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Describes the phenotypic expression of polymerase chain reaction (PCR)-generated mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation. Homologous recombination of PCR-amplified DNA fragments into a chromosomal docking site; Effectiveness of the technique.

Examines the sequence analysis of the DNA downstream of the genes RubA and rubB in Acinetobacter sp. strain ADP1. Mutations determined for alkane mutants; Detection of oxyR gene transcripts; Regulation of the peroxide stress response in Acinetobacter by oxyR.

Describes the characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413. Generation of mutants; Identification of classes of mutants and lipolytic enzymes; Involvement of Xcp-like system in translocation of lipase and Tween esterase across outer membrane.

Reports on a study which conducted a mutation analysis of the transcriptional activators, PobR and PcaU in the Acinetobacter. Similarities of the primary structures, DNA-binding sites, and physiological functions of the activators; Details on the usage of surface plasmon resonance; Methodology used to conduct the study; Results of the study.

Examines the physiological factors affecting the production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413. Measurement of the extracellular lipase activity and intracellular beta-galactosidase; Lipase production and LipA expression during the growth on hexadecane.

The extracellular lipase from Acinetobacter calco-aceticus 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 tri-butyrin 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 signal sequence at the N-terminus of the mature lipase suggests that the lipase of Acineto-bacter 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 disulphide 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... [ABSTRACT FROM AUTHOR]

Three Acinetobacter calcoaceticus transformation-deficient mutants, obtained by insertional mutagenesis with the nptll gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100-1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal ONA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA-deficient strain. [ABSTRACT FROM AUTHOR]