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PubMed1984

Control of autotrophic carbon assimilation in Alcaligenes eutrophus by inactivation and reactivation of phosphoribulokinase.

Phosphoribulokinase in Alcaligenes eutrophus was partially inactivated when an autotrophic culture was shifted to heterotrophic growth with pyruvate as the sole source of carbon and energy. A similar response was observed on addition of various organic substrates to autotrophic cultures during the transition to mixotrophic growth. The extent of inactivation depended on the added substrate. Pyruvate or lactate caused the strongest inactivation among the tested substrates. Up to 75% of the phosphoribulokinase activity found in the autotrophic cells was lost within 30 min after supplementation of the cultures with either of these two substrates. This loss of enzyme activity was not the result of degradation of enzyme protein. Inactivation of phosphoribulokinase was accompanied by a decrease in the CO2 fixation rate of the cells. Reactivation of the enzyme occurred after exhaustion of pyruvate from the medium. Neither inactivation nor reactivation required de novo protein synthesis; however, continued energy conversion was necessary for the inactivation to occur. We suggest that the pyruvate metabolism of A. eutrophus is involved in these regulatory processes which act on phosphoribulokinase. They appear to contribute to the control of autotrophic CO2 assimilation in this organism.

Alcaligenes↗
PubMed1984

Alcaligenes eutrophus hydrogenase genes (Hox).

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.

Alcaligenes↗
PubMed1984

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway.

Alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway. DNA analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pRA1000. HindIII and XhoI restriction endonuclease analysis of pRA1000 showed it to be similar to the archetypal TOL plasmid, pWWO, differing in the case of HindIII only by the absence of fragments B and D present in pWWO. In strain 345, the presence of pRA1000 prevented the expression of chromosomally encoded enzymes required for the degradation of p-cresol, whereas these enzymes were expressed in strains cured of pRA1000. On the basis of studies with an R68.45-pRA1000 cointegrate plasmid, pRA1001, we conclude that the gene(s) responsible for the effect of p-cresol degradation resides within or near the m- and p-toluate degradative region on pRA1000.

Alcaligenes↗
PubMed1984

Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.

Alcaligenes↗
PubMed1984

Regulation by molecular oxygen and organic substrates of hydrogenase synthesis in Alcaligenes eutrophus.

Chemoautotrophic growth of Alcaligenes eutrophus 17707 is inhibited by 20% oxygen in the gas phase. Lowering the oxygen concentration to 4% results in chloramphenicol-sensitive derepression of soluble and membrane-bound hydrogenase activity (and of soluble hydrogenase antigen), showing that oxygen inhibition is due at least in part to repression of hydrogenase synthesis. Mutations resulting in derepression of hydrogenase activity (and antigen) under 25% oxygen (Ose-) mobilized with a self-transmissable plasmid which is already known to carry genes necessary for hydrogenase expression. Plasmid-borne mutations resulting in loss of soluble hydrogenase activity have no effect on the Ose phenotype, but chromosomal mutations resulting in reduction or loss of both hydrogenase activities cannot be made Ose-. The Ose- mutation does not alter the thermostability of either hydrogenase, and soluble hydrogenase in the mutant reacts with complete identity with that of the wild type, indicating that the Ose- phenotype does not result from structural alterations in either enzyme. Ose- mutants are also relieved of normal hydrogenase repression by organic substrates, which aggravates hydrogenase-mediated inhibition of heterotrophic growth by hydrogen. Regulation of hydrogenase in Ose- strains of A. eutrophus 17707 is nearly identical to that of wild-type A. eutrophus strains H1 and H16.

Alcaligenes↗
PubMed1984

Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus.

Phosphoenolpyruvate (PEP) carboxykinase was identified to be the only C3-carboxylating enzyme in Alcaligenes eutrophus. The enzyme requires GDP or inosine diphosphate (GTP or inosine triphosphate) for activity. Pyruvate- and other PEP-dependent CO2-fixing enzyme activities were not detected, regardless of whether the cells were grown autotrophically or heterotrophically. It is suggested that two pathways are present in the organism for the formation of PEP from C4 dicarboxylic acids. Besides decarboxylation of oxaloacetate by PEP carboxykinase, the consecutive action of NADP+-malic enzyme and PEP synthetase can also accomplish this synthesis. An oxaloacetate decarboxylase activity observed in the cell extracts may also contribute to the latter route. The properties of a mutant deficient in PEP synthetase supported the biochemical data. This mutant was unable to grow on pyruvate or lactate and grew slower than the wild type on direct or indirect metabolites of the tricarboxylic acid cycle such as succinate, glutamate, or acetate. Growth on fructose and autotrophic growth were not affected by the enzyme defect. The findings suggest that, depending on the growth substrate utilized, PEP carboxykinase can serve a dual physiological function in A. eutrophus, an anaplerotic function in oxaloacetate synthesis from PEP, or a gluconeogenic function in PEP synthesis from oxaloacetate.

Alcaligenes↗
PubMed1984

Construction and use of a gene bank of Alcaligenes eutrophus in the analysis of ribulose bisphosphate carboxylase genes.

A gene bank of the DNA from the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was constructed in the broad host range cosmid vector pVK102 and established in Escherichia coli. A triparental replica plating procedure was developed to allow rapid screening of large numbers of isolated E. coli gene bank clones for complementation of A. eutrophus mutants. This procedure was used to identify hybrid cosmids that complemented CO2 fixation-negative (Cfx-), H2 uptake-negative (Hup-), and auxotrophic A. eutrophus mutants. The average insert DNA size in these hybrid cosmids was 22 kilobases. Nine hybrid cosmids that complemented ribulose bisphosphate carboxylase-negative (RuBPCase-) mutants were characterized. They fell into two distinct groups with respect to their restriction patterns. Complementing subclones from the two groups contained no common restriction fragments, but hybridization experiments indicated a high degree of sequence homology. Restriction fragments corresponding to one of the subclones were absent in total DNA from a cured strain that had lost plasmid pAE7, indigenous to the wild type. It is concluded that functional CO2 fixation genes in the A. eutrophus ATCC 17707 chromosome are reiterated on plasmid pAE7.

Alcaligenes↗
PubMed1985

Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals.

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

Alcaligenes↗
PubMed1985

Denitrification by Alcaligenes eutrophus is plasmid dependent.

Curing of the hydrogenase-specifying megaplasmid pHG indigenous to strains of the facultative lithoautotrophic bacterium Alcaligenes eutrophus was correlated with a loss of denitrifying ability (Nitd). The retransfer of plasmid pHG1 reconstituted the Nitd phenotype. Plasmid-free mutants were still capable of converting some nitrate to nitrite, but they did not metabolize nitrite under anaerobic conditions.

Alcaligenes↗
PubMed1985

Chromosomal and plasmid locations for phosphoribulokinase genes in Alcaligenes eutrophus.

Genes coding for phosphoribulokinase (PRK), a key enzyme of the Calvin cycle, were localized in the genome of the chemoautotroph Alcaligenes eutrophus. The NH2-terminal sequence of the PRK subunit was determined. With a synthetic oligodeoxynucleotide probe complementary to a portion of this sequence, hybridization analysis revealed PRK genes to be located on both the chromosome and the megaplasmid pHG1 of A. eutrophus H16.

Alcaligenes↗
PubMed1986

Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other gram-negative bacteria.

The Escherichia coli pfkA gene has been cloned in the non-self-transmissible vector pVK101 from hybrid plasmids obtained from the Clarke and Carbon clone bank, resulting in the plasmids pAS300 and pAS100; the latter plasmid also encoded the E. coli tpi gene. These plasmids were transferred by conjugation to mutants of Alcaligenes eutrophus which are unable to grow on fructose and gluconate due to lack of 2-keto-3-deoxy-6-phosphogluconate aldolase activity. These transconjugants recovered the ability to grow on fructose and harbored pAS100 or pAS300. After growth on fructose, the transconjugants contained phosphofructokinase at specific activities between 0.73 and 1.83 U/mg of protein, indicating that the E. coli pfkA gene is readily expressed in A. eutrophus and that the utilization of fructose occurs via the Embden-Meyerhof pathway instead of the Entner-Doudoroff pathway. In contrast, transconjugants of the wild type of A. eutrophus, which are potentially able to catabolize fructose via both pathways, grew at a decreased rate on fructose and during growth on fructose did not stably maintain pAS100 or pAS300. Indications for a glycolytic futile cycling of fructose 6-phosphate and fructose 1,6-bisphosphate are discussed. Plasmid pA 100 was also transferred to 14 different species of gram-negative bacteria. The pfkA gene was expressed in most of these species. In addition, most transconjugants of these strains and of A. eutrophus exhibited higher specific activities of triosephosphate isomerase than did the corresponding parent strains.

Alcaligenes↗
PubMed1986

Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16.

A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.

Alcaligenes↗
PubMed1987

Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus.

Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.

Alcaligenes↗
PubMed1987

Sequence analysis of the Alcaligenes eutrophus chromosomally encoded ribulose bisphosphate carboxylase large and small subunit genes and their gene products.

The nucleotide sequence of the chromosomally encoded ribulose bisphosphate carboxylase/oxygenase (RuBPCase) large (rbcL) and small (rbcS) subunit genes of the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was determined. We found that the two coding regions are separated by a 47-base-pair intergenic region, and both genes are preceded by plausible ribosome-binding sites. Cotranscription of the rbcL and rbcS genes has been demonstrated previously. The rbcL and rbcS genes encode polypeptides of 487 and 135 amino acids, respectively. Both genes exhibited similar codon usage which was highly biased and different from that of other organisms. The N-terminal amino acid sequence of both subunit proteins was determined by Edman degradation. No processing of the rbcS protein was detected, while the rbcL protein underwent a posttranslational loss of formylmethionyl. The A. eutrophus rbcL and rbcS proteins exhibited 56.8 to 58.3% and 35.6 to 38.5% amino acid sequence homology, respectively, with the corresponding proteins from cyanobacteria, eucaryotic algae, and plants. The A. eutrophus and Rhodospirillum rubrum rbcL proteins were only about 32% homologous. The N- and C-terminal sequences of both the rbcL and the rbcS proteins were among the most divergent regions. Known or proposed active site residues in other rbcL proteins, including Lys, His, Arg, and Asp residues, were conserved in the A. eutrophus enzyme. The A. eutrophus rbcS protein, like those of cyanobacteria, lacks a 12-residue internal sequence that is found in plant RuBPCase. Comparison of hydropathy profiles and secondary structure predictions by the method described by Chou and Fasman (P. Y. Chou and G. D. Fasman, Adv. Enzymol. 47:45-148, 1978) revealed striking similarities between A. eutrophus RuBPCase and other hexadecameric enzymes. This suggests that folding of the polypeptide chains is similar. The observed sequence homologies were consistent with the notion that both the rbcL and rbcS genes of the chemoautotroph A. eutrophus and the thus far characterized rbc genes of photosynthetic organisms have a common origin. This suggests that both subunit genes have a very ancient origin. The role of quaternary structure as a determinant of the rate of accepted amino acid substitution was examined. It is proposed that the sequence of the dimeric R. rubrum RuBPCase may be less conserved because there are fewer structural constraints for this RuBPCase than there are for hexadecameric enzymes.

Alcaligenes↗
PubMed1987

Plasmid pCBI carries genes for anaerobic benzoate catabolism in Alcaligenes xylosoxidans subsp. denitrificans PN-1.

Pseudomonas sp. strain PN-1 is reclassified as Alcaligenes xylosoxidans subsp. denitrificans PN-1. Strain PN-1 is a gram-negative, rod-shaped organism, is motile by means of lateral flagella, is oxidase positive, and does not ferment sugars. Plasmid pCBI, carrying genes for the anaerobic degradation of benzoate in strain PN-1, is 17.4 kilobase pairs in length and is transmissible to a number of denitrifying Pseudomonas aeruginosa and Pseudomonas stutzeri strains. A restriction endonuclease map was constructed.

Alcaligenes↗
PubMed1987

The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells.

The gene encoding a blue copper protein (a member of the pseudoazurins) of 123 amino acid residues, containing a single type I Cu2+ ion, was cloned from Alcaligenes faecalis S-6. The nucleotide sequence of the coding region, as well as the 5'- and 3'-flanking regions, was determined. The deduced amino acid sequence after Glu-24 coincided with the reported sequence of the blue protein, and its NH2-terminal sequence of 23 residues resembled a typical signal peptide. The cloned gene was expressed under the control of the tac promoter in Escherichia coli, and the correctly processed blue protein was secreted into the periplasm. The blue protein produced in E. coli possessed the activity to transfer electrons to the copper-containing nitrite reductase of A. faecalis S-6 in vitro.

Alcaligenes↗
PubMed1987

Genetic and physical mapping and expression in Pseudomonas aeruginosa of the chromosomally encoded ribulose bisphosphate carboxylase genes of Alcaligenes eutrophus.

We have previously shown that functional ribulose bisphosphate carboxylase (RuBPCase, rbc) genes in Alcaligenes eutrophus ATCC 17707 are present both on the chromosome and on the indigenous plasmid pAE7. Here we demonstrate that the chromosomal rbc locus encodes both a large (rbcL)- and a small (rbcS)-subunit gene. A 2.3-kilobase DNA fragment containing both subunit genes was subcloned into the broad-host-range vector pRK310 to yield plasmid pAE312. This plasmid was transferred into Pseudomonas aeruginosa in which expression of both the rbcL and rbcS genes took place, as demonstrated by Western blot analysis. A high level of RuBPCase activity was observed for P. aeruginosa(pAE312), suggesting that assembly of the subunits took place. Plasmid pAE312 was mutagenized with Tn5 in Escherichia coli. Complementation of A. eutrophus RuBPCase structural gene mutants with pAE312 containing mapped Tn5 insertions allowed functional analysis of the rbc gene region. The polar effect of the Tn5 insertions suggested that the two subunit genes were cotranscribed in A. eutrophus, with rbcL located promoter proximal. Northern blot analysis of total RNA from P. aeruginosa(pAE312) confirmed cotranscription of the two subunit genes. DNA probes containing both the rbcL and rbcS genes, or fragments of each gene, all hybridized to a predominant transcript about 2.1-kilobases long. These observations indicate that the chromosomally encoded rbcL and rbcS genes of A. eutrophus constitute an operon.

Alcaligenes↗
PubMed1987

Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 contains all genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). Five of these genes, tfdB, tfdC, tfdD, tfdE, and tfdF, have recently been localized and cloned (R. H. Don, A. J. Weightman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). Gene tfdA, which codes for the 2,4-D monooxygenase, has now been found by mutagenesis with transposon Tn5. A 3-kilobase fragment of pJP4 cloned in a broad-host-range vector could complement the 2,4-D-negative phenotype of two mutants which lacked 2,4-D monooxygenase activity. The cloned tfdA gene was also transferred to A. eutrophus JMP222, which is a cured derivative of JMP134. The recombinant strain could utilize phenoxyacetic acid as a sole source of carbon and energy. Pseudomonas sp. strain B13, containing the cloned tfdA, was able to degrade phenoxyacetic acid and 4-chlorophenoxyacetic acid. Gene tfdA was subcloned and analyzed by deletions. Expression of 2,4-D monooxygenase in Escherichia coli containing a 1.4-kilobase subfragment was demonstrated by radioisotopic enzyme assay, and a protein of 32,000-dalton molecular mass was detected by labeling experiments. A 2-kilobase subfragment containing tfdA has been sequenced. Sequence analysis revealed an open reading frame of 861 bases which was identified as the coding region of tfdA by insertion mutagenesis.

Alcaligenes↗