Limits...
Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

Stohl EA, Gruenig MC, Cox MM, Seifert HS - PLoS ONE (2011)

Bottom Line: Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology.Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec).This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America. e-stohl@northwestern.edu

ABSTRACT
The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

Show MeSH

Related in: MedlinePlus

Strand exchange with DNA substrates to mimic DNA transformation and antigenic variation in vitro.A. Schematic of pGEM vector with relevant restriction sites used to clone heterologous inserts (see Materials and Methods). B. Linear dsDNA heterologous inserts digested with NdeI to give medial heterology and reacted with pGEM cssDNA. C. % nicked circular product observed in strand exchange reactions promoted by RecANg and RecAEc using pGEM circular ssDNA reacted with pGEM-10 and pGEM-100 linear dsDNA (designated “10” and “100” in Figure). Error bars represent the standard error of the mean of 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg 100 compared to RecAEc 100). D. Schematic of construct pGEM 1-81-S2 containing the 1-81-S2 pilE DNA sequence and relevant restriction sites. E. Linear dsDNA of antigenic variants SV, HVL, and SV-HVL heterologies (designated with shading and cross-hatches, see Figure S3 and Materials and Methods) digested with XmnI to give medial heterology and reacted with pGEM 1-81-S2 circular ssDNA. F. % nicked circular product observed in strand exchange reactions promoted by RecAEc and RecANg using pGEM-1-81-S2 cssDNA reacted with pGEM-1-81-S2 or pGEM-SV-HVL linear dsDNA. Error bars represent the standard error of the mean of at least 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg SV-HVL relative to RecAEc SV-HVL).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040777&req=5

pone-0017101-g006: Strand exchange with DNA substrates to mimic DNA transformation and antigenic variation in vitro.A. Schematic of pGEM vector with relevant restriction sites used to clone heterologous inserts (see Materials and Methods). B. Linear dsDNA heterologous inserts digested with NdeI to give medial heterology and reacted with pGEM cssDNA. C. % nicked circular product observed in strand exchange reactions promoted by RecANg and RecAEc using pGEM circular ssDNA reacted with pGEM-10 and pGEM-100 linear dsDNA (designated “10” and “100” in Figure). Error bars represent the standard error of the mean of 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg 100 compared to RecAEc 100). D. Schematic of construct pGEM 1-81-S2 containing the 1-81-S2 pilE DNA sequence and relevant restriction sites. E. Linear dsDNA of antigenic variants SV, HVL, and SV-HVL heterologies (designated with shading and cross-hatches, see Figure S3 and Materials and Methods) digested with XmnI to give medial heterology and reacted with pGEM 1-81-S2 circular ssDNA. F. % nicked circular product observed in strand exchange reactions promoted by RecAEc and RecANg using pGEM-1-81-S2 cssDNA reacted with pGEM-1-81-S2 or pGEM-SV-HVL linear dsDNA. Error bars represent the standard error of the mean of at least 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg SV-HVL relative to RecAEc SV-HVL).

Mentions: The ability of N. gonorrhoeae to take up and incorporate environmental DNA into its genome efficiently is important for the spread of antibiotic resistance genes [39]. Therefore we asked whether RecANg is specifically adapted to allow the recombination of heterologous DNA into the gonococcal genome. We created a number of DNA substrates to mimic the DNA transformation of antibiotic resistance genes i.e. DNAs with increasing amounts of insert heterology: pGEM with either a 10 bp PacI linker (pGEM-10), a 100 bp fragment of an ermR gene (pGEM-100), or a 1000 bp fragment of the ermR gene cloned into the vector (pGEM-1000) (Figure 6A, B). To evaluate the ability of RecANg and RecAEc to promote strand exchange through DNAs with increasing heterology, pGEM circular ssDNA was isolated and reacted with the each of the heterologous dsDNA substrates. The dsDNA substrates were digested with NdeI, which places the region of DNA heterology in the middle of the linear dsDNA molecule (medial heterology) (Figure 6A,B). We observed that RecANg and RecAEc exhibited essentially identical strand exchange ability through the heterologous pGEM-10 construct. Through the construct with 100 bp of heterology, pGEM-100, RecANg exhibited slightly increased formation of NC product only at 10 minutes; however, RecAEc exhibited increased formation of NC product in later time points (time 30, 60) (Figure 6C). Neither RecA protein was able to yield the NC form when circular ss pGEM DNA was reacted with the linear ds pGEM-1000 DNA (data not shown). These data demonstrate that, despite the increased degree of strand exchange using completely homologous DNA substrates and the increased ATPase activity of RecANg, the RecAEc protein exhibits more strand exchange through substrates with a 100 bp heterologous DNA insert.


Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

Stohl EA, Gruenig MC, Cox MM, Seifert HS - PLoS ONE (2011)

Strand exchange with DNA substrates to mimic DNA transformation and antigenic variation in vitro.A. Schematic of pGEM vector with relevant restriction sites used to clone heterologous inserts (see Materials and Methods). B. Linear dsDNA heterologous inserts digested with NdeI to give medial heterology and reacted with pGEM cssDNA. C. % nicked circular product observed in strand exchange reactions promoted by RecANg and RecAEc using pGEM circular ssDNA reacted with pGEM-10 and pGEM-100 linear dsDNA (designated “10” and “100” in Figure). Error bars represent the standard error of the mean of 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg 100 compared to RecAEc 100). D. Schematic of construct pGEM 1-81-S2 containing the 1-81-S2 pilE DNA sequence and relevant restriction sites. E. Linear dsDNA of antigenic variants SV, HVL, and SV-HVL heterologies (designated with shading and cross-hatches, see Figure S3 and Materials and Methods) digested with XmnI to give medial heterology and reacted with pGEM 1-81-S2 circular ssDNA. F. % nicked circular product observed in strand exchange reactions promoted by RecAEc and RecANg using pGEM-1-81-S2 cssDNA reacted with pGEM-1-81-S2 or pGEM-SV-HVL linear dsDNA. Error bars represent the standard error of the mean of at least 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg SV-HVL relative to RecAEc SV-HVL).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040777&req=5

pone-0017101-g006: Strand exchange with DNA substrates to mimic DNA transformation and antigenic variation in vitro.A. Schematic of pGEM vector with relevant restriction sites used to clone heterologous inserts (see Materials and Methods). B. Linear dsDNA heterologous inserts digested with NdeI to give medial heterology and reacted with pGEM cssDNA. C. % nicked circular product observed in strand exchange reactions promoted by RecANg and RecAEc using pGEM circular ssDNA reacted with pGEM-10 and pGEM-100 linear dsDNA (designated “10” and “100” in Figure). Error bars represent the standard error of the mean of 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg 100 compared to RecAEc 100). D. Schematic of construct pGEM 1-81-S2 containing the 1-81-S2 pilE DNA sequence and relevant restriction sites. E. Linear dsDNA of antigenic variants SV, HVL, and SV-HVL heterologies (designated with shading and cross-hatches, see Figure S3 and Materials and Methods) digested with XmnI to give medial heterology and reacted with pGEM 1-81-S2 circular ssDNA. F. % nicked circular product observed in strand exchange reactions promoted by RecAEc and RecANg using pGEM-1-81-S2 cssDNA reacted with pGEM-1-81-S2 or pGEM-SV-HVL linear dsDNA. Error bars represent the standard error of the mean of at least 3 independent experiments. *P<0.05 by Students two-tailed t-test (RecANg SV-HVL relative to RecAEc SV-HVL).
Mentions: The ability of N. gonorrhoeae to take up and incorporate environmental DNA into its genome efficiently is important for the spread of antibiotic resistance genes [39]. Therefore we asked whether RecANg is specifically adapted to allow the recombination of heterologous DNA into the gonococcal genome. We created a number of DNA substrates to mimic the DNA transformation of antibiotic resistance genes i.e. DNAs with increasing amounts of insert heterology: pGEM with either a 10 bp PacI linker (pGEM-10), a 100 bp fragment of an ermR gene (pGEM-100), or a 1000 bp fragment of the ermR gene cloned into the vector (pGEM-1000) (Figure 6A, B). To evaluate the ability of RecANg and RecAEc to promote strand exchange through DNAs with increasing heterology, pGEM circular ssDNA was isolated and reacted with the each of the heterologous dsDNA substrates. The dsDNA substrates were digested with NdeI, which places the region of DNA heterology in the middle of the linear dsDNA molecule (medial heterology) (Figure 6A,B). We observed that RecANg and RecAEc exhibited essentially identical strand exchange ability through the heterologous pGEM-10 construct. Through the construct with 100 bp of heterology, pGEM-100, RecANg exhibited slightly increased formation of NC product only at 10 minutes; however, RecAEc exhibited increased formation of NC product in later time points (time 30, 60) (Figure 6C). Neither RecA protein was able to yield the NC form when circular ss pGEM DNA was reacted with the linear ds pGEM-1000 DNA (data not shown). These data demonstrate that, despite the increased degree of strand exchange using completely homologous DNA substrates and the increased ATPase activity of RecANg, the RecAEc protein exhibits more strand exchange through substrates with a 100 bp heterologous DNA insert.

Bottom Line: Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology.Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec).This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America. e-stohl@northwestern.edu

ABSTRACT
The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

Show MeSH
Related in: MedlinePlus