Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus

Averhoff, Beate; Gregg-Jolly, Leslie A.; Elsemore, David A.; Ornston, L N

doi:10.1128/jb.174.1.200-204.1992

Cited by 33 publications

(29 citation statements)

References 23 publications

(31 reference statements)

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“…The genes for the two convergent, biochemically and genetically homologous branches of the pathway are found in two supraoperonic clusters, the ben-cat cluster (29,32), comprising the genes for the conversion of benzoate to tricarboxylic acid cycle intermediates, and the pca-qui-pob cluster (2,8,9), comprising the genes for the catabolism of p-hydroxybenzoate and quinate. Both clusters are Ͼ20 kbp and yet are separated on the chromosome by about 290 kbp (13).…”

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mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source

Williams

Shaw

1997

J Bacteriol

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Benzyl alcohol, benzaldehyde, benzoate, and anthranilate are metabolized via catechol, cis,cis-muconate, and the ␤-ketoadipate pathway in Acinetobacter calcoaceticus ADP1 (BD413). Mutant strain ISA25 with a deletion spanning catBCIJF and unable to metabolize muconate further will not grow in the presence of an aromatic precursor of muconate. Growth on fumarate as the sole carbon source with added benzyl alcohol or benzaldehyde selected spontaneous mutants of ISA25. After repair of the cat deletion by natural transformation with linearized plasmid pPAN4 (catBCIJF) 10 mutants were unable to grow on benzoate or cis,cis-muconate but could still grow on anthranilate. Transformation with wild-type chromosomal DNA demonstrated the presence of two unlinked mutations in each strain, one in the benABCD region, encoding the conversion of benzoate to catechol, and the other in a gene determining the ability to grow on exogenous cis,cis-muconate. The wild-type gene, named mucK, was cloned into pUC18, and its nucleotide sequence was determined. It encodes a 413-residue protein of M r ‫؍‬ 45,252 which is a member of a superfamily of membrane transport proteins and which is within a subgroup involved in the uptake of organic acids. Five of the mutant alleles were cloned, and the mutations were determined by nucleotide sequencing. All the mutations were in the mucK coding region and consisted of three deletions, one duplication, and a substitution. Insertional inactivation of mucK resulted in the loss of the ability to utilize exogenous muconate. The location of mucK on the chromosome appeared to be unique for genes associated with the benzoate branch of the ␤-ketoadipate pathway in being close to the pca-qui-pob gene cluster (for p-hydroxybenzoate utilization) and distant from the functionally related ben-cat cluster. Downstream of mucK and transcribed in the same direction is an open reading frame encoding a protein of 570 residues (M r ‫؍‬ 63,002) which shows considerable homology with a mammalian electron transport protein; its insertional inactivation had no detectable phenotypic effect.

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mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source

Williams

Shaw

1997

J Bacteriol

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“…Strain BD413 is naturally competent during growth, and has often been used for transformation studies in vitro (Averhoff et al, 1992;de Vries and Wackernagel, 2002;Juni, 1972;Juni and Janik, 1969;Palmen and Hellingwerf, 1997), in soil and water microcosms (Chamier et al, 1993;Clerc and Simonet, 1998;Lorenz et al, 1992;Nielsen and van Elsas, 2001;Nielsen et al, 1997a;1997b), in a river (Williams et al, 1996), in planta (Kay et al, 2002a;2002b;Tepfer et al, 2003) and most recently in vivo in tobacco horn worm (Deni et al, 2005). Strain DB413 was originally isolated from soil.…”

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An assessment of the potential of herbivorous insect gut bacteria to develop competence for natural transformation

Ray

Andersen

Young

et al. 2007

Environ. Biosafety Res.

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Whereas the capability of DNA uptake has been well established for numerous species and strains of bacteria grown in vitro, the broader distribution of natural transformability within bacterial communities remains largely unexplored. Here, we investigate the ability of bacterial isolates from the gut of grass grub larvae (Costelytra zealandica (White); Coleoptera: Scarabaeidae) to develop natural genetic competence in vitro. A total of 37 mostly species-divergent strains isolated from the gut of grass grub larvae were selected for spontaneous rifampicin-resistance. Genomic DNA was subsequently isolated from the resistant strains and exposed to sensitive strains grown individually using established filter transformation protocols. DNA isolated from wild-type strains was used as a control. None of the 37 isolates tested exhibited a frequency of conversion to rifampicinresistance in the presence of DNA at rates that were significantly higher than the rate of spontaneous mutation to rifampicin-resistance in the presence of wild-type DNA (the limit of detection was approximately < 1 culturable transformant per 10 9 exposed bacteria). To further examine if conditions were conducive to bacterial DNA uptake in the grass grubs gut, we employed the competent bacterium Acinetobacter baylyi strain BD413 as a recipient species for in vivo studies. However, no transformants could be detected above the detection limit of 1 transformant per 10 3 cells, possibly due to low population density and limited growth of A. baylyi cells in grass grub guts. PCR analysis indicated that chromosomal Acinetobacter DNA remains detectable by PCR for up to 3 days after direct inoculation into the alimentary tract of grass grub larvae. Nevertheless, neither transforming activity of the DNA recovered from the alimentary tract of grass grubs larvae nor competence of bacterial cells recovered from inoculated larvae could be shown.

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“…It therefore is surprising that the biosynthetic AroD function in the gram-negative bacterium Acinetobacter calcoaceticus is served by a constitutively expressed enzyme that appears to be type II as judged by its thermostability (53). Catabolism of dehydroquinate in these bacteria is initiated by an inducible QuiB enzyme that, as indicated by its thermolability, appears to be type I (28,53,54).Further exploration of relationships among the A. calcoaceticus dehydratases was made possible by localization of the qui genes in a supraoperonic cluster between pca genes (associated with the catabolism of protocatechuate) and pob genes (associated with conversion of p-hydroxybenzoate to protocatechuate) (2,20,23). Engineered deletion mutations were introduced into A. calcoaceticus by natural transformation, and the properties of the resulting mutant strains allowed identification of quiA, the gene for the dehydrogenase that catalyzes the conversion of quinate and shikimate to dehydroquinate and dehydroshikimate, respectively ( Fig.…”

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“…Further exploration of relationships among the A. calcoaceticus dehydratases was made possible by localization of the qui genes in a supraoperonic cluster between pca genes (associated with the catabolism of protocatechuate) and pob genes (associated with conversion of p-hydroxybenzoate to protocatechuate) (2,20,23). Engineered deletion mutations were introduced into A. calcoaceticus by natural transformation, and the properties of the resulting mutant strains allowed identification of quiA, the gene for the dehydrogenase that catalyzes the conversion of quinate and shikimate to dehydroquinate and dehydroshikimate, respectively ( Fig.…”

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Unusual ancestry of dehydratases associated with quinate catabolism in Acinetobacter calcoaceticus

Elsemore

Ornston

1995

J Bacteriol

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Catabolism of quinate to protocatechuate requires the consecutive action of quinate dehydrogenase (QuiA), dehydroquinate dehydratase (QuiB), and dehydroshikimate dehydratase (QuiC). Genes for catabolism of protocatechuate are encoded by the pca operon in the Acinetobacter calcoaceticus chromosome. Observations reported here demonstrate that A. calcoaceticus qui genes are clustered in the order quiBCXA directly downstream from the pca operon. Sequence comparisons indicate that quiX encodes a porin, but the specific function of this protein has not been clearly established. Properties of mutants created by insertion of ⍀ elements show that quiBC is expressed as part of a single transcript, but there is also an independent transcriptional initiation site directly upstream of quiA. The deduced amino acid sequence of QuiC does not resemble any other known sequence. A. calcoaceticus QuiB is most directly related to a family of enzymes with identical catalytic activity and biosynthetic AroD function in coliform bacteria. Evolution of A. calcoaceticus quiB appears to have been accompanied by fusion of a leader sequence for transport of the encoded protein into the inner membrane, and the location of reactions catalyzed by the mature enzyme may account for the failure of A. calcoaceticus aroD to achieve effective complementation of null mutations in quiB. Analysis of a genetic site where a DNA segment encoding a leader sequence was transposed adds to evidence suggesting horizontal transfer of nucleotide sequences within genes during evolution.Dehydroquinate dehydratase (QuiB) and dehydroshikimate dehydratase (QuiC) catalyze consecutive reactions allowing growth of microorganisms with quinate by its conversion to protocatechuate ( Fig. 1) and subsequent metabolism by the ␤-ketoadipate pathway (41, 52). The enzymatic activity of QuiB is identical to that of AroD, an enzyme that catalyzes biosynthetic formation of dehydroshikimate (Fig. 1). Enzymes with similar catalytic activities often can be traced to a common ancestor (32), and the similarity of dehydratases associated with dehydroshikimate metabolism raised the possibility that a single ancestral protein was the evolutionary precursor of two or more of these enzymes.In contrast to this prediction, biosynthetic dehydroquinate dehydratases differ substantially from catabolic enzymes with this activity (18,30). With the exception of the dehydroquinate dehydratases of gram-positive bacteria (13, 15), enzymes with the biosynthetic function indicated as AroD in Fig. 1 are classified as type I and are thermolabile, a property that distinguishes them from the isofunctional type II enzymes which are heat stable and mediate the activity indicated as QuiB in Fig. 1 Some gram-negative bacteria elaborate a single dehydroquinate dehydratase associated solely with the biosynthetic function indicated as AroD in Fig. 1. Genes for this enzyme from Enterococcus faecalis (3), Salmonella typhi (51), and Escherichia coli (10) have been sequenced and shown to encode type I enzymes. These fi...

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Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus

Cited by 33 publications

References 23 publications

mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source

mucK, a gene in Acinetobacter calcoaceticus ADP1 (BD413), encodes the ability to grow on exogenous cis,cis-muconate as the sole carbon source

An assessment of the potential of herbivorous insect gut bacteria to develop competence for natural transformation

Unusual ancestry of dehydratases associated with quinate catabolism in Acinetobacter calcoaceticus

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