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

ABSTRACT: 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.

SUBMITTER: Williams PM

PROVIDER: S-EPMC197045 | biostudies-other | 1994 Nov

REPOSITORIES: biostudies-other

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

Williams P M PM Bannister L A LA Redfield R J RJ

Journal of bacteriology 19941101 22

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 thi ...[more]

PMID: 7961436

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Project description:Competence for transformation in Haemophilus influenzae is stimulated by cyclic AMP (cAMP) and requires the cAMP-dependent catabolite regulatory protein CRP. Thus, understanding the control of competence will require understanding how cAMP levels are regulated. As a first step, we have cloned the H. influenzae adenylate cyclase gene (cya) by complementing the Lac- phenotype of delta cya Escherichia coli. Its sequence specifies an 843-amino-acid protein which has significant identity to other known bacterial adenylate cyclases (41 to 43% and 61% identical to the cya genes of enteric bacteria and of Pasteurella multocida, respectively). As seen in other bacterial cya genes, there is evidence for regulation similar to that demonstrated for E. coli: the presence of a strong consensus CRP binding site within the promoter of the gene may provide feedback control of cAMP levels by repressing cya transcription, and translation may be limited by the weak ribosome binding site and by initiation of protein synthesis with GUG rather than AUG or the UUG used in other bacterial cya genes. We confirmed the essential role of cAMP in competence by constructing and characterizing H. influenzae cya mutants. This strain failed to develop competence either spontaneously or after transfer to a competence-inducing medium. However, it became as competent as its wild-type parent in the presence of exogenous cAMP. This result suggests that the failure of exogenously added cAMP to induce optimum competence in wild-type cells is not due to a limitation to the entry of cAMP into the cells. Rather, it strongly favors models in which competence induction requires both an increase in intracellular cAMP and a second as yet unidentified regulatory event. H. influenzae strains mutant in cya or crp were unable to ferment xylose or ribose. This confirms that influenzae, like E. coli, uses cAMP and CRP to regulate nutrient uptake and utilization and lends increasing support to the hypothesis that DNA uptake is mechanism of nutrient acquisition.

Project description:The occurrence of fimbria gene clusters in nonencapsulated Haemophilus influenzae strains from chronic bronchitis patients (n = 58), patients with acute otitis media (n = 13), and healthy carriers (n = 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate H. influenzae type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting of hifA to hifE inserted in the chromosome between purE and pepN, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (n = 2), leading to optimal positioning between the -10 and -35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity. hifA appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between hifA and purE has not been found in H. influenzae type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genes purE and pepN suggest that the fimbria gene cluster was originally contained on a mobile element.

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

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

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