Dependence of linkage of alleles on their physical distance in natural transformation of Acinetobacter sp. strain ADP1.

The interdependence of genetic linkage in transformation and physical distance was studied in the bacterium Acinetobacter sp. strain ADP1. Transformation experiments were performed using 17 strains containing different mutations within the 21-kb pca-qui-pob gene cluster as recipients for the DNA of one of two strains carrying a mutation causing a temperature-sensitive phenotype. The different phenotypes of the transformants (temperature-sensitive or wild-type-like) were used to evaluate linkage. Combination of the recipient and donor strains resulted in physical distances ranging from 2 bp to 10,533 bp. A logarithmic relationship of decreasing linkage and increasing distance was observed, thus leading to calibration of a system for analysis of physical distance derived from linkage data. Limitations of this application are described here: Certain mutations (3 out of 17 mutations used in this study) are an exception to the observed relationship and result in much lower linkage than expected. Observed DNA sequence repetitions leading to DNA rearrangements may be the cause of this anomaly.

Functions of the mismatch repair gene mutS from Acinetobacter sp. strain ADP1.

The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS(+) recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.

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.

Spontaneous mutations affecting transcriptional regulation by protocatechuate in Acinetobacter.

Positive selection yields Acinetobacter strains with a spontaneous mutation blocking catabolism of protocatechuate. For this study, the growth temperature during selection was lowered to 22 degrees C: growth at 37 degrees C was found to mask the role of the protocatechuate-responsive transcriptional regulator PcaU. The resulting mutants included those with amino acid substitutions useful for understanding PcaU structure and function, a 20-bp deletion whose repeated isolation suggested genetic instability of DNA in the putative PcaU operator, and a large deletion whose phenotype revealed that the supraoperonic cluster of genes for the protocatechuate branch of the beta-ketoadipate pathway extends to genes for the utilization of C(6)-C(10) straight-chain dicarboxylic acids including adipate.

Structure of Acinetobacter strain ADP1 protocatechuate 3, 4-dioxygenase at 2.2 A resolution: implications for the mechanism of an intradiol dioxygenase.

The crystal structures of protocatechuate 3,4-dioxygenase from the soil bacteria Acinetobacterstrain ADP1 (Ac 3,4-PCD) have been determined in space group I23 at pH 8.5 and 5.75. In addition, the structures of Ac 3,4-PCD complexed with its substrate 3, 4-dihydroxybenzoic acid (PCA), the inhibitor 4-nitrocatechol (4-NC), or cyanide (CN(-)) have been solved using native phases. The overall tertiary and quaternary structures of Ac 3,4-PCD are similar to those of the same enzyme from Pseudomonas putida[Ohlendorf et al. (1994) J. Mol. Biol. 244, 586-608]. At pH 8.5, the catalytic non-heme Fe(3+) is coordinated by two axial ligands, Tyr447(OH) (147beta) and His460(N)(epsilon)(2) (160beta), and three equatorial ligands, Tyr408(OH) (108beta), His462(N)(epsilon)(2) (162beta), and a hydroxide ion (d(Fe-OH) = 1.91 A) in a distorted bipyramidal geometry. At pH 5.75, difference maps suggest a sulfate binds to the Fe(3+) in an equatorial position and the hydroxide is shifted [d(Fe-OH) = 2.3 A] yielding octahedral geometry for the active site Fe(3+). This change in ligation geometry is concomitant with a shift in the optical absorbance spectrum of the enzyme from lambda(max) = 450 nm to lambda(max) = 520 nm. Binding of substrate or 4-NC to the Fe(3+) is bidentate with the axial ligand Tyr447(OH) (147beta) dissociating. The structure of the 4-NC complex supports the view that resonance delocalization of the positive character of the nitrogen prevents substrate activation. The cyanide complex confirms previous work that protocatechuate 3,4-dioxygenases have three coordination sites available for binding by exogenous substrates. A significant conformational change extending away from the active site is seen in all structures when compared to the native enzyme at pH 8.5. This conformational change is discussed in its relevance to enhancing catalysis in protocatechuate 3,4-dioxygenases.

Repression of Acinetobacter vanillate demethylase synthesis by VanR, a member of the GntR family of transcriptional regulators.

Vanillate is converted to protocatechuate by the action of vanillate demethylase encoded by vanAB. Convergent upon and overlapping Acinetobacter vanB is an open reading frame encoding a member of the gntR repressor family and designated vanR. This gene organization differs from that found in a Pseudomonas isolate. An Acinetobacter strain with a knockout mutation in vanR constitutively converted vanillate to protocatechuate. Reverse transcriptase-polymerase chain reaction was used to demonstrate that control of vanAB was exerted at the level of transcription.

Substrate range and genetic analysis of Acinetobacter vanillate demethylase.

An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs m-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type Acinetobacter bacteria. PCR mutagenesis of vanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level.

Substitution, insertion, deletion, suppression, and altered substrate specificity in functional protocatechuate 3,4-dioxygenases.

Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for Acinetobacter strains containing spontaneous mutations blocking expression of pcaH or -G, genes encoding the alpha and beta subunits of protocatechuate 3, 4-dioxygenase. The crystal structure of the Acinetobacter oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivating Acinetobacter protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate Acinetobacter strains in which mutations within pcaH or -G cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in pcaH or -G were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into the Acinetobacter chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.

Genetic analysis of a chromosomal region containing vanA and vanB, genes required for conversion of either ferulate or vanillate to protocatechuate in Acinetobacter.

VanA and VanB form an oxygenative demethylase that converts vanillate to protocatechuate in microorganisms. Ferulate, an abundant phytochemical, had been shown to be metabolized through a vanillate intermediate in several Pseudomonas isolates, and biochemical evidence had indicated that vanillate also is an intermediate in ferulate catabolism by Acinetobacter. Genetic evidence supporting this conclusion was obtained by characterization of mutant Acinetobacter strains blocked in catabolism of both ferulate and vanillate. Cloned Acinetobacter vanA and vanB were shown to be members of a chromosomal segment remote from a supraoperonic cluster containing other genes required for completion of the catabolism of ferulate and its structural analogs, caffeate and coumarate, through protocatechuate. The nucleotide sequence of DNA containing vanA and vanB demonstrated the presence of genes that, on the basis of nucleotide sequence similarity, appeared to be associated with transport of aromatic compounds, metabolism of such compounds, or iron scavenging. Spontaneous deletion of 100 kb of DNA containing this segment does not impede the growth of cells with simple carbon sources other than vanillate or ferulate. Additional spontaneous mutations blocking vanA and vanB expression were shown to be mediated by IS1236, including insertion of the newly discovered composite transposon Tn5613. On the whole, vanA and vanB appear to be located within a nonessential genetic region that exhibits considerable genetic malleability in Acinetobacter. The overall organization of genes neighboring Acinetobacter vanA and vanB, including a putative transcriptional regulatory gene that is convergently transcribed and overlaps vanB, is conserved in Pseudomonas aeruginosa but has undergone radical rearrangement in other Pseudomonas species.

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.

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation.

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.

Mutation analysis of PobR and PcaU, closely related transcriptional activators in acinetobacter.

Acinetobacter PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite p-hydroxybenzoate and activates transcription of pobA, the structural gene for the enzyme that converts p-hydroxybenzoate to protocatechuate. This compound, differing from p-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU from Acinetobacter strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which pobR function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the pobA-pobR intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on p-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing PcaU to mimic PobR activity were shown to be G222V amino acid substitutions in the C terminus of the 274-residue protein. Together, the analyses suggest that PobR and PcaU possess a linear domain structure similar to that of LysR transcriptional activators which largely differ in primary structure.

PcaU, a transcriptional activator of genes for protocatechuate utilization in Acinetobacter.

The Acinetobacter pcaIJFBDKCHG operon encodes the six enzymes that convert protocatechuate to citric acid cycle intermediates. Directly downstream from the operon are qui and pob genes encoding sets of enzymes that convert quinate and p-hydroxybenzoate, respectively, to protocatechuate. Prior to this investigation, the only known regulatory gene in the pca-qui-pob cluster was pobR, which encodes a transcriptional activator that responds to p-hydroxybenzoate and activates transcription of pobA. The pca and qui genes were known to be expressed in response to protocatechuate, but a protein that mediated this induction had not been identified. This study was initiated by characterization of a spontaneous mutation that mapped upstream from pcaI and prevented expression of the pca genes. Sequencing of wild-type DNA extending from the translational start of pcaI through and beyond the location of the mutation revealed a 282-bp intergenic region and a divergently transcribed open reading frame, designated pcaU. Downstream from pcaU are two open reading frames encoding proteins similar in amino acid sequence to those associated with the oxidation of acyl thioesters. Inactivation of pcaU reduced the induced expression of pca structural genes by about 90% and impeded but did not completely prevent growth of the mutant cells with protocatechuate. PcaU was expressed in Escherichia coli and shown to bind to a portion of the pcaI-pcaU intergenic region containing a sequence identical in 16 of 19 nucleotide residues to a segment of the pob operator. Further similarity of the two regulatory systems is indicated by 54% amino acid sequence identity in the aligned primary structures of PobR and PcaU. The pob and pca systems were shown to differ, however, in the relative orientations of transcriptional starts with respect to the site where the activator binds to DNA, the size of the intergenic region, and the tightness of transcriptional control. The spontaneous mutation blocking pca gene expression was located in the promoter for the pca operon. The 19-nucleotide residue operator sequences were shown to be parts of a consensus associated with transcriptional activation of genes associated with protocatechuate catabolism. Two different binding sites for Pseudomonas putida PcaR differ from the consensus in only a single nucleotide residue, and DNA directly downstream from Acinetobacter pcaU contains a 19-bp segment differing from the consensus in only two residues. PcaU was shown to bind to DNA containing this segment as well as to the DNA in the pcaU-pcaI intergenic region.

Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori strains tested by polymerase chain reaction or slot blotting but not in Campylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed in Escherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in different H. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits of Mr approximately 26,000 and 24, 000 that are both necessary for H. pylori CoA-transferase function. The lack of alpha-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism.

Combining localized PCR mutagenesis and natural transformation in direct genetic analysis of a transcriptional regulator gene, pobR.

We present a procedure for efficient random mutagenesis of selected genes in a bacterial chromosome. The method combines PCR replication errors with the uptake of PCR-amplified DNA via natural transformation. Cloning of PCR fragments is not required, since mutations are transferred directly to the chromosome via homologous recombination. Random mutations were introduced into the Acinetobacter chromosomal pobR gene encoding the transcriptional activator of pobA, the structural gene for 4-hydroxybenzoate 3-hydroxylase. Mutant strains with strongly reduced PobR activity were selected by demanding the inability to convert 4-hydroxybenzoate to a toxic metabolite. Of spontaneous pobR mutants, 80% carry the insertion element IS1236, rendering them inappropriate for structure-function studies. Transformation with Taq-amplified pobR DNA increased the mutation frequency 240-fold and reduced the proportion of IS1236 inserts to undetectable levels. The relative fidelity of Pfu polymerase compared with Taq polymerase was illustrated by a reduced effect on the mutation frequency; a procedure for rapid assessment of relative polymerase fidelity in PCR follows from this observation. Over 150 independent mutations were localized by transformation with DNA fragments containing nested deletions of wild-type pobR. Sequence analysis of 89 of the mutant pobR alleles showed that the mutations were predominantly single-nucleotide substitutions broadly distributed within pobR. Promoter mutations were recovered, as were two mutations that are likely to block pobR translation. One-third of the recovered mutations conferred a leaky or temperature-sensitive phenotype, whereas the remaining null mutations completely blocked growth with 4-hydroxybenzoate. Strains containing two different nonsense mutations in pobR were transformed with PCR-amplified DNA to identify permissible codon substitutions. Independently, second-site suppressor mutations were recovered within pcaG, another member of the supraoperonic pca-qui-pob cluster on the Acinetobacter chromosome. This shows that combining PCR mutagenesis with natural transformation is of general utility.

IS1236, a newly discovered member of the IS3 family, exhibits varied patterns of insertion into the Acinetobacter calcoaceticus chromosome.

Analysis of spontaneous mutations in Acinetobacter calcoaceticus revealed a 1237 bp insertion sequence named IS1236 and possessing a nucleotide sequence resembling those of members of the lS3 family. The chromosome of A. calcoaceticus strain ADP1 contains seven copies of IS1236 which appears to insert preferentially into pobR, the transcriptional activator of the structural gene for p-hydroxybenzoate hydroxylase. IS1236 creates tandem 3 bp DNA duplications flanking the sites of its insertion in pobR. Different duplication patterns are found following insertion of IS1236 into pcaH, a structural gene for protocatechuate 3,4-dioxygenase. Therefore the insertion of properties of IS1236 appear to be influenced by its DNA target. Amino acid sequences associated with the apparent transposase function have been conserved in ORFB of IS1236 whereas the presumed DNA-binding helix-turn-helix region of IS1236 ORFA exhibits substantial amino acid sequence divergence from its IS3 counterparts. IS1236 ORFA and ORFB coding sequences overlap considerably, and sequence evidence indicates mechanisms for ORFB expression in IS1236 may resemble those employed by other members of the IS3 family. Portions of the IS1236 terminal repeats exhibit substantial sequence divergence from other members of the IS3 family, but evolution appears to have conserved a mechanism preventing expression of the insertion sequence genes as a consequence of transcriptional readthrough.

Cloning of catBCIJFD genes for catechol degradation into chromosomal pobA and genetic stability of the recombinant Acinetobacter calcoaceticus.

A possible obstacle in the development of hybrid strains of Acinetobacter calcoaceticus by the introduction of a metabolic pathway into the chromosome is genetic instability of the resulting recombinant strains. Therefore, the possibility that the pobA gene can be used as a chromosomal cloning site where the transposed genes can be maintained and expressed, was explored in this study. For this purpose, two model hybrid strains of A. calcoaceticus were created, in which a DNA fragment carrying catBCIJFD genes for catabolic degradation of catechol was inserted into pobA in opposite directions of each other, and their genetic stabilities were experimentally examined. Our data demonstrated that the stability of the genes neighboring the insertions depends on the orientations of the insertions. Also, the data further indicated that the functional metabolic pathways introduced into pobA can be expressed successfully as far as the insertion is engineered in an appropriate way. Concurrently, it was proposed that the pobA can be used as a chromosomal cloning site, and that introduction of an useful metabolic pathway into pobA may offer considerable promise to the construction of a hybrid strain with improved metabolic capabilities.

Unusual ancestry of dehydratases associated with quinate catabolism in Acinetobacter calcoaceticus.

Catabolism of quinate to protocatechuate requires the consecutive action of quinate dehydrogenase (QuiA), dehydroquinate dehydratase (QuiB), and dehydroshikimate dehyratase (QuiC), Genes for catabolism of protocatechuate are encoded by the pca operon in the Acinetobacter calcoaceticus chromosome. Observations reported here demonstrate that A. calcoaceticus qui genes are clustered in the order quiBCXA directly downstream from the pca operon. Sequence comparisons indicate that quiX encodes a porin, but the specific function of this protein has not been clearly established. Properties of mutants created by insertion of omega elements show that quiBC is expressed as part of a single transcript, but there is also an independent transcriptional initiation site directly upstream of quiA. The deduced amino acid sequence of QuiC does not resemble any other known sequence. A. calcoaceticus QuiB is most directly related to a family of enzymes with identical catalytic activity and biosynthetic AroD function in coliform bacteria. Evolution of A. calcoaceticus quiB appears to have been accompanied by fusion of a leader sequence for transport of the encoded protein into the inner membrane, and the location of reactions catalyzed by the mature enzyme may account for the failure of A. calcoaceticus aroD to achieve effective complementation of null mutations in quiB. Analysis of a genetic site where a DNA segment encoding a leader sequence was transposed adds to evidence suggesting horizontal transfer of nucleotide sequences within genes during evolution.

Distance between alleles as a determinant of linkage in natural transformation of Acinetobacter calcoaceticus.

Cotransformation frequencies of 16, 39, 51, and 60% were observed when donor alleles were separated by distances of 9.2, 7.4, 6.3, and 5.1 kb, respectively, in donor Acinetobacter calcoaceticus DNA. A different and unexpected pattern was observed when the distance between recipient alleles was reduced from 9.2 to 5.1 kb. Ligation of unlinked chromosomal DNA fragments allowed them to be linked genetically through natural transformation.