Codon Usage in Acinetobacter Structural Genes

White, Peter J; Hunter, Irene; Fewson, Charles A.

doi:10.1007/978-1-4899-3553-3_17

Cited by 12 publications

(14 citation statements)

References 17 publications

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“…Codon usage and codon base composition of the esterase gene are similar to what has been calculated as the average for 20 Acinetobacter genes by White et al (1991). The esterase gene has a combined A + T composition at codon positions one, two and three of 44~9,616 and 66.3 mol %, respectively, exactly matching calculations by White et al (1991).…”

Section: Sequence Analysis Of the Esterase Encoding Regionsupporting

confidence: 73%

“…The esterase gene has a combined A + T composition at codon positions one, two and three of 44~9,616 and 66.3 mol %, respectively, exactly matching calculations by White et al (1991). The overall A + T content of the esterase gene (60 mol%), is also in agreement with the A + T content of the Acinetobacter genome (55-62 mol% ; Henriksen, 1976).…”

Section: Sequence Analysis Of the Esterase Encoding Regionsupporting

confidence: 62%

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Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases

Kok¹,

Christoffels²,

Vosman³

et al. 1993

Journal of General Microbiology

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Acinetobacter calcoaceticus BD413, when grown in batch culture in nutrient broth, produces both extracellular lipase activity and cell-bound esterase activity during and after the transition between exponential growth and the stationary phase. From a library of A. calcoaceticus DNA in Escherichia cofi, plasmids were isolated that enabled E. coli to grow on media with tributyrin as the sole carbon source. Assays with model substrates classified the product of the cloned gene as an esterase. Via deletion analysis, the esterase gene was mapped on a 1.8 kbp chromosomal DNA fragment. This fragment was sequenced and found to contain one open reading frame, termed estA, which encodes a protein of 40.0 kDa. The amino acid sequence of this protein shows homology to a number of lipolytic enzymes, most notably to esterases. Deletion of estA only partially abolished cell-bound esterase activity in A. calcoaceticus, indicating that BD413 forms at least two esterases. Both esterases show the same temporal regulation of expression. /?-Galactosidase activity was measured in strains in which a promoterless lac2 gene was inserted into estA. Induction of lac2 expression in these strains also occurred at the end of exponential growth in batch cultures, indicating that production of the esterase is regulated at the genetic level.

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Section: Sequence Analysis Of the Esterase Encoding Regionsupporting

confidence: 73%

Section: Sequence Analysis Of the Esterase Encoding Regionsupporting

confidence: 62%

Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases

Kok¹,

Christoffels²,

Vosman³

et al. 1993

Journal of General Microbiology

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“…The pcaG and -H genes from six such transformants, independently derived with six different PCRs, were sequenced, and a point mutation creating an amino acid substitution was found in pcaG in each case. In two cases, the intragenic suppressor mutation was Leu 39 to Phe 39 , created by changing TTG to TTT, and in the remaining cases, Gln 93 to His 93 was created, three times by a CAA to CAC change and once by a CAA to CAT change. In order to confirm that the sequenced second-site mutation was the cause of suppression, PCR amplification across both mutations in the suppressed strains was used to create DNA that gave rise to over 1,000 times more transformants of the parental strain than in the initial selection.…”

Section: Resultsmentioning

confidence: 99%

“…5B). Use of AGA as a codon is quite infrequent in Acinetobacter structural genes (39), and the presence of the codon in transcriptional activators might be regarded as the result of selection for reduced expression of these genes by translational control (3). Alternatively, the unusual codons may simply have been accepted in transcriptional activators because there has been no demand for elevated expression of these genes.…”

Section: Resultsmentioning

confidence: 99%

Combining localized PCR mutagenesis and natural transformation in direct genetic analysis of a transcriptional regulator gene, pobR

1997

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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-quipob cluster on the Acinetobacter chromosome. This shows that combining PCR mutagenesis with natural transformation is of general utility.Amplification of DNA by thermostable polymerases in the PCR introduces mutations (2,23,34,36,41). This is an inconvenience when the fidelity of replication is a concern, but in some cases, the randomness of nucleotide substitutions introduced by PCR can facilitate genetic analysis. For example, defects caused by a wide spectrum of independent mutations within a gene can give insight into how structure influences the function of the encoded protein. The efficiency of such structure-function studies is determined in large part by the ease with which the phenotype conferred by altered DNA can be observed. In this regard, oxygenative pathways for dissimilation of aromatic compounds by Acinetobacter calcoaceticus (18) provide a convenient genetic system allowing ready selection of defective genes (4, 8, 13) introduced by natural transformation (18).An intriguing target for genetic investigation is Acine...

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“…It contains three open reading frames (ORFs) (Fig. 1), each of which is preceded by a putative ribosome binding site, as judged by sequence comparison with other Acinetobacter genes (38). Transformation of plasmids obtained from a nested-deletion reaction of pWH1597 mapped the complementing fragment within ORF3 (Fig.…”

mentioning

confidence: 99%

Identification and characterization of xcpR encoding a subunit of the general secretory pathway necessary for dodecane degradation in Acinetobacter calcoaceticus ADP1

1997

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A mutant of Acinetobacter calcoaceticus ADP1 unable to grow on alkanes was complemented for growth on hexadecane with a DNA fragment encoding a protein with homology to XcpR, a subunit of the general secretion pathway for exoproteins in Pseudomonas aeruginosa. Insertional inactivation of xcpR in A. calcoaceticus ADP1 by transcriptional fusion to lacZ abolishes secretion of lipase and esterase and leads to lack of growth on dodecane and slower growth on hexadecane. We, therefore, propose the participation of a secreted protein in alkane degradation.Acinetobacter calcoaceticus ADP1, also called BD413 (18,28), is able to grow on n-alkanes as sole carbon and energy sources via the -hydroxylation pathway (2). Candidates (n ϭ 2,650) able to grow on minimal medium with lauric acid were obtained by ethyl methanesulfonate mutagenesis and screened for the inability to grow on dodecane, which yielded 27 candidates with a negative phenotype on solid agar plates (11). Mutant WH365 is also unable to grow on hexadecane and fails to secrete lipase and esterase, in contrast to the wild type (20)(21)(22). Extracellular lipase of A. calcoaceticus ADP1, which shows activity towards triglycerides and phospholipids, was monitored on indicator plates with NB medium (Difco) containing 2% (vol/vol) egg yolk (Oxoid), whereas esterase activity was determined on NB medium with 1% (vol/vol) Tween 80. This Tween esterase is supposed to be secreted, but the corresponding gene has not been identified and nothing is known about the biological importance of this activity.Partially Sau3A-digested total DNA from A. calcoaceticus ADP1 was inserted into BamHI-cleaved pWH1274 carrying an origin of replication for Escherichia coli and A. calcoaceticus (16). The resulting gene library was transformed into WH365 by electroporation, and transformants were selected for growth on Luria broth (LB) plates with ampicillin (300 mg/liter). Replica plating on minimal medium with metal 44 (8), hexadecane delivered through the gas phase from a droplet on a filter disk in the lid of the plate, and ampicillin (200 mg/liter) yielded seven candidates. After passage of the plasmids through E. coli DH5␣ (13) and retransformation into WH365, one plasmid, called pWH1590, conferred the ability to grow with hexadecane as the sole carbon source. It contains a 15-kb insertion of contiguous chromosomal DNA, as confirmed by Southern hybridization (data not shown). pWH1590 was digested with HindIII, resulting in nine fragments. Eight of them were ligated with HindIII-restricted pBluescript SKIIϩ (Stratagene), yielding pWH1591 to -98. pWH1597 transformed WH365 to grow on hexadecane and to secrete lipase and esterase. The 2.8-kb insert was sequenced on both strands. It contains three open reading frames (ORFs) (Fig. 1), each of which is preceded by a putative ribosome binding site, as judged by sequence comparison with other Acinetobacter genes (38). Transformation of plasmids obtained from a nested-deletion reaction of pWH1597 mapped the complementing fragment within ORF3 (Fig. 1...

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Codon Usage in Acinetobacter Structural Genes

Cited by 12 publications

References 17 publications

Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases

Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases

Combining localized PCR mutagenesis and natural transformation in direct genetic analysis of a transcriptional regulator gene, pobR

Identification and characterization of xcpR encoding a subunit of the general secretory pathway necessary for dodecane degradation in Acinetobacter calcoaceticus ADP1

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