A novel relaxase homologue is involved in chromosomal DNA processing for type IV secretion in Neisseria gonorrhoeae

Abstract

The Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA that acts in natural transformation. To examine the mechanism of DNA processing for secretion, we made mutations in the putative relaxase gene traI and used nucleases to characterize the secreted DNA. The nuclease experiments demonstrated that the secreted DNA is single-stranded and blocked at the 5' end. Mutation of traI identified Tyr93 as required for DNA secretion, while substitution of Tyr201 resulted in intermediate levels of DNA secretion. TraI exhibits features of relaxases, but also has features that are absent in previously characterized relaxases, including an HD phosphohydrolase domain and an N-terminal hydrophobic region. The HD domain residue Asp120 was required for wild-type levels of DNA secretion. Subcellular localization studies demonstrated that the TraI N-terminal region promotes membrane interaction. We propose that Tyr93 initiates DNA processing and Tyr201 is required for termination or acts in DNA binding. Disruption of an inverted-repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion. The TraI domain architecture, although not previously described, is present in 53 uncharacterized proteins, suggesting that the mechanism of TraI function is a widespread process for DNA donation.

Fig. 1

A. Fluorometric detection of secreted DNA. Piliated gonococcal strains were grown for 2.5h in liquid culture. Cell-free culture supernatants were collected, and DNA was detected with the fluorescent DNA-binding dye PicoGreen and normalized to total cell protein. MS11 was used as the wild-type (WT) strain and ND500 (ΔGGI in MS11) as the negative control. The results are an average of at least four independent experiments. *p-value<0.003 when compared to wild-type. ^†p-value<0.003 when compared to the respective mutant. ^§Not significantly different than wild type. B. Detection of TraI by Western blot. TraI was produced by wild-type (wt) and mutant strains at approximately the same levels. TraI is not detected in strain ND500, a strain in which the GGI was deleted.

Fig. 2

Amino acid sequence alignment of predicted relaxases from plasmid R27 (R27 TraI), X. fastidiosa (Xf TraI), and N. gonorrhoeae (Ng TraI). Identical amino acids are highlighted. Underlined sequences in R27 TraI were reported to correspond to the three motifs of classical relaxases. However, the first two motifs are not conserved in the Ng TraI family. The hallmarks of the Ng TraI family are a conserved tyrosine (Tyr⁹³ in Ng TraI) closely followed by a histidine (H)-rich motif, and the HD phosphohydrolase domain. The signature sequence of the H-rich motif is h(Q/H)xhPASExHHHx₃GG(L/M)h, where h is a hydrophobic residue and x is any residue. The HD domain motifs are labeled HD I to V (where HD II is the signature motif). The consensus amino acid sequence for the fifty-four proteins in the Ng TraI family of predicted relaxases is shown (the dotted lines represent no consensus). The consensus amino acid sequence for the HD domain is also shown [(b) big, (s) small, (h) hydrophobic, (c) charged, and the capital letters represent invariant amino acids]. The arrows indicate the amino acids in Ng TraI that were the targets for site-directed mutagenesis in this study. Mutations that decrease DNA secretion are indicated with a star. Mutations that have no effect on DNA secretion are indicated with a solid circle.

Fig. 3

Phylogenetic tree of proteins with the same domain architecture as Ng TraI. The phylogenetic tree was generated from the CLUSTAL W alignment of the full-length proteins. A black box highlights the cluster containing Ng TraI. The different font colors represent proteins with similar predicted amphipathic α helices at the N-terminus proximal region: yellow, orange, red, and navy blue. Black font and white font indicate proteins with unique amphipathic α helices. Ng TraI, black font (white background), contains an N-terminal amphipathic α helix. Azoarcus sp. EbN1 (purple font) contains a predicted N-terminal transmembrane domain in addition to the N-terminus proximal amphipathic α helix. Green font indicates proteins that contain two predicted N-terminus proximal amphipathic α helices. Underlined proteins do not contain predicted amphipathic α helices. H. somnus contains a charged reside on the hydrophobic side, and the Shewanella ANA-3 sequence available is truncated. Orange font indicates proteins that conserve motif I and II of relaxases. *Plasmid-encoded proteins.

Fig. 4

Map of the TraI N-terminal region. A. A SignalP 3.0 algorithm analysis of the Ng TraI amino acid sequence predicted the presence of a signal sequence (0.923 probability) with a cleavage site between Tyr²¹ and Leu²² (0.5 probability). The hydrophobic (h) region is interrupted by polar residues (P), which results in the secondary structure prediction of an amphipathic α helix (c, coil; h, helix; e, sheet). B. Helical wheel of amino acids 5 to 12. Black font, white background represents amino acids in the polar side of the helix. White font, black background represents amino acids in the hydrophobic side. *Charged residues were introduced into the hydrophobic side of the predicted amphipathic α helix at positions 6 and 12 (L6K/L12K).

Fig. 5

In vitro translocation assay with isolated inverted inner membrane vesicles of E. coli. A. FtsQ and TraI_Δ261-850 were used in a co-translational targeting/transport assay in which the translocation into inverted inner membrane vesicles was monitored by the accessibility of the transported protein to externally added proteinase K, in the presence or absence of Triton X-100. To detect even small amounts of transported protein, inverted membrane vesicles derived from a strain overexpressing the SecYEG translocase were also used. Ten (10) % of the reaction mixture before addition of proteinase K was loaded as a synthesis control. FtsQ is a membrane protein whose insertion into the vesicles is mediated by Sec proteins. Only a small domain of FtsQ is exposed on the outside of the vesicles and is accessible to degradation by proteinase K. B. proOmpA and TraI_Δ261-850 were used in a post-translation protein transport assay in the presence or absence of inner membrane vesicles, as described above, with or without ATP. One (1) % of synthesized protein before addition of proteinase K was loaded as a control. proOmpA contains a signal peptide for transport into the periplasm and is processed by leader peptidase.

Fig. 6

Subcellular localization of TraI. A. Full-length TraI overexpressed in a wild-type strain from an inducible construct on the chromosome. TraI was detected by Western blot with TraI-specific polyclonal antiserum. Periplasmic (peri), membrane (memb), and cytoplasmic (cyto) fractions are indicated. A cross-reactive protein (∼35kDa) was found in the periplasmic fractions from strains with or without the T4SS. Antiserum to the gonococcal outer membrane protein PilQ was used to detect membrane material. Chloramphenicol acetyltransferase (CAT) was detected to identify cytoplasmic material. B. Top Panels. Membrane fractions from wild-type (WT) and ΔGGI strains (left panel) and strains overexpressing (oe) traI or traIL6K/L12K from the native site (right panel). Bottom Panels. Cytoplasmic fractions from wild-type (WT) and ΔGGI strains (left panel) and strains overexpressing (oe) traI or traIL6K/L12K from the native site (right panel). TraI was detected by Western blot with TraI-specific polyclonal antiserum.

Fig. 7

Nuclease treatment of culture supernatants. Fluorometric detection of DNA in cell-free supernatants after overnight incubation with nucleases. ExoIII is a double-strand specific exonuclease that converts double-stranded DNA to single-stranded DNA, by degrading one strand. ExoI is a 3′→5′ single-strand specific exonuclease. DNaseI and BAL-31 have endonuclease activity. BAL-31 also has double-strand specific exonuclease activity. RecJ_f is a 5′→3′ single-strand specific exonuclease. Culture supernatants collected from a wild-type strain (A), culture supernatants supplemented with HindIII-digested, single-stranded λ DNA (B) or with double-stranded λ DNA (C). *p-value<0.05. ^§Not significantly different from no treatment.

Fig. 8

Co-culture transformation in the presence of nucleases. Transfer of an antibiotic resistance marker during co-culture transformation in the presence of nucleases. The transferred marker, cat, carries an internal EcoRI site. EcoRI is a restriction enzyme that cuts double-stranded DNA. ExoIII is a double-strand specific exonuclease. ExoI is a 3′→5′ single-strand specific exonuclease. RecJ_f is a 5′→3′ single-strand specific exonuclease. DNaseI has double- and single-strand endonuclease activities; BAL-31 has single-strand specific endonuclease and double-strand specific exonuclease activities. *p-value<0.05. ^§Not significantly different from no treatment.

Fig. 9

Predicted oriT sequence located in the GGI. A. Map of the GGI region predicted to contain the oriT. traI encodes the putative relaxase, yaf encodes an unknown protein, and ltgX encodes a putative lytic transglycosylase. Arrows labeled “P” mark the promoter region for the transcripts. The sequence of the yaf-ltgX inverted repeat is shown. An insertion containing a cat marker was introduced at the StuI site of the inverted repeat, deleting the sequence in between the StuI sites. B. Fluorometric detection of secreted DNA. Piliated gonococcal strains were grown for 2.5h in liquid culture. Cell-free culture supernatants were collected and DNA was detected with the fluorescent DNA-binding dye PicoGreen and normalized to total protein in the cell pellet. MS11 was used as the wild-type (WT) strain and ND500 (ΔGGI in MS11) as the negative control. The results are an average of at least four independent experiments. *p-value<0.003 when compared to wild-type. ^†p-value<0.003 when compared to its respective mutant.