Domain exchange at the 3' end of the gene encoding the fratricide meningococcal two-partner secretion protein A

Abstract

Background: Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis, several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA-related gene cassettes, called tpsC, are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be.

Results: We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination.

Conclusions: Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.

Figure 1

Comparison of the genetic organization of three TPS islands in different meningococcal strains. Each island consists from the 5’ to the 3’ end of a tpsB, a tpsA, and a variable number of tpsC cassettes interspersed with intervening ORFs (IORFs) (open arrows). The tpsA genes and tpsC cassettes can display very different domains of ~450 bp at their 3’ end as indicated by the different colors. Domains with very high sequence similarity are indicated with the same color. Also the IORFs show high heterogeneity and IORFs with high sequence similarity are indicated with the same color. Note that tpsA or tpsC genes with similar sequences at their 3’ end are always followed by IORFs with high sequence similarity. Regions with high sequence similarity between the different islands are indicated by grey shading, indicating gross rearrangements in the organization between the islands. Note that strain MC58 contains five tpsA genes, including two tpsA genes of system 1 (Figure S1 in Additional file 1). The island depicted here contains the tpsA1b gene with locus tag NMB0497.

Figure 2

Growth-inhibitory function of meningococcal TpsA. (A) Cells of an unencapsulated derivative of strain B16B6, carrying a plasmid with a chloramphenicol-resistance marker, were mixed 1:1 with cells of ΔtpsA-tpsC, ΔtpsB or ΔtpsC_2-5 mutants, all carrying a kan cassette. The suspensions were spotted on GC plates without antibiotics and incubated for various time periods. In the experiment with the ΔtpsC_2-5 mutant as the target cells, the suspensions were only spotted after 0 and 48 hours. The ratios of the ΔtpsA-tpsC (grey bars), ΔtpsB (white bars), or ΔtpsC_2-5 (hatched bars) mutants over wild-type bacteria in the spots was determined by plating on GC media containing kanamycin or chloramphenicol and counting colony-forming units after overnight incubation. (B) A rifampicin-resistant derivative of the unencapsulated B16B6 strain was mixed 1:1 with the ΔtpsA-tpsC mutant harboring plasmid pFPIORF₁, which contains the first IORF of B16B6 under the control of an IPTG-inducible promoter. The bacteria were incubated in the presence or absence of IPTG for 24 hours as above. The ratio of the ΔtpsA-tpsC mutant over the wild-type bacteria was determined as described. Results are means and s.d. of three independent experiments. All the strains tested here did not show differences in viability when grown separately.

Figure 3

Genetic organization of the deviant TPS organization in cc11 isolates 2001044 and 348 and consequences in growth-inhibition activity. (A) Comparison of the TPS organization in cc11 reference strain FAM18 and the clinical isolate 2001044. (B) Comparison of the genetic organization of the TPS island in clinical isolate 348 and those in cc11 reference strain FAM18 and cc4 reference strain Z2491. In both panels, regions of high sequence similarity between the islands are indicated by grey shading. Note that the TPS organization in isolate 2001044 could have been generated by intragenomic recombination, whilst that in isolate 348 must have implicated horizontal gene transfer. (C) Unencapsulated derivatives of strains B16B6 and its ΔtpsA-tpsC mutant, both carrying an erythromycin-resistance marker, were mixed 1:1 with an unencapsulated derivative of strain 2001044 carrying a chloramphenicol-resistance marker, spotted on GC plates and incubated as described in the legend to Figure  2. The ratios of 2001044 bacteria over B16B6 (left) or B16B6ΔtpsA-tpsC mutant (right) bacteria were determined after 0 hours (gray bars) or 24 hours (white bars) of incubation by plating on GC media containing erythromycin or chloramphenicol.

Figure 4

In vitro recombination in the TPS islands. (A) Organization of the TPS islands in strain B16B6 and its mutant derivative B16B6ΔtpsC2-5 where a kan cassette replaces several tpsC cassettes and IORFs. (B) Organization of the TPS island in recombinant strain α14-tpsA* (middle) obtained after transformation of strain α14 (top) with chromosomal DNA from strain B16B6ΔtpsC2-5 (bottom). (C) Organization of the TPS island in recombinant strain α153-tpsA* (middle) obtained after transformation of strain α153 (top) with chromosomal DNA from strain α14-tpsA*#1 (bottom). The organization of the TPS island of strain α153 is not completely depicted because it is not located on a single contig in the available genome sequence.

Figure 5

Recombination model for the TPS system. (A) Recombination model requiring double crossover. The recipient and donor DNA are indicated in the first and the second line, respectively, and carry in the depicted example a different central core region in tpsA. IORFs are not depicted. Within the 3’ region, the tpsA genes share sequences with segments at the 5’ end of tpsCs (black boxes). The minimal recombination units (indicated by double-headed arrows) are formed by the 5’ region of a tpsC, an IORF, and the 5’ region of the immediately downstream tpsC. The recombination products show the recipient DNA organization after double crossovers with donor DNA sequences. The double-headed lines show the recombination sites where the crossovers were established. (B) Recombination model requiring an intra-chromosomal single crossover and the product of such an event evolved from the organization of the recipient DNA shown in the first line of panel A.