Construction of stable lactose-utilizing Xanthomonas campestris by chromosomal integration of cloned lac genes using filamentous phage ?Lf DNA

Fu, Jen-Fen; Chang, Ruey-Yi; Tseng, Yi-Hsiung

doi:10.1007/bf00178175

Cited by 25 publications

(17 citation statements)

References 18 publications

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“…campestris 17 (Xc17) (Chang 1989). pS19 was later used as an integrative vector to insert the E. coli lacZY genes into Xc17, resulting in a stable strain that is able to utilize whey for the production of xanthan gum (Fu et al 1992). In this study, we dissected the sequences required for /Lf integration using various mutant strains.…”

Section: Introductionmentioning

confidence: 99%

Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

et al. 2001

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Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.

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Section: Introductionmentioning

confidence: 99%

Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

et al. 2001

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“…campestris (Tseng et al, 1990). Several have been shown to integrate into the host chromosomes (Dai et al, 1987;Chang, 1989;Pai, 1989;Fu et al, 1992). In this study, fragments of ~bXv and qIXo RF DNA were cloned into pOK12 (Vieira & Messing, 1991), a pACYC177 derivative which cannot be maintained in Xanthomonas, and electroporated into the respective indicator hosts, selecting for kanamycin resistance conferred by the vector.…”

mentioning

confidence: 99%

Characterization of two novel filamentous phages of Xanthomonas

Lin¹,

You²,

Huang³

et al. 1994

Journal of General Virology

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Two filamentous phages of Xanthomonas campestris pv.vesicatoria and Xanthomonas oryzae pv. oryzae were isolated and designated ~bXv and ~bXo, respectively. They were similar to other filamentous phages of Xanthomonas in (i) shape, (ii) restrictive host specificity, (iii) high stability, (iv) an ssDNA genome, (v) a dsDNA as the replicative form (RF), (vi) propagation without lysis of host cells and (vii) ability to integrate into the host chromosome. These phages showed sequence homology to filamentous phage ~bLf ofX. c. pv. campestris. ~bXv was inactivated by antisera against q~Xv, ~bXo and ~bLf, whereas ~bXo and ~bLf were inactivated only by their respective antisera and the anti-~bXv serum. Both the single-stranded phage DNAs and the RF DNAs of q~Xv, ~bXo and ~bLf were able to transfect X. c. pv. vesicatoria, X. o. pv. oryzae and X. c. pv. campestris. Physical maps of ~bXv and ~bXo were constructed for the RF DNAs.Genome sizes were estimated, based on mapping data, to be 6"8 kb for ~bXv and 7-6 kb for ~bXo, larger than that of the ~bLf genome (6.0 kb). The difference in genome sizes appeared to result from insertions of large DNA fragments. These fragments and the regions mediating integration were localized in the physical maps.

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“…Therefore, efforts have been made to isolate strains able to use lactose and whey (the lactose-containing waste product of the cheese industry) for xanthan production. Selection, mutation and recombinant DNA techniques have all been used [Fu et al, 1992;Fu and Tseng, 1990;Schwartz and Bodie, 1985;Thorne et al, 1988;Walsh et al, 1984;Yang et al, 2002]. We recently isolated Xc17L, which exhibits elevated ß-galactosidase levels, from the wild-type Xc17 by mutagenesis with nitrous acid [Yang et al, 2002].…”

Section: Introductionmentioning

confidence: 99%

Molecular Genetic Analyses of Potential β-Galactosidase Genes in <i>Xanthomonas campestris</i>

Yang

Hu²,

Hsiao

et al. 2003

Microb Physiol

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Xanthomonas campestris pv. campestris, which displays no significant β-1,4-D-galactopyranosidase activity, has three annotated β-galactosidase genes in the sequenced genome, designated galA, galB and galC herein. GalA and GalB are similar to glycosyl hydrolase (GH) family 2 enzymes, including Escherichia coli LacZ. galA and galB cannot express detectable activity even after being cloned in-frame and driven by the vector’s promoter. GalC is a GH35 enzyme homologous to the Xanthomonas axonopodis pv. manihotis Bga. The latter cleaves β,1-3-linked galactose 1,000 times faster than β,1-4-linked galactose and is not responsible for lactose utilization. In X. campestris pv. campestris cells, GalC is readily detectable by Western blotting, and the levels can be increased by cloning the gene under the control of the vector’s promoter. Results of insertional mutation, transcriptional fusion assay and Western blotting indicated that galC, clustered with several GH genes, is cotranscribed with the upstream gene(s) and is expressed constitutively. Xc17L is a previously isolated mutant with elevated β-galactosidase activity and a greatly improved ability to grow on lactose. Results of DNA sequencing of Xc17L galA, galB and galC, enzyme assays of galA, galB and galC mutants derived from Xc17L, and Western blotting of GalC in Xc17L indicated that the three β-galactosidase genes do not encode the elevated β-galactosidase activity in Xc17L. The presence of a fourth β-galactosidase gene is proposed.

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Construction of stable lactose-utilizing Xanthomonas campestris by chromosomal integration of cloned lac genes using filamentous phage ?Lf DNA

Cited by 25 publications

References 18 publications

Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Characterization of two novel filamentous phages of Xanthomonas

Molecular Genetic Analyses of Potential β-Galactosidase Genes in <i>Xanthomonas campestris</i>

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