Random Transposition by Tn916 in Desulfitobacterium Dehalogenans Allows for Isolation and Characterization of Halorespiration-deficient Mutants

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 1999 Nov 13

PMID 10559152

Citations 14

Authors

H Smidt

D Song

J van der Oost

W M de Vos

Affiliations

Soon will be listed here.

Abstract

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.

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References

Sawers G . The hydrogenases and formate dehydrogenases of Escherichia coli. Antonie Van Leeuwenhoek. 1994; 66(1-3):57-88. DOI: 10.1007/BF00871633. View

Clewell D, Flannagan S, Jaworski D . Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons. Trends Microbiol. 1995; 3(6):229-36. DOI: 10.1016/s0966-842x(00)88930-1. View

Renault P, Nogrette J, Galleron N, Godon J, Ehrlich S . Specificity of insertion of Tn1545 transposon family in Lactococcus lactis subsp. lactis. Dev Biol Stand. 1995; 85:535-41. View

Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W . Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17):3389-402. PMC: 146917. DOI: 10.1093/nar/25.17.3389. View

Berks B . A common export pathway for proteins binding complex redox cofactors?. Mol Microbiol. 1996; 22(3):393-404. DOI: 10.1046/j.1365-2958.1996.00114.x. View

Gribble G . Naturally occurring organohalogen compounds--a comprehensive survey. Fortschr Chem Org Naturst. 1996; 68:1-423. View

de Vos W, Kleerebezem M, Kuipers O . Expression systems for industrial Gram-positive bacteria with low guanine and cytosine content. Curr Opin Biotechnol. 1997; 8(5):547-53. DOI: 10.1016/s0958-1669(97)80027-4. View

Andrews S, Berks B, McClay J, Ambler A, Quail M, Golby P . A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology (Reading). 1997; 143 ( Pt 11):3633-3647. DOI: 10.1099/00221287-143-11-3633. View

El Fantroussi S, Naveau H, Agathos S . Anaerobic dechlorinating bacteria. Biotechnol Prog. 1998; 14(2):167-88. DOI: 10.1021/bp980011k. View

10.

Neumann A, Wohlfarth G, Diekert G . Tetrachloroethene dehalogenase from Dehalospirillum multivorans: cloning, sequencing of the encoding genes, and expression of the pceA gene in Escherichia coli. J Bacteriol. 1998; 180(16):4140-5. PMC: 107409. DOI: 10.1128/JB.180.16.4140-4145.1998. View

11.

Fetzner S . Bacterial dehalogenation. Appl Microbiol Biotechnol. 1999; 50(6):633-57. DOI: 10.1007/s002530051346. View

12.

van de Pas B, Smidt H, Hagen W, van der Oost J, Schraa G, Stams A . Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans. J Biol Chem. 1999; 274(29):20287-92. DOI: 10.1074/jbc.274.29.20287. View

13.

Jacob A, Hobbs S . Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J Bacteriol. 1974; 117(2):360-72. PMC: 285522. DOI: 10.1128/jb.117.2.360-372.1974. View

14.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

15.

Clewell D . Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria. J Bacteriol. 1984; 159(1):214-21. PMC: 215615. DOI: 10.1128/jb.159.1.214-221.1984. View

16.

Hill C, Venema G, Daly C, Fitzgerald G . Cloning and characterization of the tetracycline resistance determinant of and several promoters from within the conjugative transposon Tn919. Appl Environ Microbiol. 1988; 54(5):1230-6. PMC: 202631. DOI: 10.1128/aem.54.5.1230-1236.1988. View

17.

Triglia T, Peterson M, Kemp D . A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988; 16(16):8186. PMC: 338531. DOI: 10.1093/nar/16.16.8186. View

18.

Mohn W, Tiedje J . Microbial reductive dehalogenation. Microbiol Rev. 1992; 56(3):482-507. PMC: 372880. DOI: 10.1128/mr.56.3.482-507.1992. View

19.

Scott J, Bringel F, Marra D, Van Alstine G, Rudy C . Conjugative transposition of Tn916: preferred targets and evidence for conjugative transfer of a single strand and for a double-stranded circular intermediate. Mol Microbiol. 1994; 11(6):1099-108. DOI: 10.1111/j.1365-2958.1994.tb00386.x. View

20.

Flannagan S, Zitzow L, Su Y, Clewell D . Nucleotide sequence of the 18-kb conjugative transposon Tn916 from Enterococcus faecalis. Plasmid. 1994; 32(3):350-4. DOI: 10.1006/plas.1994.1077. View