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Journal of Bacteriology, May 2005, p. 3273-3276, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3273-3276.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Genes Required for Competence in Neisseria meningitidis

Yao-Hui Sun, Rachel Exley, Yanwen Li, David Goulding, and Christoph Tang*

Centre for Molecular Microbiology and Infection, Department of Infectious Diseases, Faculty of Medicine, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, United Kingdom

Received 30 November 2004/ Accepted 29 January 2005


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ABSTRACT
 
We have identified genes required for competence of Neisseria meningitidis, a naturally transformable human pathogen. Although not comprehensive, our analysis identified competence-defective mutants with transposon insertions in genes not previously implicated in this process in Neisseria.


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TEXT
 
Neisseria meningitidis is a human pathogen that colonizes the nasopharynx (5, 11, 27) but also causes septicemia and meningitis (33). The bacterium is naturally transformable, providing a mechanism for genetic heterogeneity (4, 11, 21, 42). For example, N. meningitidis sodA has been acquired from Haemophilus influenzae (18), and genes encoding housekeeping functions and targets of the immune response have a mosaic structure (9, 10, 29, 37, 38).

Transformation involves multiple steps, including DNA uptake, processing, and chromosomal integration (19). The basis of transformation in Neisseria gonorrhoeae has been intensively studied (16, 17). Uptake of genus-specific DNA (8, 13) is mediated by type IV pili (Tfp) at the bacterial surface. Next, incoming DNA is converted, in part, to a single-stranded intermediate (6), while the subsequent events are not well defined.

Little is known about transformation in N. meningitidis (1), although it expresses one of two types of Tfp, with only class I (differentiated from class II pili by binding of the monoclonal antibody SM1) conferring competence (24). We describe the use of signature-tagged mutagenesis (STM) to identify genes required for meningococcal competence (14).

The bacterial strains and plasmids used are shown in Table 1, and growth conditions and antibiotics are described elsewhere (32). A recA mutant was constructed as a strain defective for homologous recombination. Primers NG102 (5'-ATCGCTCGGATTAGACCTC-3') and NG103 (5'-ATGTCGATCAATTCGCC-3') amplified a 702-bp recA product, which was ligated into pCR2.1 TOPO; inverse PCR with oligonucleotides NG106 (5'-GTCACGCGTACTACCATATCTATG-3') and NG107 (5'-GTCACGCGTTGAGCCAGGCTTTGC-3') introduced a deletion and a MluI site (underlined) into the plasmid into which a kanamycin resistance gene was inserted. Plasmids containing an inactivated pilE allele (as a strain defective for DNA uptake) were obtained by in vitro mutagenesis. pilE was amplified using PilE1 (5'-TTTACCCTTATCGAGCTGATG-3') and PilE2 (5'-TTAGCTGGCATCACTTGCGTCG-3') and ligated into pCR2.1 TOPO, and the kanamycin resistance marker was deleted by SphI digestion and self-ligation. The resultant plasmid was subjected to Tn5 mutagenesis (Km-2; Epicenter). All N. meningitidis transformants were analyzed by Southern hybridization. For complementation, pilN was amplified (using NG310 [5'-ACGCGTCCCGGGAACAATTTAATCAAAATCAACCTC-3'] and NG311 [5'-AGGATCCGTTTGCCTCCTGTGCGTTTCCCG-3']) and introduced into the chromosome in single copy between two genes (NMB0102 and -0103) orientated in a tail-to-tail fashion (36), resulting in C311{Delta}pilN::pilNect.


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  		TABLE 1. Bacterial strains and plasmids used in this study

Mutants were grown in liquid brain heart infusion medium with 5% Levinthal's supplement in microtiter dishes, pooled, and then resuspended in phosphate-buffered saline (PBS). The cells (10 µl) were spotted to agar plates and DNA was added (1 µg in 10 µl PBS), and plates were incubated for 3 h at 37°C in 5% CO2, after which the bacteria were spread onto selective media. Transformation frequencies were calculated as the number of transformants per 108 CFU, using 1 µg of donor DNA.

For Western analysis, bacteria were resuspended in loading buffer (50 mM Tris [pH 6,8], 2% SDS, 0.1% bromophenol blue, 10% glycerol, 1010 CFU/ml) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis before transfer to polyvinylidene difluoride membranes (Imobilon-P; Millipore). Membranes were washed in 0.5% milk in PBS and incubated with monoclonal antibody SM1 (1:10,000 dilution), which recognizes Tfp (34), followed by a horseradish peroxidase-conjugated antimouse immunoglobulin antibody (1:2,000; DAKO). For electron microscopy, colonies were collected from plates into 2.5% glutaraldehyde in PBS, placed on ice for 30 min, and finally resuspended in distilled water. Ten µl was placed onto a formvar/carbon-coated grid with an equal volume of 1% ammonium molybdate (pH 6.5); grids were viewed on a Philips CM100 microscope.

The construction of a library of C311+ mutants, detection of signature tags, and isolation of insertion sites were performed as described previously (2, 30). Insertional mutants (n = 1,330) were tested in duplicate for their ability to acquire erythromycin resistance following transformation with pIP10 (35). For controls, pools of mutants were transformed with pIP10 but plated to medium without antibiotics. Competence-defective mutants were defined as mutants detectable on control blots but absent on both output blots (Fig. 1); 14 competence-defective mutants were identified.



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  		FIG. 1. Outline of the strategy used to identify transformation-defective mutants. Ninety-five tagged N. meningitidis mutants were pooled, and three aliquots were transformed with pIP10 (siaD::ery). The cells from two transformations were plated to medium containing erythromycin, while cells from the other transformation were streaked to unselective medium. Tags were amplified from bacteria harvested from the plates and then labeled and used to generate output and input blots. Transformation-defective mutants (circled) were present on the input blot but consistently absent from output blots.

To confirm that the competence defect of mutants resulted from the transposon insertion, the 14 mutations were backcrossed into C311+, and the competence of the newly constructed mutants was tested individually with (i) pIP10, (ii) another marker inserting at a different locus (pSTM208, lgtG::tet), and (iii) chromosomal DNA (C311{Delta}lgtG) (32). The results using all DNA donors were comparable (not shown). C311{Delta}recA and C311{Delta}pilE were used as negative controls. Six mutants remained competence defective after backcrossing, and the sites of transposon insertion in these mutants are shown in Fig. 2. The result of database searches for functionally defined homologues is given in Table 2.



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  		FIG. 2. Location of the transposon insertions (solid triangles) in transformation-defective mutants.


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  		TABLE 2. Transformation-defective mutantsa

pilN is located in a cluster of genes required for Tfp biogenesis, including pilQ (7), and is related to an Escherichia coli gene that encodes a lipoprotein required for the biogenesis of the R64 mating pilus (25). Complementation with pilN alone was sufficient to restore competence in 5H2 (C311{Delta}pilN), although not to wild-type levels (transformation frequencies per 108 CFU: C311, 1.06 x 10–5; C311{Delta}pilN, <10–8; C311{Delta}pilN::pilNect, 1.7 x 10–6), demonstrating that PilN itself has a role in competence. Western and electron microscopy analyses (Fig. 3) showed that although pilN is not absolutely required for pilus assembly, Tfp on the pilN mutant were less abundant and shorter than on the wild-type or complemented strains. Further experiments are required to define the precise function of PilN in Tfp synthesis.



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  		FIG. 3. Complementation of C311{Delta}pilN. For Southern analysis (A), chromosomal DNA was digested with ClaI and membranes were hybridized with a probe for pilN. For Western analysis (B), whole-cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were reacted with the monoclonal antibody SM1 to detect subunits of Tfp (B). The strains are indicated above each lane, and the sizes of molecular mass markers are shown. Electron microscopy of bacteria (C). The strains are shown on each panel. Scale bar = 1 µm.

Two mutants had insertions in NMB0116, which shares 43% amino acid identity with DprA, required for transformation of H. influenzae (15). DprA is also involved in competence of Helicobacter pylori, Bacillus subtilis, and Streptococcus pneumoniae (3, 22, 28).

One mutant (5A10) had an insertion in a homologue of recG from E. coli which encodes a helicase that rescues damaged replication forks by resolving them to Holliday junctions (21). RecG might also be involved in formation of a Holliday junction between donor and host DNA during transformation. This is also consistent with a role for RecG in pilin variation in N. gonorrhoeae (H. Seifert, presented at the 14th International Pathogenic Neisseria Conference, Milwaukee, Wis., September 2004), which is also mediated by homologous recombination (31).

Mutant 7A6 had an insertion in a homologue of ptsI from B. subtilis (12, 23). PtsI is the initial phospho-acceptor in the phospho-transfer system (PTS), which mediates the uptake of certain sugars following their phosphorylation. Thus, competence in Neisseria spp. might be regulated by the nutritional environment, similar to H. influenzae, in which a ptsI mutant also has a substantially reduced transformation frequency (20). Interestingly, the frequency of gonococcal pilin recombination is also elevated when the bacterium is starved of iron (26). Loss of Rho (mutant 5G1), the transcription termination factor, is likely to have pleiotropic effects; there are no previous reports of the influence of Rho on competence.

In conclusion, we have adapted STM to examine factors required for meningococcal competence. Our failure to identify genes known to be involved in competence (including pilE, pilT, and pilC) indicates that our analysis was not comprehensive. However, we have demonstrated that several novel genes are involved in competence, and understanding how they contribute to horizontal DNA transfer should be informative about the generation of phenotypic diversity in this important human pathogen.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases, Faculty of Medicine, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, United Kingdom. Phone: (44) 207-594-3072. Fax: (44) 207-594-3076. E-mail: c.tang{at}imperial.ac.uk. Back


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Journal of Bacteriology, May 2005, p. 3273-3276, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3273-3276.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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