Abstract

A repetitive DNA motif was used as a marker to identify novel genes in the mucosal pathogen Moraxella catarrhalis. There is a high prevalence of such repetitive motifs in virulence genes that display phase variable expression. Two repeat containing loci were identified using a digoxigenin-labelled 5′-(CAAC)6-3′ oligonucleotide probe. The repeats are located in the methylase components of two distinct type III restriction–modification (R–M) systems. We suggest that the phase variable nature of these R–M systems indicates that they have an important role in the biology of M. catarrhalis.

Introduction

Moraxella catarrhalis is a causative agent of otitis media in children and can act as an opportunistic pathogen in adults with predisposing lung disease. M. catarrhalis is also carried asymptomatically in the respiratory tract of a subset of the human population [1]. The molecular mechanisms of M. catarrhalis that enable this carriage, and those which promote the transition between commensalism and virulence, are not yet fully understood.

Phase variation, the high frequency reversible on/off switching of phenotypic expression, is a feature of many virulence determinants [2]. Phase variation may be mediated by simple repetitive DNA motifs (e.g., mono-, di-, tetra-, penta-nucleotide repeats) that exhibit high mutation rates by loss or gain of repeat units during replication [3,4]. Oligonucleotide repeats are associated with numerous phase variable virulence determinants of pathogenic bacteria, such as adhesins (e.g., Opa proteins of Neisseria spp. [5,6]), iron acquisition systems (e.g., haemoglobin binding proteins of Haemophilus influenzae[7,8]) and lipopolysaccharide (LPS) biosynthesis genes (e.g., in Neisseria spp. and Haemophilus spp. [9–11]). Indeed the presence of such oligonucleotide repeats has been used to identify novel genes encoding virulence determinants [7,12].

The tetranucleotide repeat motif 5′-(CAAC)n-3′ is associated with phase variable LPS biosynthesis genes in H. influenzae[7]. The same motif is present in M. catarrhalis, but the identity of the associated genes was not known [13]. On the basis that many virulence genes contain repetitive elements, we used the 5′-(CAAC)n-3′ motif as a marker for the identification of genes predicted to be involved in the virulence of M. catarrhalis. Here we present the identification and analysis of the genes containing these 5′-(CAAC)n-3′ repeats in M. catarrhalis.

Materials and methods

Bacterial strains and culture conditions

M. catarrhalis strains used in this study were ATCC strains 8913, 23246, 25238 and 25839, and clinical isolates IB28, IC23 and IDT5 (isolated from patients with middle ear infections from the Royal Brisbane Hospital, Qld, Australia). M. catarrhalis strains were grown overnight on brain heart infusion (Oxoid, Basingstoke, UK) 1% agar plates supplemented with 10% Levinthal's base [14] at 37°C in 5% CO2. Escherichia coli strain DH5a[15] was grown overnight in Luria–Bertani (LB) broth or on LB plates containing 1.5% bacteriological agar (Difco, Detroit, MI, USA) at 37°C. Ampicillin was used at a final concentration of 100 µg ml−1.

Southern blot analysis and hybridisation

Miniprep of bacterial genomic DNA was performed as described by Ausubel et al. [16]. Restriction endonuclease (Apo I, Cla I, Hin cII or Mfe I) digested genomic DNA was separated on 0.7% agarose gels and transferred to GeneScreen Plus® Hybridisation Transfer membrane (NEN™ Life Science Products, Boston, MA, USA) by capillary action essentially as described in Sambrook et al. [15]. Hybridisation with the digoxigenin (DIG)-labelled 5′-(CAAC)6-3′ oligonucleotide probe (Genset) was carried out for 16 h at 50°C. Washes and detection were carried out (using the DIG DNA Labelling and Detection Kit, Boehringer Mannheim, Indianapolis, IN, USA) as recommended by the manufacturer. All restriction endonucleases and ligases were obtained from New England Biolabs (NEB), Beverly, MA, USA.

Recombinant DNA library construction and screening

A plasmid library was constructed from Cla I/Mfe I digested M. catarrhalis 23246 genomic DNA cloned into Acc I/Eco RI digested pUC19 (NEB). The library was screened via colony hybridisation using the DIG-labelled 5′-(CAAC)6-3′ oligonucleotide probe. Colonies were transferred to autoclaved GeneScreen Plus® Hybridisation Transfer membrane (NEN™ Life Science) by placing the membrane disc onto the surface of the agar plate for 1 min. The membranes (colony side up) were placed on Whatman 3MM filter paper soaked with: denaturation solution (15 min), neutralisation solution (15 min), then 2×saline sodium citrate (SSC) (10 min). Cell debris was removed by gentle agitation in 2×SSC. Hybridisation, washes and detection were carried out as described above.

Recombinant DNA techniques and nucleotide sequence analysis

Most recombinant DNA techniques were as described in Ausubel et al. [16]. Plasmid DNA was isolated from clones using the Qiagen Plasmid Midi Kit (Qiagen, Chatsworth, CA, USA) and sequenced on both strands in full (Prism Dye Terminator Sequencing Kit with AmpliTaq DNA polymerase FS (Perkin Elmer, Norwalk, CT, USA) in conjunction with a model 377 automated sequencer (Applied Biosystems, Foster City, CA, USA)) using vector specific primers and by primer walking. Further sequence was obtained via inverse polymerase chain reaction (PCR) and using a degenerate primer designed from a conserved region in homologous genes. Oligonucleotide primers were synthesized by GeneWorks Pty Ltd. PCR was essentially carried out as described by Saiki et al. [17]. Nucleotide sequence analysis was carried out using MacVector (Oxford Molecular Ltd) and BLASTX [18]. For comparisons in Table 1 the entire amino acid sequence of each methylase protein was compared, and the first 475 (McaRI) and 430 (McaRII) amino acids of the restriction enzymes were compared, using the ClustalW program (Oxford Molecular Ltd).

Table 1

Comparison of (A) type III methylase (mod) genes and (B) type III restriction endonuclease (res) genes of several bacterial pathogens, shown as % similarity (identity)

 Hp Hi Nm2 Nm1 Ph McaRII 
McaRI 37 (26) 35 (22) 30 (19) 41 (27) 52 (36) 30 (19) 
McaRII 27 (17) 43 (28) 51 (36) 31 (19) 29 (17)  
Ph 35 (21) 31 (17) 29 (16) 36 (21)   
NmI 40 (25) 32 (17) 29 (16)    
NmII 26 (15) 55 (48)     
Hi 29 (15)      
McaRI 46 (28) 33 (17) 33 (17) 37 (20) 68 (49) 26 (12) 
McaRII 24 (12) 36 (19) 37 (20) 21 (10) 28 (11)  
Ph 44 (25) 32 (14) 32 (14) 35 (20)   
NmI 37 (24) 24 (13) 22 (11)    
NmII 30 (13) 92 (90)     
Hi 30 (13)      
 Hp Hi Nm2 Nm1 Ph McaRII 
McaRI 37 (26) 35 (22) 30 (19) 41 (27) 52 (36) 30 (19) 
McaRII 27 (17) 43 (28) 51 (36) 31 (19) 29 (17)  
Ph 35 (21) 31 (17) 29 (16) 36 (21)   
NmI 40 (25) 32 (17) 29 (16)    
NmII 26 (15) 55 (48)     
Hi 29 (15)      
McaRI 46 (28) 33 (17) 33 (17) 37 (20) 68 (49) 26 (12) 
McaRII 24 (12) 36 (19) 37 (20) 21 (10) 28 (11)  
Ph 44 (25) 32 (14) 32 (14) 35 (20)   
NmI 37 (24) 24 (13) 22 (11)    
NmII 30 (13) 92 (90)     
Hi 30 (13)      

McaRI, M. catarrhalis (AY049056); McaRII, M. catarrhalis (AY049057); Ph, Pasteurella haemolytica (AF060119) [48]; NmI, Neisseria meningitidis Z2491 (NMA1467, CAB84700) [49]; NmII, N. meningitidis Z2491 (CAB84817, CAB84818) [49]; Hi, H. influenzae Rd (AAC22721, AAC22720) [50]; Hp, Helicobacter pylori 26695 (AAD07659, AAD07657) [51].

The 5′-(CAAC)n-3′ associated loci of the seven M. catarrhalis strains were amplified from single colonies by PCR using the primers MCA3 (5′-AGCGGTGAAAGCAGTGCGTG-3′) and MCA4 (5′-CCTGTGGCAATTTCATAGTC-3′). Products of approximately 684 bp were sequenced to determine the number of repeat units present in each strain. Primers used for inverse PCR reactions include: MCA2 (5′-TAAGAGCCCATGTGATCGGC-3′), MCA7 (5′-TAATGGCGTTTATTGGCTAAC-3′) and MCA10 (5′-TCCTGTACAAGCATTTGAGC-3′) for Mca RI res, and MCB5 (5′-AGGTAGTGGAACAACAGCTC-3′) and MCB7 (5′-TGCCAACACTTGCTGTCAGG-3′) for Mca RII res. The degenerate primer RE1 (5′-TTGGGRTTRTCCCAGCCTTC-3′) for PCR was designed from a conserved region of type III restriction enzyme genes from Pasteurella haemolytica, Neisseria meningitidis and Helicobacter pylori.

Results

We have previously reported the presence of two loci containing the 5′-(CAAC)n-3′ tetranucleotide repeat in M. catarrhalis ATCC strain 8913 by Southern blot analysis using 32P-labelled oligonucleotide probes [13]. These probes were designed from tracts of repetitive DNA identified in the H. influenzae genome [7]. To investigate the presence of 5′-(CAAC)n-3′ repeats in other M. catarrhalis strains, a collection of strains was surveyed by Southern blot analysis using a DIG-labelled 5′-(CAAC)6-3′ oligonucleotide probe (Fig. 1). This survey revealed the presence of repeats in all seven of the M. catarrhalis strains tested, with six of the seven strains containing at least two 5′-(CAAC)n-3′ repeat associated loci.

Figure 1

Distribution of 5′-(CAAC)n-3′ repeats in various M. catarrhalis strains. Southern blot of M. catarrhalis chromosomal DNA restriction digests hybridised with the DIG-labelled oligonucleotide 5′-(CAAC)6-3′ probe. Each M. catarrhalis ATCC strain is digested with Hin cII, Cla I, Mfe I, and Apo I respectively. Lanes 1–4, strain 8913; 5–8, strain 23246; 9–12, strain 25238; 13–16, strain 25839. Molecular mass standards are indicated in kb.

Figure 1

Distribution of 5′-(CAAC)n-3′ repeats in various M. catarrhalis strains. Southern blot of M. catarrhalis chromosomal DNA restriction digests hybridised with the DIG-labelled oligonucleotide 5′-(CAAC)6-3′ probe. Each M. catarrhalis ATCC strain is digested with Hin cII, Cla I, Mfe I, and Apo I respectively. Lanes 1–4, strain 8913; 5–8, strain 23246; 9–12, strain 25238; 13–16, strain 25839. Molecular mass standards are indicated in kb.

To further investigate the repeat associated loci, a plasmid library derived from genomic DNA of M. catarrhalis strain 23246 was screened using the 5′-(CAAC)6-3 probe (see Section 2). Two hybridising clones were isolated from this library, pMcrepI and pMcrepII (see Fig. 2). The cloned regions were sequenced and homology searches were performed. Both clones contain 5′-(CAAC)n-3′ repeats within the 5′-end of open reading frames (ORFs) of two distinct genes encoding methylases of type III R–M systems (see below). Inverse PCR and PCR with degenerate primers were used to isolate additional flanking sequences required to determine the sequence of the methylase (mod) genes and associated downstream restriction endonuclease (res) genes. These ORFs were designated Mca RI mod and res, and Mca RII mod and res. The sequenced regions are displayed in Fig. 2 and are deposited in GenBank under the accession numbers AY049056 and AY049057.

Figure 2

Schematic representation of (A) the Mca RI and (B) the Mca RII type III R–M systems identified in M. catarrhalis. The lines labelled Mc 23246 represent the double stranded sequence obtained from the M. catarrhalis ATCC strain 23246 (GenBank entries AY049056, AY049057). The arrows above the line indicate the location, orientation and predicted function of the ORFs identified in the sequence. The location of the 5′-(CAAC)n-3′ repeats are shown above the methylase ORF. The pMcrepI and pMcrepII lines represent the clones obtained from the Cla I/Mfe I genomic library screened with the 5′-(CAAC)6-3′ oligonucleotide probe. The vector backbone for these plasmids (pUC19) is represented by a box. The sequence obtained by PCR and inverse PCR is indicated and the primers used are represented by arrowheads.

Figure 2

Schematic representation of (A) the Mca RI and (B) the Mca RII type III R–M systems identified in M. catarrhalis. The lines labelled Mc 23246 represent the double stranded sequence obtained from the M. catarrhalis ATCC strain 23246 (GenBank entries AY049056, AY049057). The arrows above the line indicate the location, orientation and predicted function of the ORFs identified in the sequence. The location of the 5′-(CAAC)n-3′ repeats are shown above the methylase ORF. The pMcrepI and pMcrepII lines represent the clones obtained from the Cla I/Mfe I genomic library screened with the 5′-(CAAC)6-3′ oligonucleotide probe. The vector backbone for these plasmids (pUC19) is represented by a box. The sequence obtained by PCR and inverse PCR is indicated and the primers used are represented by arrowheads.

The Mca RI mod ORF was sequenced in full and is 1911 nucleotides (nucleotide position (ntp) 1755–3665 of GenBank entry AY049056). The predicted protein (M.Mca RI) (637 amino acids (aa), calculated molecular mass of 72.7 kDa and estimated pI of 5.56) displays a high degree of similarity to the P. haemolytica type III methylase (Table 1). The P. haemolytica methylase also contains a repetitive tract (5′-(CACAG)24-3′) in its 5′-end. Immediately downstream of Mca RI mod is a second ORF, Mca RI res, that was sequenced in part (1427 nucleotides starting at ntp 3681 of GenBank entry AY049056). The predicted 475 aa translation displays similarity to the P. haemolytica type III restriction endonuclease (879 aa) (Table 1). The G+C contents of the ORFs are 33% for Mca RI mod and res, and 50% for the predicted aminotransferase located upstream of the R–M system. These figures are significantly different for adjacent genes and vary from the genome averages cited for M. catarrhalis (40–43%) [19].

Nucleotide sequence analysis of Mca RI mod from various sources (the initial clone, various subclones, and a PCR amplified section of chromosomal DNA) revealed the presence of 20–22 copies of the 5′-(CAAC)-3′ repeat unit. The predicted start codon, the 5′-(CAAC)n-3′ repeats and the methylase gene are in-frame when there are 20 repeat units; however, addition or deletion of one or two repeat units is predicted to result in premature truncation of the putative protein.

The Mca RI mod repeat region from the seven M. catarrhalis strains surveyed by Southern blot were amplified by PCR and sequenced (Table 2). The repeats are associated with a methylase in all strains and the number of repeats ranged from 19 to 44. Expression of the predicted type III methylase is possible in only four of the seven strains due to a premature stop codon in two reading frames after the repeats. Alteration in the number of repeat units will affect expression in all strains.

Table 2

Nucleotide sequence and predicted translation of the Mca RI mod 5′-(CAAC)n-3′ repetitive tract from seven M. catarrhalis strains

graphic
 
graphic
 

The second 5′-(CAAC)n-3′ repetitive tract in M. catarrhalis 23246 is also located within the 5′-end of an ORF homologous to methylases of type III R–M systems (Table 1). The Mca RII mod ORF was sequenced in full and is 1632 nucleotides (ntp 371–2002 of GenBank entry AY049057) and the predicted translation is similar to a Mycoplasma pulmonis type III restriction endonuclease over 430 aa. The repetitive DNA tract of Mca RII mod contains 18 copies of the 5′-(CAAC)-3′ unit. Addition or deletion of one or two units is predicted to result in premature truncation of the putative protein. In addition, two homopolymeric tracts containing eight deoxyadenylate residues are present, with a frameshift mutation seen after the second polyA tract. The G+C content of Mca RII mod and res is 35%.

The putative proteins described above contain regions conserved in the family of type III R–M systems (see Fig. 3). Type III methylases contain a motif, I-Y-I-D-P-P-Y, involved in the transfer of a methyl group to the N-6 adenine (the amino group at the C-6 position of adenines) in the DNA recognition sequence [20]. The predicted amino acid sequences of M.Mca RI and M.Mca RII both contain this sequence. A slight variation of the F-x-G-x-G motif, involved in binding the methyl donor S-adenosylmethionine (AdoMet) [20,21], is also found in M.Mca RI (F-A-G-S-A) and M.Mca RII (H-A-G-S-G). Mca RI and Mca RII contain the first of seven motifs, T-G-T-G-K-T, conserved in type I and type III restriction endonucleases [22]. This motif is characteristic of the helicase superfamily II and is proposed to be an ATP binding motif responsible for helicase activity [22] and/or DNA translocation [20].

Figure 3

Schematic representation of repetitive DNA within potentially phase variable type III R–M systems. The methylase (mod) genes, restriction endonuclease (res) genes and the repeat regions are indicated. McaRI, M. catarrhalis (AY049056); McaRII, M. catarrhalis (AY049057); Ph, P. haemolytica (AF060119) [48]; NmI, N. meningitidis Z2491 (NMA1467, CAB84700) [49]; NmII, N. meningitidis Z2491 (CAB84817, CAB84818) [49]; Hi, H. influenzae Rd (AAC22721, AAC22720) [50]; Hp, H. pylori 26695 (AAD07659, AAD07657) [51]. *These genes are also found in N. meningitidis MC58 (NMB1261, AAF41638; NMB1375) [52] and Neisseria gonorrhoeae (University of Oklahoma's Advanced Centre for Genome Technology, OK, USA). Conserved regions within type III R–M systems are also shown. The DPPY motif is involved in catalysis of the methylation reaction [20], the FxGxG motif is involved in binding of the methyl donor (AdoMet) [20,21], and the TGxGKT motif is involved in ATP binding [20,53].

Figure 3

Schematic representation of repetitive DNA within potentially phase variable type III R–M systems. The methylase (mod) genes, restriction endonuclease (res) genes and the repeat regions are indicated. McaRI, M. catarrhalis (AY049056); McaRII, M. catarrhalis (AY049057); Ph, P. haemolytica (AF060119) [48]; NmI, N. meningitidis Z2491 (NMA1467, CAB84700) [49]; NmII, N. meningitidis Z2491 (CAB84817, CAB84818) [49]; Hi, H. influenzae Rd (AAC22721, AAC22720) [50]; Hp, H. pylori 26695 (AAD07659, AAD07657) [51]. *These genes are also found in N. meningitidis MC58 (NMB1261, AAF41638; NMB1375) [52] and Neisseria gonorrhoeae (University of Oklahoma's Advanced Centre for Genome Technology, OK, USA). Conserved regions within type III R–M systems are also shown. The DPPY motif is involved in catalysis of the methylation reaction [20], the FxGxG motif is involved in binding of the methyl donor (AdoMet) [20,21], and the TGxGKT motif is involved in ATP binding [20,53].

The sequence similarity (Table 1) and the presence of the tetranucleotide repeats within the predicted coding regions suggest that these loci represent two novel, phase variable type III R–M systems. This is supported by the fact that the reading frame of the methylase gene is influenced by the number of repeat units present, and that the number of repeats contained in Mca RI mod varies in number both within and between strains.

Discussion

We have identified and cloned two novel, potentially phase variable type III R–M systems in M. catarrhalis. This report adds to the growing list of repeat-containing, potentially phase variable methylases within type III R–M systems (Fig. 3). In addition, P. haemolytica contains a type I R–M system with 5′-(CACAG)n-3′ repeats [23,24], and M. pulmonis has a phase variable type I system mediated by site-specific inversion [25,26].

R–M systems are ubiquitous in bacteria and are traditionally described as simple immune systems that protect the host against infection by foreign DNA [27], including phage DNA (although R–M systems may protect incompletely, or have an ‘ephemeral’ role in protection) [28–30]. The distinct methylation pattern distinguishes ‘self’ from ‘non-self’ DNA, and incoming DNA is subjected to endonucleolytic cleavage. In this context, the advantage of phase variable R–M systems is not obvious. The prevalence of phase variable methylases and R–M systems in bacteria suggests that important, hitherto unrecognised functions are being fulfilled. There are possible implications for several cellular processes such as inter- and intra-species transformation and genetic regulation. A role in pathogenicity has also been suggested [29,31].

R–M systems are classified into three groups (type I, II and III) based on differences in the subunit structures of their enzymes, cofactor requirements, recognition sites and enzymatic mechanisms (see [27,32] for reviews of R–M systems). The components of type III R–M systems catalyse two distinct reactions: (1) the modification enzyme/methylase (Mod) is required for sequence recognition in both modification and restriction reactions, and catalyses the post-replicative addition of a methyl group to an adenine residue in a specific DNA sequence; (2) the cognate restriction endonuclease (Res) recognises the same sequence and catalyses double stranded cleavage of unmethylated foreign DNA in the presence of the mod gene product [33,34]. Type I R–M systems similarly require a complex of the hsd RMS gene products (the restriction, modification and specificity subunits) for endonuclease activity. In contrast, the restriction and modification proteins of type II R–M systems act independently of each other.

The requirement for protection against autodegradation and the consequent linkage of genes of R–M systems has led to type II R–M systems being considered examples of ‘selfish genes’ [35]. Several authors have suggested that phase variation of methylases may lead to autolytic self-DNA degradation by the cognate restriction enzyme and that such systems may be suicidal [12,29]. More specifically, it has been suggested “for bacterial species possessing natural transformation systems the induction of phase variable restriction activity may be part of an autolysis process that releases DNA into the environment for uptake by other cells” [29]. Saunders et al. [12] suggest that a population gains a selective advantage through “‘bacterial suicide’ by a proportion of that population”. These observations lead to the conclusion that such a system comprises “a remarkably ‘unselfish gene’” [12] and that “the phase variable nature of the system strongly argues against the selfish behaviour hypothesis” [29]. When the known biology of restriction systems is reviewed (see above), it is obvious that type I and type III restriction subunits are not active in isolation. If a type I or type III methylase gene is not expressed (switched off by phase variation), the resulting phenotype would be a non-functional R–M system. If during replication a type III R–M system is switched on again, then cellular conditions are such that methylation is favoured over restriction [36]. Thus for type I and type III R–M systems, phase variation of a methylase subunit will not lead inevitably to suicidal self-restriction: the repeat-containing R–M systems identified to date are of the type I and type III families.

Many of the organisms in which phase variable R–M systems have been identified are naturally transformable [37]. In the case of N. meningitidis, N. gonorrhoeae and H. influenzae, DNA uptake is predominantly confined to double stranded DNA containing genus-specific uptake sequences, and is the primary route of genetic exchange. N. meningitidis and N. gonorrhoeae utilise intergenomic exchange as a mechanism to generate antigenic variation of pilin (type 4 fimbriae) [38]. In addition, the mosaic structure of many genes in these bacteria indicates a history of recombination and implies integration of short DNA fragments. Methylation states have been shown to affect transformation efficiency [39,40] and the on/off switching of methylation would ensure that at any point a proportion of the population would have a different methylation pattern to the majority. The consequent uptake of this differentially methylated DNA would result in restriction and provide substrate for one or a few small insertions, without the obvious potential consequences of large-scale integration of ‘non-self’ DNA. Similarly, DNA from heterologous sources will be restricted. Conversely, if incoming DNA contains the same methylation pattern, this DNA will not be restricted and larger fragments may be incorporated, potentially contributing to larger scale genomic recombination, and possibly rearrangement. Such large-scale rearrangement has been noted for N. gonorrhoeae[41]. By analogy to the (not naturally competent) species E. coli, R–M system differences (and presumably differential methylation) have been implicated in size of DNA replacements in P1 transduction experiments ([42] and references therein). Hence, R–M systems may operate as a mechanism for producing many ‘recombinogenic ends’ [43].

Another possible role for phase variable R–M systems is in providing an additional layer of gene regulation. Regulation of virulence determinant expression by methylation has been well documented. Dam methylation affects Pap pili [44] and Ag43 [28] expression in E. coli, virulence gene expression in Salmonella typhimurium[45] and the rate of phase variation of capsular polysaccharide expression in N. meningitidis[46] (this is not seen in all phase variable systems in N. meningitidis and is not the result of mismatch repair deficiencies [47]). It has also been suggested that phase variation of R–M activity is associated with antigenic variation in M. pulmonis[25,26].

Acknowledgements

Work in M.P.J.'s laboratory is supported by the NHMRC. K.L.S. is supported by an Australian Postgraduate Award.

References

[1]
Enright
M.C.
McKenzie
H.
(
1997
)
Moraxella (Branhamella) catarrhalis — clinical and molecular aspects of a rediscovered pathogen
.
J. Med. Microbiol.
 
46
,
360
371
.
[2]
Robertson
B.D.
Meyer
T.F.
(
1992
)
Genetic variation in pathogenic bacteria
.
Trends Genet.
 
8
,
422
427
.
[3]
Streisinger
G.
Okada
Y.
Emrich
J.
Newton
J.
Tsugita
A.
Terzaghi
E.
Inouye
M.
(
1966
)
Frameshift mutations and the genetic code
.
Cold Spring Harbor Symp. Quant. Biol.
 
31
,
77
84
.
[4]
Levinson
G.
Gutman
G.A.
(
1987
)
Slipped-strand mispairing: a major mechanism for DNA sequence evolution
.
Mol. Biol. Evol.
 
4
,
203
221
.
[5]
Stern
A.
Meyer
T.F.
(
1987
)
Common mechanism controlling phase and antigenic variation in pathogenic neisseriae
.
Mol. Microbiol.
 
1
,
5
12
.
[6]
Stern
A.
Brown
M.
Nickel
P.
Meyer
T.F.
(
1986
)
Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation
.
Cell
 
47
,
61
71
.
[7]
Hood
D.W.
Deadman
M.E.
Jennings
M.P.
Bisercic
M.
Fleischmann
R.D.
Venter
J.C.
Moxon
E.R.
(
1996
)
DNA repeats identify novel virulence genes in Haemophilus influenzae
.
Proc. Natl. Acad. Sci. USA
 
93
,
11121
11125
.
[8]
Ren
Z.
Jin
H.
Whitby
P.W.
Morton
D.J.
Stull
T.L.
(
1999
)
Role of CCAA nucleotide repeats in regulation of hemoglobin and hemoglobin–haptoglobin binding protein genes of Haemophilus influenzae
.
J. Bacteriol.
 
181
,
5865
5870
.
[9]
Weiser
J.N.
Love
J.M.
Moxon
E.R.
(
1989
)
The molecular mechanism of phase variation of H. influenzae lipopolysaccharide
.
Cell
 
59
,
657
665
.
[10]
Jarosik
G.P.
Hansen
E.J.
(
1994
)
Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide
.
Infect. Immun.
 
62
,
4861
4867
.
[11]
Jennings
M.P.
Srikhanta
Y.N.
Moxon
E.R.
Kramer
M.
Poolman
J.T.
Kuipers
B.
van der Ley
P.
(
1999
)
The genetic basis of the phase variation repertoire of lipopolysaccharide immunotypes in Neisseria meningitidis
.
Microbiology
 
145
,
3013
3021
.
[12]
Saunders
N.J.
Peden
J.F.
Hood
D.W.
Moxon
E.R.
(
1998
)
Simple sequence repeats in the Helicobacter pylori genome
.
Mol. Microbiol.
 
27
,
1091
1098
.
[13]
Peak
I.R.
Jennings
M.P.
Hood
D.W.
Bisercic
M.
Moxon
E.R.
(
1996
)
Tetrameric repeat units associated with virulence factor phase variation in Haemophilus also occur in Neisseria spp. and Moraxella catarrhalis
.
FEMS Microbiol. Lett.
 
137
,
109
114
.
[14]
Alexander
H.E.
(
1965
)
The Haemophilus group
. In:
Bacterial and Mycotic Infections of Man
  (
Dabos
R.J.
Hirsch
J.G.
, Eds.), pp.
724
741
.
Pitman
,
London
.
[15]
Sambrook
J.
Fritsch
E.F.
Maniatis
T.
(
1989
)
Molecular Cloning: A Laboratory Manual
 ,
2
nd edn.
Cold Spring Harbor Laboratory
,
Cold Spring Harbor, NY
.
[16]
Ausubel
F.M.
Brent
R.
Kingston
R.E.
Moore
D.D.
Seidman
J.G.
Smith
J.A.
Struhl
K.
(Eds.) (
1997
)
Short Protocols in Molecular Cloning
 .
John Wiley and Sons
,
New York
.
[17]
Saiki
R.K.
Gelfand
D.H.
Stoffel
S.
Scharf
S.J.
Higuchi
R.
Horn
G.T.
Mullis
K.B.
Erlich
H.A.
(
1988
)
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase
.
Science
 
239
,
487
491
.
[18]
Altschul
S.F.
Madden
T.L.
Schaffer
A.A.
Zhang
J.
Zhang
Z.
Miller
W.
Lipman
D.J.
(
1997
)
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
.
Nucleic Acids Res.
 
25
,
3389
3402
.
[19]
Catlin
B.W.
(
1990
)
Branhamella catarrhalis: an organism gaining respect as a pathogen
.
Clin. Microbiol. Rev.
 
3
,
293
320
.
[20]
Saha
S.
Ahmad
I.
Reddy
Y.V.
Krishnamurthy
V.
Rao
D.N.
(
1998
)
Functional analysis of conserved motifs in type III restriction–modification enzymes
.
Biol. Chem.
 
379
,
511
517
.
[21]
Cheng
X.
Kumar
S.
Posfai
J.
Pflugrath
J.W.
Roberts
R.J.
(
1993
)
Crystal structure of the Hha I DNA methyltransferase complexed with S-adenosyl-L-methionine
.
Cell
 
74
,
299
307
.
[22]
Gorbalenya
A.E.
Koonin
E.V.
(
1991
)
Endonuclease (R) subunits of type-I and type-III restriction–modification enzymes contain a helicase-like domain
.
FEBS Lett.
 
291
,
277
281
.
[23]
Highlander
S.K.
Garza
O.
(
1996
)
The restriction–modification system of Pasteurella haemolytica is a member of a new family of type I enzymes
.
Gene
 
178
,
89
96
.
[24]
Highlander
S.K.
Hang
V.T.
(
1997
)
A putative leucine zipper activator of Pasteurella haemolytica leukotoxin transcription and the potential for modulation of its synthesis by slipped-strand mispairing
.
Infect. Immun.
 
65
,
3970
3975
.
[25]
Bhugra
B.
Voelker
L.L.
Zou
N.
Yu
H.
Dybvig
K.
(
1995
)
Mechanism of antigenic variation in Mycoplasma pulmonis: interwoven, site-specific DNA inversions
.
Mol. Microbiol.
 
18
,
703
714
.
[26]
Dybvig
K.
(
1993
)
DNA rearrangements and phenotypic switching in prokaryotes
.
Mol. Microbiol.
 
10
,
465
471
.
[27]
Bickle
T.A.
Kruger
D.H.
(
1993
)
Biology of DNA restriction
.
Microbiol. Rev.
 
57
,
434
450
.
[28]
Henderson
I.R.
Owen
P.
(
1999
)
The major phase-variable outer membrane protein of Escherichia coli structurally resembles the immunoglobulin A1 protease class of exported protein and is regulated by a novel mechanism involving Dam and oxyR
.
J. Bacteriol.
 
181
,
2132
2141
.
[29]
Dybvig
K.
Sitaraman
R.
French
C.T.
(
1998
)
A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements
.
Proc. Natl. Acad. Sci. USA
 
95
,
13923
13928
.
[30]
Dybvig
K.
Yu
H.
(
1994
)
Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis
.
Mol. Microbiol.
 
12
,
547
560
.
[31]
Gumulak-Smith
J.
Teachman
A.
Tu
A.H.
Simecka
J.W.
Lindsey
J.R.
Dybvig
K.
(
2001
)
Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats
.
Mol. Microbiol.
 
40
,
1037
1044
.
[32]
Boyer
H.W.
(
1971
)
DNA restriction and modification mechanisms in bacteria
.
Annu. Rev. Microbiol.
 
25
,
153
176
.
[33]
Hadi
S.M.
Bachi
B.
Iida
S.
Bickle
T.A.
(
1983
)
DNA restriction–modification enzymes of phage P1 and plasmid p15B. Subunit functions and structural homologies
.
J. Mol. Biol.
 
165
,
19
34
.
[34]
Iida
S.
Meyer
J.
Bachi
B.
Stalhammar-Carlemalm
M.
Schrickel
S.
Bickle
T.A.
Arber
W.
(
1983
)
DNA restriction–modification genes of phage P1 and plasmid p15B. Structure and in vitro transcription
.
J. Mol. Biol.
 
165
,
1
18
.
[35]
Nakayama
Y.
Kobayashi
I.
(
1998
)
Restriction-modification gene complexes as selfish gene entities: roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion
.
Proc. Natl. Acad. Sci. USA
 
95
,
6442
6447
.
[36]
Meisel
A.
Mackeldanz
P.
Bickle
T.A.
Kruger
D.H.
Schroeder
C.
(
1995
)
Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis
.
EMBO J.
 
14
,
2958
2966
.
[37]
Solomon
J.M.
Grossman
A.D.
(
1996
)
Who's competent and when: regulation of natural genetic competence in bacteria
.
Trends Genet.
 
12
,
150
155
.
[38]
Gibbs
C.P.
Reimann
B.Y.
Schultz
E.
Kaufmann
A.
Haas
R.
Meyer
T.F.
(
1989
)
Reassortment of pilin genes in Neisseria gonorrhoeae occurs by two distinct mechanisms
.
Nature
 
338
,
651
652
.
[39]
Butler
C.A.
Gotschlich
E.C.
(
1991
)
High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor
.
J. Bacteriol.
 
173
,
5793
5799
.
[40]
Voelker
L.L.
Dybvig
K.
(
1996
)
Gene transfer in Mycoplasma arthritidis: transformation, conjugal transfer of Tn916, and evidence for a restriction system recognizing AGCT
.
J. Bacteriol.
 
178
,
6078
6081
.
[41]
Gibbs
C.P.
Meyer
T.F.
(
1996
)
Genome plasticity in Neisseria gonorrhoeae
.
FEMS Microbiol. Lett.
 
145
,
173
179
.
[42]
Milkman
R.
(
1997
)
Recombination and population structure in Escherichia coli
.
Genetics
 
146
,
745
750
.
[43]
DuBose
R.F.
Dykhuizen
D.E.
Hartl
D.L.
(
1988
)
Genetic exchange among natural isolates of bacteria: recombination within the pho A gene of Escherichia coli
.
Proc. Natl. Acad. Sci. USA
 
85
,
7036
7040
.
[44]
Nou
X.
Braaten
B.
Kaltenbach
L.
Low
D.A.
(
1995
)
Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli
.
EMBO J.
 
14
,
5785
5797
.
[45]
Heithoff
D.M.
Sinsheimer
R.L.
Low
D.A.
Mahan
M.J.
(
1999
)
An essential role for DNA adenine methylation in bacterial virulence
.
Science
 
284
,
967
970
.
[46]
Bucci
C.
Lavitola
A.
Salvatore
P.
Del Giudice
L.
Massardo
D.R.
Bruni
C.B.
Alifano
P.
(
1999
)
Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype
.
Mol. Cell
 
3
,
435
445
.
[47]
Richardson
A.R.
Stojiljkovic
I.
(
2001
)
Mismatch repair and the regulation of phase variation in Neisseria meningitidis
.
Mol. Microbiol.
 
40
,
645
655
.
[48]
Ryan
K.A.
Lo
R.Y.
(
1999
)
Characterization of a CACAG pentanucleotide repeat in Pasteurella haemolytica and its possible role in modulation of a novel type III restriction–modification system
.
Nucleic Acids Res.
 
27
,
1505
1511
.
[49]
Parkhill
J.
Achtman
M.
James
K.D.
Bentley
S.D.
Churcher
C.
Klee
S.R.
Morelli
G.
Basham
D.
Brown
D.
Chillingworth
T.
Davies
R.M.
Davis
P.
Devlin
K.
Feltwell
T.
Hamlin
N.
Holroyd
S.
Jagels
K.
Leather
S.
Moule
S.
Mungall
K.
Quail
M.A.
Rajandream
M.A.
Rutherford
K.M.
Simmonds
M.
Skelton
J.
Whitehead
S.
Spratt
B.G.
Barrell
B.G.
(
2000
)
Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491
.
Nature
 
404
,
502
506
.
[50]
Fleischmann
R.D.
Adams
M.D.
White
O.
Clayton
R.A.
Kirkness
E.F.
Kerlavage
A.R.
Bult
C.J.
Tomb
J.F.
Dougherty
B.A.
Merrick
J.M.
et al
(
1995
)
Whole-genome random sequencing and assembly of Haemophilus influenzae Rd
.
Science
 
269
,
496
512
.
[51]
Tomb
J.F.
White
O.
Kerlavage
A.R.
Clayton
R.A.
Sutton
G.G.
Fleischmann
R.D.
Ketchum
K.A.
Klenk
H.P.
Gill
S.
Dougherty
B.A.
Nelson
K.
Quackenbush
J.
Zhou
L.
Kirkness
E.F.
Peterson
S.
Loftus
B.
Richardson
D.
Dodson
R.
Khalak
H.G.
Glodek
A.
McKenney
K.
Fitzgerald
L.M.
Lee
N.
Adams
M.D.
Venter
J.C.
et al
(
1997
)
The complete genome sequence of the gastric pathogen Helicobacter pylori
.
Nature
 
388
,
539
547
.
[52]
Tettelin
H.
Saunders
N.J.
Heidelberg
J.
Jeffries
A.C.
Nelson
K.E.
Eisen
J.A.
Ketchum
K.A.
Hood
D.W.
Peden
J.F.
Dodson
R.J.
Nelson
W.C.
Gwinn
M.L.
DeBoy
R.
Peterson
J.D.
Hickey
E.K.
Haft
D.H.
Salzberg
S.L.
White
O.
Fleischmann
R.D.
Dougherty
B.A.
Mason
T.
Ciecko
A.
Parksey
D.S.
Blair
E.
Cittone
H.
Clark
E.B.
Cotton
M.D.
Utterback
T.R.
Khouri
H.
Qin
H.
Vamathevan
J.
Gill
J.
Scarlato
V.
Masignani
V.
Pizza
M.
Grandi
G.
Sun
L.
Smith
H.O.
Fraser
C.M.
Moxon
E.R.
Rappuoli
R.
Venter
J.C.
(
2000
)
Complete genome sequence of Neisseria meningitidis serogroup B strain MC58
.
Science
 
287
,
1809
1815
.
[53]
Walker
J.E.
Saraste
M.
Runswick
M.J.
Gay
N.J.
(
1982
)
Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold
.
EMBO J.
 
1
,
945
951
.