebsiella pneumoniae nifL gene (Kp-nifL) at the nucleotide level and a little more than 52% homology at the amino acid level. The N-terminal regions show more homology than the C-terminal regions. As is the case in K. pneumoniae, Av-nifL is located just upstream of the A. vinelandii nifA gene (Av-nifA) and both genes constitute an operon. The expression of Av-nifL, however, seems to be independent of NtrA and NtrC. Furthermore, Av-nifL expression is not autogenously regulated by NifA, unlike the case in K. pneumoniae. The expression of an Av-nifL: lacZ fusion in A. vinelandii is inhibited by novobiocin and coumermycin A, which are inhibitors of DNA gyrase.     

Chemical synthesis,cloning and expression of human preproinsulin gene

A 355 base pair DNA sequence coding for human preproinsulin has been assembled by joining 55 synthetic deoxyoligonucleotide fragments prepared by the modified phosphotriester methodology. Proinsulin was expressed underlac promoter control and truncatedΒ-galactosidase 590 amino acid long sequence. The fusedΒ-galactosidase proinsulin protein was produced in amount to 30 % of the totalEscherichia coli proteins. It was also expressed in M13 bacteriophage and yeast system.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression

Summary The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5 and 3 flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme. Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany     

Evolutionary relationships between catabolic pathways for aromatics: Conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids

Summary TOL plasmid pWW0 and plasmid NAH7 encode catabolic enzymes required for oxidative degradation of toluene and naphthalene, respectively. The gene order of the catabolic operon of NAH7 for salicylate oxidation was determined to be: promoter-nahG (the structural gene for salicylate hydroxylase)-nahH (catechol 2,3-dioxygenase)-nahI (hydroxymuconic semialdehyde dehydrogenase)-nahN (hydroxymuconic semialdehyde hydrolase)-nahL (2-oxopent-4-enoate hydratase). This order is identical to that of the isofunctional genes of TOL plasmid pWW0. The complete nucleotide sequence of nahH was determined and compared with that of xylE, the isofunctional gene of TOL plasmid pWW0. There were 20% and 16% differences in their nucleotide and amino acid sequences, respectively. The homology between the NAH7 and TOL pWW0 plasmids ends upstream of the Shine-Dalgarno sequences of nahH and xylE, but the homology continues downstream of these genes. This observation suggested that genes for the catechol oxidative enzymes of NAH7 and TOL pWW0 were derived from a common ancestral sequence which was transferred as a discrete segment of DNA between plasmids.     

The complete sequence and segregational stability analysis of a new cryptic plasmid pIGWZ12 from a clinical strain of Escherichia coli

A new cryptic plasmid from a multi-resistant, multi-plasmid clinical strain of Escherichia coli has been isolated. The sequence of the 4072-base-pair pIGWZ12 (GenBank Accession No. DQ311641) was determined and analyzed. Two open-reading frames that code for proteins involved in plasmid mobilization and initiation of replication were identified. The putative origin of replication possesses all characteristic features of the theta mechanism for replicating plasmids. pIGWZ12 is stably maintained without selective pressure in bacterial cultures (for up to 80 generations), making it a good candidate for engineering a new cloning vector.     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

The uvp1 gene of plasmid pR cooperates with mucAB genes in the DNA repair process

Summary We show that a DNA fragment that contains the uvp1 gene of the plasmid pR directs the synthesis in Escherichia coli minicells of a protein of apparent molecular weight 20 kDa. Inspection of the nucleotide sequence of the region reveals an open reading frame that has the capacity to encode a protein of 198 amino acids. The uvp1 gene product has been found, in two different systems, to enhance the recombination activity of E. coli cells. We have also observed a striking similarity to resolvase and invertase proteins. The significance of this finding for the function of the uvp1 gene product requires further investigation. We conclude that the uvp1 gene encodes a 20 kDa protein which appears to be responsible for enhancement of both UV survival and recominational activity in E. coli.     

Cloning,DNA sequence,and expression of Aeromonas caviae WS7b chitinase gene

A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeromonas caviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of the truncated version of the gene in Escherichia coli. The chitinase gene product showed amino-acid sequence similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotometrically, using colloidal chitin azure as substrate for extracellular and intracellular fractions. The ability of the chitinase cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host.     

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme