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An outer membrane phospholipase A active against phosphatidylglycerol and phosphatidylethanolamine was characterized from Acinetobacter sp. HO1-N.
The electron transport system of Acinetobacter sp. HO1-N was studied to determine the specific cytochromes and to measure changes in the composition of the respiratory system due to growth in various concentrations of oxygen or types of growth substrates. Spectrophotometric analysis revealed that the quantity and types of cytochromes changed in response to growth under various concentrations of oxygen. Growth on alkane and nonalkane substrates resulted in only minor differences in cytochrome composition or oxidase activities. Membranes prepared from cells grown under oxygen-limiting conditions contained at least one b-type cytochrome, cytochrome o, cytochrome d, and slight traces of cytochrome a1, whereas membranes prepared from cells grown in the presence of high oxygen concentrations contained only low levels of cytochromes b and o. Polarographic measurements, electron transport inhibitor studies, and photoaction spectrum analyses indicated that cytochromes o, a1, and d were potentially capable of functioning as terminal oxidases in this organism. These experiments also revealed that all three cytochromes may be involved in the oxidation of reduced nicotinamide adenine dinucleotide, succinate, or N,N,N',N'-tetramethyl-p-phenylenediamine.
The high affinity of Acinetobacter calcoaceticus RAG-1 for liquid hydrocarbons permitted the isolation of a spontaneous nonadherent mutant, MR-481. Strain MR-481 exhibited no significant affinity for three test hydrocarbons, yet resembled the wild type in many properties, including production of the extracellular emulsifying agent emulsan. To study the role of adherence in growth on hydrocarbons, RAG-1 and MR-481 were compared for growth on hexadecane under conditions of limited agitation and at low initial cell densities. Adherent RAG-1 cells were able to grow rapidly under these conditions, whereas nonadherent MR-481 cells failed to grow for at least 54 h. However, the addition of emulsan either initially or at various times after inoculation enabled the nonadherent MR-481 cells to grow on hexadecane. Growth was not the result of reversion of MR-481 from nonadherent to adherent cells. The data demonstrate that adherence is a crucial factor in the growth of A. calcoaceticus RAG-1 on hexadecane in the absence of extracellular emulsification of the substrate.
When exponentially growing cultures of Acinetobacter calcoaceticus RAG-1 or RAG-92 were either treated with inhibitors of protein synthesis or starved for a required amino acid, there was a stimulation in the production of emulsan, an extracellular polyanionic emulsifier. Emulsan synthesis in the presence of chloramphenicol was dependent on utilizable sources of carbon and nitrogen and was inhibited by cyanide or azide or anaerobic conditions. Radioactive tracer experiments indicated that the enhanced production of emulsan after the addition of chloramphenicol was due to both the release of material synthesized before the addition of the antibiotic (40%) and de novo synthesis of the polymer (60%). Chemical analysis of RAG-1 cells demonstrated large amounts of polymeric amino sugars; it was estimated that cell-associated emulsan comprised about 15% of the dry weight of growing cells. The data are consistent with the hypothesis that a polymeric precursor of emulsan accumulates on the cell surface during the exponential growth phase; in the stationary phase or during inhibition of protein synthesis, the polymer is released as a potent emulsifier.
Various immunochemical techniques were employed to probe the relationship between the extracellular emulsifying agent (emulsan) and the cell-associated form of the polymer in Acinetobacter calcoaceticus RAG-1. Using an emulsan-specific antibody preparation, immunocytochemical labeling revealed that an emulsan-like antigen is a major component of the 125-nm minicapsule which envelopes the exponential-phase cell of the parent strain. The marked reduction of this capsule in stationary-phase cells was correlated with the production of extracellular emulsifying activity. Crossed immunoelectrophoresis techniques demonstrated that the major antigenic component (S1) of the culture supernatant fluid is immunochemically identical to purified emulsan, yet electrophoretically distinct. The characteristics of the parent strain were compared with those of two phage-resistant mutant strains which are defective in extracellular emulsan production. One of these mutants, termed TR3, lacked both the emulsan-like capsule on the cell surface and the extracellular S1 component. A second phage-resistant emulsan-defective mutant (TL4) was characterized by an antigenically altered and inactive form of extracellular emulsan. A relatively small amount of emulsan-like capsular material was consistently demonstrated on the cell surface of this mutant. The correlation between phage sensitivity and extracellular emulsan production was strengthened by the fact that emulsan-specific antibodies inhibited both emulsification activity and phage adsortion onto cells of the parent strain.
An esterase activity has been found, both in the cell-free growth medium and on the cell surface of the hydrocarbon-degrading Acinetobacter calcoaceticus RAG-1. The enzyme catalyzed the hydrolysis of acetyl and other acyl groups from triglycerides and aryl and alkyl esters. Emulsan, the extracellular heteropolysaccharide bioemulsifier produced by strain RAG-1, was also a substrate for the enzyme. Gel filtration showed that the cell-free enzyme was released from the cell surface either emulsan free or associated with the bioemulsifier. The partially purified enzyme was found to interact specifically with the esterified fully active emulsan, but not with the deesterified polymer. A role for esterase in emulsan release from the cell surface was indicated when the enzyme was preferentially depleted from the cell surface under conditions in which emulsan was not released. Such cells lost the capacity to release the biopolymer.
The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.
Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH) exhibiting an apparent Km for ethanol of 512 microM and a Vmax of 138 nmol/min. An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme which exhibited an apparent Km for acetaldehyde of 50 microM and a Vmax of 183 nmol/min. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is a soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH exhibited apparent Kms for hexadecanol of 1.6 and 2.8 microM in crude extracts derived from hexadecane- and hexadecanol-grown cells, respectively. HDH was distinct from ADH-A and ADH-B, since HDH and ADH-A were not coinduced; Eth1 had wild-type levels of HDH; and HDH requires NAD, while ADH-B requires NADP. NAD- and NADP-independent HDH activity was not detected in the soluble or membrane fraction of extracts derived from hexadecane- or hexadecanol-grown cells. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.
This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed.
This article describes the cloning of the mutarotase gene from Acinetobacter calcoaceticus and its expression in Escherichia coli. Purification of mutarotase (EC 5.1.3.3) led to a single polypeptide of 40 kilodaltons. The sequences of 27 N-terminal and 76 C-terminal amino acids were determined. From six amino acids of the N-terminal and seven amino acids of the C-terminal portion of the protein, the sequences of two oligonucleotides were deduced. These were synthesized and used as gene probes. Completely restricted chromosomal DNA from A. calcoaceticus was size fractioned, and only fractions hybridizing with the gene probes were used to construct gene banks enriched for the mutarotase determinant. With the N-terminal gene probe, a bank of 6- to 7-kilobase-pair BclI fragments in pBR327 was obtained. A total of 1,200 candidates were screened by colony hybridization followed by dot-blot analysis of purified plasmids from positive candidates and subsequent Southern blot analysis of the respective restricted plasmids, and 500 base pairs (bp) from the 5' end of the mutarotase gene were isolated by this procedure. The 3' portion of the gene was isolated from a gene bank containing 1,500-bp-long HindIII fragments inserted in M13mp11. This bank was screened by dot-blot analysis of single-stranded phage DNA with the C-terminal gene probe. The isolated gene fragments were fused at a common restriction site in their overlapping region to yield the complete mutarotase gene. High-level expression of mutarotase in E. coli was achieved when the gene was placed under transcriptional control of the phage lambda promoter pL. More than 90% of mutarotase activity was found in the culture medium. The E. coli-derived mutarotase was purified and shown to be identical to the A. calcoaceticus-derived product with respect to the molecular weight and N-terminal amino acid sequence. The expression of mutarotase in E. coli was increased 200-fold in comparison to that the wild-type A. calcoaceticus.
Catechol 1,2-dioxygenase (EC 1.13.1.1), the product of the catA gene, catalyzes the first step in catechol utilization via the beta-ketoadipate pathway. Enzymes mediating subsequent steps in the pathway are encoded by the catBCDE genes which are carried on a 5-kilobase-pair (kbp) EcoRI restriction fragment isolated from Acinetobacter calcoaceticus. This DNA was used as a probe to identify Escherichia coli colonies carrying recombinant pUC19 plasmids with overlapping sequences. Repetition of the procedure yielded an A. calcoaceticus 6.7-kbp EcoRI restriction fragment which contained the catA gene and bordered the original 5-kbp EcoRI restriction fragment. When the catA-containing fragment was placed under the control of the lac promoter on pUC19 and induced with isopropylthiogalactopyranoside, catechol dioxygenase was formed in E. coli at twice the level found in fully induced cultures of A. calcoaceticus. A. calcoaceticus strains with mutations in the catA gene were transformed to wild type by DNA from lysates of E. coli strains carrying the catA gene on recombinant plasmids. Thus, A. calcoaceticus strains with a mutated gene can be used in a transformation assay to identify E. coli clones in which at least part of the wild-type gene is present but not necessarily expressed.
Mutants of Acinetobacter calcoaceticus LMD79.41 were isolated that are defective in the synthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ). A gene bank of the wild-type. A. calcoaceticus genome was constructed with the binary plasmid system pLV21-RP4 delta Km. The DNA of A. calcoaceticus LMD79.41 was partially digested with Sau3A, and fragments of about 15 kilobases were inserted into the BamHI site of pLV21. The hybrid plasmids maintained in Escherichia coli were transferred by conjugation to the PQQ- mutants of A. calcoaceticus. One hybrid plasmid was isolated that complements all isolated PQQ- mutants. Subcloning of this plasmid in the vector pRK290 resulted in an insert of 5 kilobases on which at least four different genes involved in PQQ synthesis could be indicated. With Tn5 insertions the four PQQ genes were mapped, and it was shown that these genes are most probably located in three operons.
Biosynthetic regulation of catA, the gene encoding catechol 1,2-dioxygenase (EC 1.13.1.1), was studied in an Acinetobacter calcoaceticus mutant strain unable to metabolize benzoate. Benzoate and muconate independently induced the enzyme. In glucose-grown cells, benzoate yielded higher enzyme levels than did muconate, whereas muconate was the more effective inducer in succinate-grown cells.
The catabolic genes necessary for the conversion of benzoate to catechol have been cloned from Acinetobacter calcoaceticus into Escherichia coli. The cloned genes, benABCD, encoded both a benzoate 1,2-dioxygenase system, composed of NADH-cytochrome c reductase and terminal oxygenase components, and a cis-diol dehydrogenase. The dioxygenase system appears to be encoded by three genes, benABC, whose products, 53-, 19-, and 38-kilodalton proteins, correspond in size to those of components in other bacterial dioxygenases. The cloned dioxygenase system is expressed at high level in E. coli, enabling the conversion of benzoate to a cis-diol, 2-hydro-1,2-dihydroxybenzoate, at a rate comparable to that of fully induced A. calcoaceticus cultures. A cis-diol dehydrogenase, the product of the A. calcoaceticus benD gene, when present in E. coli enables this organism to convert the cis-diol intermediate to catechol. The dehydrogenase has been partially purified and is a dimer with two identical 31-kilodalton subunits. The ben genes are clustered on the A. calcoaceticus chromosome with independently regulated genes needed for the dissimilation of catechol. In a 16-kilobase-pair region of the chromosome there are 10 genes for benzoate catabolism, organized in no fewer than three transcriptional units. This kind of arrangement, termed supraoperonic clustering, has been observed previously in pseudomonads.
The beta-ketoadipate pathway of Acinetobacter calcoaceticus comprises two parallel metabolic branches. One branch, mediated by six enzymes encoded by the cat genes, converts catechol to succinate and acetyl coenzyme A (acetyl-CoA); the other branch, catalyzed by products of the pca genes, converts protocatechuate to succinate and acetyl-CoA by six metabolic reactions analogous or identical to those of the catechol sequence. We used the expression plasmid pUC18 to construct expression libraries of DNA from an A. calcoaceticus mutant strain from which the cat genes had been deleted. Immunological screening with antiserum to the pcaE gene product, beta-ketoadipate:succinyl-CoA transferase I, resulted in the isolation of a cloned 11-kilobase-pair (kbp) fragment which inducibly expressed all six pca genes under control of the lac promoter on pUC18. The induced Escherichia coli cells formed the six pca gene products at levels 10- to 30-fold higher than found in fully induced A. calcoaceticus cultures, although protocatechuate 3,4-dioxygenase (the iron-containing product of the pcaA gene) from the recombinant strain possessed a relatively low turnover number. An E. coli culture expressing the cloned pca genes quantitatively converted protocatechuate to beta-ketoadipate; failure of the organism to metabolize the latter compound can be most readily ascribed to relatively low pool levels of succinyl-CoA, a required substrate for beta-ketoadipate:succinyl-CoA transferase, in E. coli. The gene order and direction of transcription were determined to be pcACBDFE by identification of enzymes expressed in subclones, by using natural transformation to identify subclones carrying DNA corresponding to dysfunctional alleles in mutant A. calcoaceticus strains, and by restriction mapping of both the 11-kbp fragment and derivatives of the 11-kbp fragment containing Tn5 in the pcaA, pcaB, pcaD, and pcaE genes. The fragment containing the pca gene hybridized strongly and specifically to a previously cloned fragment containing A. calcoaceticus cat genes.
Isofunctional beta-ketoadipate:succinyl coenzyme A transferases I and II are encoded by the pcaE and catE genes, respectively, of Acinetobacter calcoaceticus. The genes are under separate transcriptional control and genetically unlinked. Mutations in the pcaE gene result in a p-hydroxybenzoate-negative (POB-) phenotype, whereas catE mutations cause a benzoate-negative (Ben-) phenotype. A. calcoaceticus ADP125 carries the pcaE3125 mutation and gave rise to POB+ revertants with a frequency of 10(-4). A 5.0-kilobase-pair (kb) EcoRI restriction fragment containing the catBCDE genes possesses two SalI restriction sites separated by 1.5 kb. Removal of the DNA between the SalI sites created a deletion removing the terminal 35 base pairs of the catB gene, the 300-base-pair catC gene, and about 1.1 kb of the 1.2-kb catE gene. Transformation of strain ADP125 with the modified EcoRI fragment lacking the SalI segment produced natural transformants containing this designed deletion with a frequency of 20%. The frequency of POB+ phenotypic reversion of the pcaE3125 mutation in these transformants was more than 300-fold lower than the frequency of phenotypic reversion observed in genetic backgrounds containing the catBCE segment. Alleles created by pcaE phenotypic reversion in a wild-type cat genetic background were unlinked to the cat gene cluster, and revertant transferases were expressed inducibly with the pca genes. Alterations in the restriction pattern of the pca gene cluster in several revertants were observed, indicating that multiple sequence changes have occurred in the pca genes during reversion. Growth of the phenotypic revertants under nonselective conditions resulted in loss of either the POB+ phenotype or both the POB+ and Ben+ phenotypes at high frequency. Southern hybridizations revealed that loss of the POB+ of Ben+ phenotype was due to deletion of the entire pca or cat gene cluster, a loss of at least 16 kb in some strains. Revertants isolated in a catBCE deletion background were stable. These results suggest that enhanced phenotypic reversion of pcaE3125 in wild-type cat background is due to repair of the mutation by recombination between the catBCE and pcaE3125 sequences. Genetic instability of the phenotypic revertants may be attributed to deletion of pca and cat sequences by recombination between regions of homology created as a consequence of pcaE repair.
The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.
The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli.