Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis

Abstract

Genes required for natural transformation of Haemophilus influenzae R d were identified by a cassette mutagenesis protocol consisting of the following steps : random insertional mutagenesis, phenotypic screening, sequencing of genome sequence tags from the DNA flanking the insertion in the selected mutants and comparison of genome sequence tags to genomic sequence data. The cassette mutagenesis screen for transformation genes resulted in five distinct mutant classes, two of which have been identified in previous studies.Insertions in the three newly identified loci interrupted genes with predicted protein products homologous to a type IV pilin-like protein biogenesis operon, drug-eff lux transporters and a phospholipid-biosynthesis enzyme. The most significant finding of this screen is the requirement for type IV pilin-like proteins in genetic transformation of H. influenzae. These surface structures are utilized for DNA uptake in a number of Gram-positive and Gram-negative bacteria, and appear to be the common component among the systems for DNA binding. INTRODUCTIONHaemophilus inf'luenxae is a Gram-negative facultative anaerobe which resides in the human upper respiratory tract. Isolates are classified either as serotypes A-F or as non-typable, depending on the presence or absence, respectively, of type-specific capsular polysaccharide. H. influenxae is also capable of taking DNA from its environment and integrating it into the bacterial chromosome (Smith et al., 1981; Goodgal, 1982). This natural transformation process has been demonstrated for many strains of H . influenxae but has been best characterized using strain Rd (Alexander & Leidy, 1951 Abbreviations: sBHI, supplemented brain heart infusion media; Kan: kanamycin-resistance; GST, genome sequence tag; MIV, medium IV; Tfo-, transformation-deficient.formed cell per lo7 viable cells) in early exponential phase to about lo-* in late exponential phase. The addition of CAMP to early-phase cultures increases the frequency of transformation to lop4 (Wise et al., 1973;. Furthermore, under conditions of transient anaerobiosis (Goodgal & Herriott, 1961) or transfer to starvation medium (Herriott et al., 1970) transformation frequencies are optimized to approximately lop2. Transformation is also observed when H. influenxae is grown in diffusion chambers implanted into animals (Dargis et al., 1992), suggesting that transformation is not an artifact of in vitro cultivation.A number of genes required for DNA transformation have been identified in H . influenzae. These studies have used a variety of molecular methods to identify transformation genes, including complementation of transformation-deficient mutants derived by chemical mutagenesis , a 'poison-DNA' selection method (Beattie & Setlow, 1971), mini-Tn20 mutagenesis (Tomb et al., 1989), Tn916 mutagenesis (Gwinn et al., 1998) and other genetic selection techniques . It has been a goal of our laboratory to screens (e.g. hot-spotting) and yet retain the advantage of gene disruption with an antibiotic-resistance...

Demonstration of Type IV Pilus Expression and a Twitching Phenotype by Haemophilus influenzae

“…In P. aeruginosa, the biogenesis and function of Tfp are controlled by over 40 genes (42). To date, only a subset of the vast number of related Tfp genes (12,43) has been found in several members of the HAP (Haemophilus, Actinobacillus, and Pasteurella) family (7,13,14,40), and the expression of Tfp, or a twitching phenotype, has not been described for any H. influenzae isolate. In fact, H. influenzae is classically described as a bacterium that does not express these structures (1,8,18,19), despite the presence of a cryptic gene cluster within the strain Rd genome (16).…”

ComP, a Pilin-Like Protein Essential for Natural Competence in Acinetobacter sp. Strain BD413: Regulation, Modification, and Cellular Localization

“…Genetic and biochemical studies indicate that homologues of type IV pilus subunits are involved in natural transformation in a variety of bacteria (4,5,7,39). However, it is still an open question whether the type IV pili or a putative pilus-like structure is implicated in DNA uptake (7,44).…”

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…Several genes are known to affect transformation in H. influenzae (Caster et al, 1970;Caster and Smith, 1972;Notani et al, 1972;Concino and Goodgal, 1982;Kooistra et al, 1983;Smith, 1985, 1986;Tomb et al, 1989Tomb et al, , 1991Larson and Goodgal, 1991;Chandler, 1992;Dorocicz et al, 1993;Clifton et al, 1994;Gwinn et al, 1996Gwinn et al, , 1997Gwinn et al, , 1998Chandler and Smith, 1996;Karudapuram and Barcak, 1997;Dougherty and Smith, 1999;Dubnau, 1999;Ma and Redfield, 2000;Chen and Dubnau, 2004;VanWagoner et al, 2004;Redfield et al, 2005Redfield et al, , 2006Cameron and Redfield, 2006;Smeets et al, 2006). The cAMP-CRP (cyclic-AMP receptor protein) complex helps to promote their expression and induce competence (the ability to take up and incorporate DNA) (Chandler, 1992;Redfield et al, 2005;Cameron and Redfield, 2006).…”