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).     

Haemophilus influenzae biochemotyping

Biochemotyping was performed in 129 strains of Haemophilus influenzae. 95% of strains could be assigned to one of the five biochemotypes proposed by Kilian. Most of the serotype B strains isolated in meningitis belonged to biochemotype I. The biochemical differentiation of Haemophilus influenzae is regarded as a reliable technique deserving further application.     

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.     

Point mutations in a peptidoglycan biosynthesis gene cause competence induction in Haemophilus influenzae

We have identified three new Haemophilus influenzae mutations causing cells to exhibit extreme hypercompetence at all stages of growth. The mutations are in murE, which encodes the meso-diaminopimelate-adding enzyme of peptidoglycan synthesis. All are point mutations causing nonconservative amino acid substitutions, two at a poorly conserved residue (G(435)-->R and G(435)-->W) and the third at a highly conserved leucine (L(361)-->S). The mutant strains have very similar phenotypes and do not exhibit any defects in cell growth, permeability, or sensitivity to peptidoglycan antibiotics. Cells retain the normal specificity of DNA uptake for the H. influenzae uptake signal sequence. The mutations do not bypass genes known to be needed for competence induction but do dramatically increase expression of genes required for the normal pathway of DNA uptake. We conclude that the mutations do not act by increasing cell permeability but by causing induction of the normal competence pathway via a previously unsuspected signal.     

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.     

Time-Dependent Effects of Inosine on Competence Development in Haemophilus influenzae

The effect of inosine on the development of competence is dependent upon the time at which it is added to the competence medium. Competence is inhibited if inosine is added within 15 min after the development begins, but is stimulated if the addition is made at later times. Protein synthesis is stimulated by the addition of inosine, but the effect is quantitatively similar regardless of the time at which inosine is added.     

Automated Detection of Haemophilus influenzae

Addition of heme (X factor) and pyridine nucleotide (V factor) to the medium permits rapid growth of Haemophilus influenzae, with evolution of easily detectable amounts of (14)CO(2). Radiometric media containing X and V factor should be used when evaluating clinical specimens which might contain Haemophilus species.     

Electron microscopy of Haemophilus influenzae

The electron microscopic study of H. influenzae standard strains, serovar b, in the capsular and noncapsular forms has revealed the pronounced pleomorphism of these organisms in successive generations.     

New Bacteriophage of Haemophilus influenzae

Some properties of a newly isolated Haemophilus influenzae bacteriophage are described. The phage is morphologically distinct from previously described H. influenzae phages.