Genetic systems in Haemophilus influenzae.
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Bacteriophages↗
Biomedical subjects
R J Redfield
Publications and source records attributed to R J Redfield.
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.
Anaerobiosis↗
Organization of the Haemophilus influenzae Rd genome.
We present the first complete map of the Haemophilus influenzae genome, consisting of a detailed restriction map with a number of genetic loci. All of the ApaI, SmaI, and RsrII restriction sites (total of 45 sites) were mapped by Southern blot hybridization analysis of fragments separated by pulsed-field gel electrophoresis. Cloned genes were placed on the restriction map by Southern hybridization, and antibiotic resistance loci were also located by transformation with purified restriction fragments. The attachment site of the HP1 prophage was mapped. In addition, the number, locations, and orientations of the six rRNA operons in the H. influenzae chromosome were determined. The positions of conserved restriction sites in these rrn operons confirm that the direction of transcription is 16S to 23S, as in most other bacteria. The widely used strain BC200 appears to contain an unexpected 45-kilobase duplication.
Blotting, Southern↗
Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.
A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.
Alleles↗
Evolution of bacterial transformation: is sex with dead cells ever better than no sex at all?
Computer simulations of bacterial transformation are used to show that, under a wide range of biologically reasonable assumptions, transforming populations undergoing deleterious mutation and selection have a higher mean fitness at equilibrium than asexual populations. The source of transforming DNA, the amount of DNA taken up by each transforming cell, and the relationship between number of mutations and cell viability (the fitness function) are important factors. When the DNA source is living cells, transformation resembles meiotic sex. When the DNA source is cells killed by selection against mutations, transformation increases the average number of mutations per genome but can nevertheless increase the mean fitness of the population at equilibrium. In a model of regulated transformation, in which the most fit cells of a transforming population do not transform, transforming populations are always fitter at equilibrium than asexual populations. These results show that transformation can reduce mutation load.
Models, Biological↗
Structure of cryptic lambda prophages.
When Escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors. The lambda variant crypticogen (lambda crg) carries an insertion of the transposable element IS2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized. They all contain substitutions that replace the early segment of the prophage genome (from the IS2 to near the cos site) with a duplicate copy of a large segment of the host chromosome. The right end of the substitution always results from recombination between the nin-QSR-cos region of the prophage and the homologous incomplete lambdoid prophage Qsr' at 12.5 minutes in the E. coli chromosome. The left end of the substitution is usually a crossover that recombines the IS2 element in the prophage with an E. coli IS2 at 8.5 minutes, near the lac gene, or with a second IS2 located counterclockwise from leu at 2 minutes, generating duplications of at least 200,000 bases. Five cryptic lysogens derived from cells lysogenic for a reference strain of lambda (which lacks the IS2 present in lambda crg) have been characterized. They contain substitutions whose right termini are generated by a crossover with the Qsr' prophage. The left termini of these substitutions are formed either by a crossover between the lambda exo gene and a short exo-homologous segment of Qsr' (2/5), or by a crossover between sequences to the left of attL and an unmapped distant region of the host chromosome (3/5). The large duplications carried by these cryptic lysogens are stable, unlike tandem duplications, and so may significantly influence the cell's evolutionary potential.
Bacteriophage lambda↗