INTRODUCTION

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Many mobile DNA elements transpose from one chromosomal location to another by a fundamentally similar mechanism. They include IS elements (25), transposons (20), phages (4), and more recently the so-called pathogenicity islands (8). These elements contribute substantially to genetic diversity and genome plasticity. Particularly, in pathogenic bacteria some of these elements may contribute to the exchange of genetic material coding for virulence traits. This mechanism may increase the fitness of bacterial strains through acquisition of virulence factors. Among the mechanisms for transfer of DNA, lysogenic conversion by bacteriophages appears to be advantageous; in fact, bacteriophages can carry large blocks of DNA and can survive harsh conditions. Bacteriophages may also code for virulence factors that allow the host bacterium to enlarge its host range and provide mechanisms to evade immune response. Examples of bacterial virulence factors carried on bacteriophages include the well-studied diphtheria toxin of Corynebacterium diphtheriae (1), cholera toxin (CTX) of Vibrio cholerae (11), the pore-forming toxin CTX of Pseudomonas aeruginosa (9), the erythrogenic toxins of Streptococcus pyogenes (24), the Clostridium botulinum neurotoxin (1), and the Shiga-like toxins and enterohemolysin produced by Escherichia coli (2, 15).

Neisseria meningitidis, a gram-negative capsulated bacterium, is a major cause of septicemia and meningitis that can kill children and young adults within hours. There are five pathogenic N. meningitidis serogroups (A, B, C, Y, and W135) as determined by capsular polysaccharide typing (26). Very recently, the genomic sequences of N. meningitidis serogroup B strain MC58 (22) and serogroup A strain Z2491 (17) have been determined, showing, among other features, a number of open reading frames (ORFs) with homology to phage functions. We analyzed the chromosomal region of serogroup B strain MC58 coding for these genes and compared it to the genomes of N. meningitidis serogroup A strain Z2491 (17) and the closely related bacterium Haemophilus influenzae strain Rd (5). Our analysis indicates that these genomes contain chromosomal regions with similarities to Mu-like phages. These phage DNA regions are clearly mosaic with obvious sequence similarity to phage Mu interspersed with segments that are apparently unrelated. We show that some genes mapping within the phage regions code for surface-exposed proteins capable of eliciting serum bactericidal response. A possible role of these proteins in bacterial virulence and vaccine development is discussed.

	MATERIALS AND METHODS

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Computer analysis. The region spanning positions 1,099,626 to 1,134,164 of the serogroup B N. meningitidis genome strain MC58 (22) was analyzed for coding capacity by using databases and computer programs included in the Wisconsin Package (version 10.0; Genetics Computer Group [GCG], Madison, Wis.). We revisited each single ORF in order to assign the correct start codon on the basis of ribosomal binding sequence and promoter regions. Subsequently, the programs Psi-BLAST, FASTA, MOTIFS, FINDPATTERNS, and PSORT (http://psort.nibb.ac.jp), as well as the databases ProDom, Pfam, and Blocks were used to predict protein features and to assign putative functions. The selected region containing a hypothetical Mu-like prophage was screened for conservation against the complete genomes of N. meningitidis serogroup A available at the Sanger Center (17, http://www.genome.ou.edu/gono.html) and H. influenzae Rd available at The Institute for Genomic Research (TIGR) (5) (http://www.tigr.org/tdb/CMR/ghi/htmls/SplashPage.html) and against the partial Neisseria gonorrhoeae genomic sequences available at the Advanced Center for Genome Technology, University of Oklahoma (http://www.genome.ou.edu/gono.html). Identified prophage regions map within positions 1,768,530 to 1,807,766 (PNM1) and 1,207,176 to 1,236,496 (PNM2) of the serogroup A strain Z2491 genome and within positions 1,559,960 to 1,594,298 of the H. influenzae complete genome. The same analysis on coding capacity, ORF reassignments and functional predictions described for MuMenB has been carried out for DNA segments defining PNM1, PNM2, and MuHi.

Cloning, expression, and protein purification. ORFs were amplified by PCR on chromosomal DNA from strain 2996 (23), with synthetic oligonucleotides used as primers. The amplified DNA fragments were cloned into pGEX-KG vector (7) to express the proteins as NH₂-terminal glutathione-S-transferase fusions. Expression of recombinant proteins was evaluated according to the appearance of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant fusion proteins were purified by affinity chromatography on glutathione-Sepharose 4B resin (Pharmacia). Twenty micrograms of each purified protein was mixed with Freund's adjuvant and used to immunize mice at days 1, 21, and 35. Blood samples were taken at days 34 and 49.

Serum analysis. (i) FACScan bacterium binding assay. N. meningitidis strain M7 (acapsulated) was grown on chocolate agar plates overnight at 37°C with 5% CO₂. Bacterial colonies were collected with a sterile Dacron swab and used to inoculate four tubes (8 ml each) of Mueller-Hinton broth (Difco) containing 0.25% glucose. Cells were harvested at an optical density at 620 nm (OD₆₂₀) of 0.35 to 0.5, washed, and resuspended in blocking buffer (1% bovine serum albumin in phosphate-buffered saline, 0.4% NaN₃) at an OD₆₂₀ of 0.05. One hundred microliters of diluted sera (1:100, 1:200, 1:400) was added to 100 µl of bacterial cells in a 96-well plate (Costar), and incubated for 2 h at 4°C, washed with blocking buffer (200 µl/well), and 100 µl of 1:100 dilution of R-phycoerythrin-conjugated F(ab')₂ goat anti-mouse was added to each well and incubated for 1 h at 4°C. Cells were collected, washed, resuspended in PBS (200 µl/well)-phosphate-buffered saline 0.25% formaldehyde and transferred to FACScan tubes.

(ii) Bactericidal assay. N. meningitidis strain 2996 was cultivated overnight at 37°C on chocolate agar plates with 5% CO₂. Colonies were collected and used to inoculate 7 ml of Mueller-Hinton broth, containing 0.25% glucose, grown at 37°C with shaking to an OD₆₂₀ of 0.23 to 0.24, and diluted to 10⁵ CFU/ml in assay buffer (50 mM phosphate buffer [pH 7.2] containing 10 mM MgCl₂, 10 mM CaCl₂, and 0.5% [wt/vol] bovine serum albumin). Serum bactericidal activity determination (18) was carried out in a final volume of 50 µl with 25 µl of serial twofold dilutions of test serum, 12.5 µl of bacteria at the working dilution, and 12.5 µl of baby rabbit complement (final concentration, 25%). Controls included bacteria incubated with complement serum and immune sera incubated with bacteria and with complement inactivated by heating at 56°C for 30 min. Immediately after the addition of the baby rabbit complement, 10 µl of the controls was plated on Mueller-Hinton agar plates using the tilt method (time zero). The 96-well plate was incubated for 1 h at 37°C with rotation. Seven microliters of each sample was plated on Mueller-Hinton agar plates as spots, whereas 10 µl of the controls was plated on Mueller-Hinton agar plates using the tilt method (time one). Agar plates were incubated for 18 h at 37°C, and the colonies corresponding to time zero and time one were counted.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Identification of a Mu-like prophage in the genome of N. meningitidis serogroup B strain MC58. The annotation of the complete genome sequence of N. meningitidis serogroup B strain MC58 revealed the existence of 10 ORFs with striking amino acid similarities (identities ranging from 28.1 to 70.3%) to phage functions (22). These ORFs, interspersed within a genomic region of 20,995 bp spanning coordinates 1,101,155 to 1,131,150, include genes coding for regulatory functions of phage Mu (Ner, MuA, MuB) as well as genes coding for baseplate and tail functions of this phage (MuG, MuI, GpL, VpN, VpP, gp45, VpH). With the exception of a transposase of the IS30 family (NMB1099 in reference 22), these phage functions are surrounded by ORFs of unknown functions (Fig. 1). Moreover, two partial ORFs, NMB1077 and NMB1122, with homologies to ABC transporters map upstream and downstream of the region under study, respectively. Reunion of these truncated ORFs gives rise to a complete ORF which shows 55% amino acid identity to a hypothetical ABC transporter ATP-binding protein of H. influenzae (5). This indicates that the ABC transporter-encoding gene was split upon integration of a DNA segment. Nucleotide sequence analysis of the region flanking the split transporter gene revealed two imperfect septamer direct repeats, 5'-CTCA(A/G)CA-3'. This repeated sequence might arise from a duplication event following integration of a large DNA segment of 34,539 bp spanning positions 1,099,626 to 1,134,164 of the N. meningitidis genome strain MC58 (22). A schematic representation of this DNA region is shown in Fig. 1. This ~35-kb DNA region includes 46 ORFs, most of which have been recently annotated (22), whereas 5 additional ORFs, named NB1 to NB5 (for new in serogroup B) (Fig. 1), have been identified, including previously unseen duplicated genes (see below).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Schematic representation of gene organization in prophage MuMenB of N. meningitidis strain MC58. For each gene, arrows indicate the direction of transcription and are scaled according to gene length. Numbers above arrows correspond to TIGR annotation (the suffix NMB has been omitted for simplicity in all cases). Newly annotated ORFs are marked NB1 to NB5 (for new in serogroup B). Putative functional assignments and correspondences to Mu homologues are reported below the arrows. Hypothetical sources of genes are color coded as indicated below the map. Hatched colored arrows represent genes for which source assignment may fall into two categories.

Similarities of the deduced MuMenB gene products to known sequences and functional assignments. By comparing the genetic map of phage Mu and the genetic map of the newly identified phage MuMenB (Fig. 2), we observed a certain degree of resemblance in the number of ORFs, their amino acid length, and map positions between the two phages. Therefore, the amino acid sequences of the products deduced from the MuMenB ORFs were screened for similarities with sequences from the available databases and between each pair of the corresponding proteins from the two phages. The basic characteristics of the predicted gene products and the significant homologies found that allowed hypothetical functional assignments are described below and summarized in Table 1.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   Pairwise comparison between the structure of Mu (36,717 bp) and the indicated related Mu-like bacteriophages MuMenB (34,538 bp), PNM1 (39,237 bp), PNM2 (29,321 bp), and MuHi (34,339 bp). Green boxes highlight a group of ORFs specifically acquired by neisserial prophages, whereas yellow spaces indicate genes, which either have been inserted or differ from the corresponding region of Mu. Color codes are as described in the legend to Fig. 1 and on Fig. 1 itself.

(i) Early region. Only 4 (NMB1078, NMB1080, NMB1081, and NMB1083) out of 19 early functions are conserved between MuMenB and Mu bacteriophage genomes (Fig. 1; Table 1). Probably, the missing early functions have been lost during or after phage integration. By contrast, the map positions and orientation of transcription of the conserved genes and of the downstream genes are identical between the two phages.

(ii) Middle region. A region of about 6 kb that includes 14 ORFs (NMB1084 to NMB1094 and NB1 to NB4) separates hypothetical early and late Mu-related functions. Of these 14 ORFs, NMB1092 shows homology (34% identity on 70 amino acids) to the Sid protein from phage phi-R73 (Fig. 1; Table 1). This protein has been suggested to function as determining head size with a DNA binding activity (21). While ORF NB4 and NMB1084 show similarity to proteins encoded by plasmid-borne genes, the other ORFs detected in this DNA region show no amino acid similarity to known protein. Moreover, it is worth noting that, whereas ORFs NB1 and NB2 are identical to ORFs NMB0988 and NMB0989, respectively, NB3 has a point mutation compared to NMB0990. Therefore, these three ORFs represent duplicated genes with one copy on the bacterial genome and one copy on the phage genome.

(iii) Late region. The major number of conserved functions between the MuMenB and Mu phages are mainly related to late functions such as to head assembly and to virion morphogenesis and tail proteins (Fig. 1; Table 1). This region spans ORFs NMB1095 to NMB1115. With the exception of ORFs NMB1099, which codes for the IS1655 Tnp transposase, and NMB1107 (of unknown function), the map gene order and direction of transcription parallel those of phage Mu.

Comparison of MuMenB with prophages in group A meningococcus and H. influenzae. We compared the genetic structure of the Mu functions with those of the two major regions PNM1 and PNM2 of the N. meningitidis serogroup A strain Z2491, which encode putative phage functions (17) as well as with the region coding for phage functions in H. influenzae strain Rd (5), which we call MuHi. Surprisingly, search for a similar region on the partial genomic sequence of N. gonorrhoeae strain FA1090 (http://www.genome.ou.edu/gono.html) revealed no corresponding clusters of Mu-related functions. Accordingly, it has been recently reported (12), that a region corresponding to phage PNM1 of N. meningitidis serogroup A represents a specific genetic island missing in N. gonorrhoeae.

Horizontally acquired regions. Horizontal transfer of DNA between species is well documented and is often associated with evolution of pathogenicity and drug resistance (6, 13). Bacteriophages may play an important role in acquisition of new genetic information, acting either as carriers of DNA fragments or as specialized systems for virulence-related genes (3, 4). Regions of DNA that have been acquired by horizontal transfer are often characterized by atypical DNA composition relative to the rest of the genome. One example is the cytotoxin-converting phage phi-CTX of P. aeruginosa (9). This phage shows an extensive homology to and a gene arrangement similar to that of coliphage P2 and P2-related Mu-like phages, and it carries the cytotoxin gene coding for the pore-forming toxin inserted within a region of atypical nucleotide content (14). Therefore, G+C composition study was used to identify recently acquired regions within neisserial prophages with results reported in Fig. 3.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   G+C content plots derived for neisserial prophages. The x and y axes report base positions and percentage of G+C, respectively. Arrows indicate peaks of atypical G+C composition along with the ORFs encoded therein. The graphs were obtained by using the computer programs WINDOWS and STATPLOT of the GCG Package.

Some genes acquired by MuMenB encode surface-exposed antigens. The mosaic genetic architecture of the neisserial phages indicates the existence of acquired conserved genes as well as of genes unique to each phage with hypothetical assigned or unknown functions (Fig. 1, Fig. 2, and Table 1). We selected three ORFs mapping to different positions in the phage genome for further characterization. ORF NB3 is in common with the three phages and maps within the region of conserved ORFs into the hypervariable early-middle region (Fig. 2); it was originated by gene duplication from the genome, and it may code for an outer membrane protein (Table 2). NMB1107 is likely to represent a recently acquired gene (Fig. 3), it is MuMenB specific (Fig. 2), and it may code for a lipoprotein (Table 1). NMB1119 is in common to MuMenB and PNM1 and has a homolog in PNM2 (27% amino acid identity); it maps within the 3' end of the genomes, with features similar to a phage function different from that of Mu, and it may code for a tail assembly protein. These proteins are likely to be membrane associated on the bacterial envelope.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   FACS analyses showing binding of polyclonal NB3, NMB1107, and NMB1119 antisera to the ethanol-treated homologous strain 2996. Gray profiles show binding of preimmune sera; white profiles show binding of immune sera.

Conclusions. We have reported the identification of chromosomal DNA regions of N. meningitidis strains that represent remnants of a Mu-like phage infection. Likely, this phage originally infected the bacterium and subsequently acquired specific genes to spread itself among a population of different strains. This is supported by the observation that a few of these genes are duplicated genes with one copy still residing within the bacterial chromosome. By contrast, some of the acquired genes seem to be unique to a given strain, thus suggesting a peculiar function in the host strain. Interestingly, computer search and comparison analyses suggest that both specific and common genes might code for membrane-associated proteins. This suggests that these proteins contribute to the variability in envelope structure and composition and may influence virulence and pathogenicity. Also, three of these proteins can be added to the list of vaccine candidates recently discovered among the meningococcal genome by Pizza and coworkers (19).