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PubMed1988

Cloning of the gene encoding quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus: evidence for the presence of a second enzyme.

We cloned the gene coding for the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. This clone complements gdh mutations in A. calcoaceticus, Pseudomonas aeruginosa, and Escherichia coli. The gene codes for a protein with an Mr of 83,000. Evidence is presented for the presence of two different glucose dehydrogenase enzymes in A. calcoaceticus: a protein with an Mr of 83,000 and a dimer of two identical subunits with an Mr of 50,000.

Acinetobacter↗
PubMed1989

Characterization of Acinetobacter calcoaceticus catM, a repressor gene homologous in sequence to transcriptional activator genes.

Two structural genes needed for catechol degradation, catA and catB, encode the respective enzymes catechol 1,2-dioxygenase (EC 1.13.11.1) and muconate cycloisomerase (EC 5.5.1.1). Catechol is an intermediate in benzoate degradation, and the catA and catB genes are clustered within a 17-kilobase-pair (kbp) region of Acinetobacter calcoaceticus chromosomal DNA containing all of the structural genes required for the conversion of benzoate to tricarboxylic acid cycle intermediates. catA and catB were transcribed in the same direction and were separated by 3.8 kbp of DNA. The 3.8-kbp sequence revealed that directly downstream from catA and potentially transcribed in the same direction were two open reading frames encoding polypeptides of 48 and 36 kilodaltons (kDa). Genetic disruption of these open reading frames did not discernably alter either catechol metabolism or its regulation. A third open reading frame, beginning 123 bp upstream from catB and transcribed divergently from this gene, was designated catM. This gene was found to encode a 28-kDa trans-acting repressor protein that, in the absence of cis,cis-muconate, prevented expression of the cat structural genes. Constitutive expression of the genes was caused by a mutation substituting Arg-156 with His-156 in the catM-encoded repressor. The repressor protein proved to be a member of a diverse family of procaryotic regulatory proteins which, with rare exception, are transcriptional activators. Repression mediated by catM was not the sole transcriptional control exercised over catA in A. calcoaceticus. Expression of catA was elicited by either benzoate or cis,cis-muconate in a genetic background from which catM had been deleted. This induction required DNA in a segment lying 1 kbp upstream from the catA gene. It is likely that an additional gene, lying outside the region containing the structural genes necessary for benzoate metabolism, contributes to this control.

Acinetobacter↗
PubMed1990

Molecular cloning, nucleotide sequence, and promoter structure of the Acinetobacter calcoaceticus trpFB operon.

The trpFB operon from Acinetobacter calcoaceticus encoding the phosphoribosyl anthranilate isomerase and the beta-subunit of tryptophan synthase has been cloned by complementation of a trpB mutation in A. calcoaceticus, identified by deletion analysis, and sequenced. It encodes potential polypeptides of 214 amino acids with a calculated molecular weight of 23,008 (TrpF) and 403 amino acids with a molecular weight of 44,296 (TrpB). The encoded TrpB sequence shows striking homologies to those from other bacteria, ranging from 47% amino acids identity with the Brevibacterium lactofermentum protein and 64% identity with the Caulobacter crescentus protein. The encoded TrpF sequence, on the other hand, is much less homologous to the ones from other species, ranging between 27% identity with the Bacillus subtilis enzyme and 36% identity with the C. crescentus enzyme. The homologies of both polypeptides are evenly distributed over the entire sequences. The codon usage shows the strong preference for A and T in the third positions typical for A. calcoaceticus genes. The trpFB operon appears to be unlinked to trpA. The trpFB promoter has been determined by primer extension analysis of RNA synthesized from the chromosomally and plasmid-encoded trpFB operons. The starting nucleotides are identical in both cases and define the first promoter from A. calcoaceticus. Potential regulatory features are implied by a palindromic element overlapping the -35 consensus box of the promoter.

Acinetobacter↗
PubMed1990

Selection of Acinetobacter calcoaceticus mutants deficient in the p-hydroxybenzoate hydroxylase gene (pobA), a member of a supraoperonic cluster.

p-Hydroxybenzoate hydroxylase, the product of the pobA gene, gives rise to protocatechuate, which is metabolized by enzymes encoded by the pca operon in Acinetobacter calcoaceticus. Mutations in pcaD prevented growth of A. calcoaceticus with succinate in the presence of p-hydroxybenzoate. Mutants selected on this medium contained the original mutation in pcaD and also carried spontaneous mutations in pobA. These independently expressed genes were cotransformed with a frequency of 15% and thus are components of a supraoperonic cluster.

Acinetobacter↗
PubMed1990

Recovery of DNA from the Acinetobacter calcoaceticus chromosome by gap repair.

A strain of Acinetobacter calcoaceticus that demonstrates unusually high competence for natural transformation by linear DNA has proven valuable for analysis of genes and gene clusters associated with aromatic catabolism. The transformation system allowed gap repair to be used to recover mutant chromosomal DNA within recombinant plasmids. The sizes of the recovered fragments, 5 and 7 kilobase pairs in length, indicate that gap repair will be a useful procedure for isolation of wild-type and modified gene clusters from the A. calcoaceticus chromosome.

Acinetobacter↗
PubMed1990

DNA sequences of genes encoding Acinetobacter calcoaceticus protocatechuate 3,4-dioxygenase: evidence indicating shuffling of genes and of DNA sequences within genes during their evolutionary divergence.

The DNA sequence of a 2,391-base-pair HindIII restriction fragment of Acinetobacter calcoaceticus DNA containing the pcaCHG genes is reported. The DNA sequence reveals that A. calcoaceticus pca genes, encoding enzymes required for protocatechuate metabolism, are arranged in a single transcriptional unit, pcaEFDBCHG, whereas homologous genes are arranged differently in Pseudomonas putida. The pcaG and pcaH genes represent separate reading frames respectively encoding the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.1.3); previously a single designation, pcaA, had been used to represent DNA encoding this enzyme. The alpha and beta protein subunits appear to share common ancestry with each other and with catechol 1,2-dioxygenases from A. calcoaceticus and P. putida. Marked conservation of amino acid sequence is observed in a region containing two histidyl residues and two tyrosyl residues that appear to ligate iron within each oxygenase. In some regions within the aligned oxygenase sequences, DNA sequences appear to be conserved at a level beyond the extent that might have been demanded by selection at the level of protein. In other regions, divergence of DNA sequences appears to have been achieved by substitution of DNA sequence from one genetic segment into another. The results are interpreted to be the consequence of sequence exchange by gene conversion between slipped strands of DNA during evolutionary divergence; mismatch repair between slipped strands may contribute to the maintenance of DNA sequence in divergent genes.

Acinetobacter↗
PubMed1991

Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases.

The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.

Acinetobacter↗
PubMed1991

Properties of polyphosphate: AMP phosphotransferase of Acinetobacter strain 210A.

Polyphosphate:AMP phosphotransferase, an enzyme which catalyzes the phosphorylation of AMP to ADP at the expense of polyphosphate, was purified more than 1,500-fold from Acinetobacter strain 210A by streptomycin sulfate precipitation and by Mono-Q, Phenyl Superose, and Superose column chromatography. Streptomycin sulfate precipitation appeared to be an effective step in the purification procedure. During the following chromatographic steps, there was a 29-fold increase in specific activity but the yield was low (0.3%). Kinetic studies showed apparent Km values of 0.26 mM for AMP and 0.8 microM for polyphosphate with an average chain length of 35 phosphate groups. The highest activities were found with polyphosphate molecules of 18 to 44 phosphate residues. The polyphosphate chain was degraded completely to ADP. The mechanism of degradation is processive. No activity was obtained with ortho-, pyro-, tri-, and tetraphosphate. The enzyme was inhibited by pyro-, tri-, and tetraphosphate. The inhibition by tri- and tetraphosphate was mixed with polyphosphate as a substrate. The inhibition constants for the dissociation of the enzyme-inhibitor complex and for the enzyme-inhibitor-substrate complex were 0.9 and 6.5 mM, respectively, for triphosphate and 0.7 and 1.5 mM, respectively, for tetraphosphate.

Acinetobacter↗
PubMed1991

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.

Acinetobacter calcoaceticus↗
PubMed1991

Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases.

The xylXYZ DNA region is carried on the TOL pWW0 plasmid in Pseudomonas putida and encodes a benzoate dioxygenase with broad substrate specificity. The DNA sequence of the region is presented and compared with benABC, the chromosomal region encoding the benzoate dioxygenase of Acinetobacter calcoaceticus. Corresponding genes from the two biological sources share common ancestry: comparison of aligned XylX-BenA, XylY-BenB, and XylZ-BenC amino acid sequences revealed respective identities of 58.3, 61.3, and 53%. The aligned genes have diverged to assume G+C contents that differ by 14.0 to 14.9%. Usage of the unusual arginine codons AGA and AGG appears to have been selected in the P. putida xylX gene as it diverged from the ancestor it shared with A. calcoaceticus benA. Homologous A. calcoaceticus and P. putida genes exhibit different patterns of DNA sequence repetition, and analysis of one such pattern suggests that mutations creating different DNA slippage structures made a significant contribution to the evolutionary divergence of xylX.

Acinetobacter calcoaceticus↗
PubMed1992

Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus.

DNA within Escherichia coli colonies carrying cloned Acinetobacter calcoaceticus genes transforms mutant A. calocaceticus cells with high efficiency. Therefore, E. coli colonies containing such cloned genes can be identified by replica plating onto a lawn of A. calcoaceticus mutant cells. Transformation of A. calcoaceticus also facilitates gap repair and thus allows recovery of specified chromosomal segments in recombinant plasmids. These procedures were used to demonstrate the clustering of A. calcoaceticus genes required for utilization of p-hydroxybenzoate. Chromosomal linkage of the bacterial genes, contained in different operons separated by about 10 kbp of DNA, may have been selected on the basis of their physiological interdependence.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1992

Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.

Acinetobacter calcoaceticus↗
PubMed1993

Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A.

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.

Acinetobacter↗
PubMed1993

Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus.

We have identified pobR, a gene encoding a transcriptional activator that regulates expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase (PobA) in Acinetobacter calcoaceticus ADP1. Inducible expression of cloned pobA in Escherichia coli depended upon the presence of a functional pobR gene, and mutations within pobR prevented pobA expression in A. calcoaceticus. A pobA-lacZ operon fusion was used to demonstrate that pobA expression in A. calcoaceticus is enhanced up to 400-fold by the inducer p-hydroxybenzoate. Inducer concentrations as low as 10(-7) M were sufficient to elicit partial induction. Some structurally related analogs of p-hydroxybenzoate, unable to cause induction by themselves, were effective anti-inducers. The nucleotide sequence of pobR was determined, and the activator gene was shown to be transcribed divergently from pobA; the genes are separated by 134 DNA base pairs. The deduced amino acid sequence yielded a polypeptide of M(r) = 30,764. Analysis of this sequence revealed at the NH2 terminus a stretch of residues with high potential for forming a helix-turn-helix structure that could serve as a DNA-binding domain. A conservative amino acid substitution (Arg-61-->His-61) in this region inactivated PobR. The primary structure of PobR appears to be evolutionarily distinct from the four major families of NH2-terminal helix-turn-helix containing bacterial regulatory proteins that have been identified thus far.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1994

RpoN (sigma 54) is required for conversion of phenol to catechol in Acinetobacter calcoaceticus.

Members of the sigma 54 protein family, encoded by rpoN, are required for the transcription of genes associated with specialized metabolic functions. The ability to grow with phenol appears to be a specialized trait because it is expressed by few of the microorganisms that grow with catechol, the metabolic product of phenol monooxygenase. A mutation preventing the expression of phenol monooxygenase in the bacterial strain Acinetobacter calcoaceticus NCIB8250 was complemented by wild-type DNA segments containing an open reading frame encoding a member of the sigma 54 protein family. DNA sequencing revealed a second open reading frame, designated ORF2, directly downstream of A. calcoaceticus rpoN. The locations of both ORF2 and the 113-residue amino acid sequence of its product are highly conserved in other bacteria. The mutation preventing the expression of rpoN results in an opal codon that terminates the translation of RpoN at a position corresponding to Trp-91 in the 483-residue amino acid sequence of the wild-type protein. Negative autoregulation of rpoN was suggested by the fact that the mutation inactivating RpoN enhanced the transcription of rpoN. Primer extension revealed independent transcription start sites for rpoN and ORF2.

Acinetobacter calcoaceticus↗
PubMed1994

Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus.

PobR is a transcriptional activator required for the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase. The pobA and pobR genes are divergently transcribed and separated by 134 bp in the Acinetobacter calcoaceticus chromosome. Primer extension analysis revealed that the pobA transcript begins 22 bp upstream from the structural gene and the pobR transcript begins 69 bp upstream from the regulatory gene. This arrangement requires superimposition of the -10 base pair and -35 base pair RNA polymerase-binding sites for the respective genes. Expression of a pobR-lacZ fusion was found to be repressed three- to fourfold by pobR when the functional gene was carried in trans on a plasmid. The pobR gene was placed under control of a lac promoter in an expression vector, and the recombinant plasmid inducibly expressed high levels of PobR in Escherichia coli. Cell extracts containing this protein were used to conduct gel mobility shift analyses. PobR binds specifically to DNA in the pobA-pobR intergenic region, and this binding does not appear to be influenced by p-hydroxybenzoate, the inducer of pobA expression. DNase I footprinting indicates that the DNA-binding site for PobR extends from about 10 bp to about 45 bp downstream from the site of the beginning of the pobR transcript. Within this putative operator is a region of inverted symmetry. Evidently, interaction of the inducer with the PobR-operator complex triggers elevated expression of pobA, beginning at a position separated by 55 bp of DNA. The general mechanisms by which PobR exerts transcriptional control resemble those that typify the LysR family of transcriptional activators, a group from which PobR is evolutionarily remote.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1994

The pca-pob supraoperonic cluster of Acinetobacter calcoaceticus contains quiA, the structural gene for quinate-shikimate dehydrogenase.

An 18-kbp Acinetobacter calcoaceticus chromosomal segment contains the pcaIJFBDKCHG operon, which is required for catabolism of protocatechuate, and pobSRA, genes associated with conversion of p-hydroxybenzoate to protocatechuate. The genetic function of the 6.5 kbp of DNA between pcaG and pobS was unknown. Deletions in this DNA were designed by removal of fragments between restriction sites, and the deletion mutations were introduced into A. calcoaceticus by natural transformation. The mutations prevented growth with either quinate or shikimate, growth substrates that depend upon qui gene function for their catabolism to protocatechuate. The location of quiA, a gene encoding quinate-shikimate dehydrogenase, was indicated by its expression in one of the deletion mutants, and the position of the gene was confirmed by determination of its 2,427-bp nucleotide sequence. The deduced amino acid sequence of QuiA confirmed that it is a member of a family of membrane-associated, pyrrolo-quinoline quinone-dependent dehydrogenases, as had been suggested by earlier biochemical investigations. Catabolism of quinate and skikimate is initiated by NAD(+)-dependent dehydrogenases in other microorganisms, so it is evident that different gene pools were called upon to provide the ancestral enzyme for this metabolic step.

Acinetobacter calcoaceticus↗
PubMed1995

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase.

Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A. calcoaceticus BD413 is proposed.

Acinetobacter calcoaceticus↗