Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland
Correspondence
Andrzej Piekarowicz
anpiek{at}biol.uw.edu.pl
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ABSTRACT |
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INTRODUCTION |
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A characteristic feature of H. influenzae is the ability to switch between genetic variants of alternative phenotypes with high frequency (Hood et al., 1996b; Bayliss et al., 2002
, 2004
). The phase variation (PV) of the R-M system and resistance/sensitivity to phage infection could modulate the defence against phage infection without loss of the ability to acquire foreign DNA through horizontal transfer mediated by transformation or transfection. Previously it was shown (Glover & Piekarowicz, 1972
) that the type I R-M phenotype of H. influenzae and the gene mod, a part of the type III R-M system of H. influenzae, are subject to reversible, high-frequency ON/OFF PV (De Bolle et al., 2000
). However, nothing is known about the PV of resistance/sensitivity to phage infection.
We do not know the cell structures of H. influenzae responsible for adsorption of HP1c1 or other H. influenzae phages. Bacterial phage receptors are present in the outer-membrane proteins, or the lipopolysaccharide (LPS) (Nesper et al., 2000; Kutter et al., 2005
). In H. influenzae the particular surface-exposed epitopes of LOS are subject to high-frequency PV (Hood et al., 1996a
; Patrick et al., 1987
; Weiser et al., 1989
; Bayliss et al., 2002
; De Bolle et al., 2000
), as are the outer-membrane proteins (Bayliss et al., 2004
). PV is an adaptive mechanism that is advantageous for survival of bacteria confronted by differing microenvironments and immune responses of the host (Hood et al., 2004
).
The process of switching surface antigens (PV) is driven by highly mutable loci, called contingency loci, which generate repertoires of phenotypic variants. There are many mechanisms of hypermutation in contingency loci, but one of the commonest is mediated by changes in the number of tetranucleotide repeats within the coding sequence of a gene (Hood et al., 1996b; Moxon et al., 1994
; Bayliss et al., 2001
, 2002
). PV also involves alterations in the number of 5'-TA repeats located between the 10 and 35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate the biosynthesis and assembly of H. influenzae pili (Bayliss et al., 2004
). The rate of PV of genes containing tetranucleotide repeats is influenced by mutations in polI but not genes responsible for mismatch repair, MMR (i.e. mutS, mutL, mutH, dam), or other DNA repair pathways (i.e. mfd and recA) (Bayliss et al., 2002
). On the other hand, it was shown that the MMR system is active on dinucleotide repeat tracts on artificially constructed repeat regions connected to reporter genes, but does not affect 5'-TA-mediated pilin PV in vivo (Bayliss et al., 2004
).
We have shown previously (Zaleski & Piekarowicz, 2004) that MMR is necessary to repair DNA damage caused by oxidative compounds and that Dam methylation plays an important role in the infectivity of phage HP1c1. Thus, the major role of Dam methylation in different aspects of DNA stability and phage/host relations prompted us to investigate its role in other phage/host relations such as the activity of the R-M system and resistance and sensitivity to phage infection. In this paper, we present results indicating that Dam methylation changes the PV frequency of resistance/sensitivity to phage infection, which is correlated with the PV frequency of one of the genes involved in LOS expression. This allowed us to determine the role of LOS as a receptor for HP1c1 phage.
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METHODS |
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PV frequencies of the R-M properties and resistance or sensitivity were determined as follows. The frozen stock culture of the strain to be tested was spread on BHI plates to obtain single colonies. After 18 h growth at 37 °C, single colonies were resuspended in 2 ml BHI. These cultures were tested for restriction proficiency and/or resistance to HP1c1 phage infection to establish parental phenotype and for preparation of serial dilutions, subsequently spread on BHI plates to obtain single colonies. Material from each colony served as an inoculum for liquid cultures grown in BHI at 37 °C for 18 h and then tested for restriction and phage-resistance phenotype. For each experiment several hundred colonies were screened by these methods. At least four colonies of each variant or construct were tested.
PV frequencies of the lic1 gene (Hood et al., 2001) were tested in a similar way, except that after obtaining colonies from serial dilutions, they were transferred to nitrocellulose filters, and probed with mAb TEPC 15 to detect the presence of a phosphocholine residue on the LOS of bacterial cells. The mutation rates were determined by the fluctuation method (Lea & Coulson, 1949
) as described by De Bolle et al. (2000)
. Alterations in repeat tracts were determined by PCR amplification of the DNA sequence that contained the repeat tract from the parental and variant bacterial cells, and by direct sequencing of the products.
DNA manipulations and cloning.
All general techniques were used according to protocols described for the two host organisms E. coli (Sambrook et al., 1989) and H. influenzae (Barcak et al., 1991
). Isolation of plasmids and chromosomal DNA, restriction analysis of DNA, cloning of DNA fragments and PCR were done by standard procedures (Sambrook et al., 1989
).
Transformation and transfection assays.
Competent cells of H. influenzae were prepared by the anaerobicaerobic method and were transformed with 0·1 µg chromosomal DNA as described by Barcak et al. (1991). For transfection, competent cells were exposed to 0·1 µg HP1c1 phage DNA according to Beattie & Setlow (1971)
. The efficiency of transfection is expressed as number of p.f.u. per concentration of DNA.
Chemicals, reagents and enzymes.
Restriction enzymes and T4 DNA ligase were from Fermentas. All chemicals used for this study were reagent-grade or from Sigma unless otherwise specified. Tris-Tricine gels (15 %) were prepared according to Schagger & von Jagow (1987). mAb TEPC 15 was obtained from Sigma.
LOS purification.
Quick preparations of Haemophilus LOS were made from plated cultures as described by Jones et al. (1992). LOS were diluted 1 : 25 in lysing buffer (Hitchcock & Brown, 1983
). The suspension was boiled for 10 min before loading. Approximately 0·1 µg LOS was subjected to SDS-PAGE on 15 % Tris-Tricine gel in Tris-Tricine running buffer, following the protocol suggested by Bio-Rad. The gel was fixed overnight in 40 % ethanol/5 % acetic acid, and the LOS was visualized by silver staining (Tsai & Frasch, 1982
).
Immunological methods.
Purified LOS was transferred onto nitrocellulose membrane (Schleicher & Schuell). After air drying for 1 h, the membrane was processed in buffer [20 mM Tris/HCl, pH 8·0, 150 mM NaCl, 2 % casein (fat free)] to block all nonspecific binding sites, and then screened for reactivity with mAb TEPC 15. Bound mAb was detected by covering the nitrocellulose filter with alkaline-phosphatase-labelled goat anti-mouse IgG, and tested for the presence of bound alkaline phosphatase using BCIP/NBT solution (Sigma). When colony blotting was performed, overnight colonies were transferred to a nitrocellulose membrane (Schleicher & Schuell) and processed as described above.
PCR.
Primers used in this study are listed in Table 2. PCRs were performed using Pfu polymerase (Fermentas) and carried out according to the manufacturer's specifications. Primers were purchased from IBB Warsaw, Poland. PCR reactions were resolved on 1 % agarose gels in Tris/borate/EDTA running buffer (Sambrook et al., 1989
). Sequencing of the DNA was performed at IBB Warsaw, Poland.
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RESULTS |
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According to Roberts & Macelis (2001), the HindI hsdM gene starts at position 1367826 and spans up to 1369157 bp of the genome sequence, encoding a protein of 443 amino acids. However, analysis of the DNA sequence of this region as well as the gene annotations (accession no. NC_000907), indicate that hsdM may begin 400 bp upstream and that the protein consists of 559 amino acids (Fig. 1
).
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The H. influenzae Rd30 dam mutant is sensitive to HP1c1 infection although the phage burst in this strain is decreased 10-fold compared to the wild-type strain (Zaleski & Piekarowicz, 2004). Wild-type H. influenzae Rd shows PV of resistance/sensitivity to phage infection (Table 3
) that is 10-fold higher in the dam strain. The variation frequency from the HP1c1S (ON) to HP1c1R (OFF) phenotype in the Dam strain was 810-fold higher than that in the opposite direction.
The phage-resistant variants of H. influenzae Rd wild-type and dam strains showed about 710-fold less ability to adsorb phage particles. Phage DNA introduced by transfection into resistant and sensitive wild-type variants gave the same number of progeny phage particles, indicating that resistance to phage infection was due to changes in the cells' receptors (data not shown).
Changes in the LOS of H. influenzae are responsible for the PV of resistance and sensitivity to phage infection
For a number of phages the LPS (LOS) structure or its components (Nesper et al., 2000; Kutter et al., 2005
), are the phage receptor(s). It was therefore reasonable to check whether there is a difference between the LOS structure of the resistant and sensitive variant derivatives from both wild-type and dam cells. H. influenzae LOS is a complex glycolipid composed of a membrane-anchoring lipidA portion linked by a single 2-keto-3-deoxyoctulosonic acid (Kdo) molecule to a heterogeneous oligosaccharide composed of neutral heptose and hexose sugars (Zamze & Moxon, 1987
; Hood et al., 1996a
). Detailed analysis of LOS from H. influenzae Rd strain RM118 showed that this strain produces LOS with a globotetraose [
-D-galpnac-(1
3)-
-D-galp-(1
4)-
-D-galp-(1
4)-
-D-Glcp] extension from the third heptose (HepIII) and a single glucose as an extension from the first heptose (HepI) (Fig. 2
). This single glucose is decorated by the addition of a phosphocholine residue (Hood et al., 1996b
; Risberg et al., 1999
). Several of the surface-exposed epitopes of H. influenzae are subject to high-frequency PV (Patrick et al., 1987
; Weiser et al., 1989
). The addition of the globotetraose extension to HepIII is dependent on three phase-variable genes, lgtC, lic2A and lex2A (lpsA) (Hood & Moxon, 1999
; Bayliss et al., 2002
; Hood et al., 2004
). The PV of these genes is related to the differences in the number of the tetranucleotide repeats, which is not influenced by the MMR system except for the polI gene (Bayliss et al., 2002
, 2004
).
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The H. influenzae Rd HP1c1 phage receptor is localized in the Galp-(14)-
-D-Glcp part of the globotetraose extension from the HepIII of the LOS molecule
In the LOS structure of H. influenzae Rd the second heptose (HepII) does not contain any extension. This leads us to assume that the main role of the HP1c1 phage receptor is played either by the globotetraose [-D-galpnac-(1
3)-
-D-galp-(1
4)-
-D-galp-(1
4)-
-D-Glcp], extending from HepIII, or by the glucose attached to HepI of the LOS molecule, or by both these extensions. The fact that the majority of the phage-resistant/sensitive variants showed reactivity to TEPC 15 antibodies could suggest that the glucose residue extending from HepI (Fig. 2
) is probably not a part of the phage receptor. To confirm this assumption we constructed an lgtF mutant of the phage-sensitive H. influenzae Rd30 strain (see Methods) and tested for phage resistance and sensitivity. All 50 colonies of this mutant tested were still phage-sensitive, which indicates that lack of this glucose residue does not play a role in phage adsorption.
The addition of sugars to HepIII is dependent on four genes, lgtD, lgtC, lic2A and lpsA (Hood et al., 2001, 2004
; Bayliss et al., 2002
) (see Fig. 2
). In order to test which sugars are necessary to retain the sensitivity to phage infection, lgtC and lic2A mutants of phage-sensitive H. influenzae Rd30 were constructed (see Methods) and 20 single colonies of each mutant were tested for phage infectivity and LOS migration. The lic2A mutants were always resistant to phage infection and their LOS migrated in the same way as that of HP1c1-resistant variants of H. influenzae Rd30 (Fig. 4b
, lanes 4 and 5). The lgtC mutants were HP1c1-sensitive and their LOS migrated faster than LOS of the wild-type cells but slower than LOS from lic2A mutant (Fig. 4a
, lanes 4 and 5). This would be in accordance with the loss of the digalactoside residue in the globotetraose extension from HepIII of the LOS molecule.
As an additional test to see whether lic2A mutation is responsible for the resistance to phage HP1c1 infection, chromosomal DNA isolated from H. influenzae Rd30 lic2A was used to transform wild-type H. influenzae Rd30. All the chloramphenicol-resistant transformants were resistant to HP1c1 phage infection and showed changes in LOS migration (data not shown).
The PV of resistance/sensitivity to phage infection is related to the changes in the number of tetranucleotide repeats
The above results indicate that the LOS molecule of H. influenzae Rd plays the role of the phage receptor and that while the digalactoside [-D-galpnac-(1
3)-
-D-Galp] residue (see Fig. 2
) is not needed for phage adsorption, the loss of the third galactose residue results in the loss of ability to adsorb the phage. According to Hood et al. (2001)
, the addition of all these sugar residues depends on the lgtC and lic2A genes, which in strain H. influenzae Rd and strain KW-20 contain 22 5'-GACA and 22 5'-CAAT repeats respectively (Fleischmann et al., 1995
). To check whether the PV of the lgtC and lic2A genes observed in this paper is related to the changes in the number of the tetranucleotide repeats we tested these repeats in the original H. influenzae Rd30 strain and its spontaneous resistant/sensitive and LOS variants. Table 4
shows that in all tested cases a change from phage sensitivity to resistance was accompanied by a change in the number of 5'-CAAT repeats from 22 to 21 in the lic2A gene (i.e. a switch from ON to OFF). The phage-resistant lic2A variant (strain Rd30dam4#16) also showed a change in the number of 5'-CAAT repeats from 22 to 21. A change in the number of repeats (5'-GACA) was also observed in the lgtC gene present in strains Rd30dam4#1 and Rd30dam4#2, which were HP1c1 phage sensitive and showed a change in the LOS migration, confirming the assumption that this gene and its PV does not play a role in phage sensitivity.
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DISCUSSION |
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The discrepancy between the HindI hsdM structures presented in the literature (Roberts & Macelis, 2001; Fleischmann et al., 1995
) makes it difficult to interpret the mechanism of PV of the HindI R-M system. It seems that the long HsdM represents an active form of the enzyme. The N-terminal 123 amino acid end of this protein contains domains that are required for the normal activity of the DNA MTases (data not presented). The lack of these domains seems to preclude the possibility that the short HsdM would be an active enzyme. If the long HsdM represents an active form of the enzyme then it can be synthesized only in the presence of the four pentanucleotide repeats. In the presence of three or five repeats it would be synthesized out of frame as an inactive protein. If the synthesis in the presence of three or five repeats starts from another start codon, the short inactive form of the enzyme will be formed.
In this report we also show that the resistance/sensitivity to phage infection is changed by PV. The PV of this phenotype correlates with the PV of one of the genes engaged in LOS biosynthesis, lic2A. Analysis of the resistant/sensitive PV variants and insertion mutants made in lic2A, lgtC and lgtF clearly indicated that in the LOS structure of H. influenzae Rd, phage adsorption (receptor) is mediated by the Glc-Gal extending from the third heptose residue. The loss of the terminal digalactoside, as an extension of the Glu-Gal at the third heptose, or the Glc extending from the first heptose and/or phosphocholine residue decorating this Glc, does not influence the ability to adsorb HP1c1 phage. In H. influenzae, LOS is a principal component of the outer membrane, mediating interactions between the bacterium and the host's immune system, and contributing to its pathogenicity. The use of the LOS as a receptor for phage HP1c1 increases the probability that it will always be present on the surface of bacterial cells and increases the chance of the bacteriophage surviving in the environment.
In H. influenzae, PV is mediated mainly by long tetranucleotide or dinucleotide repeat tracts (Bayliss et al., 2002, 2004
; Hood et al., 2004
). The frequency of PV of the gene containing tetranucleotide and dinucleotide repeats strongly depends on the number of such repeats. This becomes visible when this number is greater than 10 (De Bolle et al., 2000
). Although the presence of penta- or heptanucleotide repeats was reported (van Belkum et al., 1997
), their influence on PV in H. influenzae was not known. Here we show for the first time that pentanucleotide repeats are responsible for PV of the HindI R-M system. However, the striking difference between PV mediated by tetranucleotide and dinucleotide tracts, compared to pentanucleotide tracts, is that the same frequency of PV is observed for the genes containing 35 pentanucleotide repeats as for genes containing more than 20 tetranucleotide or dinucleotide repeats (De Bolle et al., 2000
; Bayliss et al., 2002
).
All the phage-resistant variants that were tested demonstrate the lack of one of the 5'-CAAT repeats in the lic2A gene, while reversion to phage sensitivity restores the original number of repeats. Similarly, the difference between the variants that are able or unable to restrict the HP1c1 phage is related to the change of one or two pentanucleotide repeats. The same results observed by De Bolle et al. (2000) showed that 90 % of ON-to-OFF phase variants in the mod : : lacZ reporter H. influenzae Rd strain exhibited a single repeated unit change.
A striking observation in this paper is that the lack of Dam methylation in H. influenzae increases the rate of PV of sensitivity/resistance to phage infection, which correlates with the PV of lic2A activity. Since the resistance to phage infection is a consequence of an inactive lic2A gene, this indicates that the lack of Dam methylation increases the frequency of PV of this gene. Although not so rigorously tested, our data also indicate that the lack of Dam methylation increases the PV frequency of the lgtC gene. Our results are in contrast with the observation of Bayliss et al. (2002), who showed that among seven MMR genes of H. influenzae, none, except for polI, influenced PV mediated by tetranucleotide repeats. The increase of PV of phage resistance/sensitivity phenotype due to switching ON or OFF of expression of lic2A in a dam mutant is a spontaneous process not influenced by any selection. The test for phage resistance/sensitivity was a tool that allowed the detection of such events. The production of HP1c1 phage in the dam mutant is only 10-fold lower than in the wild-type cells (Zaleski & Piekarowicz, 2004
) and cannot be responsible for the observed resistance to phage infection (since the resistance in such a case means a complete loss of the ability to adsorb phage and not its production inside the cell). However, Bayliss et al. (2002)
tested the influence of mutations in the MMR system using artificial constructs introduced into the chromosome of H. influenzae, while in our experiments tests for PV were carried out in in vivo conditions.
Our experiments have shown that the lack of Dam methylation does not change the frequency of PV of the lic1 gene, responsible for the addition of the phosphocholine residue to the glucose extended from the first heptose. The PV of this property is governed by the instability of the 5'-CAAT within the licA gene (Weiser et al., 1989). Although it was suggested that Dam methylation may play a role in the regulation of PV in Neisseria meningitidis (Bucci et al., 1999
), further studies have shown that it does not change the frequency of PV of several tested loci in which PV was due to the presence of poly(G) tracts (Alexander et al., 2004
). All these results suggest that the effect of Dam on PV may depend on the particular gene tested and whether it was tested in vivo or using artificial constructs.
In conclusion, our results, together with those of other groups, suggest that the H. influenzae MMR pathway can destabilize in vivo the PV mediated by tetranucleotide and pentanucleotide repeats. Dam methylation, as a part of the MMR system, is responsible for the correction of single- and double-stranded DNA breakage in H. influenzae (Zaleski & Piekarowicz, 2004) and could be responsible for correction of slippage mutations generated in particular tetranucleotide and pentanucleotide repeat tracts. This paper shows that dam mutation does not change the PV rates of lic1A (i.e. TEPEC 15 reactivity), and Bayliss et al. (2002)
showed that this mutation does not affect a reporter in the mod locus. Therefore, the dam effect must be acting in a locus-specific manner.
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ACKNOWLEDGEMENTS |
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Received 10 May 2005;
revised 17 June 2005;
accepted 5 July 2005.
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