Gene_locus Report for: acica-estB

Polyphosphate kinase (Ppk) catalyzes the formation of polyphosphate from ATP. We cloned the ppk gene (2,073 bp) from Acinetobacter sp. strain ADP1; this gene encodes a putative polypeptide of 78.6 kDa with extensive homology to polyphosphate kinase from Escherichia coli and other bacteria. Chromosomal disruption of ppk by inserting a transcriptionally fused lacZ does not affect growth under conditions of phosphate limitation or excess. beta-Galactosidase activity expressed from the single-copy ppk::lacZ fusion is induced 5- to 15-fold by phosphate starvation. An increased amount of ppk transcript (2.2 kb) was detected when cells were grown at a limiting phosphate concentration. Primer extension analysis revealed a regulated promoter located upstream of a second, constitutive promoter. Potential similarities of this regulation with the effects of PhoB and PhoR of E. coli are discussed.

We determined 5.8 kilobases of nucleotide sequence upstream of the rubredoxin encoding rubA gene of Acinetobacter calcoaceticus (Ac) ADP1. Sequence analysis revealed four open reading frames named cysD', cobQ, sodA and lysS, coding for proteins with high similarity to known sulfate adenylate transferases (partial), cobyric acid synthases, superoxide dismutases (Sod) and lysyl tRNA synthetases, respectively. Out of a large number of bacterial Sod sequences SodA of Ac ADP1 is the first member of the Fe/Mn Sod family apparently located in the periplasmic space.

Mutants of Acinetobacter calcoaceticus ADP1 unable to grow on dodecane, but retaining the ability to grow on lauric acid were isolated after ethylmethanesulphonate (EMS) treatment. This growth deficiency was complemented by a clone from a gene library constructed from chromosomal DNA of the wild-type strain. The complementing DNA mapped in a gene encoding a polypeptide with homology to rubredoxins. The deduced putative rubredoxin amino acid sequence is more similar to related proteins from Gram-positive bacteria than to the Pseudomonas oleovorans rubredoxin involved in alkane oxidation. An adjacent gene encodes a protein with similarity to rubredoxin reductase from Pseudomonas oleovorans and related NAD(P)-dependent reductases. Disruption of the rubredoxin-encoding gene by insertion of a KmR/lacZ cassette rendered the resulting strain unable to grow on dodecane or hexadecane. This demonstrates that these genes are necessary for alkane degradation. Transcriptional fusion of lacZ to the rubredoxin-encoding gene led to low level constitutive beta-galactosidase expression, whereas the fusion oriented in the opposite direction was not expressed.

Acinetobacter sp. strain ADP1 is a nutritionally versatile soil bacterium closely related to representatives of the well-characterized Pseudomonas aeruginosa and Pseudomonas putida. Unlike these bacteria, the Acinetobacter ADP1 is highly competent for natural transformation which affords extraordinary convenience for genetic manipulation. The circular chromosome of the Acinetobacter ADP1, presented here, encodes 3325 predicted coding sequences, of which 60% have been classified based on sequence similarity to other documented proteins. The close evolutionary proximity of Acinetobacter and Pseudomonas species, as judged by the sequences of their 16S RNA genes and by the highest level of bidirectional best hits, contrasts with the extensive divergence in the GC content of their DNA (40 versus 62%). The chromosomes also differ significantly in size, with the Acinetobacter ADP1 chromosome <60% of the length of the Pseudomonas counterparts. Genome analysis of the Acinetobacter ADP1 revealed genes for metabolic pathways involved in utilization of a large variety of compounds. Almost all of these genes, with orthologs that are scattered in other species, are located in five major 'islands of catabolic diversity', now an apparent 'archipelago of catabolic diversity', within one-quarter of the overall genome. Acinetobacter ADP1 displays many features of other aerobic soil bacteria with metabolism oriented toward the degradation of organic compounds found in their natural habitat. A distinguishing feature of this genome is the absence of a gene corresponding to pyruvate kinase, the enzyme that generally catalyzes the terminal step in conversion of carbohydrates to pyruvate for respiration by the citric acid cycle. This finding supports the view that the cycle itself is centrally geared to the catabolic capabilities of this exceptionally versatile organism.

Alkanes are oxidized in Acinetobacter sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase. rubA and rubB encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions in rubA or rubB lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. Differences in the degradation capacity for hexadecane and dodecane in these mutants are discussed. Two genes, estB and oxyR, are located downstream of rubB, but are not necessary for alkane degradation. estB encodes a functional esterase. oxyR encodes a LysR-type transcriptional regulator, conferring resistance to hydrogen peroxide. rubA, rubB, estB, and oxyR constitute an operon, which is constitutively transcribed from a sigma70 promoter, and an estB-oxyR containing message is also transcribed from an internal promoter.

Polyphosphate kinase (Ppk) catalyzes the formation of polyphosphate from ATP. We cloned the ppk gene (2,073 bp) from Acinetobacter sp. strain ADP1; this gene encodes a putative polypeptide of 78.6 kDa with extensive homology to polyphosphate kinase from Escherichia coli and other bacteria. Chromosomal disruption of ppk by inserting a transcriptionally fused lacZ does not affect growth under conditions of phosphate limitation or excess. beta-Galactosidase activity expressed from the single-copy ppk::lacZ fusion is induced 5- to 15-fold by phosphate starvation. An increased amount of ppk transcript (2.2 kb) was detected when cells were grown at a limiting phosphate concentration. Primer extension analysis revealed a regulated promoter located upstream of a second, constitutive promoter. Potential similarities of this regulation with the effects of PhoB and PhoR of E. coli are discussed.

We determined 5.8 kilobases of nucleotide sequence upstream of the rubredoxin encoding rubA gene of Acinetobacter calcoaceticus (Ac) ADP1. Sequence analysis revealed four open reading frames named cysD', cobQ, sodA and lysS, coding for proteins with high similarity to known sulfate adenylate transferases (partial), cobyric acid synthases, superoxide dismutases (Sod) and lysyl tRNA synthetases, respectively. Out of a large number of bacterial Sod sequences SodA of Ac ADP1 is the first member of the Fe/Mn Sod family apparently located in the periplasmic space.

Mutants of Acinetobacter calcoaceticus ADP1 unable to grow on dodecane, but retaining the ability to grow on lauric acid were isolated after ethylmethanesulphonate (EMS) treatment. This growth deficiency was complemented by a clone from a gene library constructed from chromosomal DNA of the wild-type strain. The complementing DNA mapped in a gene encoding a polypeptide with homology to rubredoxins. The deduced putative rubredoxin amino acid sequence is more similar to related proteins from Gram-positive bacteria than to the Pseudomonas oleovorans rubredoxin involved in alkane oxidation. An adjacent gene encodes a protein with similarity to rubredoxin reductase from Pseudomonas oleovorans and related NAD(P)-dependent reductases. Disruption of the rubredoxin-encoding gene by insertion of a KmR/lacZ cassette rendered the resulting strain unable to grow on dodecane or hexadecane. This demonstrates that these genes are necessary for alkane degradation. Transcriptional fusion of lacZ to the rubredoxin-encoding gene led to low level constitutive beta-galactosidase expression, whereas the fusion oriented in the opposite direction was not expressed.