Donor DNA processing is blocked by a mutation in the com101A locus of Haemophilus influenzae.

Evidence is presented indicating that a donor DNA processing step of the Haemophilus influenzae transformation pathway is blocked in the Com-101 mutant. Additional data are presented suggesting that, as in the Rec-2 strain, the donor DNA remains associated with the H. influenzae envelope.

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.

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.

Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.

The identification of the bacteriophage HP1c1 and S2 integration sites in Haemophilus influenzae Rd by field-inversion gel electrophoresis of large DNA fragments.

The resolution of high molecular weight DNA fragments by field-inversion gel electrophoresis (FIGE) demonstrate the presence of two phage (S2 and HP1c1) integration sites (attB) in the Haemophilus influenzae Rd chromosome. In a population of wild-type cells either prophage site appears to be occupied in a single cell by one to at least three, tandemly repeated, amplified phage DNA molecules. The attL of the second bacterial attachment site present in the host SmaI fragment 7 and the leftmost part of phage S2 type B DNA of its genome organization (Piekarowicz et. al., 1986) have been sequenced. A comparison of the two bacterial att sites demonstrated that their homology is limited to the core region. A comparison of the DNA sequences of phage S2 type B and HP1c1 type C revealed a 530-bp insertion in the HP1c1 type C (not present in S2 type B) in addition to DNA variants due mostly to single-base mismatches. We postulate that phage S2 and HP1c1 genome variants (A, B, and C) evolved from a single phage origin and might stem from passage history arisen through accumulation of mutations.

Sequence and uptake specificity of cloned sonicated fragments of Haemophilus influenzae DNA.

Our data support the conclusion that all tight binding of DNA by competent cells of Haemophilus influenzae is due to 9 base pairs (bp) of the 11-bp motif 5'-AAGTGCGGTCA or its inverse complement, TGACCGCACTT. Sonicated chromosomal DNA of H. influenzae was absorbed and extracted from competent cells to prepare a subpopulation of uptake fragments enriched for single uptake (binding) sequences. This DNA was inserted into the plasmid pUC18 and cloned into Escherichia coli. Whole sonicated chromosomal DNA was also cloned in pUC18 as a population of control fragments. Seventy-one plasmids were labeled with 3H and tested for DNA binding, and the inserts of 28 of them were sequenced. The control plasmid pUC18 was absorbed to competent H. influenzae cells at low levels of 5 to 10% of DNA added. The plasmids containing uptake inserts were mostly absorbed at levels of 70 to 80%, but a significant number contained inserts with an intermediate level of binding, 20 to 30%. The inserts of 15 plasmids were excised and tested for DNA uptake to demonstrate good agreement between uptake of these plasmids and their insert fragments. Sequencing of inserts revealed that the presence of 9 bp of the 11-bp motif was associated with tight binding. Some inserts with intermediate levels of binding had no significant similarities to the 9-bp sequence. The 9-bp sequence appeared to account for most of the binding to competent cells, but appreciable binding occurred with fragments without 9-bp homology. About one-third of the 9-bp uptake sequences were found as inverted repeats that could form strong stem-loop structures. It has been suggested by Goodman and Scocca (Proc. Nal. Acad. Sci. USA 85:6982-6986, 1988) that in Neisseria gonorrhoeae, uptake sequences occur as palindromes and act as transcription terminators. Although consistent, the data are insufficient to make this conclusion for uptake sequence palindromes in H. influenzae.

The genome of Haemophilus influenzae Rd has a unique NotI site.

Previous analysis of physical maps of Haemophilus influenzae, which is circular and 1.9 Mb in length [Lee and Smith, J. Bacteriol. 170 (1988) 4402-4405; Kauc et al., J. Bacteriol. 171 (1989) 2474-2479], did not detect any NotI (GCGGCCGC) restriction sites. A transposon, Tn916, was constructed to contain a NotI linker cloned into its NciI site and introduced into the H. influenzae chromosome. NotI digestion of chromosomes containing a Tn916-associated NotI site followed by separation of fragments by field-inversion gel electrophoresis revealed the presence of two fragments obtained by two NotI cuts, one in Tn916 and the other, a unique, 'natural' NotI site in the original chromosomal DNA. The examination of other Haemophilus strains demonstrated the presence of one or more NotI sites in all of those tested.

Bacterial expression of immunoglobulin VH proteins.

A bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the VH protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-2-sulfonyl (Dns) group. This system has been applied to the expression of the VH genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F. Studier) utilize a T7 expression cassette whose activity is initiated by infection with a lambda phage derivative carrying the T7 RNA polymerase gene. The VH proteins were extracted from the bacterial pellet in 8 M urea and purified by chromatography in 8 M urea. Recombinants with the homologous light (L) chains were prepared to yield VHL molecules. These were used to measure intrinsic affinity for Dns-lysine by resonance energy transfer. The association constants were 7 x 10(6) M-1 and 7 x 10(9) M-1 for the low- and high-affinity systems, respectively. These values are not significantly different from those observed with monoclonal antibodies secreted from the corresponding cell lines. This system lends itself to the quantitative evaluation of the binding properties of the VH protein itself as well as the modulation of affinity by site-directed mutagenesis.

Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae.

Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.

The size and a physical map of the chromosome of Haemophilus parainfluenzae.

The physical map of the Haemophilus parainfluenzae chromosome is circular and approx. 2340 kb in circumference. The size of the map was determined by digesting agarose-immobilized chromosomes with the restriction enzymes, NotI (GCGGCCGC), RsrII (CGGATCCG) and ApaI (GGGCCC), and using field-inversion gel electrophoresis to resolve the resulting fragments. The enzymes digest the H. parainfluenzae genome into 7, 10, and 18 fragments, respectively. The map order of the fragments was obtained by using Southern-blot hybridization to establish overlapping regions.

Size and physical map of the chromosome of Haemophilus influenzae.

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.

Amplification of DNA at a prophage attachment site in Haemophilus influenzae.

The Escherichia coli plasmids pBR322 and pBR327 can be taken up by Haemophilus influenzae but do not replicate in this organism; however, integration of pBR into the H. influenzae chromosome was achieved by ligation to a fragment of the Haemophilus phage S2 that carried a phage attachment site (attP). Once these sequences were integrated, they could serve as sites of recombination and amplification for homologous (pBR or phage) DNA. Amplification appeared to occur in one of two prophage sites (attB) present in the H. influenzae chromosome. The extent of amplification was different in different cells and reflected the ability of these sequences to undergo rearrangement leading to the formation of a DNA ladder. The ladder was obtained by treatment of DNA with restriction enzymes that cut outside of the inserted DNA, i.e., did not cut in the repeat sequence, and represented different numbers of repeat elements. Reversed-field gel electrophoresis was instrumental in resolving amplified structures. Inasmuch as single-cell isolates gave rise to the same ladder structure, it was assumed that amplification was under regulatory control and that it reproduced the same equilibrium of repeat structures. Transformation of E. coli with the amplified H. influenzae DNA resulted in precise excision and replication of the original monomeric plasmids. This excision was independent of the recA and recBC genes.

Germ-line affinity and germ-line variable-region genes in the B cell response.

The predominance of germ-line genes in IgM expression was evaluated from the nucleotide sequences of mRNA, derived from 10 hybridoma cell lines, coding for the VH and VL regions of anti-5-dimethylaminonaphthalene-1-sulfonyl (anti-Dns) IgM antibody. At least six germ-line VH gene segments distributed among four families are used in this response. Seven of the 10 independently rear-ranged VH genes were identified as germ line, with the other three possibly germ line. In all of them the D and JH portions retained the germ-line sequences of the D and JH segments from which they were derived. Maximum diversity was found in the D segments and the use of noncoded nucleotides at the VH-D and D-JH junctions. Of the eight cell lines expressing the lambda light chains, all were germ line and involved the three subtypes. Maximum affinity for the homologous ligand was found among the seven cell lines identified as expressing germ-line gene segments. Thus any somatic mutation among the remaining 3 cell lines did not provide enhanced affinity and the observed affinity of each cell line can be described as germ-line affinity. It is further suggested that the anti-Dns selectivity of the IgM antibodies is associated primarily with the CDR3 regions.

Uptake of heterologous DNA by Haemophilus influenzae.

With the use of highly competent Haemophilus influenzae cells, it was possible to demonstrate the uptake of heterologous DNAs. However, these DNAs, as expected, were only 1% or less as effective when competing for uptake with Haemophilus DNA. Escherichia coli DNA was removed from solution by competent cells to the extent expected if all the E. coli DNA particles contained at least one uptake recognition signal. The data were consistent with a model in which there was one uptake signal per 20 X 10(6) to 30 X 10(6) daltons of E. coli DNA. Since H. influenzae DNA has many more recognition signals, approximately one per 2 X 10(6) daltons (Danner et al., Gene 77:311-318, 1980; K. Vogt and S. H. Goodgal, submitted for publication), it has been suggested that the slower rate of E. coli DNA binding and the so-called specificity of Haemophilus DNA binding are due to the number of recognition signals per molecule of DNA as well as the nature of the DNA receptor (Vogt and Goodgal, submitted for publication). The specificity of native H. influenzae DNA binding does not apply to the uptake of denatured DNA in the transforming system (low pH) for denatured DNA.

Possible mechanism for donor DNA binding and transport in Haemophilus.

Morphological differences were observed in competent and noncompetent Haemophilus parainfluenzae and Haemophilus influenzae when thin sections of these cells were examined by electron microscopy. The membranous extensions present on the surface of competent H. parainfluenzae cells disappeared on treatment with transforming DNA, while vacuole-like structures appeared in the periplasm. Noncompetent cells had 1/5th as many extensions on their surface as competent cells, and no vacuoles were observed after treatment with homologous DNA. Competent cells treated with radiolabeled DNA were disrupted and the clarified lysate was centrifuged on CsCl density gradients. Material having a density of 1.34 g/ml was found to contain the majority of the DNase-resistant radioactive DNA recovered from the bacteria and was shown by electron microscopy to be composed of membrane vesicles. The polypeptide composition of this dense membrane fraction was similar to that of H. parainfluenzae outer membrane.

DNA-binding vesicles released from the surface of a competence-deficient mutant of Haemophilus influenzae.

Haemophilus influenzae com-51, a mutant deficient in DNA uptake, produces an extracellular DNA-binding activity. The activity was specific for Haemophilus DNA and was isolated from cell-free competence medium after incubation for 100 to 130 min. Initial steps in the purification procedure resulted in the loss of detectable binding activity, but activity was restored by the addition of a nonionic detergent. The active fractions contained vesicles derived from the outer membrane of the cells. The vesicles were produced only under conditions that normally lead to competence development. The lack of competence of com-51 cells was not due to loss of protein synthesis in M-IV competence medium or to competition of extracellular protein for exogenous DNA. Results suggest that the inability of cells to bind DNA was due in part to the loss of DNA receptors that are released into the medium in membrane fragments.

Haemophilus influenzae polypeptides involved in deoxyribonucleic acid uptake detected by cellular surface protein iodination.

Polypeptides that appear to be involved in competence development and deoxyribonucleic acid (DNA) uptake by Haemophilus influenzae were detected with a surface-specific iodinating reagent 1,3,4,6,-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. As shown on electrophoretograms, a number of polypeptides became sensitive to 125I protein labeling with the ability of these cells to bind DNA. Of these polypeptides, nine were reduced in their ability to be labeled (ral polypeptides) extensively after the incubation of competent cells with homologous, but not with heterologous, DNA. Iodination of many of these ral polypeptides was reduced in competence-deficient mutants compared with wild-type competent cells. One 125I-labeled polypeptide corresponding to a molecular weight of 29,000 was present at reduced levels in mutants reduced in the ability to bind DNA. Our results suggest that the 29,000-molecular-weight polypeptide corresponds with the ability of H. influenzae to take up DNA and that a complex of proteins is involved in DNA uptake and transformation.

A technique for the rapid selection of drug-sensitive and auxotrophic mutants of Phycomyces blakesleeanus.

An auxotroph enrichment procedure has been developed for Phycomyces blakesleeanus which has proven useful in obtaining both auxotrophs and drug-sensitive mutants. The technique is based on the differential heat sensitivity between ungerminated auxotrophic spores and germinated prototrophic spores. Germinated spores and mycelia die when left at temperatures higher than about 35 degrees C for 16 to 24 h but ungerminated spores survive this treatment and subsequently germinate if transferred to complete medium. With the dwarf colonial strain, replica plating permits the rapid characterization of these newly selected auxotrophs.