Transformation In Haemophilus Influenzae Research Articles

Natural competence in Histophilus somni strain 2336

Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases of bovines. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. However, to identify the genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult, likely due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and shows a strong bias for fragments containing specific uptake signal sequences (USS) within the genome. We hypothesized that natural transformation may also be possible in H. somni strain 2336 because its genome is over-represented with H. influenzae USS (5′-AAGTGCGGT-3′) and contains most of the genes necessary for competence. H. somni strain 2336 was successfully transformed and mutated with genomic linear DNA from an H. somni mutant (738Δlob2a), which contains a kanamycin-resistance (KanR) gene and the USS within lob2A. Although most of the competence genes found in H. influenzae were present in H. somni, comD and the 5′ portion of comE were absent, which may account for the low transformation efficiency. The transformation efficiency of strain 2336 was greatest during mid-log growth phase and when cyclic adenosine monophosphate was added to the transformation medium. However, mutants were not isolated when strain 2336 was transformed with genomic DNA containing the same KanR gene from H. somni luxS or uspE mutants, which lack the USS in these specific genes. Shuttle vector pNS3K was also naturally transformed into strain 2336, though at a lower efficiency. However, natural transformation with either H. somni linear DNA (2336Δlob2A) or pNS3K was unsuccessful with H. somni commensal strain 129Pt and several other disease isolates.

Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans.

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.

The Haemophilus influenzae dprABC genes constitute a competence-inducible operon that requires the product of the tfoX (sxy) gene for transcriptional activation.

We previously showed that dprA is required for efficient processing of linear DNA during cellular transformation in Haemophilus influenzae. In this study the transcriptional regulation of dprA and two downstream genes, dprB and dprC, is examined. We demonstrate by Northern blot analysis that the dprABC genes are transcriptionally coregulated and competence inducible. We used primer extension analysis to map the transcriptional start site of dprA and of rec-2, another transformation gene involved in DNA processing. Based upon these results, we were able to identify a 26-bp dyad symmetry element immediately upstream of the -35 regions of the predicted promoters of dprA, rec-2, and two other transformation genes, comA and pilA. Finally, using transcriptional fusions of dprA to the Escherichia coli lacZ gene, we show that expression of dprA::lacZ requires tfoX and that the presence of multiple copies of tfoX abolishes the temporal regulation of dprA, resulting in its constitutive expression.

DNA sequence and characterization of Haemophilus influenzae dprA+, a gene required for chromosomal but not plasmid DNA transformation

Natural genetic transformation in Haemophilus influenzae involves DNA binding, uptake, translocation, and recombination. In this study, we cloned and sequenced a 3.8-kbp H. influenzae DNA segment capable of complementing in trans the transformation defect of an H. influenzae strain carrying the tfo-37 mutation. We used subcloning, deletion analysis, and in vivo protein labeling experiments to more precisely define the gene required for efficient DNA transformation on the cloned DNA. A novel gene, which we called dprA+, was shown to encode a 41.6-kDa polypeptide that was required for efficient chromosomal but not plasmid DNA transformation. Analysis of the deduced amino acid sequence of DprA suggested that it may be an inner membrane protein, which is consistent with its apparent role in DNA processing during transformation. Four other open reading frames (ORFs) on the cloned DNA segment were identified. Two ORFs were homologous to the phosphofructokinase A (pfkA) and alpha-isopropyl malate synthase (leuA) genes of Escherichia coli and Salmonella typhimurium, respectively. Homologs for the two other ORFs could not be identified.

The Haemophilus influenzae adenylate cyclase gene: cloning, sequence, and essential role in competence

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.

DNA repair and the evolution of transformation in Haemophilus influenzae.

Under certain environmental conditions, naturally transforming bacteria are induced to pick up DNA released into the environment by other cells of the same or closely related species and, by homologous recombination, integrate that DNA into their chromosome. The selective pressures responsible for the evolution and maintenance of this form of genetic outcrossing, or sex, in bacteria are not known. A prominent hypothesis is that transformation, and sex in general, evolved as a means of obtaining DNA templates to repair damaged regions of the chromosome. Previous results obtained with Bacillus subtilis were consistent with the repair hypothesis. In an effort to explore the generality of those results, I have tested the repair hypothesis with Haemophilus influenzae, a naturally transforming, gram-negative species of bacteria. The results of UV damage-survivorship experiments with H. influenzae were also consistent with that hypothesis. However, additional experiments demonstrate that the higher survival of transformed cultures cannot be accounted for by use of the transforming DNA as templates for repair. I consider alternative hypotheses for the means by which transformation can increase cell survival following UV exposure and discuss the implications of these results with respect to the DNA repair hypothesis and the evolution of transformation.

Transformation ofHaemophilus influenzae following electroporation with plasmid and chromosomal DNA

Naturally transformable species, such asHaemophilus influenzae, have evolved systems for the efficient uptake and integration of DNA from the surrounding environment. We compared this competence-dependent DNA uptake system to electroporation, which has been widely used in the past few years to introduce DNA into cells, for transformingHaemophilus influenzae. Electroporation improved transformation efficiency when noncompetent cells were used and when DNA lackingHaemophilus-specific uptake sequences was used for transformation of competent cells. An increase in plasmid-to-chromosome recombination was seen when plasmid DNA containing chromosomal inserts was introduced into competent cells by electroporation, as observed previously for natural transformation.

Sequence and transcriptional regulation of com101A, a locus required for genetic transformation in Haemophilus influenzae

A 2.8-kb EcoRI-BglII fragment cloned from the wild-type Haemophilus influenzae Rd chromosome is shown to increase the transformability of the Com-101 mutant through trans complementation. Deletion and sequence analyses indicate that the active region of the clone carries a 687-bp open reading frame. A 0.3-kb insertion in the corresponding EcoRI-BglII fragment of the Com-101 chromosome is shown to be a partial (331-bp) duplication of this open reading frame. The wild-type sequence produces a peptide of a size that is consistent with the sequence data when this sequence is expressed in Escherichia coli with a T7 promoter-based transcription vector. RNA hybridization analysis using a DNA probe derived from the open reading frame suggests that the sequence is transiently expressed during competence development. On the basis of these observations, it is proposed that the open reading frame corresponds to the com101A gene.

Transformation of Haemophilus influenzae by plasmid RSF0885 containing a cloned segment of chromosomal deoxyribonucleic acid.

A plasmid containing a single cloned insertion of Haemophilus influenzae chromosomal deoxyribonucleic acid that carried a novobiocin resistance marker was 2.6 times larger than the parent plasmid, RSF0885, which conferred ampicillin resistance. The most frequent type of transformation by this plasmid (designated pNov1) was the transfer of novobiocin resistance to the chromosome, with the loss of the plasmid from the recipient. In accord with this observation, after radioactively labeled pNov1 entered a competent cell, it lost acid-insoluble counts, as well as biological activity. The level of ampicillin transformation, which involved establishment of the plasmid, was almost two orders of magnitude lower than the level of novobiocin transformation. Both types of transformation were depressed profoundly in rec-1 and rec-2 mutants. Ampicillin transformants of wild-type cells always contained plasmids that were the same size as pNov1, although most of these transformants were not novobiocin resistant. Plasmid pNov1 in wild-type cells but not in rec-1 or rec-2 cells often recombined with the chromosome, causing a homologous region of the chromosome to be substituted for part of the plasmid, as shown by restriction and genetic analyses. Our data suggested that plasmid-chromosome recombination took place only around the time when the plasmid entered a cell, rather than after it became established.

Transformation of Haemophilus influenzae by plasmid RSF0885

Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximum, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.

Genetic and molecular events in transformation ofHaemophilus influenzae with plasmid RSF 0885 carrying cloned segments of chromosomal DNA

InHaemophilus influenzae genetic transformation for a plasmid marker is significantly increased when recombinant plasmid RSF 0885 DNA carrying chromosomal DNA segments is used instead of the plasmid DNA alone. Chromosomal DNA by itself, added even a few minutes after the addition of plasmid DNA to competent cells, stopped further uptake of the plasmid DNA. These observations are consistent with the idea that plasmid RSF 0885 contains a ‘degenerate’ version of the required eleven base-pair ‘uptake sequence’ inHaemophilus. The transformation activity of the recombinant plasmid DNA is recoverable after its entry into cells, although the specific biological activity of the re-isolated plasmid DNA is less than that of the parental recombinant plasmid DNA. Therec 1 gene function of the host is necessary for obtaining higher transformation frequencies with recombinant DNA from five different clones. The reduced transformation frequencies seen inrec 1 - strain is not all due to a permanent damage to the donor DNA since the recovered recombinant plasmid DNA from such cells can increase the transformation efficiency onrec 1 + strain.

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.

Mechanism of additive genetic transformation in Haemophilus influenzae

Transforming deoxyribonucleic acid (DNA) preparations from Haemophilus influenzae Rd strains carrying a chromosomally integrated, conjugative, antibiotic resistance transfer (R) plasmid were exposed to ultraviolet radiation and then assayed for antibiotic resistance transfer on sensitive wild-type Rd competent suspensions and on similar suspensions of a uvr-1 mutant unable to excise pyrimidine dimers. No host cell reactivation of resistance transfer (DNA repair) was observed. Parallel experiments with ethanol-precipitated, heated, free R plasmid DNA preparations gave much higher survival when assayed on the wild-type strain compared to the survival on the uvr-1 strain. These observations indicate that additive genetic transformation (in this case, the addition of the integrated R plasmid to the recipient genome) involves single-strand insertion.

Relationship between prophage induction and transformation in Haemophilus influenzae.

The interaction between transformation and prophages of HP1c1, S2, and a defective phage of Haemophilus influenzae has been investigated by measurement of (i) the effect of prophage on transformation frequency and (ii) the effect of transformation on phage induction. The presence of any of the prophages does not appreciably alter transformation frequencies in various Rec(+) and Rec(-) strains. However, exposure of competent lysogens to transforming deoxyribonucleic acid (DNA) may induce phage but only in Rec(+) strains, which are able to integrate transforming DNA into their genome. Transformation of Rec(+) lysogens with DNA irradiated with ultraviolet (UV) light causes the production of even more phage than results from unirradiated DNA, but this indirect UV induction is not as effective as direct induction by UV irradiation of lysogens. Both types of UV induction are influenced by the repair capacity of the host. Wild-type cells contain a prophage and can be induced by transformation to produce a defective phage, which kills a small fraction of the cells. Defective phage in wild-type cells are also induced by H. parainfluenzae DNA, and a much larger fraction of the cells is killed. Strain BC200, which is highly transformable but is not inducible for defective phage, is not killed by H. parainfluenzae DNA, suggesting that wild-type cells are killed by killed by this DNA because of phage induction. A minicell-producing mutant, LB11, has been isolated. Some phage induction occurs in this strain when the cells are made competent, unlike the wild type. A large majority of LB11 cells surviving the competence regime are killed by exposure to transforming DNA.

Fate of recipient deoxyribonucleic acid during transformation in Haemophilus influenzae.

During genetic transformation of Haemophilus influenzae, segments of the host deoxyribonucleic acid (DNA) corresponding to the integrating donor DNA were degraded and liberated into the medium. This degradation was detected by the release of the radioactive label from host DNA during a time period matching the time of development of maximal linkage between donor and host markers. The host label released above that released from nontransformed, control cultures was equivalent to about 2% of the host genome or 16 x 10(6) daltons of DNA. The released, labeled material was acid-soluble and dialyzable. The label release from control cultures was unaffected at 30 C; at this temperature, the recombination-specific release from transformed cells was suppressed. High molecular weight fragments of host DNA corresponding in size to the donor fragments could not be found free within the cell, weakly bound to other host DNA, or bound to non-integrated donor DNA by a reciprocal cross mechanism.