The Neisseria gonorrhoeae photolyase orthologue phrB is required for proper DNA supercoiling but does not function in photo-reactivation

Summary

Neisseria gonorrhoeae (Gc) is an obligate human pathogen and the causative agent of the sexually transmitted infection, gonorrhea. Despite the fact that the gonococcus is not normally exposed to UV irradiation or visible light, the bacterium expresses a phrB orthologue which in other organisms encodes a DNA photolyase that repairs UV-induced pyrimidine dimers with energy provided by visible light. We show that a Gc phrB mutant is not more sensitive to UV irradiation, independent of visible light exposure, and that the Gc phrB cannot complement an Escherichia coli phrB mutant strain. The Gc phrB mutant had a reduced colony size that was not a result of a growth defect and the mutant cells exhibited an altered morphology. Although the phrB mutant exhibited increased sensitivity to oxidative killing; it showed increased survival on media containing nalidixic acid or rifampicin, but did not have an increased mutation rate to these antibiotics or spectinomycin and kasugamycin. The Gc phrB mutant showed increased negative DNA supercoiling, but while the protein bound double-stranded DNA, it did not express topoisomerase activity. We conclude that the Gc PhrB has a previously unrecognized role in maintaining DNA supercoiling that is important for normal cell physiology.

Introduction

The genus Neisseria consists of both pathogenic and commensal species (Aho et al., 1987, Aho et al., 2005, Knapp, 1988). The pathogenic species, Neisseria gonorrhoeae (the gonococcus or Gc) and Neisseria meningitidis, exist solely in humans while most commensal Neisseria species have been found in both humans and animals (Aho et al., 1987, Aho et al., 2005, Knapp, 1988). Whole genome sequence analysis has revealed that the Neisseria species genomes consist of between 2,000 and 2,842 predicted genes, and the set of core genes present in all Neisseria species consists of 896 genes (Marri et al., 2010). Of the pathogenic species, Gc is the sole causative agent of the sexually transmitted infection gonorrhea which usually presents as urethritis in men and cervicitis in woman (Edwards & Apicella, 2004). Interpretation of Egyptian and Biblical writings imply that gonorrhea has plagued humans for thousands of years (Mortan, 1977 ed., Wain, 1947), suggesting that both the pathogenic and commensal Neisseria have co-evolved within humans over all recorded history (Kline et al., 2003, Toleman et al., 2001).

Since there are no known free-living members of this genus, the Neisseria are not expected to have experienced UV irradiation, desiccation, or other extreme environments. Consequently, the Neisseria repair capabilities are predicted to be specialized for damage that might occur within the host. For example, the pathogenic Neisseria possess a diverse range of mechanisms for coping with reactive oxygen species (ROS) which the bacteria encounter during aerobic respiration and interactions with phagocytic cells (Seib et al., 2004, Seib et al., 2006). Mechanisms for coping with ROS are crucial for survival of these mucosal organisms (Seib et al., 2004, Seib et al., 2006, Stohl & Seifert, 2006).

Gc encodes components of many of the known DNA repair pathways including nucleotide and base excision repair, very short repair, methyl-dependent mismatch repair, replication restart and recombinational repair (Criss et al., Helm & Seifert, 2009, Kline et al., 2003, Kline & Seifert, 2005a, Kline & Seifert, 2005b, LeCuyer et al., Mehr & Seifert, 1998, Sechman et al., 2006, Skaar et al., 2002, Stohl & Seifert, 2001, Stohl & Seifert, 2006). However, Gc lacks homologues of the genes involved in alkylation repair suggesting that the organism does not encounter alkylating agents in the host and that selective pressure failed to maintain these genes (Kline et al., 2003). Interestingly, despite the fact that Gc does not normally experience UV irradiation, the pathogen encodes a DNA photolyase orthologue, phrB suggesting that a selective pressure exists to maintain this gene (Kline et al., 2003).

DNA photolyases have been characterized in all three kingdoms of life (Goosen & Moolenaar, 2008). The Gc phrB orthologue is most similar to the cyclobutane pyrimidine dimer (CPD) photolyases which repair the most frequently UV-induced DNA lesions, CPDs (Goosen & Moolenaar, 2008). CPD photoloyases are considered to be ancient DNA repair enzymes that evolved in the first forms of life on the surface of primordial Earth, when the UV flux was at least three orders of magnitude higher than today (Essen, 2006, Muller & Carell, 2009). CPD photolyases are monomeric DNA binding proteins that have two non-covalently bound chromophores, flavin adenine dinucleotide (FADH) and methenyltetrahydrofolate (MTHF) (Goosen & Moolenaar, 2008, Sancar, 2004). To repair CPD DNA lesions, the antenna pigment, MTHF absorbs a blue-light photon and subsequently transfers the excitation energy to FADH (Goosen & Moolenaar, 2008). The excited flavin then transmits an electron to the CPD lesion which reverses the cyclobutane ring to repair the damage, with the released electron restoring the functional form of FADH (Goosen & Moolenaar, 2008).

In this work, we show that the gonococcal PhrB does not function in photo-reactivation but instead is a DNA binding protein required for proper negative DNA supercoiling. Interestingly, inactivation of phrB rendered gonococci more sensitive to ROS, and oxidative damage also alters Gc DNA supercoiling. These results support the hypothesis that the maintenance of proper DNA supercoiling is important for DNA repair and pathogenesis in Neisseria.

Results

Phylogeny of the gonococcal PhrB orthologue

In eubacterial species PhrB orthologues function as DNA photolyases, repairing UV induced pyrimidine dimers with the energy provided by visible light (Goosen & Moolenaar, 2008, Sancar, 2004). To determine the Gc FA1090 PhrB orthologue (NGO1707) protein conservation and the evolutionary relationship of PhrB orthologues amongst the Neisseria species and other bacterial species, we performed an alignment of protein sequences using blastp and CLUSTALW (Altschul et al., 1990, Thompson et al., 1994). The PhrB sequence is conserved in both pathogenic and commensal Neisseria species but shares little identity to other bacterial species (Fig. 1). The pathogenic Neisseria, Gc and N. meningitidis strains are most closely related to Gc FA1090 PhrB with 93–100% identity followed by the commensal Neisseria species with 44–93% identity (Fig. 1). Interestingly, N. meningitidis strain MC58 has a frame-shift 120 base pairs from the start codon of the gene suggesting that PhrB is not functional in this strain (Davidsen & Tonjum, 2006). The evolutionary relatedness of the Neisserial PhrB orthologues follows the same phylogenetic relationship of the Neisseria species after whole genome comparison and analysis (Marri et al., 2010). The closest related PhrB orthologue to Gc FA1090 outside of the Neisseria species was Eikenella corrodens with 36% identity followed by Yersinia pestis and Escherichia coli with 32% identity (Fig. 1).

Figure 1. The evolutionary relationship and protein conservation of the N. gonorrhoeae PhrB orthologue compared to other PhrB orthologues.

Phylogenetic tree of PhrB orthologues from the pathogenic and commensal Neisseria species and other bacterial species created by CLUSTAL analysis. The percent protein identity of each PhrB orthologue compared to the Gc FA1090 sequence is shown in parenthesis. (A) Represents the PhrB sequence of two N. menigitidis strains FAM18 and 053442 which share 100% identity.(B) Represents the PhrB sequence of seven N. gonorrhoeae strains FA1090, 35/02, FA19, PID332, SK-92-679, SK-93-1035, and F62 which share 100% identity. (C) Represents the PhrB sequence of two N. gonorrhoeae strains PID18, and DGI2 which share 100% identity. (D) Represents the PhrB sequence of six N. gonorrhoeae strains NCCP11945, DGI18, FA6140, PID1, PID24-1, and 1291 which share 100% identity.

The gonococcal PhrB gene is not a functional orthologue of the E. coli PhrB

Since a PhrB orthologue is conserved in almost all pathogenic and commensal sequenced Neisseria species, none of which are routinely exposed to either UV irradiation or visible light, we asked whether the Gc PhrB acts as a photolyase or expresses an alternate function. In Gc FA1090, NGO1707 is most similar to deoxyribodipyrimidine photolyase COG 0415 having both a DNA photolyase domain and a flavin adenine dinucleotide (FAD)-binding domain (Fig. 2A) (Weber, 2005). Since photolyase orthologues from all three kingdoms of life (archaea, bacteria, and eukarya) have been found to complement phrB-deficient E. coli (Essen, 2006, Goosen & Moolenaar, 2008, Muller & Carell, 2009, Sancar, 2008, Sancar & Rupert, 1978a), we asked whether the Gc FA1090 PhrB orthologue could complement the E. coli phrB mutant strain CSR603(Sancar & Rupert, 1978b). Plasmids expressing either the E. coli or Gc phrB genes were constructed and quantitative RT-PCR measurements showed that the Gc phrB message was expressed at 75% of the E. coli phrB message (data not shown). E. coli CSR603 (parental) or CSR603 harboring either E. coli phrB or Gc phrB were UV irradiated, and immediately following irradiation, bacteria were either exposed to visible light (L) to allow photo-reactivation or left in the dark (D) for 1 hour and then grown overnight in the dark before the relative survival was calculated (Fig. 2B). None of the strains kept in the dark following UV irradiation showed increased survival, however, the strain expressing the E. coli phrB gene showed about 500-fold increased survival after exposure to visible light following UV irradiation (Fig. 2B). These results show that the Gc phrB cannot complement the E. coli phrB mutant and suggest that the Gc PhrB does not function as a DNA photolyase.

Figure 2. Comparison of N. gonorrhoeae PhrB and E. coli PhrB.

A. Amino acid sequence alignment of N. gonorrhoeae (Gc) strain FA1090 PhrB and E. coli (Ec) PhrB. Identical residues are indicated by white letters boxed in grey while conserved residues are indicated by black letters boxed in grey. A black line indicates gonococcal residues 18–171 that are most similar (E-value of 2e⁻⁰⁸) to the DNA photolyase superfamily pfam00875 and a grey line indicates gonococcal residues 206–426 that are most similar (E-value of 4e⁻⁴⁷) to the flavin adenine dinucleotide (FAD) binding domain superfamily 7 pfam03441. Asterisks indicate identical or conserved residues that are involved in DNA binding (Weber, 2005). B. E. coli survival after UV irradiation. E. coli phrB strain CSR603 (Ec PhrB−) was complemented with either E. coli phrB (Ec PhrB−/pBlunt-phrB_Ec) or the gonococcal phrB (Ec PhrB−/pBlunt-phrB_Gc) on a high-copy plasmid (Sancar & Rupert, 1978b). Immediately following 0, 20, 40, and 60 joules/m² UV irradiation, bacteria were either exposed to light (L) for 1 hour or kept in the dark (D). Survival relative to cells not treated with UV irradiation was calculated after growth at 37°C in the dark for 16–18 hours. Error bars represent the standard error of the mean of four independent experiments. Asterisks indicate P<0.05 as compared to exposed to light show as measured by two-tailed Student’s t-test.

Inactivation of phrB affects colony and cell morphology but does not affect Gc growth

Since the Gc phrB gene was unable to complement an E. coli phrB mutant, we were interested in determining the role of phrB in Gc strain FA1090. Expression of the Gc phrB gene was confirmed by RT-PCR (data not shown), and an insertional phrB loss-of-function mutant was constructed to determine what role this gene plays in Gc genetics or physiology (Fig. 3A). To rule out polarity or second-site mutation as a basis for a phenotype, a functional complement with phrB under control of an inducible lac promoter inserted at an ectopic locus was created (Fig. 3B) (Mehr & Seifert, 1998).

Figure 3. Genome organization of tthe phrB locus, mutant and complement.

A. Chromosomal map of the phrB locus. In Gc strain FA1090 chromosome, phrB (black arrow) is located between NGO1706 and NGO1708 (large grey arrows). The phrB mutant strain contains a kanamycin transposon insertion (Km^R, triangle) 227 bps from the 5′ end of the 1296 bp orf. B. The phrB complement strain. The phrB gene was cloned downstream of a dual taclac promoter (hatched box) (Mehr & Seifert, 1998). This construct was transformed into the phrB mutant and inserted into the neisserial intergenic complementation site (NICS) (Mehr & Seifert, 1998).

Growth of the phrB mutant on solid media resulted in smaller colonies than the parental strain (Fig. 4A), and this phenotype was complemented by expressing the gene from the ectopic locus (Fig. 4A & B) (Mehr & Seifert, 1998). Interestingly, even though the phrB colonies were smaller than the parental strain, inactivation of phrB did not alter the number of colony forming units per colony (Fig. 4C). Moreover, the phrB mutant small colony phenotype remained both when bacteria were incubated in an anaerobic environment, as well as when cells were UV irradiated followed by an hour of light or dark incubation and subsequent aerobic overnight growth (data not shown). We conclude that the phrB small colony size phenotype was not due to a growth defect and was not altered by oxygen availability or exposure to UV irradiation or visible light.

Figure 4. Inactivation of phrB effects colony size and cell morphology.

A. Colony phenotypes. Shown are representative stereo micrographs of the parental, phrB mutant (PhrB−) and complement colonies grown at 37^°C in 5 % CO₂ for 22 hours. B. Colony diameters. Shown is the average colony diameter of the parental, phrB mutant (PhrB−), and complement strains. Error bars represent the standard error of the mean of 20 colonies. The diameter of the phrB mutant is less than the parental strain as measured by Student’s t-test, P<0.05 (asterisk). C. Growth quantification. The average colony forming units (CFU) per colony for 6 independent colonies of the parental, phrB mutant (PhrB−), and complement strains is shown after 22 hours of growth at 37^°C in 5 % CO₂. Error bars represent the standard error of the mean. D. Thin section transmission electron microscopy of cellular morphology. Representative micrographs of the parental strain and phrB mutant (PhrB−) are shown. The average cross-section diameter of 100 (79 monococci and 21 diplococci) parental strain cells was 0.86 +/− 0.01 μm while the average cross-section diameter of 100 (84 monococci and 16 diplococci) phrB mutant cells was 0.99 +/− 0.02 μm. Cell cross-section diameter was measured from two independent experiments. The average cross-section diameter of the parental strain is less than the phrB mutant as measured by Student’s t-test, P<0.05. Of the cells measured, 21 and 69 were not uniformly coccalcoccal for the parental strain and phrB mutant, respectively.

Since the smaller-sized colonies were not the result of a growth defect, we visualized gonococcal cells by thin section transmission electron microscopy (TEM) (Kellenberger et al., 1958, Silva et al., 1968). The phrB mutant cells exhibited a misshapen morphology which was not observed with the parent strain, which was more uniformly coccal. The phrB mutant cells had an average cross-section diameter of 0.99 +/− 0.02 μm while the parental cells had an average cross-section diameter of 0.86 +/−0.01 μm (Fig. 4D). Therefore, loss of phrB alters cell shape and size and this overall change in cell physiology is the likely cause of the smaller colonies.

PhrB does not function as a photolyase and loss of phrB does not result in a mutator phenotype

To test whether phrB encodes a functional photolyase, the parental, phrB, and complement strains were exposed to UV irradiation with subsequent 1 hour incubation with or without light exposure followed by overnight growth in the dark. There were no differences in survival of any of the tested strains independent of light exposure (Fig. S1A). These data are consistent with the observations made by Campbell and Yasbin (Campbell & Yasbin, 1979), and conclusively demonstrate that phrB does not encode a functional photolyase nor is photo-reactivation provided by an alternative process.

Since PhrB orthologues in other organisms participate in DNA repair, we asked whether loss of the gene would result in a mutator phenotype (Sancar, 2003). We measured the spontaneous mutation frequency of the parental, phrB mutant, and complement strains when exposed to three antibiotics: spectinomycin (Spec), rifampicin (Rif), and kasugamycin (KSG) (Fig. S1B & C, data not shown). The parental, phrB mutant, and complement strains showed no difference in spontaneous mutation frequency when exposed to Spec, Rif, or KSG (Fig. S1B & C, data not shown). Thus, inactivation of phrB does not cause a mutator phenotype.

Inactivation of phrB affects survival upon exposure to oxidative damage, nalidixic acid, and spectinomycin

Since the gonococcal phrB orthologue is expressed and does not encode a DNA photolyase, we asked whether phrB has a role in resisting reactive oxygen species (ROS) by measuring survival after exposure to hydrogen peroxide (Fig. 5A). We found that the phrB mutant showed a statistically significant 4-fold increase in sensitivity to hydrogen peroxide (Fig. 5A). This sensitivity to ROS was complemented by expression of phrB from an ectopic locus (Fig. 5A) (Mehr & Seifert, 1998). These results show that PhrB aids gonococcal survival to the oxidative damaging agent hydrogen peroxide.

Figure 5. Gonococcal survival assays.

A. Survival after exposure to H₂O₂. The parental, phrB mutant (PhrB−) and complement strains were exposed to 0, 10, 20, 50 mM H₂O₂. Error bars represent the standard error of the mean of five-six independent experiments. The phrB mutant (PhrB−) is more sensitive than the parental strain when exposed to 10 and 20 mM H₂O₂ as measured by Student’s t-test, P<0.05 (asterisks). B. Nalidixic acid survival assay. Nalidixic acid is a compound that inhibits DNA gyrase and causes DNA double strand breaks (Zhao et al., 1997, Cozzarelli, 1980). The parental, phrB mutant (PhrB−), and complement strains were grown on media containing 0.8 ug/ml nalidixic acid. Bars represent the median of seven independent experiments where each experiment had five replicates. The phrB mutant is different from the parental strain as measured by Student’s t-test, P<0.05 (asterisks). C. Rifampicin survival assay. Rifampicin inhibits transcription by binding to the β-subunit of DNA-dependent RNA polymerase (Nolte et al., 2003). The parental, phrB mutant (PhrB−), and complement strains were grown on media containing a level of rifampicin (30 ng/ml), which allows survival to be measured. Bars represent the median of five independent experiments where each experiment had five replicates. The phrB mutant is different from the parental strain as measured by Student’s t-test, P<0.05 (asterisks).

ROS can damage many essential molecules, but one result of ROS exposure is DNA damage that leads to double strand breaks (Demple & Harrison, 1994, Galhardo et al., 2000). We therefore measured survival of the parental strain, phrB mutant, and complement strain on media containing bactericidal levels of nalidixic acid (Nal). Nal is a quinolone type antibiotic that causes DNA double strand breaks by inhibiting the ligation activity of DNA gyrase (Zhao et al., 1997). The parental and complement strains showed similar levels of survival to Nal exposure, but the phrB mutant showed greater than 7,000-fold increase in survival (Fig. Consistant5B). Consistent with the lack of a mutator phenotype, the the survival to Nal-induced killing was not the result of mutation to Nal resistance assince the resulting colonies had not acquired increased resistance to Nal. Also, the phrB mutant showed no differential resistance to killing by the gyrase inhibitor novobiocin (Gellert et al., 1976), which inhibits gyrase prior to cleavage and thus does not produce double strand breaks (data not shown).

We also examined survival of the phrB mutant on media containing Rif. The phrB mutant showed greater than 13,000-fold increase in survival on media containing Rif as compared to the parental and complement strains, which was not the result of mutation to Rif resistance suggesting that mutation of phrB alters the sensitivity of Gc to Rif mediated killing (Fig. 5C).

Expression of phrB affects DNA supercoiling

Since the phrB mutant showed increased survival on media containing Nal and Rif, two antibiotics that can influence DNA supercoiling, we asked whether mutation of phrB might alter gonococcal DNA supercoiling. Changes to DNA supercoiling levels were assayed using chloroquine-agarose gel electrophoresis of the gonococcal cryptic plasmid as a surrogate measure for total cellular DNA supercoiling (Clark & Leblanc, 2009, Goldstein & Drlica, 1984, Korch et al., 1985,). The gonococcal cryptic plasmid is a natural low-copy plasmid which is maintained as an episome and exhibited a range of topoisomers on the chloroquine gel reflecting the influence of transcription and replication on supercoiling density. Surprisingly, inactivation of phrB increased the average negative DNA supercoiling of the cryptic plasmid relative to the parental strain and this phenotype was complemented by phrB expression at an ectopic locus (Fig. 6A) (Mehr & Seifert, 1998).

Figure 6. Analysis of DNA supercoiling by chloroquine-agarose gel electrophoresis of the gonococcal cryptic plasmid.

Extracted plasmid DNA from the parental, phrB mutant (PhrB−), and complement strains were analyzed by agarose-gel electrophoresis in the presence of 2.5 μg/ml chloroquine. The direction of migration of less negatively supercoiled DNA (+) and more negatively supercoiled DNA (−) is indicated next to each chloroquine gel image. Plots show the relative density of the parental (black), phrB mutant (red), and complement (blue) strains at the relative distance from the nicked circular plasmid DNA (arrowed). A. Shows untreated gonococci. B. Shows gonococci treated with the DNA gyrase inhibitor, nalidixic acid. C. Shows gonococci treated with the oxidative damaging agent, H₂O₂.

Since, the phrB mutant showed increased survival to Nal killing, we tested how negative DNA supercoiling of the mutant was affected by Nal. As expected, Nal treatment of the parental Gc, phrB mutant or complement strain showed decreased average negative DNA supercoiling when compared to the untreated bacteria (Fig. 6A & B). However, the Nal-treated phrB mutant showed increased average negative DNA supercoiling when compared to the Nal-treated parental and complement strains (Fig. 6B), suggesting that PhrB is not working in conjunction with DNA gyrase.

This increased negative supercoiling of the phrB mutant suggested that some or all of the phenotypes expressed by the mutant could be the result of altered supercoiling. Since the phrB mutant showed changes in cell morphology by thin section TEM, we tested whether changes in DNA supercoiling would result in altered cell morphology (Fig. S2). First, we treated gonococci with Nal and then visualized the cells by thin section TEM (Fig. S2). We found that changes in DNA supercoiling induced by Nal inhibition of DNA gyrase caused a significant decrease in cellular size of the parental strain (Fig. 4D & S2). In addition, the Nal treated phrB mutant showed cells that were less irregularly shaped when compared to the untreated phrB mutant cells (Fig. 4D & S2). Taken together, these data suggest that changes in DNA supercoiling induced by Nal inhibition of DNA gyrase may be responsible for some of the changes in Gc cellular morphology.

Since we had demonstrated that the phrB mutant showed increased survival to Nal killing, we tested how negative DNA supercoiling of the mutant was affected by Nal. As expected, Nal-treatment of the parental Gc or complemented strain showed decreased average negative DNA supercoiling when compared to the untreated bacteria (Fig. 7A & B). The Nal-treated phrB mutant also showed increased average negative DNA supercoiling when compared to the untreated phrB strain (Fig. 7B), suggesting that PhrB is not working in conjunction with DNA gyrase

Figure 7. Analysis of PhrB DNA binding and bending in vitro.

A. Fluorescence anisotropy of PhrB plotted at increasing protein concentrations per amount of 30 bp dsDNA oligonucleotide substrate (o30dsDNA). Error bars represent the standard error of the mean for 3 independent experiments with a total of 6 replicates. B. Fluorescence anisotropy of PhrB plotted at increasing protein concentrations per amount of UV irradiated (1000 joules/m²) o30dsDNA oligonucleotide substrate. Error bars represent the standard error of the mean for 3 independent experiments with a total of 6 replicates. C. Protein duplex DNA bending assay. Shown is a (2.5 μg/ml) chloroquine gel of plasmid DNA (pCRBlunt2bp) incubated with topoisomerase I, DNA gyrase with or without the addition of topoisomerase I, and increasing concentrations of PhrB to DNA (1:1, 10:1, 100:1) with or without the addition of topoisomerase I. The direction of migration of less negatively supercoiled DNA (+) and more negatively supercoiled DNA (−) is indicated.

Due to the sensitivity of the phrB mutant to hydrogen peroxide killing, we tested whether ROS might also affect DNA supercoiling. Hydrogen peroxide-treated Gc showed decreased average negative DNA supercoiling when compared to untreated bacteria (Fig. 6A & C). However, the phrB mutant still showed increased average negative DNA supercoiling compared to the parental strain after hydrogen peroxide treatment (Fig. 6C). The effect of these different DNA damaging agents on supercoiling suggests that some of the phenotypes associated with the phrB mutant are a direct result of the role PhrB has in maintaining proper supercoiling within gonococci.

PhrB binds DNA and causes topological strain in vitro

Since photolyases are DNA binding proteins and the Gc PhrB has conserved residues that are important for DNA binding in other organisms (Goosen & Moolenaar, 2008, Sancar, 2004, Weber, 2005), we tested directly whether the Gc PhrB could bind DNA (Fig. 2B, 7A & C). We used fluorescence anisotropy, to determine the equilibrium DNA binding behavior of purified PhrB in vitro (Favicchio et al., 2009, Anderson et al., 2008). In these experiments, we used FAM-labeled dsDNA, UV treated dsDNA, oligo dT30, and hairpin DNA (Fig. 7A & B, data not shown). PhrB bound dsDNA with an apparent K_d of 65.1 +/− 6.3 nM whereas UV irradiated dsDNA was bound with weaker affinity having an apparent K_d of 118.5 +/− 63.7 nM (Fig. 7A & B). These data suggest that PhrB is not acting like a photolyase to bind pyrimidine dimers. We also determined that PhrB bound hairpin DNA with an apparent K_d similar to dsDNA but was unable to bind oligo dT30, suggesting that the protein is unable to bind ssDNA.

We tested whether purified PhrB protein showed any topoisomerase activity on DNA. There was no change in DNA supercoiling when plasmid DNA was incubated with PhrB in while. In contrast, topoisomerase I completely relaxed the supercoiled plasmid while DNA gyrase slightly increased the negative supercoiling density of this plasmid (Fig. 7C). We additionally tested whether PhrB was able to induce DNA bending or torsional strain upon DNA binding in vitro. PhrB was incubated with a circular dsDNA molecule (plasmid DNA) and topoisomerase I was added to the protein-DNA complex. In this assay, if a protein has caused the DNA to bend, topoisomerase I dissipates the topological strain by changing the linking number of the dsDNA molecule which can be visualized by chloroquine gel electrophoresis (Clark & Leblanc, 2009). As little as a 1:1 ratio of PhrB to plasmid DNA caused DNA topological strain indicating that PhrB bends DNA upon binding (Fig. 7C).

Discussion

We have demonstrated that the gonococcal PhrB orthologue does not function in photo-reactivation but has a novel role in maintaining proper negative DNA supercoiling and cellular physiology. The gonococcal phrB mutant exhibited several interesting phenotypes: small colonies, larger misshapen cells, peroxide sensitivity, resistance to nalidixic acid and rifampicin killing, and increased negative DNA supercoiling. Most of these phenotypes are likely to be directly or indirectly linked to the supercoiling changes in the mutant. In bacteria, a homeostatic balance between DNA gyrase and topoisomerase I is thought to maintain negative DNA supercoiling at levels that are beneficial for the cell (Dorman, 1991). Moreover, alterations in DNA supercoiling has been found to modulate gene expression (Dorman, 2006), and therefore it is possible that some or all of the phenotypes recorded for the phrB mutant are the result of changes in gene expression.

There are three main models that could explain the requirement of PhrB for proper negative DNA supercoiling. First, since the phrB mutant has increased negative supercoiling and the PhrB protein bends DNA upon binding in vitro, it is possible that PhrB acts directly on DNA to remove negative DNA supercoils. We consider this scenario to be unlikely since there are no conserved residues or domains in PhrB that suggest it has topoisomerase activity and we did not see any evidence of topoisomerase activity in vitro (Fig. 7C). Alternatively, it is possible that PhrB is required for topoisomerase I to relax DNA supercoiling. The interaction of PhrB with topoisomerase I could be direct or indirect, but topoisomerase I loss-of-function mutants are non-viable, and while there are no known commercially available compounds that specifically inhibit bacterial topoisomerase I in vivo, the phospholipid cardiolipin has been suggested to inhibit E. coli topoisomerase I in vivo (Mizushima et al., 1992, Tse-Dinh, 2009). However, treatment of Gc with cardiolipin did not alter supercoiling in the wild-type strain, which suggests that either cardiolipin has no affect on Gc topoisomerase I or other mechanisms compensate for the affect of cardiolipin on Gc topoisomerase I (Fig. 6A & S3) (Mizushima et al., 1992). Interestingly, cardiolipin-treatment of the phrB mutant decreased the average amount of DNA supercoiling causing the mutant to resemble the untreated parental strain (Fig. 6A & S3). This indicates that cardiolipin affects DNA supercoiling only in the absence of PhrB and since wild-type bacteria treated with cardiolipin have no phenotype, this suggests that PhrB compensates for the affect of the compound. Thus, we cannot definitively rule in or out a model where PhrB interacts directly indirectly through topoisomase I. Finally, PhrB could influence global transcription or DNA replication both of which alter negative DNA supercoiling and also rely on proper negative DNA supercoiling. The lack of a growth defect for the phrB mutant argues against there being a replication effect, but the change in cellular morphology may indicate a change in global transcription or perhaps DNA condensation.

The larger misshapen cells observed by EM of thin sectioned bacteria suggest that phrB is required for proper cellular physiology. We assume that the smaller colonies of the phrB mutant are a reflection of this change in physiology. Since the phrB mutant showed both changes in cell physiology and increased negative DNA supercoiling, we tested whether changes in DNA supercoiling affects cellular morphology. We treated parental and phrB mutant cells with Nal and found that parental cells showed a significant decrease in size when compared to untreated parental bacteria (Fig. 7B, & S2). Although, we did not see a decrease in size of the Nal-treated phrB mutant cells, we observed that the cell shape was more coccoid and less misshapen than the untreated phrB mutant cells (Fig. 7B, & S2). While an alteration of transcription could account for this change in physiology, it is as likely that alterations in supercoiling density could directly alter the bacterial nucleoid to affect cellular physiology. Surprisingly, this drastic change in physiology and colony size was not reflected in a change in the number of CFUs per colony which shows that even with these drastic changes, the growth of the bacteria is not altered in the phrB mutant.

The phrB mutant showed divergent phenotypes. The phrB mutant was very sensitive to hydrogen peroxide, but insensitive to UV, and spontaneous mutation assays showed that inactivation of phrB does not result in a mutator phenotype which rules out a general DNA repair problem. The enhanced sensitivity to hydrogen peroxide, Nal and Rif is probably not the result of a porous membrane since there was no change in many other antibiotic resistance levels (data not shown). While we have not determined why the phrB mutant is more sensitive to hydrogen peroxide, it is possible that the altered cellular physiology of the mutant creates this increased sensitivity. To determine whether the restoration of supercoiling to parental levels in the phrB mutant could reverse its sensitivity to hydrogen peroxide, we used a level of Nal which restored supercoiling of the phrB mutant to near wild-type levels, and then treated these bacteria with hydrogen peroxide (data not shown). There was no change in hydrogen peroxide sensitivity of the Nal-treated phrB mutant (data not shown). Additionally, while there were changes in gonococcal supercoiling in response to ROS and Nal that were reversed in the phrB mutant (Fig. 6B & C), the differential sensitivity of the mutant to these toxic chemicals (Fig. 5A & B), prevents a causal relationship between the supercoiling alterations and the survival phenotypes from being made.

The PhrB orthologue is one of the most highly conserved proteins among the pathogenic Neisseria, which suggests that PhrB has the identical function in meningococci (Fig. 1). Although the MC58 N. meningitidis strain has a confirmed frame-shift within the phrB coding sequence, this stain has undergone multiple laboratory passages and could carry a second site mutation. Since the evolutionary relatedness of the Neisserial PhrB orthologues follows the same phylogenetic relationship of the Neisseria species, the more divergent PhrB orthologues encoded in the commensal strains also suggests that this gene and its function are well conserved amongst the Neisseria. (Marri et al., 2010). It has previously been established that growth temperature, oxygen availability, osmolarity, and oxidative stress can all alter DNA supercoiling and the changes in supercoiling we observed are similar to these described in other bacterial species, but the effect of a phrB mutation on these global changes in supercoiling has not been previously described (Dorman, 1991, Dorman & Corcoran, 2009, Lossius et al., 1981, Morikawa et al., 2007, Weinstein-Fischer et al., 2000). It will be interesting to determine whether other PhrB orthologues also influence supercoiling is addition to their well established roles in photo-reactivation. Regardless of how generalizable this role for PhrB becomes, this work establishes how a UV DNA lesion repair enzyme can be utilized by a strict human pathogen to presumably ensure proper cellular genetics and physiology in response to ROS during infection.

Experimental Procedures

Bacterial Growth Conditions

E. coli TOP10 competent cells (Invitrogen) were grown in Luria-Bertani (LB) broth or solid media containing 15 g/L agar at 37°C and used to propagate plasmids. E. coli selected for plasmids containing kanamycin, chloramphenicol, or erythromycin resistance were selected on media containing 50 μg/ml, 20 μg/ml or 100 μg/ml of the respective antibiotic. Gonococcal strains were grown on GC Medium Base (Difco) plus Kellogg supplements (GCB) [22.2 mM glucose, 0.68 mM glutamine, 0.45 mM co-carboxylase, 1.23 mM Fe(NO₃)₃; all from Sigma] at 37°C in 5% CO₂. For gonococcal anaerobic growth, bacteria were grown at 37°C for 72 hours (hrs) on GCB with 8 mM NaNO₂ in an anaerobic jar using the GasPak Plus anaerobic system with palladium catalyst (BD) and indicator strip (BBL).

Construction of the gonococcal phrB and complement strains

A portion of phrB was amplified from genomic DNA with PFU polymerase using primers phrBR2 and phrBL2 and cloned into pSMART (Lucigen) (Table S1). After sequencing the construct for accuracy, the EZ::TN transposition system (Epicentre) was used for in vitro transposition and the plasmid selected had a kanamycin transposon insertion 227 base pairs (bps) from the 5′ end of phrB. Then gonococcal strain FA1090 1-81-S2 was transformed as described previously and selected on media containing 50 μg/ml kanamycin (Stohl & Seifert, 2001). This strain was verified by PCR and Southern blot analysis

To study inactivation of phrB in a nucleotide excision repair (NER) deficient background, an uvrA mutant was created. First, a portion of uvrA was amplified with KOD polymerase (Novagen) from genomic DNA using primers uvrAup and uvrAdown and cloned into pcR2.1 (Invitrogen), then sequenced for accuracy (Table S1). Next, a chloramphenicol resistance cassette was cloned into in the uvrA fragment containing plasmid at a unique uvrA SalI restriction site 2082 bps from the 5′ end of the gene. Subsequently, gonococcal strains FA1090 1-81-S2 and phrB FA1090 1-81-S2 were transformed as previously described and selected on media containing 0.5 μg/ml chloramphenicol (Stohl & Seifert, 2001). These strains were verified by PCR and Southern blot analysis.

To create the IPTG inducible phrB complement strain, we used KOD polymerase (Novagen) and primers phrBF1comp engineered to have a Pac I site and phrBR1comp to amplify the region between 63 bps upstream of the phrB ATG and 75 bps downstream of the stop codon and subcloned into pCRBluntII-Topo (Invitrogen), then sequenced for accuracy (Table S1). Subsequently, the phrB containing plasmid (pBlunt-phrB_Gc) was restriction digested with PacI and EcoRV and cloned into pGCC4 downstream of a dual taclac promoter that allows induction with IPTG (Mehr et al., 2000, Mehr & Seifert, 1998). This construct was sequenced for accuracy and transformed into strains phrB FA1090 1-81-S2 and phrB uvrA FA1090 1-81-S2 which inserted into the neisserial intergenic complementation site (NICS), linked to a erythromycin resistance cassette allowing for selection on media containing 2 μg/ml erythromycin (Mehr et al., 2000, Mehr & Seifert, 1998). These strains were verified by PCR and Southern blot analysis.

Construction of the E. coli phrB complement strains

We used pBlunt-phrB_Gc (described above) which has phrB expressed from a constitutive lac promoter. To create pBlunt-phrB_Ec, E. coli phrB was amplified with KOD polymerase using primers EcphrBcompF1 and EcphrBcompR1 from plasmid DNA isolated from E. coli strain MS09 (Sancar et al., 1984) (Table S1). The region amplified begins 33 bps upstream of the ATG and ends 37 bps downstream of the stop codon. This PCR product was cloned into pCRBluntII-Topo and sequenced for accuracy. The construct selected has E. coli phrB expressed from the constitutive lac promoter. Then pBlunt-phrB_Gc and pBlunt-phrB_Ec were transformed into E. coli phrB NER deficient strain CSR603 and selected for kanamycin resistance (Sancar & Rupert, 1978b).

Determination of relative expression level of E. coli expressing E. coli phrB or Gc phrB

E. coli harboring pBlunt-phrB_Gc or pBlunt-phrB_Ec were grown over night, diluted 1/100 and grown to OD₅₅₀ 0.6 at 37°C and then total RNA was isolated (Qiagen RNeasy Kit) and checked for purity, then treated with DnaseI. cDNA was synthesized using Superscript III (Invitrogen) from two independent cultures expressing either the E. coli phrB or Gc phrB with 20 pmol of primers EcPhrBRT or phrBL2, respectively (Table S1). We also synthesized cDNA with primer TopoKANcDNA specific for the kanamycin gene expressed on the same plasmid as an internal reference (Table S1). For PCR of the cDNA and determination of primer efficiency, we used primers EcF1 and EcR1 for E. coli phrB, primers phrBR2 and phrBL1 for Gc phrB, and primers TopoKanF1 and TopoKanR1 for the kanamycin gene (Table S1).

cDNA synthesis of the gonococcal phrB

Gonococci were grown from freezer stocks for approximately 20 hrs then 20 colonies were passaged onto GCB. After 9 hrs, colonies were swabbed and resuspended to OD₅₅₀ 0.1 in liquid media with supplements (GCBL) with 0.042 % NaHCO₃, then grown 16 hrs at 30°C in 15-ml conical tubes in a drum rotator. Then diluted in GCBL with 0.042 % NaHCO₃ to an OD₅₅₀ 0.3, grown 2.5–3 hrs at 37°C, and diluted to OD₅₅₀ 0.06 in GCBL with 0.042 % NaHCO₃ and grown at 37°C to harvest log phase cells. Total RNA was extracted from these log phase cells (Qiagen RNeasy Kit) and checked for purity, then treated with DnaseI. cDNA was synthesized using Superscript III and 20 pmol of primer phrBL2 (Table S1). To confirm expression, cDNA was used as a template for PCR with GoTaq polymerase (Promega) and primer pairs, phrBR2 and phrBL1 (Table S1). To quantitate expression we used Sybr green and the Roche light cycler system as described previously (Rohrer et al., 2005).

E. coli UV Sensitivity Assay

E. coli were grown over night, diluted 1/100 and grown to OD₅₅₀ 0.9–1.1, then serially diluted 10⁻¹ to 10⁻⁹ and plated on LB agar. Subsequently, E. coli were UV irradiated with 0, 20, 40, and 60 joules/m² (Stratagene Stratalinker). Immediately following UV irradiation, bacteria were incubated in the light or dark for 1 hr and then grown in the dark for 16–18 hrs, subsequently colonies were counted and their percent survival was calculated.

Gonococcal UV Sensitivity Assay

Gonococci were grown to 20 generations, swabbed from a confluent lawn and resuspended in liquid media, then serially diluted 10⁻¹ to 10⁻⁹ and plated on solid media. Then bacteria were exposed to 0, 3, 4, 5, and 7 joules/m² UV irradiation (Stratagene Stratalinker). Immediately after UV irradiation, bacteria were incubated in the light or dark for 1 hr, and grown 20–22 hrs, then colonies were counted and their percent survival was calculated. Strains used in this assay were NER deficient.

H₂O₂ Sensitivity Assay

Gonococci were grown to 20 generations, swabbed from a confluent lawn and resuspended in liquid media to OD₅₅₀ 0.1 in GCBL with supplements and 0.042 % sodium bicarbonate, then grown OD₅₅₀ 0.3, where the complement strain had 1 mM IPTG for induction of phrB expression. These cultures were diluted 1:10 in GCBL containing IPTG where appropriate, and 10 ml aliquots were placed into 15-ml conical tubes. H₂O₂ (Sigma-Aldrich) was added to a final concentration of 0, 10, 20, or 50 mM. Culture tubes were placed in a drum rotor at 37°C for 15 min and immediately serially diluted in liquid media, after the 15 min incubation in a drum rotator at 37°C, catalase (Sigma-Aldrich) was added to a final concentration of 10 μg/ml. Serial dilutions were plated onto GCB. Piliated colonies were counted after 20 hrs growth, and their relative survival was calculated.

Survival and Spontaneous Mutation Assays

Gonococci were grown to 20 generations, swabbed from a confluent lawn and resuspended in liquid media to OD₅₅₀ 0.300. Then 5 replicates per strain of 100 μl where the phrB mutant was first diluted 1:10000 were plated on GCB containing 0.8 μg/ml Nal acid or 30 ng/ml Rif for survival assays, or 25 ng/ml Spc or 60ng/ml Rif for spontaneous mutation assays. Colonies were counted and their percent survival was calculated after 44–48 hrs growth. The total CFU/ml was determined by plating serial dilutions on GCB and counting colonies after 22–24 hrs growth.

Thin Section Electron Microscopy

Gonococci were grown as described above for the H₂O₂ sensitivity assay, where liquid cultures were harvested at early-log phase (OD₆₀₀ 0.4) with or without 40 ug/ml nalidixic acid for 30 min. Cells were fixed for 30 min in 0.1 % OsO₄ RK (Ryter and Kellenberger) vernal acetate buffer centrifuged at 2000 rpm for 5 min and resuspended in 2 % agar, 1 % Tryptone in RK vernal acetate buffer (Kellenberger et al., 1958). After agar solidification <1 mm³ cubes were cut and emerged in 1 % OsO₄ RK vernal acetate buffer for 1 hr, then samples were washed 3x in RK buffer, then stained with 0.5 % uranyl acetate in RK buffer for 2 hrs then resuspended in RK buffer and further processed and embedded by the Northwestern University Cell Imaging Facility.

Chloroquine Gel Electrophoresis

Gonococci were grown to 20 generations, swabbed from a confluent lawn and resuspended in liquid media to OD₅₅₀ 0.200. Bacteria were treated with 40 μg/ml nalidixic acid for 30 min, 20 mM H₂O₂ for 15 min with 10 μg/ml catalase added after incubation, 0.1 % MMS for 15 min, or 0.04 mM cardiolipin (Sigma-Aldrich) for 30 min in a 15-ml conical tube in a drum rotor at 37°C. Plasmid DNA was extracted (Qiagen) from the parental strain, phrB mutant, and complement strain without induction or induced for phrB expression with 1 mM IPTG. 500 μg of plasmid DNA was electrophoresed in a 0.8 % agarose gel with 2.5 μg/ml chloroquine at 40V for 20 hrs in 1x TBE with 2.5 μg/ml chloroquine. Gels were rinsed with dH₂O then stained with in 1x TBE with ethidium bromide for 2 hrs, then rinsed with dH₂O and destained ½ hr in 1x TBE before visualization. Quantity one software (Bio-Rad) was used for density traces.

Fluorescence Anisotropy

Concentrated stocks of Gc PhrB in storage buffer (10 mM Tris, 500 mM NaCl and 5 mM β-me) (a kind gift from the Wayne Andersen laboratory at Northwestern University) was serially diluted in DNA binding buffer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM β-me, 1 mM MgCl₂, 0.1 g/L BSA, 4 % glycerol) and incubated with 10 nM 3′FAM-labeled o30 dsDNA, 3′FAM-labeled o30 dsDNA irradiated with 1000 joules/m², 3′FAM-labeled hairpin DNA, or 5′FAM-oligo dT30 at room temperature for 30 min (Table S1). Then the fluorescence anisotropy for each sample was measured on a Molecular Devices SpectroMax M5 plate reader at 495 nm excitation and 540 nm emission wavelengths. Apparent K_d, average, and standard error values were calculated using Origin software.

DNA Bending Assay

Plasmid DNA [100 ng of pCRBluntII-Topo (Invitrogen) with a 2 bp insertion (pBlunt-2bp)] was untreated or incubated with Topoisomerase I or DNA gyrase as indicated by the manufacture (NEB), or with increasing amounts of PhrB to DNA (1:1, 10:1, and 100:1) at 37°C for 30 min. In parallel, plasmid DNA was incubated with DNA gyrase or increasing amounts of PhrB to DNA (1:1, 10:1, and 100:1) at 37°C for 20 min, then Topoisomerase I was added to the reactions as indicated by the manufacture (NEB) and incubated for an additional 10 min at 37°C. All reactions indicated above contain 1x BSA, 1x NEB Buffer 2, and 1.25 mM ATP. Stop buffer (2 % SDS, 25 % glycerol, 0.05 % bromophenol blue, and 0.05 % xylene cyanol) was then added to the reactions followed by chloroquine gel electrophoresis as describe above.