Phylogenetic trees based on ClustalX analysis, showing the evolutionary relationships between amino acid sequences encoded by tfc14 (a), tfc16 (b), and tfc6 (c) of the ICEHin1056 T4SS and homologues from a set including the previously described T4SSs and homologues identified by bioinformatic analysis from a wide range of proteobacteria. The topology of the trees resulting from alignment of amino acid sequences encoded by tfc6, tfc14, and tfc16 and their homologues revealed that the topology was conserved only for tfc14 and tfc16. S Typhi, Salmonella enterica serovar Typhi; V cholerae, Vibrio cholerae; P rettgeri, Proteus rettgeri; P vulgaris, Proteus vulgaris; N aromaticivorans, Neisseria aromaticivorans; N gonorrhoeae, Neisseria gonorrhoeae; A rhizogenes, Agrobacterium rhizogenes.

Transmission electron micrographs of negatively stained samples, showing the presence of pili dependent on the T4SS (arrows) in H. influenzae strain Rd harboring ICEHin1056 (a), the tfc1 mutant (b), the tfc6 mutant (c), and the tfc24 mutant (d), B, bacterium. Bars = 100 nm.

(a) Schematic diagram of the ICEHin1056 genomic island and localization of the putative type IV secretion system (red). This T4SS is encoded by 24 genes, 7 of which exhibit homology to known T4SS components (components a to g). Seven of 24 gene products of the ICEHin1056 putative T4SS have transmembrane helices (indicated by plus signs), suggesting membrane-bound localization of the relevant proteins. Moreover, 10 of 24 genes of this gene cluster have signal peptide sequences (indicated by plus signs) that are typical for genes involved in T4SSs. a, TraX homologue (F plasmid); b, PilL homologue (plasmid pKLC102); c, TraD homologue (F plasmid); d, TraB homologue (F plasmid); e, TraC/VirB4 homologue (F plasmid); f, TraG homologue (F plasmid); g, PilT homologue (L. pneumophila str. Philadelphia 1). (b) Transcriptional mapping by RT-PCR of the ICEHin1056 T4SS gene cluster. The T4SS gene cluster is expressed in the form of two polycistronic transcripts. Cotranscription of neighboring genes is indicated by solid boxes. Neighboring genes which are not transcribed together are indicated by open boxes. (c) Gel showing that no mRNA transcripts were detected between tfc19 and tfc20 and between the first gene (tfc1) and the last gene (tfc24) of the ICEHin1056 T4SS and the neighboring genes in the rest of the ICEHin1056 sequence (ORF ICEHin1056). Lane 1, ORF ICEHin1056-tfc1 transcript; lane 2, tfc1-tfc2 transcript; lane 3, tfc2-tfc3 transcript; lane 4, tfc3-tfc4-tfc5-tfc6 transcript; lane 5, tfc6-tfc7-tfc8 transcript; lane 6, tfc8-tfc9 transcript; lane 7, tfc9-tfc10-tfc11 transcript; lane 8, tfc10-tfc11-tfc12-tfc13 transcript; lane 9, tfc13-tfc14 transcript; lane 10, tfc14-tfc15 transcript; lane 11, tfc15-tfc16 transcript; lane 12, tfc16-tfc17-tfc18-tfc19 transcript; lane 13, tfc19-tfc20 transcript; lane 14, tfc20-tfc21 transcript; lane 15, tfc21-tfc22 transcript; lane 16, tfc22-tfc23-tfc24 transcript; lane 17, tfc24-ORF ICEHin1056 transcript.

Phylogenetic tree based on ClustalX analysis, showing the evolutionary relationship between concatenated amino acid sequences of tfc14 and tfc16 of the ICEHin1056 T4SS and the previously described T4SSs. The ICEHin1056 T4SS does not belong to any of the previously described groups and is evolutionarily distant. Moreover, syntenic T4SSs located on a broad spectrum of genomic islands present in a wide variety of bacterial species belong to this evolutionarily distant group. For an explanation of abbreviations see the legend to Fig. 2.

RT-PCR showing transcription levels of genes of the ICEHin1056 T4SS in H. influenzae strain Rd harboring ICEHin1056 (Rd11) and in the tfc24 mutant (Rd11tfc24). (a) Expression of the majority of the genes of the T4SS examined is downregulated in the tfc24 mutant. The results also demonstrate that there is cotranscription of T4SS genes. Lane 1, tfc1 transcript; lane 2, tfc3-tfc4-tfc5-tfc6 transcript; lane 3, tfc6 transcript; lane 4, tfc6-tfc7-tfc8 transcript; lane 5, tfc9-tfc10-tfc11 transcript; lane 6, tfc12-tfc13 transcript; lane 7, tfc13 transcript; lane 8, tfc14 transcript; lane 9, tfc14-tfc15 transcript; lane 10, tfc16 transcript; lane 11, tfc17-tfc18-tfc19 transcript; lane 12, tfc22 transcript; lane 13, tfc23-tfc24 transcript. (b) Quantification of the tfc16 and tfc17 mRNA transcripts in strains Rd11 and Rd11tfc24 using the ABI Prism 7000 sequence detection system. Interpolating from the standard curve, the number of copies of the tfc16 gene transcript was reduced from 530 × 10² in strain Rd11 to 263 × 10² in mutant Rd11tfc24 and the number of copies of the tfc17 gene transcript was reduced from 537 × 10² in strain Rd11 to 411 × 10² in mutant Rd11tfc24.