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Infection and Immunity, November 2004, p. 6743-6747, Vol. 72, No. 11
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.11.6743-6747.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia
Received 8 February 2004/ Returned for modification 21 April 2004/ Accepted 4 August 2004
| ABSTRACT |
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One of the mechanisms believed to be involved in neisserial transmissibility and virulence is that of phase variation, which has been postulated to occur in over 60 N. meningitidis genes (21, 31, 34). This regulation of gene expression typically involves slippage events in repeated nucleotide tracts (slipped-strand mispairing) during DNA replication, altering reading frames or promoter strength (27). The frequency with which meningococci change the expression state of phase-variable genes differs greatly among clinical isolates, and it has been proposed that increased frequencies may augment fitness (2, 3, 10, 20, 25). In this study, we utilized in vitro Himar1 mariner transposition to identify previously unidentified genetic loci involved in the regulation of strain-specific frequencies of phase variation within mononucleotide repeat tracts.
Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. It has been shown that N. meningitidis is capable of altering the expression state of two outer membrane receptors involved in the uptake of iron from hemoglobin (HmbR and HpuAB) and that these phase variation events occur at strain-specific frequencies via slipped-strand mispairing of poly(G) tracts within the hmbR and hpuA coding regions (6, 7, 18, 19, 23, 24, 33). Because we chose to screen mutants for augmented hmbR switching frequencies, the meningococcal strain selected for mutagenesis (IR4162) is an HpuAB mutant (hpuB::Em). Thus, we could be confident that our assays were specific to hmbR phase variation. This strain was also chosen because we have previously determined that the phase variation frequency of this highly transformable serogroup A clone falls within the "slow" category (<2 x 105 CFU1) (2, 24), which is advantageous when attempting to identify Himar1 insertions leading to increased phase variation frequencies. Neisseria cells were routinely cultured on either GCB agar or broth (Difco Laboratories, Detroit, Mich.) containing Kellogg's supplements I and II (14) and incubated at 37°C with 5% (vol/vol) CO2 or brain heart infusion media supplemented with 2.5% heat-inactivated fetal bovine serum unless otherwise noted.
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6,700 to 10,200 mutants for a confidence level of 95 to 99% (26). Therefore, 500 resultant Kmr colonies were picked from each of 20 transposition reactions and stored, creating a library of
10,000 insertion mutants.
Identification of mutants with increased phase variation frequencies.
Significant alterations in HmbR switching frequencies can be rapidly detected with hemoglobin (Hb) utilization disk assays (Fig. 1). In these experiments,
107 Kmr N. meningitidis cells were plated onto GC agar containing Kellogg's supplement I and the iron chelator deferoxamine mesylate (Desferal) (50 µM; Ciba Geigy, Toms River, N.J.). Filter disks (diameter, 1/4 in) were soaked with 10 µl of 5-mg/ml human Hb (Sigma Chemical Co., St. Louis, Mo.) and added to the inoculated plates, which were then incubated overnight at 37°C with 5% (vol/vol) CO2. The HmbR phase variation frequency of the parental strain (IR4162) on solid media is 1.9 x 106 CFU1. Therefore, by plating 107 bacteria, approximately 5 CFU of the initial inoculum will be HmbR phase "on." Such phase "on" bacteria will form single colonies around the Hb-soaked filter disks, as will those cells that vary from phase "off" to "on" during the incubation period (Fig. 1) (23). Thus, clones with augmented phase variation frequencies will have more single colonies around the filter disk than will the parental strain (Fig. 1). These assays were performed for each of the 10,000 transposon-containing clones, and assays for clones determined to have significantly increased phase variation frequencies were repeated a second time, finally resulting in 181 positive clones.
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Mapping and analysis of loci involved in phase variation modulation. Himar1 insertion sites in the 33 positive clones were mapped by a ligation-mediated PCR technique developed specifically for this Himar1 element by Pelicic et al. (22). Linkage of the increased-phase-variation phenotype to the insertion was confirmed by amplifying each unique mariner insertion by PCR and transforming these aphA3-containing products into the parental strain, IR4162. Phase variation frequencies of the resultant clones were then measured a minimum of six times each, and the statistical significance of these data was determined by the Mann-Whitney test (P < 0.05). Using this approach, we linked phenotypes of increased hmbR phase variation frequency to single Himar1 mariner insertions in nine independent genes or intergenic regions (Table 2 and Fig. 2). The presence of a single Himar1 mariner insertion in each of these nine mutants was further confirmed by Southern blot hybridization analysis (Fig. 3). As expected, three of the identified genes, mutS, mutL, and uvrD, are the known components of the neisserial mismatch repair system, which has been shown to modulate phase variation frequencies (2, 24, 25). This provided confirmation that our mutagenesis and screening methods were effective in identifying loci involved in the regulation of switching frequencies.
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Two of the Himar1 insertions mapped to intergenic regions. The transposon in mutant 820B inserted in a noncoding region 3' of two divergently transcribed hypothetical protein-encoding open reading frames (ORFs), designated NMA1060 and NMA1059, and caused a 20-fold increase in hmbR phase variation (Fig. 2). Due to this location, it is likely that the insertion does not disrupt the function of either gene product. However, it may exert a polar effect on downstream genes. Three ORFs with unknown or putative functions are located just downstream of NMA1059. NMA1058 encodes a hypothetical protein, NMA1057 encodes a putative glycosyltransferase, and the NMA1056 product appears to be a PhoP-like protein. The other intergenic insertion, in mutant 698C, had a much smaller effect on hmbR phase variation frequency (fourfold), but this effect was significant (P < 0.001). Unlike that of 820B, this locus is between the 5' ends of two ORFs, NMA0829 and NMA0830, with unknown function and may affect either or both gene product(s) as well as downstream loci.
Mutant 759C also provides little insight into the mechanism by which the gene NMA1233 modulates phase variation. NMA1233 is homologous to part of the N. gonorrhoeae putative sucAB-lpd operon designated orfA but has no known function. Additionally, there do not appear to be any nearby ORFs that this insertion would affect.
Perhaps the most interesting transposon insertions were within the ferric binding protein operon (fbpABC). This locus encodes an ABC transporter that is reported to be responsible for the transport of ferric iron across the periplasmic membrane (1). One Himar1 element (that of 629B) was located just upstream of the first gene in the operon, fbpA, which encodes the iron binding protein (1, 9). The other two insertions (those of 739C and 943H) were within the proposed cytoplasmic membrane protein-encoding locus, fbpB (1). It is not surprising that we did not isolate a mutant element within the putative ATPase-encoding gene, fbpC, as it has been reported that this protein is not required for iron acquisition in Neisseria gonorrhoeae (1, 28). In addition to its role in iron assimilation, the fbp operon, like hmbR and numerous other genes, is regulated by iron concentration through the ferric uptake regulator protein (Fur) (4, 13, 29, 35).
The involvement of Fbp in transporting ferric iron across the periplasmic membrane led us to hypothesize that inactivation of the fbpABC operon could limit iron availability in the cytosol, thereby relieving Fur repression of iron-regulated genes. One or more of these derepressed genes, in turn, could impact phase variation. It has been reported, however, that, while an fbpABC mutant cannot utilize nonheme iron, it grows normally in the presence of heme or Hb (15). Thus, iron availability should not be affected in such a mutant when grown in the presence of Hb, as is the case with the hmbR phase variation assays (23). Conversely, an earlier study suggested that Fbp does in fact transport iron from heme and Hb (11). Due to these conflicting reports, we determined which iron sources two additional fbpA insertion mutants could assimilate. We constructed the first, IR5647, by disrupting the fbpA locus of IR4162 with a SP adenyltransferase AAD (9) cassette (fbpA::Sp) (36). The second consisted of IR4162 containing fbpA insertionally inactivated by a cat cassette (fbpA::Con) (15). The latter construct was reported by Khun et al. to render Neisseria incapable of utilizing nonheme iron (15) (IR5650). Both of these Fbp mutants displayed increased hmbR phase variation phenotypes similar to that produced by the Himar1 insertion that was just upstream of fbpA (629B) (Fig. 4). These clones were tested for their ability to utilize ferric nitrate, transferrin, heme, and Hb with filter disk assays on solid media (37) and growth assays in liquid culture (15). Under all conditions tested, the fbpA mutants, as well as the wild type, grew normally, indicating that they could use any of these iron sources (data not shown), further adding to the conflicting reports about the role of the Fbp in neisserial iron assimilation. At this point, we cannot rule out the possibility that another, functionally redundant ferric iron transport system(s) exists in Neisseria spp. It may be that the inactivation of Fbp restricts iron to such as small extent that another system is capable of relieving any potential growth defects. There may, however, be sufficient iron restriction to affect gene expression, leading to alterations in phase variation frequencies. It is interesting that this certainly is not the first report of iron availability impacting antigenic variation. Not only is hmbR both phase variable and iron regulated, but also iron availability has also been linked to DNA recombination (pilin antigenic variation), transformation efficiency, and DNA repair in the gonococcus (30).
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Screening this Himar1 mariner mutant library for alterations in phase variation frequencies yielded some interesting findings. We have identified six genetic loci that modulate N. meningitidis phase variation frequencies. Three were in regions of unknown function, including two intergenic sequences and one putative ORF. Additionally, we linked phase variation frequency regulation to type IV pili (pilP) as well as iron transport and utilization (fbpA and -B), which may not be mutually exclusive (8). Determining the mechanisms by which the genes identified in this report modulate phase variation will lead to a better understanding of how pathogenic Neisseria regulates gene expression to enhance transmissibility and invasiveness.
| ACKNOWLEDGMENTS |
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This work was supported by Public Health Service Grant AI42870 (I.S.). H.L.A. was supported by National Institutes of Health Training Grant 2T32 AI07470.
| FOOTNOTES |
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