Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

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.     

Concerning the sensitivity of Acinetobacter calcoaceticus

The strains of Acinetobacter calcoaceticus, var. anitratus (A. c. a.) were isolated in the nosocomial environment as an opportune pathogen. The therapy of choice may be determined after in vitro tests. Our results show following therapeutical possibilities: beta-lactam antibiotics--cephalosporins of IIIrd generation (cefotaxime), also combinations of antimicrobials have shown good results: amoxycillin or ticarcillin with clavulanic acid. Best synergistic effect was found in combination ticarcillin-amikacin.     

Lipase activity and gene cloning of Acinetobacter calcoaceticus LP009

The production of lipase from Acinetobacter calcoaceticus LP009, a bacterium isolated from raw milk, was found to be best induced by Tween-80 at 1.0% concentration. It was efficiently secreted, and only a minute amount of activity was detected at the cell surface and intracellularly. A. calcoaceticus LP009 lipase exhibited maximum activity at pH 7.0 and 50 degrees C, and was relatively stable upon storage at pH 5.0 to 7.0 and at 4, 30, or 37 degrees C. The enzyme was found to be inactivated by EDTA suggesting that it was a metalloenzyme. Its activity was reduced by less than 20% with the addition of various ions to reaction mixtures, but long storage with them caused approximately 50% reduction in subsequent reactions under standard conditions. By contrast, the addition of Fe(3+) enhanced activity. The enzyme was highly stable upon storage with 0.1% of Triton X-100, Tween-80 or Tween-20, but highly unstable with various organic solvents tested. PMSF, a serine enzyme inhibitor, and 2-mercaptoethanol, a reducing agent, did not affect enzyme activity. After extraction and transfer, the lipase gene was efficiently expressed in recombinant Aeromonas sobria. This recombinant strain was shown to have increased hydrolyzing efficiency and have high potential for lipid-rich wastewater treatment.     

Genome sequence of Acinetobacter calcoaceticus PHEA-2, isolated from industry wastewater

Genome analysis of Acinetobacter calcoaceticus PHEA-2 was undertaken because of the importance of this bacterium for bioremediation of phenol-polluted water and because of the close phylogenetic relationship of this species with the human pathogen Acinetobacter baumannii. To our knowledge, this is the first strain of A. calcoaceticus whose genome has been sequenced.     

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.     

Nucleotide sequence of the head assembly gene cluster of bacteriophage L and decoration protein characterization

The temperate Salmonella enterica bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene, dec, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.     

Nucleotide sequence of the aknA region of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus.

A 3.4-kb BamHI fragment that is assumed to be a part of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus 3AR-33 and contains the genes required for the early stage of polyketide biosynthesis was sequenced. The nucleotide sequence of the region that hybridizes to the actIII probe reveals the presence of a gene, aknA, whose deduced protein product is very similar to the ActIII protein and other known oxidoreductases. The predicted AknA protein is believed to be responsible for catalyzing the reduction of the keto group at the ninth carbon from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The predicted AknA protein has a calculated molecular mass of 27,197 Da (261 amino acids) and the highly conserved sequence Gly-Xaa-Gly-Xaa-Xaa-Ala commonly seen in oxidoreductases. Cloning and sequence analysis of the aknA region of the 2-hydroxyaklavinone-producing strain S. galilaeus ANR-58 identified an alteration in the gene, confirming that the aknA gene is essential for aklavinone biosynthesis.     

Acinetobacter calcoaceticus encoded mutarotase: nucleotide sequence analysis of the gene and characterization of its secretion in Escherichia coli.

The nucleotide sequence of the mutarotase gene from Acinetobacter calcoaceticus has been determined. It reveals an open reading frame of 381 amino acids. The codon usage of A. calcoaceticus for this gene is similar to E. coli except for the amino acids Leu, Ala, Glu, and Arg where major differences exist. This did not interfere drastically with high level expression in E. coli. The regulatory sequences for the initiation of translation are similar to the ones described for E. coli. The N-terminal 20 amino acids, which are not found in the mature enzyme, show homology to signal sequences of exported proteins. In A. calcoaceticus and E. coli mutarotase is specifically secreted into the periplasmic space. Processing of the signal sequence occurs at identical sites in both organisms. The mature mutarotase consists of 361 amino acids and has a calculated molecular weight of 38457 Da. Expression of mutarotase at a high level in a recombinant E. coli destabilizes the outer membrane. This results in coordinated leakage of mutarotase and beta-lactamase into the culture broth.     

Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene

The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signai sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disutphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.     

Pyrimidine metabolism in Acinetobacter calcoaceticus

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5'-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5'-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5'-monophosphate which is then phosphorylated to uridine-5'-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.     

Growth of Acinetobacter calcoaceticus on Ethanol

A soil microorganism, identified as Acinetobacter calcoaceticus, was cultivated on ethanol as a sole source of carbon. This organism grew with a maximum specific growth rate of 0.7/h. The pH optimum for growth was between 6.5 and 7.5, and the temperature optimum was between 32 and 35 C. Ethanol metabolism by this organism was inducible by ethanol, and the presence of acetate led to the repression of ethanol dehydrogenase. At higher cell densities the cessation of growth on ethanol was accompanied by the accumulation of acetate or acetaldehyde, or both. These accumulations were attributed to a reduction in the magnesium or sulfur content of the medium and a lack of feedback inhibition by acetate of alcohol dehydrogenase.