The Nucleotide Excision Repair System of Borrelia burgdorferi Is the Sole Pathway Involved in Repair of DNA Damage by UV Light

Hardy, Pierre-Olivier; Chaconas, George

doi:10.1128/jb.00043-13

Cited by 20 publications

(29 citation statements)

References 111 publications

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“…In previous studies, we obtained similar results when the recA gene was inactivated, leading us to propose that the recombinational DNA repair pathway plays a minor role in M. genitalium (32). Interestingly, recent studies performed in B. burgdorferi have led to similar conclusions (62). These findings may be explained by the apparent lack of an SOS response in these microorganisms.…”

Section: Fig 5 Frequency and Accumulation Of Hasupporting

confidence: 68%

Characterization of the Operon Encoding the Holliday Junction Helicase RuvAB from Mycoplasma genitalium and Its Role in mgpB and mgpC Gene Variation

Burgos

Totten

2014

J Bacteriol

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Mycoplasma genitalium is an emerging sexually transmitted pathogen associated with reproductive tract disease in men and women, and it can persist for months to years despite the development of a robust antibody response. Mechanisms that may contribute to persistence in vivo include phase and antigenic variation of the MgpB and MgpC adhesins. These processes occur by segmental recombination between discrete variable regions within mgpB and mgpC and multiple archived donor sequences termed MgPa repeats (MgPars). The molecular factors governing mgpB and mgpC variation are poorly understood and obscured by the paucity of recombination genes conserved in the M. genitalium genome. Recently, we demonstrated the requirement for RecA using a quantitative PCR (qPCR) assay developed to measure recombination between the mgpB and mgpC genes and MgPars. Here, we expand these studies by examining the roles of M. genitalium ruvA and ruvB homologs. Deletion of ruvA and ruvB impaired the ability to generate mgpB and mgpC phase and sequence variants, and these deficiencies could be complemented with wild-type copies, including the ruvA gene from Mycoplasma pneumoniae. In contrast, ruvA and ruvB deletions did not affect the sensitivity to UV irradiation, reinforcing our previous findings that the recombinational repair pathway plays a minor role in M. genitalium. Reverse transcription-PCR (RT-PCR) and primer extension analyses also revealed a complex transcriptional organization of the RuvAB system of M. genitalium, which is cotranscribed with two novel open reading frames (ORFs) (termed ORF1 and ORF2 herein) conserved only in M. pneumoniae. These findings suggest that these novel ORFs may play a role in recombination in these two closely related bacteria.W ith a genome of only 580 kb encoding 482 predicted proteins, Mycoplasma genitalium is considered the organism with the smallest known genome capable of self-replication (1). Consequently, M. genitalium has become a model organism for understanding basic biological processes (2-4) and has been used as a platform to identify a minimal gene set required for autonomous cellular life (5). M. genitalium is also a sexually transmitted human pathogen with an implicated reproductive tract disease spectrum very similar to that of Chlamydia trachomatis, including urethritis in men (6) and cervicitis, endometritis, pelvic inflammatory disease (PID), and tubal factor infertility in women (7). M. genitalium infections are often asymptomatic and persistent (8-10), possibly increasing the risk for sexual transmission and sequelae such as PID and infertility. Despite the significance of primary disease and its sequelae, the molecular pathogenesis of M. genitalium has been understudied. Barriers to such investigations include the fastidious nature and slow growth of this bacterium, difficulty of isolating contemporary strains from human specimens, and the limited genetic tools available for molecular investigations (11).In previous studies, we and others have shown that M. genitalium strains gen...

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Section: Fig 5 Frequency and Accumulation Of Hasupporting

confidence: 68%

Characterization of the Operon Encoding the Holliday Junction Helicase RuvAB from Mycoplasma genitalium and Its Role in mgpB and mgpC Gene Variation

Burgos

Totten

2014

J Bacteriol

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“…Although the Tn library does not contain mutants with insertions in many of the known B . burgdorferi ROS and RNS resistance genes, it does contain mutants with insertions in uvrB , uvrC , and uvrD , all of which encode components of the NER pathway that are required for repairing DNA damage after NO exposure [12, 34]. These mutants were used as internal controls.…”

Section: Resultsmentioning

confidence: 99%

“…Approximately 20% of the genes that were identified in the DEA/NO screen encode DNA repair enzymes, consistent with previous reports that DNA repair enzymes are involved in NO resistance in B . burgdorferi (Fig 2B) [12, 34]. In contrast, approximately 40% of the genes identified in the TBHP and H 2 O 2 screens encode predicted inner membrane proteins or transporters (Fig 2C and 2D).…”

Section: Resultsmentioning

confidence: 99%

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A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species

et al. 2017

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Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism.

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“…B. burgdorferi was grown in BSK II medium (44) prepared in-house and supplemented with 5% rabbit serum (Cedar Lane). B. burgdorferi transformations were performed as described previously (45,46). PCR was performed on genomic DNA to screen for the presence of the insert.…”

Section: Methodsmentioning

confidence: 99%

Construction and Characterization of a Borrelia burgdorferi Strain with Conditional Expression of the Essential Telomere Resolvase, ResT

2014

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Borrelia species are unique in the bacterial world in possessing segmented genomes which sometimes contain over 20 genetic elements. Most elements are linear and contain covalently closed hairpin ends requiring a specialized process, telomere resolution, for their generation. Hairpin telomere resolution is mediated by the telomere resolvase, ResT. Although the process has been studied extensively in vitro, the essential nature of the resT gene has precluded biological studies to further probe the role of ResT. In this work, we have generated a B. burgdorferi strain that carries an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible resT gene controlled by a tightly regulated promoter. ResT is expressed in this strain at ϳ14,000 monomers per cell, similar to the ϳ15,000 monomers observed for the parental strain. We demonstrate ResT depletion with a half-life of 16 h upon IPTG washout. ResT depletion resulted in arrested growth 48 h after washout. Interestingly, not all spirochetes died after ResT washout, and at least 15% remained quiescent and could be resuscitated even at 2 weeks postwashout. Significant levels of DNA synthesis were not observed upon growth arrest, suggesting that ResT might interact directly or indirectly with factors controlling the initiation or elongation of DNA synthesis. Analysis of the linear plasmids lp17 and lp28-2 showed that the linear forms of these plasmids began to disappear and be replaced by higher-molecular-weight forms by 24 h post-IPTG washout. Treatment of DNA from the ResT-depleted strain with ResT in vitro revealed the presence of replicated telomeres expected in replication intermediates. L yme disease, caused by the bacterium Borrelia burgdorferi and related species (1-4), is the most commonly reported vectorborne illness in North America and Europe (5), with a significant presence in other regions of the Northern Hemisphere (6, 7). A unique feature of B. burgdorferi is its segmented genome. The prototype B31 genome consists of a single linear chromosome of approximately 1 Mb, as well as a mixture of approximately 20 linear and circular plasmids (8, 9). To overcome the end replication problem, the ends of the linear replicons are terminated by covalently closed hairpin telomeres (10-12). Replication of the linear elements is believed to proceed bidirectionally (Fig. 1) from an internal origin of replication (13-15), resulting in a circular, head-to-head, tail-to-tail dimer intermediate. The dimer intermediate is processed by telomere resolution, a DNA breakage and reunion reaction that results in cleavage at the dimer junctions followed by ligation of complementary strands to generate the hairpin telomeres (16-19).Telomere resolution is carried out by the telomere resolvase, ResT, encoded by the circular plasmid cp26 (20). ResT has been extensively characterized, and its mechanism is well defined (12, 21-32). The enzyme is mechanistically similar to type IB topoisomerases and tyrosine recombinases. It promotes telomere resolution through a two-step transesterification i...

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The Nucleotide Excision Repair System of Borrelia burgdorferi Is the Sole Pathway Involved in Repair of DNA Damage by UV Light

Cited by 20 publications

References 111 publications

Characterization of the Operon Encoding the Holliday Junction Helicase RuvAB from Mycoplasma genitalium and Its Role in mgpB and mgpC Gene Variation

Characterization of the Operon Encoding the Holliday Junction Helicase RuvAB from Mycoplasma genitalium and Its Role in mgpB and mgpC Gene Variation

A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species

Construction and Characterization of a Borrelia burgdorferi Strain with Conditional Expression of the Essential Telomere Resolvase, ResT

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