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.

Purification and characterization of Acinetobacter calcoaceticus 4-hydroxybenzoate 3-hydroxylase after its overexpression in Escherichia coli.

4-Hydroxybenzoate 3-hydroxylase [EC 1.14.13.2] from Acinetobacter calcoaceticus was purified to homogeneity following the 40-fold overexpression of this gene (pobA) in Escherichia coli. Overexpression was accomplished by placing the folA gene (encoding trimethoprim-resistant dihydrofolate reductase) directly downstream of the pobA gene, and demanding growth of recombinants on elevated concentration of trimethoprim. Presumably, the surviving variants have undergone a genetic alteration which allowed the overexpression of both folA and pobA. 4-Hydroxybenzoate 3-hydroxylase was purified in two chromatographic steps, characterized biochemically, and its properties were compared to those of its homolog from Pseudomonas fluorescens. The two enzymes differ in their response to Cl- ion inhibition. A single amino acid change in the putative NADPH-binding site is proposed to account for this difference. The inhibitory and catalytic properties of substrate analogs were also examined.

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.

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.

Evolutionary divergence of pobA, the structural gene encoding p-hydroxybenzoate hydroxylase in an Acinetobacter calcoaceticus strain well-suited for genetic analysis.

The pobA gene encoding p-hydroxybenzoate hydroxylase (PobA) from Acinetobacter calcoaceticus has been developed as a genetic tool for the analysis of structure-function relationships in this enzyme. By exploiting the favorable genetic system of A. calcoaceticus strain ADP1, it is possible both to select and to map mutations which disturb PobA activity; characterization and sequence determination of mutants derived in this manner may complement site-directed studies with the homologous Pseudomonas aeruginosa gene. We have determined the nucleotide (nt) sequence of A. calcoaceticus pobA and performed a systematic comparison of the deduced amino acid (aa) sequence with that of the PobA enzyme from Pseudomonas fluorescens, for which the three-dimensional structure is known. Despite a 26% difference in the G+C content of the homologous genes, constraints against structural divergence of the proteins were revealed by an overall identity of 62.4% in the aligned aa sequences of PobA. Clusters of identical sequence occur at previously identified sites of ligand binding and at regions associated with subunit-subunit interaction. Based on the conservation of specific residues involved in flavin binding, we have assembled a consensus sequence for nicotinamide-flavoprotein monooxygenases which differs from that of the oxidoreductase class of flavoproteins. In addition to the conserved regions shared by the two PobA homologs, there are isolated pockets of divergence. The nt sequence divergence in one such region within the A. calcoaceticus gene can be attributed to the acquisition of short nt sequence repetitions.

Formyl-methanofuran synthesis in Methanobacterium thermoautotrophicum.

The initial step of methanogenesis from CO2 is the formation of formyl-methanofuran (formyl-MFR) from methanofuran (MFR), CO2 and H2. The enzymology of this novel type of CO2 fixation reaction has been difficult to study because formyl-MFR synthesis is subject to a complex activation. Recently, however, a number of advances have made questions regarding formyl-MFR synthesis more approachable.

Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.

The formylmethanofuran:tetrahydromethanopterin formyltransferase from Methanobacterium thermoautotrophicum delta H. Nucleotide sequence and functional expression of the cloned gene.

The formylmethanofuran:tetrahydromethanopterin formyltransferase (FTR) from Methanobacterium thermoautotrophicum delta H was cloned and its sequence was determined. The clone was contained on a 4.8-kilobase BamHI fragment of M. thermoautotrophicum DNA ligated into pBR329. When this fragment was subcloned into the phagemid pTZ18R, a functional enzyme was synthesized under control of the lac promoter. Sequence analysis revealed the presence of a ribosome binding site and a possible terminator structure. The absence of an identifiable promoter lends credibility to the open reading frame which is present 5' to ftr. The ftr gene encodes an acidic protein with a calculated molecular weight of 31,401. The sequence of FTR does not appear to be homologous to any other sequenced proteins, including proteins which use pterin substrates.

Purification and properties of the 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum.

The 5,10-methenyltetrahydromethanopterin cyclohydrolase of Methanobacterium thermoautotrophicum was purified 128-fold to homogeneity. The enzyme had a subunit Mr of 41,000 as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From high-performance size exclusion chromatography of the native protein, an Mr of 82,000 was determined, suggesting a dimer of identical subunits. The enzyme was inhibited by 10-formyltetrahydromethanopterin and stimulated by Mg2+. Evaluation of the reaction equilibrium indicated that the methenyl derivative was favored over 5-formyltetrahydromethanopterin, with a much higher equilibrium constant than for the analogous reaction of tetrahydrofolate derivatives. Folate derivatives did not serve as substrates for this enzyme.

d-Glucose Transport System of Zymomonas mobilis.

The properties of the d-glucose transport system of Zymomonas mobilis were determined by measuring the uptake of nonmetabolizable analogs (2-deoxy-d-glucose and d-xylose) by wild-type cells and the uptake of d-glucose itself by a mutant lacking glucokinase. d-Glucose was transported by a constitutive, stereospecific, carrier-mediated facilitated diffusion system, whereby its intracellular concentration quickly reached a plateau close to but not above the external concentration. d-Xylose was transported by the d-glucose system, as evidenced by inhibition of its uptake by d-glucose. d-Fructose was not an efficient competitive inhibitor of d-glucose uptake, indicating that it has a low affinity for the d-glucose transport system. The apparent K(m) of d-glucose transport was in the range of 5 to 15 mM, with a V(max) of 200 to 300 nmol min mg of protein. The K(m) of Z. mobilis glucokinase (0.25 to 0.4 mM) was 1 order of magnitude lower than the K(m) for d-glucose transport, although the V(max) values for transport and phosphorylation were similar. Thus, glucose transport cannot be expected to be rate limiting at concentrations of extracellular glucose normally used in fermentation processes, which greatly exceed the K(m) for the transport system. The low-affinity, high-velocity, nonconcentrative system for d-glucose transport described here is consistent with the natural occurrence of Z. mobilis in high-sugar environments and with the capacity of Z. mobilis for rapid conversion of glucose to metabolic products with low energetic yield.