Institute of Microbiology, University of Warsaw, 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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Recent studies using a high-density DNA microarray of 4097 E. coli genes showed that the expression, as measured by the level of transcription, of the majority of genes was affected by dam deficiency (Oshima et al., 2002). Genes involved in aerobic respiration, stress and SOS responses, and amino acid and nucleic acid metabolism were expressed at higher levels in the mutant cells. In contrast, transcription of genes participating in anaerobic respiration, flagellar biosynthesis, chemotaxis and motility was decreased in the dam mutant strain under both aerobic and microaerobic conditions. It was proposed that Dam-mediated methylation plays an important role in the global regulation of genes, particularly those for fumarate nitrate reduction (Fnr) and catabolite activator protein binding site (CRP). These observations could explain previous observations that the Dam DNA MTase regulates such cell functions as chromosome replication (Messer et al., 1985
; Russell & Zinder, 1987
), phase variation (Bucci et al., 1999
; Cantalupo et al., 2001
) or synthesis of fimbriae (Blyn et al., 1990
).
Methyl-directed mismatch repair in E. coli (MMR, MutHLS system) appears to remove mispaired and unpaired bases from newly synthesized DNA strands, thus correcting replication errors (Modrich, 1987; Hsieh, 2001
). The discrimination between the parental and newly synthesized strand is based on the transient undermethylation of GATC sequences in nascent strands (Low et al., 2001
, Friedberg, 2003
). In the absence of the Dam methylation an increased rate of spontaneous and induced mutation is observed (Modrich, 1987
). MMR requires the presence of MutH, MutL, MutS and MutU (helicase DNA II) proteins. MutS recognizes the replication error in the DNA and binds to it. MutL helps to make the complex between MutH and MutS, and tracks this complex to the nearest 5'-GATC-3' sequence. MutH makes a scision in the newly replicated unmethylated DNA strand within the 5'-GATC-3' sequence, helicase separates the two strands while one of the four possible exonucleases removes nucleotides from the point of scission beyond the mismatch. Finally, the polymerase synthesizes the missing nucloetides and the ligase joins the strand (Modrich, 1987
; Yang, 2000
; Hsieh, 2001
; Friedberg, 2003
). Recently it was also shown that the MMR system of E. coli can prevent oxidative mutagenesis either by removing 7,8-dihydro-8-oxoguanine (8-oxoG) directly or by removing adenine misincorporated opposite 8-oxoG, or both (Wyrzykowski & Volkert, 2003
). It was also shown that Dam methylation plays a role in the integrity of the bacterial chromosome, regulating transposition of insertion elements (Roberts et al., 1985
), conjugation (Camacho & Casadesus, 2002
) and recombination (Stambuk & Radman, 1998
).
Haemophilus influenzae is an obligate commensal of the upper respiratory tract that has the potential to cause diseases such as otitis and meningitis. The H. influenzae strain Rd genome sequence contains homologues (6784 % similarity) of all of the known E. coli MMR genes (see The Institute for Genomic Research microbial database at http://www.tigr.org), indicating that H. influenzae has a fully functional MMR system. Although a genome-scale analysis for identification of genes required for H. influenzae Rd growth and survival suggested that the dam gene could be essential for cell viability (Akerley et al., 2002), a viable dam mutant was obtained (Bayliss et al., 2002
, 2004
). It was shown that this mutant, depending on marker tested, either does not exhibit higher spontaneous mutation rates, or exhibits only a three- to fourfold increase compared to the wild-type. These authors have also shown that although lack of Dam methylation destabilizes the 5'-AT tracts (Bayliss et al., 2004
), it does not influence the phase variation of the pilin. In E. coli, Dam methylation alone and as a part of the MMR system plays a much broader role than guarding against the introduction of mutations into the genome and in stabilizing dinucleotide tracts. To learn more about the role of Dam methylation in H. influenzae we first tested its effect on gene expression and its role in the maintenance of chromosome integrity. Assuming that changes of the phenotypic properties of H. influenzae dam mutant should reflect changes in the expression of the genes, we then tested some of the phenotypic properties that are governed by a wide spectrum of genes, such as sensitivity to different antibacterial agents. We were also interested to know whether, as in E. coli, the MMR system plays a role in defence against DNA damage by oxidative agents. Our results extend our understanding of the influence exerted by Dam methylation on gene expression and as a defence system against such damage.
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METHODS |
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Inactivation of DNA metabolism genes.
Cloning of the gene encoding the Dam MTase into pMPMT4INT was described previously (Bujnicki et al., 2001). The R.NdeI-R.SmaI fragment encoding this gene was introduced into pUC19 plasmid DNA on the same restriction sites. This plasmid was linearized with R.EcoRV and ligated to an R.SmaI fragment carrying a chloramphenicol-resistance cassette (derived from plasmid pK19). The R.EcoRV site within the cloned dam gene is located 220 bp from the start codon. The plasmid construct was checked by restriction digestion and sequencing the ends of the inserts, using primers that generate sequences across the multiple cloning site. This plasmid was linearized by digestion with R.Cfr10I and used to transform competent H. influenzae Rd30 cells. Transformants were selected on sBHI plates containing 2 µg chloramphenicol ml1. One dam : : CamR strain, designated H. influenzae Rd30dam4, was chosen for further studies. The dam mutH double mutant of H. influenzae Rd30 was constructed by transforming H. influenzae Rd30dam4 with chromosomal DNA prepared from H. influenzae Rd mutH : : TetR (Bayliss et al., 2002
), and selecting transformants on sBHI plates containing 3 µg tetracycline ml1. One of the TetR CamR transformants, designated H. influenzae Rd30dam4mutH, was used for further studies.
DNA manipulations and cloning.
All general techniques followed 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 were prepared by the anareobicaerobic method and transformed with 1 µg chromosomal DNA as described by Barcak et al. (1991). Transformation frequency was calculated as the number of novobiocin-resistant colonies divided by the total number of colonies. For transfection, competent cells were exposed to 0·1 µg HP1 phage DNA according to Jablonska & Piekarowicz (1976)
. The efficiency of transfection is expressed as number of plaque-forming units per 0·1 µg HP1 DNA.
Treatment of cells with antibiotics, dyes, detergents, 2-aminopurine (2-AP), ethyl methanesulfonate (EMS), hydrogen peroxide and nitrofurazone
The following experiments were done at least in triplicate with cultures that each started from a single colony.
(i) Determination of sensitivity to antibiotics, dyes and detergents.
MICs were determined by serial twofold dilution in BHI medium, using 5x104 late-exponential-phase cells, grown in sBHI, as the inoculum. The concentration at which there was no visually detectable bacterial growth was taken as the MIC.
(ii) Treatment of cells with EMS and hydrogen peroxide.
Frozen stocks were streaked onto sBHI plates containing, when necessary, 2 µg chloramphenicol ml1, and were grown overnight at 37 °C. Single colonies were then inoculated into sBHI medium. Cultures were grown with gentle aeration overnight and then diluted 1 : 50 in fresh sBHI containing, when necessary, 2 µg chloramphenicol ml1, and grown with aeration to mid-exponential phase. Samples (5 ml) were withdrawn and EMS or hydrogen peroxide was added to an appropriate final concentration. After 60 or 15 min of incubation, respectively, at 37 °C in a shaking incubator, the cells were centrifuged, washed with sBHI and suspended in the 5 ml sBHI. The samples were withdrawn and plated after appropriate dilution on sBHI agar containing, when necessary, 2 µg chloramphenicol ml1, and incubated for 24 h at 37 °C to determine survival. Of the remaining suspension treated with EMS, 2 ml was added to 5 ml pre-warmed sBHI and incubated for 4 h at 37 °C. The cells were then centrifuged, suspended in 1 ml BHI and appropriately diluted samples were plated on selective media (sBHI with antibiotics) and nonselective (viable count) media to determine the frequencies of resistant mutants.
(iii) Treatment of cells with 2-AP.
Frozen stocks were streaked onto sBHI plates containing, when necessary, 2 µg chloramphenicol ml1 and were grown overnight at 37 °C. Single colonies were then inoculated into sBHI medium. Cultures were grown with gentle aeration overnight, when necessary in the presence of 2 µg chloramphenicol ml1. Samples of 0·1 ml were withdrawn and placed in 5 ml sBHI in fresh culture vessels, and 2-AP from an aqueous stock solution (5 mg ml1) was added to a final concentration of 0, 10, 50 and 100 µg ml1. The cultures were aerated at 37 °C until the sample without 2-AP reached a titre of 2x109 (about 4 h). The cells of all samples were then harvested by centrifugation, washed twice in sBHI, and an appropriate dilution was plated on selective and nonselective media to determine the survival and frequencies of resistant mutants. Mutation frequencies were determined for at least five colonies of mutants. Mutation rates were estimated from these frequencies using the median value by the method of Drake (1991).
(iv) Treatment with nitrofurazone.
Frozen stocks were streaked onto sBHI plates containing, when necessary, 2 µg chloramphenicol ml1 and grown overnight at 37 °C. Single colonies were then inoculated into 5 ml sBHI medium. Cultures were grown with gentle aeration overnight, when necessary in the presence of 2 µg chloramphenicol ml1. Samples of 0·1 ml were withdrawn and placed in 5 ml sBHI in fresh culture vessels, and nitrofurazone from an aqueous stock solution (1 mg ml1) was added to a final concentration of 0, 0·25, 0·5 and 0·75 µg ml1. The cultures were aerated at 37 °C for 6 h. The cells of all samples were then harvested by centrifugation, washed twice in BHI, and suspended in 1 ml BHI. Appropriate dilutions were then plated on sBHI plates to determine survival.
Determination of nitroreductase activity.
Frozen stocks were streaked onto sBHI plates containing, when necessary, 2 µg chloramphenicol ml1 and were grown overnight at 37 °C. Single colonies were then inoculated into sBHI medium. Cultures were grown with gentle aeration overnight and then centrifuged (Sorvall SS-34, 6000 r.p.m., 15 min), washed and suspended in 1x PBS buffer prepared according to Sambrook et al. (1989). Cultures were centrifuged (6000 r.p.m., 30 min, 4 °C), then resuspended in 5 ml 100 mM Tris/HCl pH 7·5 and kept on ice. Cells were disrupted by sonication and the cell debris was removed by centrifugation at 10 000 r.p.m. for 30 min at 4 °C. The protein concentration was determined by the Lowry method (Smith et al., 1987
). The enzyme activity was determined spectrophotometrically at 365 nm. The sample for reaction assay contained 800 µl protein suspension at the same concentration, 100 µl 1 mM nitrofurazone and 100 µl 2 mM NADPH. The reaction was measured as absorbance decrease every 5 s for 1 min.
Determination of DNA degradation after treatment of cells with hydrogen peroxide and nitrofurazone.
This was done according to Sisson et al. (2000). Overnight cultures of H. influenzae strains were diluted 1 : 50 in 5 ml fresh sBHI and grown to mid-exponential phase. The cultures were then treated with hydrogen peroxide (0·1 %, v/v) for different times or with 5·0 µg nitrofurazone ml1 for 6 h. After treatment the cells were harvested by centrifugation, washed with fresh sBHI and resuspended in 200 µl TE buffer to give an OD600 value of 7·0 for all samples. Lysis of bacterial cells in low-melting-point agarose, DNA agarose gel electrophoresis under alkaline conditions, staining and visualization of the gel were done according to Sisson et al. (2000)
, using the same volume of prepared bacterial suspension.
One-step growth of phage HP1.
The procedure for a single cycle of growth of HP1 in H. influenzae Rd30 was described previously (Jablonska & Piekarowicz, 1976).
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RESULTS |
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In E. coli the expression of most of the genes is changed in dam mutant cells (Oshima et al., 2002), which can influence their phenotypic properties. Thus, the observed changes of phenotypic properties of a dam mutant should reflect the changes in transcription level of particular genes. To test the influence of the lack of Dam methylation in H. influenzae we examined changes in some of the phenotypic properties regulated by a broad spectrum of gene products.
Effect of dam mutation on the drug-efflux transporter system
Genomic sequencing of H. influenzae has identified a three-gene complex that is homologous to the acrRAB multidrug-resistance efflux transporters of E. coli (Fleischmann et al., 1995). The disruption of these multidrug-efflux genes causes hypersusceptibility of H. influenzae to several antibiotic and dyes (Sanchez et al., 1997
). The data presented in Table 1
indicate that the presence of a dam mutation made H. influenzae Rd30dam4 more susceptible to several compounds tested by Sanchez et al. (1997)
: antibiotics (erythromycin, tetracycline, kanamycin, spectinomycin), dyes (crystal violet, Congo red) and a detergent (SDS). The dam mutant showed in most cases not only a lower MIC, but also increased susceptibility to higher concentrations of the compounds. For example, the efficiency of plating (EOP) of the wild-type strain on medium with 2 µg kanamycin ml1 was 104 while that of the mutant strain was 108.
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DISCUSSION |
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H. influenzae has three different genes homologous to E. coli acrRAB, as well as homologues for emrAB and bcr, disruption of which leads to changes in the structure of the multidrug efflux pumps resulting in hypersusceptibility to different antibiotics and dyes (Sanchez et al., 1997; Ma et al., 1993
). In an E. coli dam mutant strain the acrRAB genes showed a decreased level of transcription (Oshima et al., 2002
). The change in the expression of these genes in the H. influenzae dam mutant could lead to different levels of the particular gene products building the drug efflux complex. Different levels of these proteins could result in changes of the structure of the drug efflux complex and the sensitivity to the same group of antibiotics and dyes tested previously (Sanchez et al., 1997
). The observed decrease in efficiency of transformation and transfection could also be explained by changes in the expression of many genes involved in DNA binding and uptake in H. influenzae (Dougherty & Smith, 1999
). It was observed that the deletion of one gene from among mtrR, acrA and acrB results in a relatively less drastic effect on transformation (Dougherty & Smith, 1999
) than changes in the other genes responsible for DNA binding and uptake and recombination. The much higher effect of the dam mutation on transfection than on transformation cannot be explained by the higher susceptibility of the transfecting phage DNA to the action of MutH nuclease since the efficiency of transfection is the same in dam and dam mutH mutants. It can be explained, however, by the fact that phage HP1 gives about 10 times fewer progeny phages in the dam mutant than in the wild-type. Why the phage growth in the dam mutant is affected by the lack of Dam methylation is not known at present. All these results suggest then that in H. influenzae, as in E. coli, the Dam methylation alters the expression of a great variety of genes.
The principal effect of Dam MTase activity in the subdivision of Proteobacteria is to prevent spontaneous and induced mutation. This implies that the Dam enzyme plays a role in differential strand tagging at the replication fork for methyl-instructed mismatch repair of newly synthesized DNA (Hsieh, 2001
). The mutation frequency in our mutant is similar to the frequency in the H. influenzae dam mutant isolated by Bayliss et al. (2004)
. The dam mutant of H. influenzae described here shows only a moderate increase of spontaneous mutability and mutability after treatment with EMS. This phenotype is very similar to that of dam mutants of E. coli (Palmer & Marinus, 1994
), S. typhimurium (Torreblanca & Casadesus, 1996
) or Serratia marcescens (Ostendorf et al., 1999
), suggesting a similar role.
Our mutant is also very sensitive to nitrosubstituted compounds, whose mutagenic and antimicrobial activity is caused by short-lived intermediates formed during their reduction by nitroreductases (Whiteway et al., 1998), and to hydrogen peroxide, which produces free radicals. It was observed (Wyrzykowski & Volkert, 2003
) that in an E. coli dam mutant sensitivity to hydrogen peroxide is due to conversion of oxidative lesions to strand breaks by MMR since mutation in the mutH or mutS genes restores peroxide resistance. Similarly, the MMR system of H. influenzae seems also to be responsible for the action on DNA containing oxidized bases. This conclusion is based on the fact that hydrogen peroxide induces breaks in a dam mutant and that mutants carrying dam and mutH mutations restore the wild-type level of sensitivity to this compound.
H. influenzae encodes only one oxygen-insensitive nitroreductase, a homologue of E. coli nfnB gene. Upregulation of the activity of this nitroreductase in the dam mutant could be responsible for its extreme sensitivity to nitrofurazone. However, the fact that the level of the enzyme in the dam mutant is the same as in the wild-type cells argues against this explanation. Moreover, the disruption of the nfnB (also called nfsB) gene in E. coli results in only a slight increase (619 %) in susceptibility to nitrofurantoin or nitrofurazone, while the dam mutant of H. influenzae is several orders of magnitude more sensitive. Thus, it seems that the extreme sensitivity of the dam mutant of H. influenzae to nitrofurazone is not mediated through changes in the regulation of nfnB.
Similar to the situation in E. coli and Campylobacter jejuni (Sisson et al., 2002), nitrofurazone does not produce DNA single- or double-stranded breaks in the chromosome of H. influenzae and is not mutagenic. We do not know what type of lesions are induced in the DNA by nitrofurazone or what type of nucleotide is affected by its action. Independently of the type of lesions produced by nitrofurazone, sensitivity of the dam mutant is at least partially due to the MMR system. This conclusion is based on the observation that the double mutant dam mutH, which has an inactive MMR system, is much more resistant to nitrofurazone than the dam single mutant. Since the action of MMR on DNA damaged by the oxidative product derived from nitrofurazone is influenced by the dam mutation, and the hemimethylated state required for MMR (Palmer & Marinus, 1994
), it is possible that the role of MMR is to immediately repair the products of misreplication past oxidative lesions. Recently, it has been shown that incorporated 8-oxo-dGMP derived from the dNTP pool is effectively removed by the yeast MMR system (Colussi et al., 2002
) from the newly synthesized DNA strand. These authors suggested that the role of MMR is to immediately repair the products of misreplication past oxidative lesions and to remove 8-oxoG incorporated by the replication machinery. In this process the yeast MMR initiates correction of the A-containing daughter strand at 8-oxoG : A pairs formed during replication. A similar system may operate in H. influenzae. We propose that in wild-type H. influenzae the MMR recognizes the mispaired bases caused by the treatment of cells with nitrofurazone. MutH makes scissions in the non-methylated newly replicated strand containing, for example, 8-oxoG, incorporated into the daughter strand opposite template A. After the scission is completed, the MMR removes 8-oxoG incorporated during replication, which involves a relatively short patch within the replication fork. Due to such a mechanism we cannot observe the DNA degradation products. In the absence of Dam methylation MutH makes scissions not precisely in proximity to mispaired bases but more randomly, which in consequence blocks the replication of DNA and causes the cell's death.
In conclusion, the presence of a dam mutation in the H. influenzae genome is not lethal under laboratory conditions. However, the resulting sensitivity to hazardous compounds, and increased sensitivity to antibiotics and dyes, may make the effect of this mutation lethal under natural conditions, as this pathogen colonizes the upper respiratory tract and invades the respiratory mucous membranes.
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ACKNOWLEDGEMENTS |
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Received 8 April 2004;
revised 16 July 2004;
accepted 22 July 2004.
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