Defined Nongrowth Media for Stage II Development of Competence in Haemophilus influenzae

The composition of a defined nongrowth medium used in stage II development of competence of Haemophilus influenzae affects the course of this development. The development of competence in two nongrowth media, M-IV and M-V, is rapid, logarithmic, and independent of the cell concentration. This last property indicates that there is probably no transfer of a competence factor from competent to noncompetent cells, in contrast to results reported for other organisms. Levels of competence reached in these completely defined media are such that 1 to 5% of the cells are transformed in the presence of an excess of marked deoxyribonucleic acid. The method of evaluating competence, which depends on the frequency of multiple independent transformations, has been reexamined. This and other methods are compared on samples taken from a culture during development of competence.     

Synthesis of envelope polypeptides by Haemophilus influenzae during development of competence for genetic transformation.

Six polypeptides with apparent molecular weights of 95,000, 90,000, 80,000, 67,000, 64,000, and 43,000 were found to be characteristic of the cell envelopes of competent Haemophilus influenzae, and were synthesized entirely during the period of competence development. Two polypeptides with apparent molecular weights of 58,500 and 40,500 were synthesized during growth as well as during competence development, but were only associated with the envelope fraction of cells that had developed competence. The kinetics of synthesis of the competence-related envelope polypeptides showed a lag period of approximately 20 min. The observation of this lag period raises the question as to whether some of these competence-related polypeptides might be involved in the process of deoxyribonucleic acid uptake, since the development of this property also exhibits a sigmoid time course during competence development.     

In vitro Tn7 mutagenesis of Haemophilus influenzae Rd and characterization of the role of atpA in transformation.

Haemophilus influenzae Rd is a gram-negative bacterium capable of natural DNA transformation. The competent state occurs naturally in late exponential growth or can be induced by a nutritional downshift or by transient anaerobiosis. The genes cya, crp, topA, and sxy (tfoX) are known to function in the regulation of competence development. The phosphoenolpyruvate:carbohydrate phosphotransferase system functions to maintain levels of cyclic AMP necessary for competence development but is not directly involved in regulation. The exact signal(s) for competence and the genes that mediate the signal(s) are still unknown. In an effort to find additional regulatory genes, H. influenzae Rd was mutated by using an in vitro Tn7 system and screened for mutants with a reduced ability to induce the competence-regulatory gene, comA. Insertions in atpA, a gene coding for the alpha subunit of the F1 cytoplasmic domain of the ATP synthase, reduce transformation frequencies about 20-fold and cause a significant reduction in expression of competence-regulatory genes, while the expression of constitutive competence genes is only minimally affected. In addition, we found that an insertion in atpB, which encodes the a subunit of the F0 membrane-spanning domain, has a similar effect on transformation frequencies.     

Physical size of the donor locus and transmission of Haemophilus influenzae ampicillin resistance genes by deoxyribonucleic acid-mediated transformation.

The properties of donor deoxyribonucleic acid (DNA) from three clinical isolates and its ability to mediate the transformation of competent Rd strains to ampicillin resistance were examined. A quantitative technique for determining the resistance of individual Haemophilus influenzae cells to ampicillin was developed. When this technique was used, sensitive cells failed to tolerate levels of ampicillin greater than 0.1 to 0.2 mug/ml, whereas three resistant type b beta-lactamase-producing strains could form from the colonies in 1- to 3-mug/ml levels of the antibiotic. DNA extracted from the resistant strains elicited transformation of the auxotrophic genes in a multiply auxotrophic Rd strain. For two of the donors, transformation to ampicillin resistance occurred after the uptake of a single DNA molecule approximately 104-fold less frequently than transformation of auxotrophic loci and was not observed to occur at all with the third. The frequency of transformation to ampicillin resistance was two- to fivefold higher in strain BC200 (Okinaka and Barnhart, 1974), which was cured of a defective prophage. All three clinical ampicillin-resistant strains were poor recipients, but the presence of the ampicillin resistant genes in strain BC200 did not reduce its competence. Sucrose gradients of DNA from ampicillin-resistant transformants of BC200 and from the original ampicillin-resistant strains showed that: (i) all the DNA preparations had high molecular weights; (ii) donor activity for ampicillin resistance sedimented heterogeneously and in parallel with genome DNA up to the highest molecular weights observed (100 x 106 to 200 x 106); and (iii) genetic transformation of ampicillin resistance from strain BC200-amp90383 required the physical integrity of a linearly integrated segment of DNA having a molecular weight of about 25 x 106 to 30 x 106.     

Identification of Competence-Repressing Factors During Log-Phase Growth of Haemophilus influenzae

Metabolic factors that suppress development of competence in Haemophilus influenzae during growth in the synthetic medium MI(c) have been identified. These include inosine, nicotinamide adenine dinucleotide, glycerol, and uracil. It is also possible to initiate competence in the presence of these substances if the oxygen tension in the culture is temporarily reduced.     

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.     

Constitution of the cell envelope of Haemophilus influenzae in relation to competence for genetic transformation.

Cell envelopes of Haemophilus influenzae have been prepared by breakage in a French pressure cell followed by differential centrifugation. The envelope fraction may be resolved into an inner-membrane (light) and an outer-membrane (heavy) fraction on density gradients. Envelopes from competent cells possess elevated levels of lipopolysaccharide with a composition different from that of log-phase cell envelopes. Three apparently new polypeptides have been observed in envelopes from competent cells by gel electrophoresis in sodium dodecyl sulfate; additional quantitative alterations in the profiles of membrane polypeptides also company the development of the capacity to transport deoxyribonucleic acid. Most of the polypeptide changes are confined to the outer membrane; one new polypeptide is associated with the inner cytoplasmic membrane of competent cells. Protein synthesis during competence developement is rquired for the change in lipopolysaccharides and in the envelope polypeptides to occur.     

Role of the electrochemical proton gradient in genetic transformation of Haemophilus influenzae.

The uptake of homologous DNA by Haemophilus influenzae was studied as a function of the proton motive force in completely competent cultures in the pH range of 6 to 8. The composition and magnitude of the proton motive force were varied by using the ionophores valinomycin and nigericin (in the presence of various potassium ion concentrations) and by using protonophores. No interaction of the ionophores with the DNA transformation system itself was observed. Either component of the proton motive force, the electrical potential or the pH gradient, can drive the uptake of DNA, and the extent of the uptake of DNA is ultimately determined by the total proton motive force. The transformation frequency increases with the proton motive force, which reaches a maximum value at around -130 mV. These results are consistent with an electrogenic proton-DNA symport mechanism, but direct evidence for such a system is not available. The proton motive force was followed during competence development of H. influenzae at pH 8. In the initial phase (up to 50 min), the proton motive force remained constant at about -90 mV, whereas the transformation frequency rose steeply. In the second phase, the proton motive force increased. The transformation frequency in this phase increased with the proton motive force, as in completely competent cultures. These observations and the observed inhibition by NAD of both the proton motive force and the transformation frequency indicate that structural components of the competent state are formed in the initial phase of competence development, whereas the second phase is characterized by an increase of the proton motive force.     

Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.     

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.     

Development of mouse embryos in media containing lectins

Lectins known to stimulate mitosis in cultured cells were evaluated for effects on development of mouse embryos in vitro. Two-cell mouse embryos were cultured in one of the following treatments: Whitten's medium as the control medium; Whitten's medium with 1, 10 or 100 mug/ml concanavalin A; Whitten's medium with 1, 10 or 100 mug/ml leucoagglutinin; Whitten's medium with 1, 10 or 100 mug/ml phytohemagglutinin; Whitten's medium with 1, 10 or 100 mug/ml pokeweed-mitogen; and Whitten's medium with 1, 10 or 100 mug/ml wheat germ agglutinin. Development to the morula stage was blocked in media with 100 mug/ml concanavalin A and 10 and 100 mug/ml wheat germ agglutinin, whereas blastocyst formation was blocked in all pokeweed-mitogen supplemented media. Embryos incubated in 10 and 100 mug/ml wheat germ agglutinin underwent premature cavitation or vacuolation at 24 to 48 h of culture. More embryos formed blastocysts in media with 1 and 100 mug/ml phytohemagglutinin and 10 mug/ml leucoagglutinin than in Whitten's medium (P<0.05). The percentage of embryos hatching was greatest in 1 mug/ml phytohemagglutinin (P<0.05), but it was the same in Whitten's medium, 1 mug/ml concanavalin A and 1 mug/ml leucoagglutinin (P>0.05). Cell division was not stimulated by the lectins; however, it was significantly suppressed in media with 10 and 100 mug/ml concanavalin A, 100 mug/ml phytohemagglutinin, 1, 10 and 100 mug/ml pokeweed-mitogen, and 10 and 100 mug/ml wheat germ agglutinin. Solubility of the zona pellucida in sodium isothicyanate (NaSCN) was reduced in 100 mug/ml phytohemagglutinin, 100 mug/ml leucoagglutinin and 1 mug/ml wheat germ agglutinin media (P<0.05) when compared to Whitten's medium and may have accounted for the reduced hatching observed in these treatments. Development of isolated blastomeres into blastocysts was reduced in media with 1 mug/ml wheat germ agglutinin, 1 mug/ml concanavalin A, and 10 and 100 mug/ml leucoagglutinin (P<0.05) but was similar in media with 1 mug/ml leucoagglutinin and 1, 10 and 100 mug/ml phytohemagglutinin when compared to Whitten's medium (P>0.05). The extent of embryo development in media with lectins depended upon the degree of cytotoxicity and potential biochemical modifications induced in the zona pellucida. Greatest embryo development took place in medium with 1 mug/ml phytohemagglutinin; however, the mechanism was not that of stimulation of cell division or a change in zona pellucida solubility.     

Bacterial pericarditis and tamponade due to nonencapsulated Haemophilus influenzae complicating a case of adult community-acquired pneumonia

We report a case of bacterial pericarditis in an immunologically competent adult female caused by nonencapsulated Haemophilus influenzae (H influenzae) that was complicated by the acute development of life-threatening pericardial tamponade. H influenzae is a gram-negative coccobacillus, a pathogen most frequently associated with childhood exanthema (otitis media, meningitis) and, less frequently, adult pneumonia. Encapsulated, type b, or typable H influenzae is the strain implicated in childhood infections. On the other hand, nonencapsulated or nontypable H influenzae is the specific strain most often associated with exacerbation of chronic obstructive airway disease. Bacterial pericarditis caused by either subtype of H influenzae is exceedingly rare. We have located only 15 previously reported cases of H influenzae pericarditis occurring in adults in the world medical literature, the majority of which date back to the pre-antibiotic era. In 12 of these 15 cases (the only cases in which typing could be accomplished), the encapsulated strain of H influenzae was cultured from the pericardial fluid. Thus, to the best of our knowledge, we are reporting here the first case of bacterial pericarditis caused by nonencapsulated H influenzae in an immunologically competent adult.     

Periodate inhibition of transformation and competence development in Haemophilus influenzae

Ranhand, Jon M. (University of Cincinnati, Cincinnati, Ohio), and Herman C. Lichstein. Periodate inhibition of transformation and competence development in Haemophilus influenzae. J. Bacteriol. 92:956-959. 1966.-Periodate treatment of competent cells reduced the frequency of transformation to streptomycin resistance about 90% while reducing cell viability about 30% or less. Moreover, when periodate was added to cells early in the competence-development phase, these, too, were unable to develop maximal competence. Periodate inhibition was dependent on time and concentration as well as on the composition of the suspending menstruum. Periodate had no effect on transforming deoxyribonucleic acid (DNA), nor did it prevent transformation when added to competent cells which had already reacted with DNA. Furthermore, the progeny from cells inactivated 90% could be made fully competent, showing that the inhibition was not genetic. It was concluded that the periodate-sensitive substrate may involve the DNA binding site(s).     

Synchronous division and rates of macromolecular synthesis in Haemophilus influenzae competent for genetic transformation.

When competent Haemophilus influenzae were returned to complete growth medium, they resumed growth at once and underwent a synchronous division after 20 to 25 min. The rate of DNA synthesis increased to the definitive rate very quickly after resumption of growth. In contrast, the rate of RNA synthesis remained low until the time of division, at which time it accelerated dramatically. Changes in the rate of protein synthesis were similar in form to those of RNA synthesis. The kinetics of loss of competence upon resumption of growth had a pattern similar to the rate of RNA synthesis, and both processes accelerated markedly at the time of cell division, suggesting an inverse relationship between RNA synthesis and the ability to take up DNA.     

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence.

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.     

Electron microscopy of single-stranded structures in the DNA of competent Haemophilus influenzae cells.

Chromosomal DNAs from exponential-phase and competent cells of Haemophilus influenzae were examined by electron microscopy to determine whether the chromosome undergoes structural changes during competence development. Single-stranded gaps and single-stranded tails formed in chromosomal DNA during competence development. The generation of gaps was dependent on the rec-2 function. Since the rec-2 mutant is defective in the translocation of donor DNA, it was inferred that the gaps were involved in the translocation step of transformation. The generation of single-stranded tails was independent of the rec-1 and rec-2 genes. Therefore, these structures were assumed to play no direct role in the interaction of donor and recipient DNAs during transformation. Gaps were preferentially associated with a readily denaturable, possibly A + T-rich fraction of the genome. This finding raised the possibility that hot spots for transformation might be associated with A + T-rich DNA.     

Prevention of cell agglutination and competence in a genetically transformablc strain ofPneumococcus byd-glucosamine andd-galactosamine

In the presence of amino sugars D-glucosamine and D-galactosamine no spontaneous competence could be observed in the highly transformable R6bd strain of Pneumococcus or it was decreased by several orders of magnitude. The highest inhibition of competence was detected when the amino sugar at a concentration 5 mg/ml of the medium was added not only to the transformation but also to the pretransformation medium. After a 150 min growth in the transformation medium in the presence of the amino sugar a 3--4-fold greater number of cells (as a viable count) could be detected as compared with the control without the amino sugar. It was found microscopically that the amino sugar prevents natural agglutination, which normally occurs in the competent culture. The role of specific amino sugar determinants for binding of the competence factor on the cell surface and the resulting inhibitory effect of these sugars on the development of competence are discussed.     

Deoxyribonucleic acid-binding properties and membrane protein composition of a competence-deficient mutant of Haemophilus influenzae.

A mutant of Haemophilus influenzae was isolated which was completely unable to take up double-stranded homologous deoxyribonucleic acid (DNA) at normal physiological conditions but which took up DNA equally as well as the wild type at low pH (pH 4.4). The properties of the mutant provide evidence for the existence of two different mechanisms for DNA entry in the H. influenzae transformation system. With the aid of the mutant the optimal conditions for entry of DNA by these two mechanisms were determined, and the dependence of entry and the specific transforming activity of the entered DNA on competence was examined. The mechanism of entry of DNA at neutral pH, which is not functioning in the mutant, effected entry of homologous DNA only, whereas the mechanism involved in entry of DNA at low pH also effected entry of heterologous DNA. This suggests that the mutant is lacking a protein which recognizes the specific base sequence(s) required for entry. Comparison of the protein composition of the membranes of mutant cells subjected to a growth regimen provoking competence in wild-type cells with that of competent wild-type cells revealed that the mutant is impaired in the synthesis of a protein with a molecular weight of 22,500.