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PubMed1989

Detection of Acinetobacter spp. in rural drinking water supplies.

A bacteriological survey was conducted of untreated, individual groundwater supplies in Preston County, W.Va. Nearly 60% of the water supplies contained total coliforms in excess of the U.S. Environmental Protection Agency maximum contaminant level of 1 CFU/100 ml. Approximately one-third of the water systems contained fecal coliforms and/or fecal streptococci. Acinetobacter spp. were detected in 38% of the groundwater supplies at an arithmetic mean density of 8 CFU/100 ml and were present in 16% of the water supplies in the absence of total coliforms, posing some concern about the usefulness of total coliforms as indicators of the presence of this opportunistic pathogen. Slime production, a virulence factor for A. calcoaceticus, was not significantly different between well water isolates and clinical strains, suggesting some degree of pathogenic potential for strains isolated from groundwater. In addition, several Acinetobacter isolates were able to interfere with sheen production by some coliform bacteria on M-Endo medium, adding further to the possible significance of Acinetobacter spp. in groundwater supplies.

Acinetobacter↗
PubMed1993

Isolation, characterization, and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in the construction of Escherichia coli shuttle plasmids.

Three cryptic plasmids have been discovered in Acinetobacter calcoaceticus BD413. These three plasmids, designated pWM10 (7.4 kb), pWM11 (2.4 kb), and pWM12 (2.2 kb), exhibited extensive homology to one another, as shown by Southern blot hybridization and restriction site analysis data, and also hybridized with three plasmids having slightly different sizes detected in a second strain, A. calcoaceticus BD4. Plasmid pWM11 and a fragment of pWM10 were each subcloned into pUC19, yielding plasmids pWM4 and pWM6, respectively, and were used in a series of inter- and intraspecies transformation experiments. Both plasmids replicated as high-copy-number plasmids in A. calcoaceticus BD413, as well as in strains of Escherichia coli. However, when transformed into the oil-degrading strain Acinetobacter lwoffii RAG-1, both plasmids were maintained at low copy numbers. No modification of the plasmids was detected after repeated transfers between hosts. An analysis of a series of deletions demonstrated that (i) a 185-bp fragment of pWM11 was sufficient to permit replication of the shuttle plasmid in A. calcoaceticus BD413, (ii) the efficiency of transformation of A. calcoaceticus BD413 decreased according to the size of the deletion in the insert by up to 4 orders of magnitude, and (iii) the entire insert was required for transformation and replication in A. lwoffii RAG-1. The sequence of pWM11 contained several small (150- to 300-bp) open reading frames, none of which exhibited any homology to known DNA or protein sequences. In addition, a number of inverted and direct repeats, as well as six copies of the consensus sequence AAAAAAATA previously described for a cryptic plasmid from A. lwoffii (M. Hunger, R. Schmucker, V. Kishan, and W. Hillen, Gene 87:45-51, 1990), were detected. Cloning and expression of the alcohol dehydrogenase regulon from A. lwoffii RAG-1 were accomplished by using the Acinetobacter shuttle plasmid.

Acinetobacter calcoaceticus↗
PubMed1997

A novel competence gene, comP, is essential for natural transformation of Acinetobacter sp. strain BD413.

Acinetobacter sp. strain BD413 (= ATCC 33305), a nutritionally versatile bacterium, has an extremely efficient natural transformation system. Here we describe the generation of eight transformation-affected mutants of Acinetobacter sp. strain BD413 by insertional mutagenesis. These mutants were found by Southern blot analysis and complementation studies to result from single nptII marker insertions at different chromosomal loci. DNA binding and uptake studies with one mutant, T205, revealed that the transformation deficiency of this mutant results from a complete lack of DNA binding and, therefore, uptake activity. A novel competence gene essential for natural transformation, named comP, was cloned by complementation of mutant T205. The nucleotide sequence of comP was determined, and its deduced 15-kDa polypeptide displays significant similarities to type IV pilins. Analysis of the ultrastructure of a transformation-deficient comP mutant and the transformation-competent wild-type strain revealed that both are covered with bundle-forming thin fimbriae (3 to 4 nm in diameter) and individual thick fimbriae (6 nm in diameter). These results provide evidence that the pilinlike ComP is unrelated to the piluslike structures of strain BD413. Taking all data into account, we propose that ComP functions as a major subunit of an organelle acting as a channel or pore mediating DNA binding and/or uptake in Acinetobacter sp. strain BD413.

Acinetobacter↗
PubMed1998

Antibiotic resistance in Acinetobacter spp. isolated from sewers receiving waste effluent from a hospital and a pharmaceutical plant.

The possible increase of antibiotic-resistant bacteria in sewage associated with the discharge of wastewater from a hospital and a pharmaceutical plant was investigated by using Acinetobacter species as environmental bacterial indicators. The level of susceptibility to six antimicrobial agents was determined in 385 Acinetobacter strains isolated from samples collected upstream and downstream from the discharge points of the hospital and the pharmaceutical plant. Results indicated that while the hospital waste effluent affected only the prevalence of oxytetracycline resistance, the discharge of wastewater from the pharmaceutical plant was associated with an increase in the prevalence of both single- and multiple-antibiotic resistance among Acinetobacter species in the sewers.

Acinetobacter↗
PubMed1999

Polyphosphate kinase of Acinetobacter sp. strain ADP1: purification and characterization of the enzyme and its role during changes in extracellular phosphate levels.

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl(2). The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40 degrees C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per microg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P(i)) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P(i)-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P(i) conditions. This activity declined when phosphate was added.

Acinetobacter↗
PubMed2000

Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants.

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10(9) CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.

Acinetobacter↗
PubMed2000

Long-chain aldehyde dehydrogenase that participates in n-alkane utilization and wax ester synthesis in Acinetobacter sp. strain M-1.

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C(4) to C(14)), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Delta) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Delta strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.

Acinetobacter↗
PubMed2001

Cloning and genetic characterization of dca genes required for beta-oxidation of straight-chain dicarboxylic acids in Acinetobacter sp. strain ADP1.

A previous study of deletions in the protocatechuate (pca) region of the Acinetobacter sp. strain ADP1 chromosome revealed that genes required for utilization of the six-carbon dicarboxylic acid, adipic acid, are linked to the pca structural genes. To investigate the genes involved in adipate catabolism, a 33.8-kb SacI fragment, which corrects a deletion spanning this region, was cloned. In addition to containing known pca, qui, and pob genes (for protocatechuate, quinate, and 4-hydroxybenzoate dissimilation), clone pZR8000 contained 10 kb of DNA which was the subject of this investigation. A mutant strain of Escherichia coli DH5alpha, strain EDP1, was isolated that was able to utilize protocatechuate and 4-hydroxybenzoate as growth substrates when EDP1 cells contained pZR8000. Sequence analysis of the new region of DNA on pZR8000 revealed open reading frames predicted to be involved in beta-oxidation. Knockouts of three genes implicated in beta-oxidation steps were introduced into the chromosome of Acinetobacter sp. strain ADP1. Each of the mutants was unable to grow with adipate. Because the mutants were affected in their ability to utilize additional saturated, straight-chain dicarboxylic acids, the newly discovered 10 kb of DNA was termed the dca (dicarboxylic acid) region. Mutant strains included one with a deletion in dcaA (encoding an acyl coenzyme A [acyl-CoA] dehydrogenase homolog), one with a deletion in dcaE (encoding an enoyl-CoA hydratase homolog), and one with a deletion in dcaH (encoding a hydroxyacyl-CoA dehydrogenase homolog). Data on the dca region should help us probe the functional significance and interrelationships of clustered genetic elements in this section of the Acinetobacter chromosome.

Acinetobacter↗
PubMed2001

Natural transformation in mesophilic and thermophilic bacteria: identification and characterization of novel, closely related competence genes in Acinetobacter sp. strain BD413 and Thermus thermophilus HB27.

The mesophile Acinetobacter sp. strain BD413 and the extreme thermophile Thermus thermophilus HB27 display high frequencies of natural transformation. In this study we identified and characterized a novel competence gene in Acinetobacter sp. strain BD413, comA, whose product displays significant similarities to the competence proteins ComA and ComEC in Neisseria and Bacillus species. Transcription of comA correlated with growth phase-dependent transcriptional regulation of the recently identified pilin-like factors of the transformation machinery. This finding strongly suggests that comA is part of a competence regulon. Examination of the genome sequence of T. thermophilus HB27 led to detection of a comA/comEC-like open reading frame (ORF) which is flanked by an ORF whose product shows significant similarities to the Bacillus subtilis competence protein ComEA. To examine whether these two ORFs, designated comEC and comEA, are implicated in natural transformation of T. thermophilus HB27, both were disrupted by using a thermostable kanamycin resistance marker. Natural transformation in comEC mutants was reduced 1,000-fold, whereas in comEA mutants the natural transformation phenotype was completely eliminated. These results strongly suggest that both genes, comEC and comEA, are required for natural transformation in T. thermophilus HB27. Several transmembrane alpha-helices are predicted based on the amino acid sequences of ComA in Acinetobacter sp. strain BD413 and ComEC in T. thermophilus HB27, which suggests that ComA and ComEC are located in the inner membrane and function in DNA transport through the cytoplasmic membrane.

Acinetobacter↗
PubMed2003

Hydroxycinnamate (hca) catabolic genes from Acinetobacter sp. strain ADP1 are repressed by HcaR and are induced by hydroxycinnamoyl-coenzyme A thioesters.

Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Km(r) genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR.

Acinetobacter↗
PubMed1984

Immunologically related ketodeoxyoctonate-containing structures in Chlamydia trachomatis, Re mutants of Salmonella species, and Acinetobacter calcoaceticus var. anitratus.

The lipopolysaccharides (LPS) of Chlamydia trachomatis, Acinetobacter calcoaceticus var. anitratus, and Re mutants of Salmonella sp. were shown to share related immunodeterminants , as demonstrated by double immunodiffusion and immunoblotting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The cross-reactive material in the extracellular slime of A. calcoaceticus var. anitratus was shown to be released LPS. The Acinetobacter LPS was found to separate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three fractions. The cross-reactive component was the fraction migrating fastest, at a rate identical to Re-type LPS of Salmonella sp. The Acinetobacter LPS could be used as antigen in complement fixation assays performed on paired sera of patients with chlamydial pneumonia; it gave results identical to those of the chlamydial complement fixation glycolipid antigen conventionally used in such assays in 9 of 10 patients.

Acinetobacter↗
PubMed1984

Insertional specificity of transposon Tn5 in Acinetobacter sp.

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced from Escherichia coli 1830 into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413. Kanamycin-resistant (Kmr) exconjugants of HO1-N and BD413, isolated on complex medium, were screened for auxotrophic requirements. Over 10,000 Kmr clones were examined, but no auxotrophs were detected. Several Kmr exconjugants of BD413 and HO1-N, obtained from independent matings, were chosen for further study. All Tn5-containing strains exhibited kanamycin phosphotransferase activity. Kmr strains lacked plasmid DNA as determined by three plasmid screening procedures, and the Kmr phenotype was not transferable by conjugal matings to kanamycin-sensitive BD413, HO1-N, or E. coli HB101. The chromosomal location of Tn5 insertions in independently isolated Kmr exconjugants of BD413 and HO1-N was characterized by restriction endonuclease mapping and hybridization studies. Results obtained from Southern hybridization studies were consistent with a single Tn5-specific insertion site in HO1-N and two such sites in BD413. Phage Mu sequences were not detected in Tn5-containing Acinetobacter sp.

Acinetobacter↗
PubMed1993

Transposon mutagenesis in Acinetobacter calcoaceticus RAG-1.

Molecular genetic studies of Acinetobacter spp. have been greatly limited by the lack of a method for transposon mutagenesis. In this study, a genetically engineered derivative of Tn10, mini-Tn10PttKm, was conjugally transferred in plate matings from Escherichia coli SM10[(lambda pir)(pLOFPttKm)] to Acinetobacter calcoaceticus RAG-1. Transfer frequencies were dependent on mating ratios and varied from 7.9 x 10(-8) to 3.4 x 10(-7) per recipient cell. The 27 lipase-deficient transconjugants which were isolated exhibited several different phenotypes, including gelatinase mutants, esterase mutants, and putative auxotrophs. Southern blot analysis confirmed the insertion of the mini-Tn10PttKm transposon in single, unique sites in five transconjugants. Four of five lipase mutants contained single insertions of mini-Tn10PttKm in the same chromosomal restriction fragments. To our knowledge, this is the first report of the use of a transposon for direct, generalized mutagenesis in Acinetobacter spp.

Acinetobacter calcoaceticus↗
PubMed1995

Phosphate concentration regulates transcription of the Acinetobacter polyhydroxyalkanoic acid biosynthetic genes.

The polyhydroxyalkanoic acid (PHA) biosynthetic gene locus was cloned and characterized from an Acinetobacter sp. isolated from activated sludge. Nucleotide sequence analysis identified three clustered genes, phaAAc (encoding a beta-ketothiolase), phaBAc (encoding an acetoacetyl coenzyme A reductase), and phaCAc (encoding a PHA synthase). In addition, an open reading frame (ORF1) with potential to encode a 13-kDa protein was identified within this locus. The sequence of the putative translational product of ORF1 does not show significant similarity to any sequences in the database. A plasmid containing the Acinetobacter pha locus conferred the ability to accumulate poly-beta-hydroxybutyrate on its Escherichia coli host. These genes appear to lie in an operon transcribed by two promoters upstream of phaBAc, an apparent constitutive promoter, and a second promoter induced by phosphate starvation and under pho regulon control. These as well as a number of additional potential transcription start points were identified by a combination of primer extension and promoter-chloramphenicol acetyltransferase gene fusion studies carried out in Acinetobacter or E. coli transformants.

Acetyl-CoA C-Acyltransferase↗
PubMed1995

Digital image analysis of growth and starvation responses of a surface-colonizing Acinetobacter sp.

Surface growth of an Acinetobacter sp. cultivated under several nutrient regimens was examined by using continuous-flow slide culture, phase-contrast microscopy, scanning confocal laser microscopy, and computer image analysis. Irrigation of attached coccoid stationary-phase Acinetobacter sp. cells with high-nutrient medium resulted in a transition from coccoid to bacillar morphology. Digital image analysis revealed that this transition was biphasic. During phase I, both the length and the width of cells increased. In contrast, cell width remained constant during phase II, while both cell length and cell area increased at a rate greater than in phase I. Cells were capable of growth and division without morphological transition when irrigated with a low-nutrient medium. Rod-shaped cells reverted to cocci by reduction-division when irrigated with starvation medium. This resulted in conservation of cell area (biomass) with an increase in cell number. In addition, the changes in cell morphology were accompanied by changes in the stability of cell attachment. During phase I, coccoid cells remained firmly attached. Following transition in high-nutrient medium, bacillar cells displayed detachment, transient attachment, and drifting behaviors, resulting in a spreading colonization pattern. In contrast, cells irrigated with a low-nutrient medium remained firmly attached to the surface and eventually formed tightly packed microcolonies. It is hypothesized that the coccoid and bacillar Acinetobacter sp. morphotypes and associated behavior represent specialized physiological adaptations for attachment and colonization in low-nutrient systems (coccoid morphotype) or dispersion under high-nutrient conditions (bacillar morphotype).

Acinetobacter↗
PubMed1996

Isolation and characterization of a novel oxygenase that catalyzes the first step of n-alkane oxidation in Acinetobacter sp. strain M-1.

In the Finnerty pathway for n-alkane, oxidation in Acinetobacter sp., n-alkanes are postulated to be attacked by a dioxygenase and the product, n-alkyl hydroperoxide, is further metabolized to the corresponding aldehyde via the peroxy acid [W. R. Finnerty, P. 184-188, in A. H. Applewhite (ed.), Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry, 1988]. However, no biochemical evidence regarding the first-step reaction is available. In this study, we found a novel n-alkane-oxidizing enzyme that requires only molecular oxygen, i.e., not NAD(P)H, in our isolate, Acinetobacter sp. strain M-1, and purified it to apparent homogeneity by gel electrophoresis. The purified enzyme is a homodimeric protein with a molecular mass of 134 kDa, contains 1 mol of flavin adenine dinucleotide per mol of subunit, and requires CU2+ for its activity. The enzyme uses n-alkanes ranging in length from 10 to 30 carbon atoms and is also active toward n-alkenes (C12 to C20) and some aromatic compounds with substituted alkyl groups but not toward a branched alkane, alcohol, or aldehyde. Transient accumulation of n-alkyl hydroperoxide was detected in the course of the reaction, and no oxygen radical scavengers affected the enzyme activity. From these properties, the enzyme is most probably a dioxygenase that catalyzes the introduction of two atoms of oxygen to the substrate, leading to the formation of the corresponding n-alkyl hydroperoxide. The enzymatic evidence strongly supports the existence of an n-alkane oxidation pathway, which is initiated by a dioxygenase reaction, in Acinetobacter spp.

Acinetobacter↗
PubMed1999

The physiological contribution of Acinetobacter PcaK, a transport system that acts upon protocatechuate, can be masked by the overlapping specificity of VanK.

VanK is the fourth member of the ubiquitous major facilitator superfamily of transport proteins to be identified that, together with PcaK, BenK, and MucK, contributes to aromatic catabolism in Acinetobacter sp. strain ADP1. VanK and PcaK have overlapping specificity for p-hydroxybenzoate and, most clearly, for protocatechuate: inactivation of both proteins severely impairs growth with protocatechuate, and the activity of either protein alone can mask the phenotype associated with inactivation of its homolog. Furthermore, vanK pcaK double-knockout mutants appear completely unable to grow in liquid culture with the hydroaromatic compound quinate, although such cells on plates convert quinate to protocatechuate, which then accumulates extracellularly and is readily visible as purple staining. This provides genetic evidence that quinate is converted to protocatechuate in the periplasm and is in line with the early argument that quinate catabolism should be physically separated from aromatic amino acid biosynthesis in the cytoplasm so as to avoid potential competition for intermediates common to both pathways. Previous studies of aromatic catabolism in Acinetobacter have taken advantage of the ability to select directly strains that contain a spontaneous mutation blocking the beta-ketoadipate pathway and preventing the toxic accumulation of carboxymuconate. By using this procedure, strains with a mutation in structural or regulatory genes blocking degradation of vanillate, p-hydroxybenzoate, or protocatechuate were selected. In this study, the overlapping specificity of the VanK and PcaK permeases was exploited to directly select strains with a mutation in either vanK or pcaK. Spontaneous mutations identified in vanK include a hot spot for frameshift mutation due to contraction of a G6 mononucleotide repeat as well as point mutations producing amino acid substitutions useful for analysis of VanK structure and function. Preliminary second-site suppression analysis using transformation-facilitated PCR mutagenesis in one VanK mutant gave results similar to those using LacY, the prototypic member of the major facilitator superfamily, consistent with the two proteins having a similar mechanism of action. The selection for transport mutants described here for Acinetobacter may also be applicable to Pseudomonas putida, where the PcaK permease has an additional role in chemotaxis.

Acinetobacter↗
PubMed2000

Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. Strain SE19 by in vitro transposition.

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.

Acinetobacter↗