Cloning and characterization of the Haemophilus influenzae Rd rec-1+ gene

Stuy, Johan H.

doi:10.1128/jb.171.8.4395-4401.1989

Cited by 5 publications

(6 citation statements)

References 40 publications

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“…We have been unable to obtain a stable clone carrying the 3.1-kb EcoRI fragment from sxy-1 cells. Stuy described extensively his difficulties in obtaining clones of the same wild-type fragment (17). Part of the problem may be the presence of the nmA promoter in this fragment (19), but we have observed frequent rearrangements, substitutions, and deletions even when the sxy-1 mutant fragment was placed in a vector containing a strong terminator.…”

Section: Resultsmentioning

confidence: 85%

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The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence

1994

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The sxy-1 mutation of Haemophilus influenzae causes a 100- to 1,000-fold increase in spontaneous natural competence. We have used mapping and sequencing to identify this mutation as a G-to-A transition in an open reading frame adjacent to the rec-1 locus. This mutation substitutes valine for isoleucine at amino acid 19 of the protein specified by this gene (now named sxy). A multicopy plasmid containing the wild-type sxy gene confers constitutive competence on wild-type cells. Cells carrying this plasmid exhibit, in all stages of growth, DNA uptake levels and transformation frequencies as high those normally seen only after full induction of competence by starvation; deletion of part of the sxy gene from the plasmid abolishes this effect. In contrast, a transposon insertion in sxy entirely prevents both DNA uptake and transformation, indicating that sxy encodes a function essential for competence. These findings suggest that sxy may act as a positive regulator of competence. However, because cells carrying the transposon-inactivated sxy::Tn allele grow slowly under conditions that do not induce competence, sxy may also have a role in noncompetent cells.

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Section: Resultsmentioning

confidence: 85%

“…Constitutive competence caused by pHKrec. Stuy reported that a plasmid containing the H. influenzae rec-1 gene enhances the competence of wild-type cells (17). Because this phenotype resembles the hypercompetence caused by the sxy-1 mutation, we characterized it in detail.…”

Section: Resultsmentioning

confidence: 96%

The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence

1994

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“…Haemophilus influenzae is a natural process that results in the homologous recombination of donor DNA into the host genome, a process dependent upon the product of the rec-1+ gene (4,53,55). To better understand the role of Rec-1 in DNA transformation, we and others have cloned and characterized rec-1 + on the basis of the ability to complement in trans the pleiotropic defects of H. influenzae rec-1 (2,55,59) and Escherichia coli recA (2, 55) strains. The rec-1+ gene is located on a 3.15-kbp EcoRI fragment (2,59) and encodes a 38,000molecular-weight (MW) protein (2,29).…”

Section: Genetic Transformation In the Gram-negative Bacteriummentioning

confidence: 99%

Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene

Zulty

Barcak

1993

J Bacteriol

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The Haemophilus influenzae rec-1+ protein plays a central role in DNA metabolism, participating in general homologous recombination, recombinational (postreplication) DNA repair, and prophage induction. Although many H. influenzae rec-1 mutants have been phenotypically characterized, little is known about the rec-1+ gene at the molecular level. In this study, we present the genetic organization of the rec-1+ locus, the DNA sequence of rec-1+, and studies of the transcriptional regulation of rec-1+ during cellular assault by DNA-damaging agents and during the induction of competence for genetic transformation. Although little is known about promoter structure in H. influenzae, we identified a potential rec-1+ promoter that is identical in 11 of 12 positions to the bacterial sigma 70-dependent promoter consensus sequence. Results from a primer extension analysis revealed that the start site of rec-1+ transcription is centered 6 nucleotides downstream of this promoter. We identified potential DNA binding sites in the rec-1+ gene for LexA, integration host factor, and cyclic AMP receptor protein. We obtained evidence that at least one of the proposed cyclic AMP receptor protein binding sites is active in modulating rec-1+ transcription. This finding makes rec-1+ control circuitry novel among recA+ homologs. Two H. influenzae DNA uptake sequences that may function as a transcription termination signal were identified in inverted orientations at the end of the rec-1+ coding sequence. In addition, we report the first use of the Escherichia coli lacZ operon fusion technique in H. influenzae to study the transcriptional control of rec-1+. Our results indicate that rec-1+ is transcriptionally induced about threefold during DNA-damaging events. Furthermore, we show that rec-1+ can substitute for recA+ in E. coli to modulate SOS induction of dinB1 expression. Surprisingly, although 5% of the H. influenzae genome is in the form of single-stranded DNA during competence for genetic transformation, an event that could be a potent SOS-inducing signal, we failed to detect significant changes in rec-1+ transcription during the induction of genetic competence.

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“…This observation may not be surprising, because other H. influenzae genes have also been found to function in E. coli (rec-1 gene [3,16,21] and mutB [25]). We were not able to show that the H. influenzae strAl gene was similarly expressed.…”

Section: Methodsmentioning

confidence: 98%

“…Standard methods have been described (18)(19)(20)(21)23). E. coli cells were made competent by the CaCl2 technique (12).…”

Section: Methodsmentioning

confidence: 99%

Cloning, characterization, and DNA base sequence of the high-level streptomycin resistance gene strA1 of Haemophilus influenzae Rd

Stuy

Walter

1992

J Bacteriol

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The high-level streptomycin resistance strAl gene ofHaemophilus influenzae Rd was cloned in plasmid pAT4 as a 2.1-kbp EcoRI insert. It was later replaced in pAT4 by the wild-type strA+ gene. Plasmid pAT4 carrying the strA+ gene is highly unstable and renders chromosomally resistant recipients sensitive to streptomycin. The strA+ gene and the instability factor both reside on a 500-base HindIII-EcoRI subfragment. The two biological activities are also expressed in Escherachia coli. Both wild-type (strA+) and mutant (strAl) genes were sequenced. They show considerable nucleotide homology with the E. coli strA+ gene and its product.

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Cloning and characterization of the Haemophilus influenzae Rd rec-1+ gene

Cited by 5 publications

References 40 publications

The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence

The Haemophilus influenzae sxy-1 mutation is in a newly identified gene essential for competence

Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene

Cloning, characterization, and DNA base sequence of the high-level streptomycin resistance gene strA1 of Haemophilus influenzae Rd

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