Nucleotide sequence of the tna insert region in Hib (Eagan), with open reading frames and deduced amino acid sequences. (a) Sequence of a 3,456-bp BamHI/PstI fragment with its 5′ end in nlpD and its 3′ end in mutS. All genes in this region are transcribed to the right. Underlined sequences (bp 1 to 84 and 3192 to 3456) are aligned with the matching sequence in Rd (12) (bp 1 to 84 and 85 to 350 on the corresponding BamHI/PstI fragment). Base sequence differences between Rd and Hib (Eagan) are indicated by nucleotides above the Eagan genetic sequence, which refer to the Rd sequence. They include (numbering Rd positions from the BamHI site) C→T substitutions at Rd positions 65 and 191, a T→C substitution at position 73, G→A substitutions at positions 89, 118, 307, and 328, an A→G substitution at 187, and deletion of a T (indicated as ^T) between 92 and 96. The paired USS repeats at either end of the insert are indicated by arrows. Putative regulatory elements (consensus CAP binding and promoter sites) are indicated with dotted lines, and potential ribosome binding sites (Shine-Dalgarno) are in boldface. The coding region for NlpD ends at 31; the open reading frame for putative TnaC extends from 232 to 312, that for TnaA extends from 400 to 1818, and that for TnaB extends from 1895 to 3112. The initiating ATG for MutS is at 3270. The locations of a conserved tryptophan residue in TnaC and of a conserved lysine residue at position 270 in TnaA are marked with asterisks; in E. coli tryptophanase, this lysine forms a Schiff base with the coenzyme pyridoxal 5′-phosphate. ▿, insertion sites for IS1 (between 112 and 113) and IS5 (between 254 and 255) in subclones of pE6 which relieve growth inhibition in E. coli (see Materials and Methods). (b) Potential RNA stem-loop structures of the 43-bp repeat sequences corresponding to USS plus-minus pairs. ΔG values were −18.2 kcal/mol for the Rd structure and −12.3 kcal/mol for each of the Hib structures.

Amino acid sequence comparisons of the inferred TnaB homolog from Hib (Eagan) with TnaB from E. coli (8, 15) and P. vulgaris (21) and Mtr from E. coli (39) and H. influenzae (12). (a) Alignment by the Clustal method; filled residues match the consensus. (b) Phenogram of TnaB and Mtr amino acid sequences.

Southern blot of genomic DNAs with a tna-specific probe. Genomic DNAs from various H. influenzae strains and from H. haemoglobinophilus (H. hb) were digested with BamHI and probed with a SacII/XbaI fragment (Fig. 1) internal to the Hib (Eagan) tna cluster, and blots were washed under stringent conditions. Strain designations are indicated above lanes; a to f indicate reference strains of serotypes a to f, respectively. The predicted 5.8-kb fragment from Hib (Eagan) is indicated with an arrow.

Prevalence of indole-positive (▪) strains among H. influenzae isolates from various sources. Data were compiled from Kilian (25; Tables 1 and 18; 175 strains, including 45 from normal oral and respiratory tract flora), from Kawakami et al. (23; 27 strains from normal throat flora), and from the present study (6 strains, described in Table 1). Numbers in parentheses indicate the total number of strains from each source. Respiratory infections included chronic bronchitis, sinusitis, pneumonia, and unspecified respiratory infections; severe disease included meningitis or CSF infection, blood infections, epiglottitis, and pneumonia. Open bars show indole-negative strains.

Map of tna insert region showing positions on Rd genome at which tna DNA is inserted and of unique restriction sites. The scale of the insert (Hib) map is 50% that of Rd. The primers used to amplify the insert region are depicted with solid arrows; F, nlpD-F; R, mutS-R. Open arrows depict paired USSs. The position of a SacII/XbaI fragment (bp 705 to 2680) used as a tna-specific probe is indicated.

Amino acid sequence comparisons of the inferred TnaA polypeptide from Hib (Eagan) with tryptophanases from E. coli (8, 15), P. vulgaris (21), E. aerogenes (24), S. thermophilum (gene 1) (18), and the tyrosine phenol-lyase from Citrobacter freundii (3). (a) Alignment by the Clustal method; filled residues are identical to those of E. coli TnaA. (b) Phenogram of TnaA amino acid sequences.

Amino acid sequence alignment (Clustal) of the putative TnaC peptide from Hib (Eagan) with TnaC peptides from E. coli (15), P. vulgaris (21), and E. aerogenes (24). Filled residues match the consensus.

PCR amplification of genomic DNA between nlpD and mutS. (a) Primers nlpD-F and mutS-R were used to amplify the intervening sequences in genomic DNA from various H. influenzae strains by long PCR, and the products were resolved by agarose gel electrophoresis. Arrows indicate the positions of the 3.6-kb amplification product from Hib (Eagan) and the 0.45-bp product from Rd. Lane designations are as in Fig. 6; M, λ DNA HindIII markers. (b) Southern blot of the same gel with a tna-specific probe.