NreB from Achromobacter xylosoxidans 31A Is a nickel-induced transporter conferring nickel resistance.

There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre. Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A. xylosoxidans 31A and Escherichia coli. E. coli cells expressing nreB showed reduced accumulation of Ni(2+), suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni(2+) resistance.

Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs.

The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera Alcaligenes, Ralstonia, and Burkholderia was evaluated using ARDRA (Amplified Ribosomal DNA Restriction Analysis). The analysis included 44 strains isolated from harsh industrial environments in sediments, soils and wastes with high content of heavy metals. These strains were selected by their ability to grow in the presence of high concentrations of multiple heavy metals and to hybridise with czc or ncc probes. The czc operon confers resistance to cadmium, zinc and cobalt in strain Ralstonia eutropha CH34. The ncc operon confers resistance to nickel, cobalt and cadmium in strain 31A known as Alcaligenes xylosoxidans. The analysis showed a close phylogenetic clustering of the czc+ strains inside the Ralstonia genus despite of their different origins and that the Ralstonia genus contained also the hydrogenotrophs and some catabolic strains assigned to the genus Ralstonia eutropha, strains up to now registrated as CDC IV c-2 strains as well as reference strains belonging to Ralstonia solanacearum and Ralstonia pickettii. The ncc+ strains are phylogenetically less related to each other compared to the czc+ strains. This suggests that the tested czc+ strains and some of the ncc+ strains may be considered as belonging to the genus Ralstonia. Inside this major Ralstonia cluster, a subcluster gathers most of the czc+ isolates maybe giving a clue to define a new species. Besides, from 30 tested strains, 15 metal resistant strains of this subcluster proved to display the unusual mutator phenotype characteristic of the representative strain CH34.

Phylogenetic identification of two major nitrogen-fixing bacteria associated with sugarcane.

Acetobacter diazotrophicus and Herbaspirillum seropedicae were identified by genetic methods based on 16S rRNA sequences. A specific PCR method in combination with probing was developed for A. diazotrophicus. The PCR system includes four primers, of which the primers named AC (CTGTTTCCCGCAAGGGAC) and DI (GCGCCCCATTGCTGGGTT) generated an 445 bp amplicon in all of the 11 A. diazotrophicus strains tested. The phylogenetic position of H. seropedicae was determined. H. seropedicae forms with Oxalobacter formigenes a separate lineage in the beta-subclass of Proteobacteria.

Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22.

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (betadelta) and an NADH:acceptor oxidoreductase (alphagamma)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed.

Biodegradation of polyhydroxyalkanoic acids.

Stimulated by the commercial availability of bacteriologically produced polyesters such as poly[(R)-3-hydroxybutyric acid], and encouraged by the discovery of new constituents of polyhydroxyalkanoic acids (PHA), a considerable body of knowledge on the metabolism of PHA in microorganisms has accumulated. The objective of this essay is to give an overview on the biodegradation of PHA. The following topics are discussed: (i) general considerations of PHA degradation, (ii) methods for identification and isolation of PHA-degrading microorganisms, (iii) characterization of PHA-degrading microorganisms, (iv) biochemical properties of PHA depolymerases, (v) mechanisms of PHA hydrolysis, (vi) regulation of PHA depolymerase synthesis, (vii) molecular biology of PHA depolymerases, (viii) influence of the physicochemical properties of PHA on its biodegradability, (ix) degradation of polyesters related to PHA, (x) biotechnological aspects of PHA and PHA depolymerases.

Nickel-resistant bacteria from anthropogenically nickel-polluted and naturally nickel-percolated ecosystems.

DNA fragments harboring the nickel resistance determinants from bacteria isolated from anthropogenically polluted ecosystems in Europe and Zaire were compared with those harboring the nickel resistance determinants from bacteria isolated from naturally nickel-percolated soils from New Caledonia by DNA-DNA hybridization. The biotinylated DNA probes were derived from the previously described Alcaligenes eutrophus CH34, Alcaligenes xylosoxidans 31A, Alcaligenes denitrificans 4a-2, and Klebsiella oxytoca CCUG 15788 and four new nickel resistance-determining fragments cloned from strains isolated from soils under nickel-hyperaccumulating trees. Nine probes were hybridized with endonuclease-cleaved plasmid and total DNA samples from 56 nickel-resistant strains. Some of the New Caledonian strains were tentatively identified as Acinetobacter, Pseudomonas mendocina, Comamonas, Hafnia alvei, Burkholderia, Arthrobacter aurescens, and Arthrobacter ramosus strains. The DNA of most strains showed homologies to one or several of the following nickel resistance determinants: the cnr and ncc operons of the strains A. eutrophus CH34 and A. xylosoxidans 31A, respectively, the nre operon of strain 31A, and the nickel resistance determinants of K. oxytoca. On the basis of their hybridization reactions the nickel resistance determinants of the strains could be assigned to four groups: (i) cnr/ncc type, (ii) cnr/ncc/nre type, (iii) K. oxytoca type, and (iv) others. The majority of the strains were assigned to the known groups. Among the strains from Belgium and Zaire, exclusively the cnr/ncc and the cnr/ncc/nre types were found. Among the New Caledonian strains all four types were represented. Homologies to the nre operon were found only in combination with the cnr/ncc operon. The homologies to the cnr/ncc operon were the most abundant and were detected alone or together with homologies to the nre operon. Only the DNA of the strains isolated from soil in Scotland and the United States and that of five of the New Caledonian strains did not show any detectable homologies to any of our probes. The nickel resistance fragment isolated from Burkholderia strain 32W-2 was studied in some detail. This 15-kb BamHI fragment conferred resistance to 1 to 5 mM NiCl(inf2) to Escherichia coli and resistance to up to 25 mM NiCl(inf2) to A. eutrophus. It showed strong homologies to both the cnr/ncc operon and the nre operon and conferred strictly regulated (inducible) nickel resistance to A. eutrophus.

Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system.

Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.

The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer, expression, regulation and DNA homologies to various nickel-resistant bacteria.

Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mM NiCl2 in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mM NiCl2. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mM Ni2+) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5-5) isolated from nickel-rich soil in New Caledonia.

Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A.

The nickel-cobalt-cadmium resistance genes carried by plasmid pTOM9 of Alcaligenes xylosoxidans 31A are located on a 14.5-kb BamHI fragment. By random Tn5 insertion mutagenesis, the fragment was shown to contain two distinct nickel resistance loci, ncc and nre. The ncc locus causes a high-level combined nickel, cobalt, and cadmium resistance in strain AE104, which is a cured derivative of the metal-resistant bacterium Alcaligenes eutrophus CH34. ncc is not expressed in Escherichia coli. The nre locus causes low-level nickel resistance in both Alcaligenes and E. coli strains. The nucleotide sequence of the ncc locus revealed seven open reading frames designated nccYXHCBAN. The corresponding predicted proteins share strong similarities with proteins encoded by the metal resistance loci cnr (cnrYXHCBA) and czc (czcRCBAD) of A. eutrophus CH34. When different DNA fragments carrying ncc genes were heterologously expressed under the control of the bacteriophage T7 promoter, five protein bands representing NccA (116 kDa), NccB (40 kDa), NccC (46 kDa), NccN (23.5 kDa), and NccX (16.5 kDa) were detected.

Degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by aerobic sewage sludge.

The degradation of sheets of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (BIOPOL) by aerobic sewage sludge was analyzed. Degradation of the polymer was highly dependent on the pH of the culture medium and was maximal between pH 7 and pH 8.5. Below pH 6 and above pH 9 the degradation rate was very low. Agitation of the culture fluid had relatively little influence on the rates of degradation. 1.2 x 10(5) aerobic polymer-degrading bacteria per ml sewage sludge were identified by halo formation on solid poly(3-hydroxybutyrate) (PHB)-containing media. The number of PHB-degrading bacteria in other ecosystems amounted to 3.8 x 10(3) per ml sludge of a fresh-water lake, 9.2 x 10(5) per g garden-soil, 1.3 x 10(6) per g field-soil and 4.3 x 10(6) per g compost.

Cloning and characterization of the poly(hydroxyalkanoic acid)-depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product.

Four different DNA fragments each coding for poly(hydroxyalkanoic acid) depolymerase (phaZ1-phaZ4) were isolated in pUC plasmids from a genomic library of Pseudomonas lemoignei in Escherichia coli. All recombinant strains secreted a highly active poly(3-hydroxybutyric acid) depolymerase and produced large translucent halos on an opaque medium containing poly(3-hydroxybutyric acid) granules. One DNA region (phaZ1) was present in seven independently isolated clones. Three other cloned DNA fragments were different from phaZ1 and from each other (phaZ2-phaZ4). In phaZ1, an open-reading frame of 1245 bp was identified from the nucleotide sequence of a 5435-bp MboI fragment (57 mol G + C/100 mol) of this region and encoded a novel poly(hydroxyalkanoic acid) depolymerase of P. lemoignei, poly(3-hydroxybutyric acid) depolymerase C. A leader-sequence peptidase-cleavage site was predicted from the deduced amino acid sequence between Ala37 and Leu38. The calculated relative molecular masses of the precursor and the putative mature protein were 43468 and 39581, respectively. The polypeptide contains a lipase consensus sequence (Gly-Xaa-Ser-Xaa-Gly) and an unusually high proportion of threonine residues (22 of 36 amino acids) near the C-terminus. The N-terminus of the deduced amino acid sequence of PhaZ1 differed from that of the purified poly(3-hydroxybutyric acid) depolymerases A, B and the poly(3-hydroxyvaleric acid) depolymerase of P. lemoignei. The phaZ1 gene product, poly(3-hydroxybutyric acid) depolymerase C, was partially purified from recombinant E. coli (pUC91::phaZ1). The purified protein was specific for poly(hydroxyalkanoic acid) consisting of monomers of four or five carbon atoms and for p-nithrophenylbutyrate as substrates. The polymer-hydrolyzing activity, but not the p-nitrophenylate esterase activity, was inhibited by complex media such as Luria-Bertani medium and by soluble E. coli proteins. The enzyme protein did not cross-react with antibodies raised against purified poly(3-hydroxyvaleric acid) depolymerase of P. lemoignei.

The giant linear plasmid pHG207 from Rhodococcus sp. encoding hydrogen autotrophy: characterization of the plasmid and its termini.

As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MR11 and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids--on pHG201 (270 kb) in strain MR11 and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possible functions.

Microbial synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate.

A microbial route was explored for the synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate (2-keto-3-deoxy-6-phosphogluconate, KDPG). Two strains of bacteria, Alcaligenes eutrophus H16 F34 (DSM 529) and Escherichia coli DF 71 (CGSC 4880), lacking in KDPG-aldolase activity were tested for excretion of KDPG. Using pyruvate and gluconate as carbon sources, Alcaligenes eutrophus H16 F34 accumulated and excreted 3-deoxy-D-erythro-hexulosonic acid 6-phosphate into the culture broth, while the E. coli strain, using pyruvate and glucuronate, failed. KDPG was isolated from the culture supernatant of Alcaligenes eutrophus H16 F34 in 78% yield and 5 g scale with respect to the consumed gluconate.

Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13.

Twenty-five gram-negative bacteria and one gram-positive bacterium capable of growing on poly(3-hydroxyoctanoic acid) [P(3HO)] as the sole source of carbon and energy were isolated from various soils, lake water, and activated sludge. Most of the isolates degraded only P(3HO) and copolymers of medium-chain-length (MCL) hydroxyalkanoic acids (HA). Except for the gram-positive strain, which was able to hydrolyze P(3HO) and poly(3-hydroxybutyric acid) [P(3HB)], no isolate was able to degrade polymers of short-chain-length HA, such as P(3HB) or poly(3-hydroxyvalerate) [P(3HV)]. All strains utilized a large variety of monomeric substrates for growth. All gram-negative strains, but not the gram-positive strain, accumulated poly(hydroxyalkanoic acids) (PHA), consisting of MCL HA, if they were cultivated under accumulation conditions. One strain, which was identified as Pseudomonas fluorescens GK13 (biovar V), was selected and the extracellular P(3HO) depolymerase of this strain was purified from the culture medium of P(3HO)-grown cells by chromatography with Octyl-Sepharose CL4B and by gel filtration with Superose 12. The relative molecular weights of the native and sodium dodecyl sulfate-treated enzymes were 48,000 and 25,000, respectively. The purified enzyme hydrolyzed P(3HO), copolymers of MCL HA, and para-nitrophenyl esters of fatty acids. P(3HB), P(3HV), and characteristic substrates for lipases, such as Tween 80 or triolein, were not hydrolyzed. The P(3HO) depolymerase of P. fluorescens GK13 was insensitive to phenylmethylsulfonyl fluoride and dithioerythritol, unlike other PHA depolymerases. The dimeric ester of 3-hydroxyoctanoic acid was identified as the main product of enzymatic hydrolysis of P(3HO).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus.

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).

High-Level Nickel Resistance in Alcaligenes xylosoxydans 31A and Alcaligenes eutrophus KTO2.

Two new nickel-resistant strains of Alcaligenes species were selected from a large number (about 400) of strains isolated from ecosystems polluted by heavy metals and were studied on the physiological and molecular level. Alcaligenes xylosoxydans 31A is a heterotrophic bacterium, and Alcaligenes eutrophus KTO2 is an autotrophic aerobic hydrogen-oxidizing bacterium. Both strains carry-among other plasmids-a megaplasmid determining resistance to 20 to 50 mM NiCl(2) and 20 mM CoCl(2) (when growing in defined Tris-buffered media). Megaplasmids pTOM8, pTOM9 from strain 31A, and pGOE2 from strain KTO2 confer nickel resistance to the same degree to transconjugants of all strains of A. eutrophus tested but were not transferred to Escherichia coli. However, DNA fragments carrying the nickel resistance genes, cloned into broad-hostrange vector pVDZ'2, confer resistance to A. eutrophus derivatives as well as E. coli. The DNA fragments of both bacteria, TBA8, TBA9, and GBA (14.5-kb BamHI fragments), appear to be identical. They share equal size, restriction maps, and strong DNA homology but are largely different from fragment HKI of nickel-cobalt resistance plasmid pMOL28 of A. eutrophus CH34.

Physiology and molecular genetics of poly(beta-hydroxy-alkanoic acid) synthesis in Alcaligenes eutrophus.

The Alcaligenes eutrophus genes for beta-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly(beta-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.

Differential stability of mRNA species of Alcaligenes eutrophus soluble and particulate hydrogenases.

The functional half-lives of Alcaligenes eutrophus hydrogenase mRNAs were determined by physiological studies. Evidence was obtained for a functional half-life of about 1 h for the soluble NAD-linked hydrogenase (HoxS) mRNA and 14 min for the particulate hydrogenase (HoxP) mRNA. The synthesis of active HoxS continued for about 4 h, albeit at a decreasing rate after inhibition of transcription, e.g., by rifampin. In this strain, the mRNA of HoxS appeared to be stable, while the mRNA of HoxP did not. Different species of hoxS mRNA were detected by the Northern (RNA) hybridization technique using as a probe plasmid pCH139 carrying hoxS structural genes. The sizes of the major hoxS mRNA species were 7.6, 6.2, 5.0, and 0.9 kb. The chemical half-lives of these species ranged from 1 h (5.0-kb mRNA) to 7 h (0.9-kb mRNA). Evidence for a specific cleavage of the 6.2-kb transcript yielding the 0.9-kb species was obtained from RNA-DNA hybridizations with subcloned hoxS DNA. The chemical half-life of total hoxP mRNA was 8 min.