2016
Felczak, M. M.; Sage, J. M.; Hupert-Kocurek, K. T.; Aykul, S.; Kaguni, J. M.
Substitutions of conserved residues in the c-terminal region of DnaC cause thermolability in helicase loading Journal Article
In: Journal of Biological Chemistry, vol. 291, no. 9, pp. 4803-4812, 2016, ISSN: 00219258, (4).
@article{2-s2.0-84964483982,
title = {Substitutions of conserved residues in the c-terminal region of DnaC cause thermolability in helicase loading},
author = { M.M. Felczak and J.M. Sage and K.T. Hupert-Kocurek and S. Aykul and J.M. Kaguni},
url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-84964483982&doi=10.1074%2fjbc.M115.708586&partnerID=40&md5=6d13be99774d88237272ccb5c1150eb7},
doi = {10.1074/jbc.M115.708586},
issn = {00219258},
year = {2016},
date = {2016-01-01},
journal = {Journal of Biological Chemistry},
volume = {291},
number = {9},
pages = {4803-4812},
publisher = {American Society for Biochemistry and Molecular Biology Inc.},
abstract = {The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity inDNAbinding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and byUVcross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaBDnaCcomplex at aDnaAbox. Because a decreasedATPconcentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.},
note = {4},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity inDNAbinding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and byUVcross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaBDnaCcomplex at aDnaAbox. Because a decreasedATPconcentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
2009
Duda, K. A.; Duda, K. T.; Beczała, A.; Kasperkiewicz, K.; Radziejewska-Lebrecht, J.; Skurnik, M.
ECA-immunogenicity of Proteus mirabilis strains Journal Article
In: Archivum Immunologiae et Therapiae Experimentalis, vol. 57, no. 2, pp. 147-151, 2009, ISSN: 0004069X, (13).
@article{2-s2.0-64649091101,
title = {ECA-immunogenicity of Proteus mirabilis strains},
author = { K.A. Duda and K.T. Duda and A. Beczała and K. Kasperkiewicz and J. Radziejewska-Lebrecht and M. Skurnik},
url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-64649091101&doi=10.1007%2fs00005-009-0018-9&partnerID=40&md5=10d4f74bf467437bc14d60290eeb04f5},
doi = {10.1007/s00005-009-0018-9},
issn = {0004069X},
year = {2009},
date = {2009-01-01},
journal = {Archivum Immunologiae et Therapiae Experimentalis},
volume = {57},
number = {2},
pages = {147-151},
abstract = {Introduction: Bacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core. Materials and Methods: Rabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECA PG ) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECA PG is co-extracted with LPS and by the phenol/chloroform/ petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECA LPS ) free of ECA PG . The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies. Results: The results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECA LPS , suggesting that it induced the anti-ECA antibody responses. Only the presence of ECA PG could be demonstrated in the Rc mutant strain R4/O28. Conclusions: These results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECA LPS . Since ECA PG is not immunogenic unless combined with some proteins, it is likely that ECA PG -protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28. © 2009 L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland.},
note = {13},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Introduction: Bacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core. Materials and Methods: Rabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECA PG ) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECA PG is co-extracted with LPS and by the phenol/chloroform/ petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECA LPS ) free of ECA PG . The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies. Results: The results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECA LPS , suggesting that it induced the anti-ECA antibody responses. Only the presence of ECA PG could be demonstrated in the Rc mutant strain R4/O28. Conclusions: These results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECA LPS . Since ECA PG is not immunogenic unless combined with some proteins, it is likely that ECA PG -protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28. © 2009 L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland.
2007
Hupert-Kocurek, K. T.; Sage, J. M.; Makowska-Grzyska, M.; Kaguni, J. M.
Genetic method to analyze essential genes of Escherichia coli Journal Article
In: Applied and Environmental Microbiology, vol. 73, no. 21, pp. 7075-7082, 2007, ISSN: 00992240, (9).
@article{2-s2.0-35949003233,
title = {Genetic method to analyze essential genes of Escherichia coli},
author = { K.T. Hupert-Kocurek and J.M. Sage and M. Makowska-Grzyska and J.M. Kaguni},
url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-35949003233&doi=10.1128%2fAEM.01756-07&partnerID=40&md5=6832cbd7330c06f16ceaad709ed7bc08},
doi = {10.1128/AEM.01756-07},
issn = {00992240},
year = {2007},
date = {2007-01-01},
journal = {Applied and Environmental Microbiology},
volume = {73},
number = {21},
pages = {7075-7082},
abstract = {The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counter-selected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β-D-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes. Copyright © 2007, American Society for Microbiology. All Rights Reserved.},
note = {9},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counter-selected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β-D-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes. Copyright © 2007, American Society for Microbiology. All Rights Reserved.