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Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor

Authors: Stephanie Beltz
Stephanie BeltzAffiliation: Pharmazeutisches Institut der Universität Tübingen, Pharmazeutische Biochemie, Tübingen, Germany
For correspondence: stephanie.beltz@uni-tuebingen.de
Bio-protocol author page: a4015
 and Joachim E. Schultz
Joachim E. SchultzAffiliation: Pharmazeutisches Institut der Universität Tübingen, Pharmazeutische Biochemie, Tübingen, Germany
For correspondence: joachim.schultz@uni-tuebingen.de
Bio-protocol author page: a4016
,  date: 1/20/2017, 941 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2112.

[Abstract] The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins....

A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii

Authors: Alberto J. Martín-Rodríguez
Alberto J. Martín-RodríguezAffiliation 1: Institute for Bioorganic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, San Cristóbal de La Laguna, Spain
Affiliation 2: Oceanic Platform of the Canary Islands (PLOCAN), Telde, Spain
Affiliation 3: Present address: Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
For correspondence: ajmartinr@ull.es
Bio-protocol author page: a3312
 and José J. Fernández
José J. FernándezAffiliation: Institute for Bioorganic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, San Cristóbal de La Laguna, Spain
Bio-protocol author page: a3313
,  date: 7/20/2016, 1449 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1866.

[Abstract] Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important ...

[14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae

Authors: Sarah C. Plecha
Sarah C. PlechaAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
Bio-protocol author page: a2783
 and Jeffrey H. Withey
Jeffrey H. WitheyAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
For correspondence: jwithey@wayne.edu
Bio-protocol author page: a2784
,  date: 12/20/2015, 1242 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1682.

[Abstract] The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able ...

A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins

Authors: Disha Srivastava
Disha SrivastavaAffiliation: Department of Microbiology and Microbial Genetics, Michigan State University, East Lansing, USA
Bio-protocol author page: a1981
 and Christopher M. Waters
Christopher M. WatersAffiliation: Department of Microbiology and Microbial Genetics, Michigan State University, East Lansing, USA
For correspondence: watersc3@msu.edu
Bio-protocol author page: a1982
,  date: 2/5/2015, 3020 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1394.

[Abstract] Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP ...

Infant Rabbit Colonization Competition Assays

Authors: Yang Fu
Yang FuAffiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
For correspondence: yang_fu@hms.harvard.edu
Bio-protocol author page: a1395
 and John J. Mekalanos
John J. MekalanosAffiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
For correspondence: john_mekalanos@hms.harvard.edu
Bio-protocol author page: a1396
,  date: 6/5/2014, 3058 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1147.

[Abstract] Enteric pathogenic bacteria such as Vibrio cholerae and enteropathogenic Escherichia coli (E. coli) cause life-threatening diarrheal diseases that have afflicted humans for centuries. Understanding the effectors required for intestinal colonization is very important to research on bacteria pathogenesis, ...

Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines

Authors: Amit K. Dey
Amit K. DeyAffiliation: Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
Bio-protocol author page: a1296
, Abha Bhagat
Abha BhagatAffiliation: Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
Bio-protocol author page: a1297
 and Rukhsana Chowdhury
Rukhsana ChowdhuryAffiliation: Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
For correspondence: rukhsana@iicb.res.in
Bio-protocol author page: a1298
,  date: 4/20/2014, 3691 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1105.

[Abstract] Vibrio cholerae (V. cholerae) colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes severe fluid loss characteristic of the disease cholera. V. cholerae is a non-invasive Gram-negative bacterium that adheres to intestinal cells as well as a variety of ...

Measurement of Haemolysin Activities in Vibrio vulnificus

Authors: Hyun-Jung Lee
Hyun-Jung LeeAffiliation: National Cancer Institute, National Institutes of Health, Bethesda, USA
For correspondence: hyun-jung.lee@nih.gov
Bio-protocol author page: a1197
, Jeong-A Kim
Jeong-A KimAffiliation: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea
Bio-protocol author page: a1198
, Mi-Ae Lee
Mi-Ae LeeAffiliation: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea
Bio-protocol author page: a1199
, Soon-Jung Park
Soon-Jung ParkAffiliation: Department of Environmental Medical Biology and Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, South Korea
Bio-protocol author page: a1200
 and Kyu-Ho Lee
Kyu-Ho LeeAffiliation: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea
Bio-protocol author page: a1201
,  date: 3/5/2014, 2472 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1062.

[Abstract] VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling ...

Differential in vivo Thiol Trapping with N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS)

Author: Wei-Yun (Winnie) Wholey
Wei-Yun (Winnie) WholeyAffiliation: Pediatrics and Communicable Diseases, University of Michigan, Medical School, Ann Arbor, MI, USA
For correspondence: weiyun@umich.edu
Bio-protocol author page: a126
,  date: 10/20/2012, 6879 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.272.

[Abstract] This protocol is used to compare the in vivo redox status of Escherichia coli and Vibrio cholerae protein before and after HOCl treatment. For example, I examined whether the EF-Tu protein is reduced or oxidized in the referenced publication. This protocol should work for other proteins and other oxidative ...

Preparation of Genomic Overexpression Library

Author: Wei-Yun (Winnie) Wholey
Wei-Yun (Winnie) WholeyAffiliation: Pediatrics and Communicable Diseases, University of Michigan, Medical School, Ann Arbor, MI, USA
For correspondence: weiyun@umich.edu
Bio-protocol author page: a126
,  date: 10/5/2012, 4619 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.271.

[Abstract] This protocol is used to identify/select for Escherichia coli genes that, when overexpressed in Vibrio cholerae Hsp33 (hslO gene) deletion mutant, protect against oxidative heat stress and, by extension, against HOCl-mediated protein damage. In the referenced publication, I found that V. cholerae mutant ...
1 

Differential in vivo Thiol Trapping with N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS)

Author: Wei-Yun (Winnie) Wholey
Wei-Yun (Winnie) WholeyAffiliation: Pediatrics and Communicable Diseases, University of Michigan, Medical School, Ann Arbor, MI, USA
For correspondence: weiyun@umich.edu
Bio-protocol author page: a126
,  date: 10/20/2012, 6879 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.272.

[Abstract] This protocol is used to compare the in vivo redox status of Escherichia coli and Vibrio cholerae protein before and after HOCl treatment. For example, I examined whether the EF-Tu protein is reduced or oxidized in the referenced publication. This protocol should work for other proteins and other oxidative ...

Preparation of Genomic Overexpression Library

Author: Wei-Yun (Winnie) Wholey
Wei-Yun (Winnie) WholeyAffiliation: Pediatrics and Communicable Diseases, University of Michigan, Medical School, Ann Arbor, MI, USA
For correspondence: weiyun@umich.edu
Bio-protocol author page: a126
,  date: 10/5/2012, 4619 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.271.

[Abstract] This protocol is used to identify/select for Escherichia coli genes that, when overexpressed in Vibrio cholerae Hsp33 (hslO gene) deletion mutant, protect against oxidative heat stress and, by extension, against HOCl-mediated protein damage. In the referenced publication, I found that V. cholerae mutant ...

Assay for Adherence of Vibrio cholerae to Eukaryotic Cell Lines

Authors: Amit K. Dey
Amit K. DeyAffiliation: Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
Bio-protocol author page: a1296
, Abha Bhagat
Abha BhagatAffiliation: Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
Bio-protocol author page: a1297
 and Rukhsana Chowdhury
Rukhsana ChowdhuryAffiliation: Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
For correspondence: rukhsana@iicb.res.in
Bio-protocol author page: a1298
,  date: 4/20/2014, 3691 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1105.

[Abstract] Vibrio cholerae (V. cholerae) colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes severe fluid loss characteristic of the disease cholera. V. cholerae is a non-invasive Gram-negative bacterium that adheres to intestinal cells as well as a variety of ...

Infant Rabbit Colonization Competition Assays

Authors: Yang Fu
Yang FuAffiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
For correspondence: yang_fu@hms.harvard.edu
Bio-protocol author page: a1395
 and John J. Mekalanos
John J. MekalanosAffiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
For correspondence: john_mekalanos@hms.harvard.edu
Bio-protocol author page: a1396
,  date: 6/5/2014, 3058 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1147.

[Abstract] Enteric pathogenic bacteria such as Vibrio cholerae and enteropathogenic Escherichia coli (E. coli) cause life-threatening diarrheal diseases that have afflicted humans for centuries. Understanding the effectors required for intestinal colonization is very important to research on bacteria pathogenesis, ...

A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins

Authors: Disha Srivastava
Disha SrivastavaAffiliation: Department of Microbiology and Microbial Genetics, Michigan State University, East Lansing, USA
Bio-protocol author page: a1981
 and Christopher M. Waters
Christopher M. WatersAffiliation: Department of Microbiology and Microbial Genetics, Michigan State University, East Lansing, USA
For correspondence: watersc3@msu.edu
Bio-protocol author page: a1982
,  date: 2/5/2015, 3020 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1394.

[Abstract] Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP ...

Measurement of Haemolysin Activities in Vibrio vulnificus

Authors: Hyun-Jung Lee
Hyun-Jung LeeAffiliation: National Cancer Institute, National Institutes of Health, Bethesda, USA
For correspondence: hyun-jung.lee@nih.gov
Bio-protocol author page: a1197
, Jeong-A Kim
Jeong-A KimAffiliation: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea
Bio-protocol author page: a1198
, Mi-Ae Lee
Mi-Ae LeeAffiliation: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea
Bio-protocol author page: a1199
, Soon-Jung Park
Soon-Jung ParkAffiliation: Department of Environmental Medical Biology and Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, South Korea
Bio-protocol author page: a1200
 and Kyu-Ho Lee
Kyu-Ho LeeAffiliation: Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea
Bio-protocol author page: a1201
,  date: 3/5/2014, 2472 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1062.

[Abstract] VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling ...

A Bioassay Protocol for Quorum Sensing Studies Using Vibrio campbellii

Authors: Alberto J. Martín-Rodríguez
Alberto J. Martín-RodríguezAffiliation 1: Institute for Bioorganic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, San Cristóbal de La Laguna, Spain
Affiliation 2: Oceanic Platform of the Canary Islands (PLOCAN), Telde, Spain
Affiliation 3: Present address: Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
For correspondence: ajmartinr@ull.es
Bio-protocol author page: a3312
 and José J. Fernández
José J. FernándezAffiliation: Institute for Bioorganic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, San Cristóbal de La Laguna, Spain
Bio-protocol author page: a3313
,  date: 7/20/2016, 1449 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1866.

[Abstract] Quorum Sensing (QS), or bacterial cell-to-cell communication, is a finely-tuned mechanism that regulates gene expression on a population density-dependent manner through the production, secretion and reception of extracellular signaling molecules termed autoinducers (AIs). Given that QS plays an important ...

[14C] Linoleic Acid Uptake and Fractionation Assay in Vibrio cholerae

Authors: Sarah C. Plecha
Sarah C. PlechaAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
Bio-protocol author page: a2783
 and Jeffrey H. Withey
Jeffrey H. WitheyAffiliation: Department of Immunology and Microbiology, Wayne State University School of Medicine, Michigan, USA
For correspondence: jwithey@wayne.edu
Bio-protocol author page: a2784
,  date: 12/20/2015, 1242 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1682.

[Abstract] The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able ...

Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor

Authors: Stephanie Beltz
Stephanie BeltzAffiliation: Pharmazeutisches Institut der Universität Tübingen, Pharmazeutische Biochemie, Tübingen, Germany
For correspondence: stephanie.beltz@uni-tuebingen.de
Bio-protocol author page: a4015
 and Joachim E. Schultz
Joachim E. SchultzAffiliation: Pharmazeutisches Institut der Universität Tübingen, Pharmazeutische Biochemie, Tübingen, Germany
For correspondence: joachim.schultz@uni-tuebingen.de
Bio-protocol author page: a4016
,  date: 1/20/2017, 941 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2112.

[Abstract] The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins....
1 
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