Characterization of a conditionally transformation-deficient mutant of Haemophilus influenzae that carries a mutation in the rec-1 gene region.

A mutant of Haemophilus influenzae, designated HM5, carrying a mutation in the rec-1 gene region, is described. This mutant transformed approximately 100-fold less well than does the wild type, but approximately 100-fold better than rec1 mutants. The mutant was less sensitive to UV irradiation and less "reckless" than rec1 mutants. In contrast to rec1 lysogens, HP1c1 lysogens of the mutant were inducible, and during transformation, recombinant-type activity was formed to the same extent as in the wild type. Although the integration of donor DNA was complete, the integrated DNA was not replicated at 36 degrees C. Both the inhibition of replication of the donor-recipient DNA complex and the transformation deficiency could be suppressed when, after DNA entry, the cells were incubated under suboptimal conditions. The loss of colony formation after UV irradiation was suppressible by the same conditions.     

Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1.

Two Haemophilus influenzae Rd genes each complemented the pleiotropic defects of the recA-like mutation rec-1. One gene, fec, was isolated on a 3.6-kilobase-pair EcoRI restriction fragment by complementation of the Fec- phenotype of bacteriophage lambda. The other gene, rec, was identified on a 3.1-kilobase-pair EcoRI fragment by Southern hybridization by using recA-like gene probes from Erwinia carotovora and Pseudomonas aeruginosa PAO. In a rec-1 strain of H. influenzae, the cloned genes restored resistance to UV irradiation, transformation by chromosomal DNA, and spontaneous release of HP1 prophage to wild-type levels. The fec and rec genes were located on the cloned segments by insertion and deletion mutagenesis and subcloning. The restriction endonuclease cleavage maps of the two DNAs were similar but not identical. Southern hybridization demonstrated that the two EcoRI restriction fragments contained homologous DNA sequences, but a fec gene-specific probe was prepared. Each gene encoded a 38,000-dalton polypeptide.     

Reexamination of phenotypic defects in rec-1 and rec-2 mutants of Haemophilus influenzae Rd.

Radiolabeled donor DNA is efficiently taken up into competent H. influenzae Rd rec-2 mutant cells but does not undergo the rapid degradation observed in wild-type cells. Furthermore, donor label is not recovered in the chromosome even after 1 h. The donor DNA appears to remain in a protected state in a compartment that can be separated from the rest of the cell. We interpret this as a failure of the donor DNA to be translocated out of the transformasome. In contrast, rec-1 cells translocate labeled donor DNA normally. The donor label accumulates in the recipient chromosome, but, as expected for cells with a recombination defect, there is no preferential localization of the label in sites homologous to the donor DNA. In addition, we have observed two enzymatic activities that act on transformasome-associated DNA of rec-2 cells, an endonuclease which may play a role in the translocation of closed circular DNA and a phosphatase.     

rec-2-dependent phage recombination in Haemophilus influenzae.

The genetic transformation mutant Rd(DB117)rec- has a pleiotropic phenotype that includes reduced levels of phage recombination. Physical mapping experiments showed that this strain has a 78.5-kbp insertion in the rec-2 gene. The rec-2 dependence of phage recombination was reexamined to determine whether the defective phenotype in Rd(DB117)rec- was due to the simple disruption of the rec-2 gene or whether trans-acting factors from the inserted DNA were responsible. Analysis of strains with transposon insertions in the rec-2 gene showed that they were also defective for phage recombination. Therefore, the phage recombination defect was due solely to the disruption of the rec-2 gene. Strain KB6 is proficient for phage recombination but has a defect in genetic transformation resembling that of Rd(DB117)rec-. The transformation defect of KB6 could be complemented by the wild-type rec-2 gene, showing that the rec-2 contributions to genetic transformation and phage recombination were uncoupled in this strain. The rec-2-dependent phenotype of KB6 suggests that the rec-2 gene participates in genetic transformation and phage recombination in different ways.     

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

The Haemophilus influenzae Rd rec-1+ gene was cloned from a partial chromosomal digest into a plasmid vector as a 20-kilobase-pair (kbp) BstEII fragment and then subcloned. The smallest subclone with rec-1+ activity carried a 3.1-kbp EcoRI fragment. The identity of the rec-I gene in these clones was confirmed by transforming an Rd strain carrying a leaky rec-1 mutation (recA4) to resistance to methyl methanesulfonate (MMS) by using whole or digested plasmids. It was demonstrated that the Rec+ phenotype of the MMSr transformants was linked to the strA, novAB, and mmsA loci, as expected if the recA4 allele had been replaced by rec-1+. In growing cultures (rec-1 or rec+), all rec-1+-carrying plasmids induced near-maximal levels of transformability when their hosts reached stationary phase; these levels are 100 to 1,000 times higher than the values seen with strains not carrying a Rec plasmid. Transfer of the 3.1-kbp subclone was greatly reduced compared with transfer of similarly sized vector plasmids, and the resulting transformants grew slowly; this suggests an explanation of my failure to directly clone this fragment from chromosomal DNA digests. Transfer of a rec-1+ plasmid to a very poorly genetically transformable H. influenzae Rb strain resulted in greatly increased transformability. Transfer of such plasmids to a noncompetent H. influenzae Rc strain did not render this strain competent. It is suggested that transformability of Rd and Rb strains is limited by rec-1 expression but that the noncompetence of Rc has some other basis.     

Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli.

The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein.     

Genetics and complementation of Haemophilus influenzae mutants deficient in adenosine 5''-triphosphate-dependent nuclease.

Eight different mutations in Haemophilus influenzae leading to deficiency in adenosine 5'-triphosphate (ATP)-dependent nuclease have been investigated in strains in which the mutations of the originally mutagenized strains have been transferred into the wild type. Sensitivity to mitomycin C and deoxycholate and complementation between extracts and deoxyribonucleic acid (DNA)-dependent ATPase activity have been measured. Genetic crosses have provided information on the relative position of the mutations on the genome. There are three complementation groups, corresponding to three genetic groups. The strains most sensitive to mitomycin and deoxycholate, derived from mutants originally selected on the basis of sensitivity to mitomycin C or methyl methanesulfonate, are in one group. Apparently all these sensitive strains lack DNA-dependent ATPase activity, as does a strain intermediate in sensitivity to deoxycholate, which is the sole representative of another group. There are four strains that are relatively resistant to deoxycholate and mitomycin C, and all of these contain the ATPase activity. Three of these are in the same genetic and complementation group, whereas the other incongruously belongs in the same group as the sensitive strains. It is postulated that there are three cistrons in H. influenzae that code for the three known subunits of the ATP-dependent nuclease.     

Effect of ethylenediaminetetraacetic acid on deoxyribonucleic acid entry and recombination in transformation of a wild-type strain and a rec-1 mutant of Haemophilus influenzae

In transformation of Haemophilus influenzae, donor deoxyribonucleic acid (DNA) enters into competent cells in the presence of ethylenediaminetetraacetic acid (EDTA), which prevents the formation of single stranded regions in the donor DNA that has entered. If after entry of DNA the recipient cells were first incubated at 17 degrees C and then at 37 degrees C in the continuous presence of EDTA, almost no integration occurred. On the other hand, if after entry of DNA the cells were incubated first at 17 degrees C in the absence of EDTA, allowing the generation of single-stranded regions (integration is blocked at this temperature), and then at 37 degrees C in the presence of EDTA, donor-recipient DNA complexes were formed. These results suggest that single-stranded regions are required for integration. Integration to completion was strongly inhibited by EDTA. In a rec-1 mutant of H. influenzae no donor-recipient DNA complexes carrying recombinant-type activity were formed during incubation at 37 degrees C in the absence of EDTA. If rec-1 cells were incubated at 37 degrees C in the presence of EDTA, which strongly inhibited breakdown of DNA, donor-recipient DNA complexes were formed if previously single-stranded regions in the donor DNA that had entered were generated by incubation at 17 degrees C in the absence of EDTA. This suggests that the rec-1 protein protects the initial donor-recipient DNA complex against degradation, so that further steps in the recombination process can proceed.     

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

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.     

rpe, a cis-acting element from the strA region of the Haemophilus influenzae chromosome that makes plasmid establishment independent of recombination.

Plasmids that share homology with the Haemophilus influenzae chromosome transform wild-type cells more efficiently than they transform recombination-defective mutants. A 5.2-kilobase-pair chromosomal fragment containing the strA gene of H. influenzae was found to promote efficient plasmid establishment in recombination-defective mutants. A cis-acting element in the insert, called rpe for rec-less plasmid establishment, promoted plasmid transformation in rec-1 and rec-2 mutants without suppressing the recombination defects of these strains. The rpe locus increased plasmid transformation in wild-type cells without interfering with the pathway of plasmid establishment that is dependent on recombination functions.     

Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5''-triphosphate-dependent deoxyribonuclease activity.

By a direct assay approach, mutants of Haemophilus influenzae Rd that are deficient in adenosine 5'-triphosphate-dependent deoxyribonuclease activity (add-) were isolated and characterized. A large proportion (50 to 90%) of the cells in cultures of these mutants failed to produce visible colonies when plated. An extensive analysis of the recombination proficiency of these strains revealed that the transformation frequency (transformants per competent cell) in the mutants was similar to that found in the wild type, but that the transformation efficiency (transformants per microgram of irreversibly bound deoxyribonucleic acid [DNA]) was reduced approximately fourfold. Sensitivities of the mutants to gamma rays, ultraviolet radiation, and methyl methane sulfonate were only slightly greater than wild-type levels. The rate of degradation of host DNA after ultraviolet irradiation was significantly reduced in the mutants. It is suggested that the adenosine 5'-triphosphate-dependent deoxyribonuclease in H. influenzae plays a nonessential role in DNA recombination and repair.     

sxy-1, a Haemophilus influenzae mutation causing greatly enhanced spontaneous competence.

A Haemophilus influenzae strain carrying a competence-enhancing mutation (sxy-1) was selected by transformation of a mutagenized culture in exponential growth at low cell density, where spontaneous competence is very rare. Under these conditions, sxy-1 cells spontaneously transformed 100 to 1,000 times more efficiently than wild-type cells. Moreover, sxy-1 cells responded to all known competence-inducing treatments with further increases in transformation frequency. At high cell densities, sxy-1 cells spontaneously developed the level of competence reached by wild-type cells only after maximal induction by transfer to starvation medium. The sxy-1 mutation appears to act early in the sequence of events leading to competence; it increased the competence of cells carrying the early-acting transformation-defective (Tfo-) mutation tfo-98 by as large a factor as it did the competence of wild-type cells, but it had no effect when combined with another early-acting Tfo- mutation (tfo-87) or with the late-acting Tfo- mutation rec-2.     

Antibiotic-tolerant mutants of Streptococcus pneumoniae that are not deficient in autolytic activity.

Several mutants of Streptococcus pneumoniae were isolated that appeared tolerant, to varying extents, to the lytic and bactericidal effects of some antibiotics that inhibit peptidoglycan synthesis, but were not deficient in autolytic activity. The method used to select the mutants was based on the survival of tolerant mutants during treatment with either bacitracin, benzylpenicillin, D-cycloserine plus beta-chloro-D-alanine, or vancomycin. Most (60 to 80%) of the surviving isolates were found to be deficient in autolytic activity, and these were rejected. The smaller proportion that had wild-type sensitivity to deoxycholate-induced lysis was studied further with respect to tolerance to the other antibiotics used in the selection procedures. Two of these mutants (selected by treatment with benzylpenicillin) were tolerant to either benzylpenicillin or D-cycloserine plus beta-chloro-D-alanine, but were supersusceptible, in terms of initiation of lysis, to either bacitracin or vancomycin. The minimal inhibitory concentration values of several antibiotics for these two mutants were identical to those for the wild-type strain. Moreover, the interaction of radioactive benzylpenicillin with the penicillin-binding proteins, examined in whole organisms, also appeared the same as previously found for either wild-type or autolytic-deficient strains of S. pneumoniae.     

Molecular cloning of two linked loci that increase the transformability of transformation-deficient mutants of Haemophilus influenzae.

A plasmid containing a 13.3-kb insert (pER194) was isolated from an EcoRI genomic library of Haemophilus influenzae on the basis of its ability to increase the transformability of the transformation-deficient mutants Com-78 and Com-101. The plasmid failed to increase the transformability of the Rec-1 and Rec-2 mutants, indicating that the mutations producing the Com-78 and Com-101 phenotypes are distinct from those giving rise to the Rec-1 and Rec-2 phenotypes. The physical mapping of the cloned fragment on the H. influenzae chromosome was found to be consistent with the genetic mapping of the Com-101 trait. A 2.8-kb EcoRI-BglII subfragment, representing one end of the 13.3-kb clone, was found to increase the transformation frequency of the Com-78 and Com-101 mutants when supplied in trans, indicating that the subfragment carries one or more loci required for chromosomal transformation. The corresponding region of the Com-101 chromosome was determined by hybridization analysis to contain a 0.3-kb insertion, suggesting that the Com-101 strain may contain an insertion mutation at this locus. A 3.0-kb EcoRI-MluI subfragment, representing the other end of the 13.3-kb EcoRI fragment, was found to increase the transformation frequency of the Com-101 mutant but not of the Com-78 mutant, suggesting that the Com-101 phenotype results from a complex genotype involving mutations at two or more transformation-related loci. This conclusion is consistent with data indicating that the Com-101 trait can be genetically separated into at least two components.     

Cloning and characterization of the Haemophilus influenzae mutB gene.

The Haemophilus influenzae mutB+ gene complements Escherichia coli uvrD mutants. The E. coli uvrD+ gene complements H. influenzae mutB1 mutants.