Expression of polyhydroxyalkanoic-acid-biosynthesis genes in methylotrophic bacteria relying on the ribulose monophosphate pathway

Four representatives of methylotrophic bacteria relying on the ribulose monophosphate (RMP) pathway were investigated for their capability to synthesize polyhydroxyalkanoic acids (PHA). In Methylophilus methylotrophus B115, Methylobacillus glycogenes strains B121 and B53 and Acetobacter methanolicus B58 no \-ketothiolase, acetoacetyl-coenzyme A (CoA) reductase or PHA synthase could be detected, and hybridization experiments using heterologous DNA probes derived from PHA-biosynthesis genes of Methylobacterium extorquens or Alcaligenes eutrophus gave no evidence for the presence of the corresponding genes in these PHA-negative methylotrophic bacteria. Fragments harbouring a cluster of PHA-biosynthesis genes of A. eutrophus or Chromatium vinosum or isolated PHA synthase structural genes of M. extorquens, Rhodospirillum rubrum or Rhodobacter sphaeroides were mobilized into the RMP pathway bacteria mentioned above. Only transconjugants, which harboured the PHA-biosynthesis genes of A. eutrophus or C. vinosum, expressed active \-ketothiolase, acetoacetyl-CoA reductase and PHA synthase and accumulated poly(3-hydroxybutyric acid) (PHB). Highest amounts of PHB (up to 15% of the cellular dry weight) were accumulated in transconjugants of Methylophilus methylotrophus B115 or of Methylobacillus glycogenes strains B121 and B53 harbouring the PHA-biosynthesis genes of C. vinosum. Correspondence to: A. Steinbüchel     

A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I

Using a 30-mer oligonucleotide probe highly specific for polyhydroxyalkanoic acid (PHA) synthase genes, the respective genes of Pseudomonas citronellolis, P. mendocina, Pseudomonas sp. DSM 1650 and Pseudomonas sp. GP4BH1 were cloned from genomic libraries in the cosmid pHC79. A 19.5-kbp and a 22.0-kbp EcoRI restriction fragment of P. citronellolis or Pseudomonas sp. DSM 1650, respectively, conferred the ability to accumulate PHA of medium-chain-length 3-hydroxyalkanoic acids (HA _mcl ) from octanoate as well as from gluconate to the PHA-negative mutant P. putida GPp104. An 11.0-kbp EcoRI fragment was cloned from P. mendocina, which restored in GPp104 the ability to synthesize PHA from octanoate but not from gluconate. From Pseudomonas sp. GP4BH1 three different genomic fragments encoding PHA synthases were cloned. This indicated that strain GP4BH1 possesses three different functionally active PHA synthases. Two of these fragments (6.4 kbp and 3.8 kbp) encoded for a PHA synthase, preferentially incorporating hydroxyalkanoic acids of short chain length (HA _scl ), and the synthases were expressed in either GPp104 and Alcaligenes eutrophus H16-PHB^–4, respectively. The PHA synthase encoded by the third fragment (6.5 kbp) led to the incorporation of HA _mcl and was expressed in GPp104 but not in PHB^–4. Correspondence to: A. Steinbüchel     

Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme

The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (M _r 40,950) and PhaC (M _r 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed. Received: 9 January 2000 / Received revision: 20 February 2000 / Accepted: 25 February 2000     

Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene

A cosmid gene bank of partially EcoRI-digested genomic DNA from Methylobacterium extorquens IBT no. 6 was screened for DNA fragments restoring polyhydroxyalkanoic-acid (PHA) accumulation in the PHA-negative mutant Alkaligenes eutrophus H16 PHB^–4. The M. extorquens PHA-synthase structural gene phaC _Mex was mapped on a 23-kbp EcoRI fragment by complementation studies, by hybridization experiments with heterologous DNA probes from A. eutrophus H16 encoding for phaA, phaB and phaC and by nucleic acid sequence analysis. Evidence for the presence of genes for a -ketothiolase or an acetoacetyl-coenzyme A reductase on this fragment was not obtained. The nucleotide sequence of a 3.7-kbp region was obtained. It contained the entire 1.815-kbp phaC _Mex plus approximately each 900-bp upstream and downstream of phaC _Mex. PhaC _Mex encoded a protein of 605 amino acods with a relative molecular mass (M_r) of 66742, which exhibited 38.1% amino acid identity with the A. eutrophus PHA synthase. Determination of the N-terminal amino acid sequence of an M_r 65 000 protein, which was enriched concomitantly with the purification of PHA granules in sucrose gradients, revealed a sequence that was identical with the amino acid sequence deduced from the most probable translation start codon except for a valine, which was obviously removed post-translationally. Enzyme analysis, which was done with the native gene and a phaC _Mex -lacZ fusion gene, gave no evidence for expression of phaC _Mex in Escherichia coli.     

Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea

From a genomic library of Thiocystis violaceae strain 2311 in L47, two adjacent EcoRI restriction fragments of 5361 base pairs (bp) and of 1978 bp were cloned. The 5361-bp EcoRI restriction fragment hybridized with a DNA fragment harbouring the Alcaligenes eutrophus poly(3-hydroxyalkanoate) (PHA) synthase operon (phbCAB) and restored the ability to synthesize and accumulate PHA in PHA-negative mutants derived from A. eutrophus. The nucleotide sequence analysis of both fragments revealed five open-reading frames (ORFs); at least three of them are probably relevant for PHA biosynthesis. The amino acid sequences of the putative proteins deduced from these genes indicate that they encode a -ketothiolase [phbA _Tv, relative molecular mass (M_r) 40850], which exhibited 87.3% amino acid identify with the -ketothiolase from Chromatium vinosum. The amino acid sequences of the putative proteins deduced from ORF2_Tv (M_r 41 450) and phbC _Tv (M_r 39 550), which were located upstream of and antilinear to phbA _Tv, exhibited 74.7% and 87.6% amino acid identify, respectively, with the corresponding gene products of C. vinosum. Downstream of and antilinear to phbC _Tv was located ORF5, which encodes for a protein of high relative molecular mass (M_r 76428) of unknown function. With respect to the divergent organisation of ORF2_Tv and phbC _Tv on one side and of phbA _Tv on the other side and from the homologies of the putative gene products, this region of the T. violaceae genome resembled very much the corresponding region of C. vinosum, which was identified recently. Correspondence to: A. Steinbüchel     

Identification of 4-hydroxyhexanoic acid as a new constituent of biosynthetic polyhydroxyalkanoic acids from bacteria

Various aerobic Gram-negative bacteria were analysed for utilizing 4-hydroxyhexanoic acid (4HHx) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA). Although many wild types grew on 4HHx, only recombinant strains of the PHA-negative mutants Pseudomonas putida GPp104 and Alcaligenes eutrophus PHB^–4, which harboured plasmid pHP1014::E156 with the PHA-biosynthesis genes of Thiocapsa pfennigii, incorporated 4HHx up to a molar fraction of 47 or 1.4%, respectively, into PHA if the cells were cultivated in the presence of 4HHx as sole carbon source and under nitrogen starvation. A terpolyester consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyhexanoic acid (3HHx) and 4HHx was synthesized, as revealed by gas chromatographic analysis of the accumulated polyester and as confirmed by nuclear magnetic resonance spectroscopic analysis of the isolated polyester. 4HHx was also detected in PHA accumulated by Rhodococcus ruber if 4HHx was used as a carbon source. However, it occurred at a molar fraction of maximally 1.3 mol% only beside 3HB, 3-hydroxyvaleric acid and 3HHx. Correspondence to: A. Steinbüchel     

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB⁻4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4–5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200–400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains. Received: 3 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

Genetic Analysis of Comamonas acidovorans Polyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaC_Ca) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp β-ketothiolase gene (phaA_Ca). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaA_Ca. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB⁻4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism’s PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.     

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB^–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction. Received: 11 March 1998 / Accepted: 2 June 1998     

Expression of the Alcaligenes eutrophus poly(β-hydroxybutyric acid)-synthetic pathway in Pseudomonas sp.

The 12.5-kb EcoRI restriction fragment PP1 of Alcaligenes eutrophus strain H16, which encodes for -ketothiolase, NADP-dependent acetoacetyl-CoA reductase and poly(-hydroxybutyric acid)-synthase was mobilized to six different species of the genus Pseudomonas belonging to the rRNA homology group I. Pseudomonas aeruginosa, P. fluorescens, P. putida, P. oleovorans, P. stutzeri and P. syringae, which are unable to synthesize and accumulate poly(-hydroxybutyric acid), PHB, were employed as recipients. Whereas the A. eutrophus PHB-synthetic enzymes were only marginally expressed in P. stutzeri, they were readily expressed in the other species. For example, the specific activity of PHB-synthase was 1.8 U/g protein in transconjugants of P. stutzeri but was between 21 and 77 U/mg protein in transconjugants of the other species. All recombinant strains harboring plasmid pVK101::PP1 except those of P. stutzeri accumulated PHB; the PHB content of the cells grown on gluconate under nitrogen limitation varied between 8 and 24.3% of the cellular dry mass.Abbreviations PHB poly(-hydroxybutyric acid) - PHA poly(hydroxyalkanoic acid)     

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids

Summary We have constructed /SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.Part of this work was taken from the doctoral thesis of E.P.A. submitted to the Freie Universität, Berlin 1979     

The Autotrophic Synthesis of Polyhydroxyalkanoate by Alcaligenes eutrophusin the Presence of Carbon Monoxide

The CO-resistant strain B5786 of the hydrogen-oxidizing bacterium Alcaligenes eutrophuswas found to be able to synthesize polyhydroxyalkanoates (PHAs) under the conditions of growth limitation by nitrogen deficiency (the factor that promotes PHA synthesis) and growth inhibition by carbon monoxide. The gas mixtures that contained from 5 to 20 vol % CO did not inhibit the key enzymes of PHA synthesis–-ketothiolase, acetoacetyl-CoA reductase, hydroxybutyrate dehydrogenase, and PHA synthase. In the presence of CO, cells accumulated up to 70–75 wt % PHA (with respect to the dry biomass) without any noticeable increase in the consumption of the gas substrate. Chromatographic–mass spectrometric analysis showed that the PHA synthesized by A. eutrophusis a copolymer containing more than 99 mol % -hydroxybutyrate and trace amounts of -hydroxyvalerate. The PHA synthesized under the conditions described did not differ from that synthesized by A. eutrophuscells from electrolytic hydrogen.     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary Using a novel positive selection method for G3P transport activity, phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.Abbreviations DHBP 3,4-dihydroxibutyl-1-phosphonate - G3P sn-glycerol-3-phosphate - G3PBP glycerol-3-phosphate binding protein - IPTG isopropyl--d-thiogalactopyranoside - XG 5-bromo-4-chloro-3-indolyl--d-galactopyranoside     

Cloning of the GSH1 and GSH2 Genes Complementing the Defective Biosynthesis of Glutathione in the Methylotrophic Yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh⁺ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the -glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh^– phenotype of the gsh2 mutant. The Gsh⁺ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme -glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase. Received: 16 March 1999 / Received revision: 24 August 1999 / Accepted: 24 September 1999     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.     

Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutropbus pbbB and to Rbizobium meliloti nodG

Summary A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. mehloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the pbbB gene and involved in poly--hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli ⁷⁰ consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.     

Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl--D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - Sm^r resistance to streptomycin - Km^r resistance to kanamycin - Ap^r resistance to ampicillin - Tc^r resistance to tetracycline - Cm^r resistance to chloramphenicol - CMCase carboxymethylcellulase