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.

Drosophila as a model host for Pseudomonas aeruginosa infection.

Using the fruit fly Drosophila melanogaster as model host, we have identified mutants of the bacterium Pseudomonas aeruginosa with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in pilGHIJKL chpABCDE, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The pil chp genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.

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.

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.

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.

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.

Comparative and functional analysis of the AP2 promoter indicates that conserved octamer and initiator elements are critical for activity.

AP-2 is a developmentally-regulated transcription factor expressed in ectodermal cell lineages. The AP-2 protein is essential for neural tube, craniofacial and body wall morphogenesis and has been implicated in oncogenesis. Here we report the isolation of the AP-2 promoter from human, mouse and chicken. The initiation sites for the human gene have been mapped in a variety of cell lines, including several derived from breast tumours. Initiation occurs just upstream of an IR3-like repetitive element, present in the human and mouse genes, but absent in chicken. The cis-acting elements responsible for promoter activity in human HeLa cells have been mapped both in vivo and in vitro. The proximal promoter contains binding sites for transcription factors AP-2, NF-1 and octamer proteins, but lacks a TATA box motif. Functional analysis demonstrates that the octamer binding site is the critical component of basal promoter activity. In addition, the promoter relies on an initiator element for efficient start site utilization. There is an excellent correlation between the requirement for the initiator and octamer elements in transcription assays and the conservation of these cis-acting sequences between chicken, mouse and human.