Berkmen

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'''My lab focuses on two broad questions in biology:'''<br>
'''My lab focuses on two broad questions in biology:'''<br>
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'''1. How do proteins come together to form a complex molecular machine, capable of such tasks like DNA transport through a membrane (e.g. in bacterial mating)?<br>'''
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1. How do proteins come together to form a complex molecular machine, capable of such tasks like DNA transport through a membrane (e.g. in bacterial mating)?<br>
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2. How are the proteins that make up a complex molecular machine targeted to the correct location in the bacterial cell?
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'''2. How are the proteins that make up a complex molecular machine targeted to the correct location in the bacterial cell?'''
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[[Image:YddE_GFP.jpg|250px|thumb|''B. subtilis cells'' with ConE-GFP (green), membrane (red), and DNA (blue) shown.|left]]
[[Image:YddE_GFP.jpg|250px|thumb|''B. subtilis cells'' with ConE-GFP (green), membrane (red), and DNA (blue) shown.|left]]
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Bacterial mating or conjugation is the transfer of DNA from one bacterium to another via direct cell-to-cell contact through a mating pore. We use the genetically-tractable bacterium ''Bacillus subtilis'' as a model system to explore the function and subcellular localization of a putative component of the bacterial mating pore apparatus. We have been characterizing the protein ConE (formerly YddE) which is encoded on the ''B. subtilis'' conjugative element ICE''Bs1''. ConE is related to proteins encoded on conjugative elements in numerous bacteria, including the Gram-positive pathogens ''S. aureus, C. difficile'', and ''L. monocytogenes''. ConE belongs to a large superfamily of ATP-dependent pumps, such as VirB4, FtsK, and SpoIIIE, involved in the extrusion of proteins and DNA through membrane pores. We have shown that ConE and its ATPase domain are essential for mating of  ICE''Bs1''. In addition, ConE-GFP localizes at the membrane, predominantly at the cell poles (see Figure). Given ConE’s localization, ATPase domain, and essentiality in conjugation, we propose that ConE and its homologs are the essential membrane-associated ATPase component of the Gram-positive mating pore apparatus. We are analyzing the role of ConE in conjugation, exploring its functional domains, and investigating its subcellular localization through a combination of bioinformatics, molecular, cellular, and biochemical techniques. Our research is funded through Suffolk University and an NSF-RUI grant from 2012-2015.
Bacterial mating or conjugation is the transfer of DNA from one bacterium to another via direct cell-to-cell contact through a mating pore. We use the genetically-tractable bacterium ''Bacillus subtilis'' as a model system to explore the function and subcellular localization of a putative component of the bacterial mating pore apparatus. We have been characterizing the protein ConE (formerly YddE) which is encoded on the ''B. subtilis'' conjugative element ICE''Bs1''. ConE is related to proteins encoded on conjugative elements in numerous bacteria, including the Gram-positive pathogens ''S. aureus, C. difficile'', and ''L. monocytogenes''. ConE belongs to a large superfamily of ATP-dependent pumps, such as VirB4, FtsK, and SpoIIIE, involved in the extrusion of proteins and DNA through membrane pores. We have shown that ConE and its ATPase domain are essential for mating of  ICE''Bs1''. In addition, ConE-GFP localizes at the membrane, predominantly at the cell poles (see Figure). Given ConE’s localization, ATPase domain, and essentiality in conjugation, we propose that ConE and its homologs are the essential membrane-associated ATPase component of the Gram-positive mating pore apparatus. We are analyzing the role of ConE in conjugation, exploring its functional domains, and investigating its subcellular localization through a combination of bioinformatics, molecular, cellular, and biochemical techniques. Our research is funded through Suffolk University and an NSF-RUI grant from 2012-2015.
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== Current Members of the Berkmen Lab ==
== Current Members of the Berkmen Lab ==
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'''Matt Broulidakis''' - Biochemistry Major, Honors Program<br>
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[[Image:Berkmen_lab_summer_2014.jpg|600px|thumb|Melanie Berkmen with research assistants Artemisa Bulku, Omar Pinkhasov, Matt Broulidakis, and Naira Aleksanyan.|left]]
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Matt joined out lab under the NSF grant in the summer of 2012 as a research assistant. He has explored the role of ''yddF'' in mating and will soon be exploring the determinants of localization of ConE-GFP.  <br>
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'''Anastasia Murthy''' - Biochemistry Major, Honors Program, May 2015 <br>
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Anastasia joined the lab under the NSF grant in the summer of 2013. Since then, she has explored the localization of several ConE mutants along with optimizing the purification of MBP-tagged ConE.  <br>
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'''Omar Pinkhasov''' - Biochemistry Major, Honors Program, May 2015<br>
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Omar joined the lab under the NSF grant in the summer of 2014. He has purified full length ConB and a truncated version lacking its transmembrane segments, and explored the oligomerization of these proteins using Blue Native PAGE.  <br>
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'''Artemisa Bulku''' - Biochemistry Major, Honors Program, May 2016<br>
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Artemisa joined the lab under the NSF grant in the summers of 2012 and 2013. She has cloned His10-ConE, along with several mutants of ConE. In addition, she has optimized the purification of His10-ConE, providing a better yield and purity.  <br>
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'''Naira Aleksanyan''' - Biology Major, McNairs Scholars Program, May 2016<br>
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Naira joined the lab under the NSF grant in the summer of 2014. She has created several site-directed mutants of ConE and is exploring their effects on mating.  <br>
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== Former Members of the Berkmen Lab ==
== Former Members of the Berkmen Lab ==
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[[Image:533393 10150880300287619 646892850 n.jpg|300px|thumb|Melanie Berkmen with research assistants Kyle Swerdlow, Stephanie Laurer, Gianna Mancuso, and Azul Pinochet Barros.|Melanie Berkmen with research assistants Kyle Swerdlow, Stephanie Laurer, Gianna Mancuso, and Azul Pinochet Barros]]
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[[Image:533393 10150880300287619 646892850 n.jpg|500px|thumb|Melanie Berkmen with research assistants Kyle Swerdlow, Stephanie Laurer, Gianna Mancuso, and Azul Pinochet Barros.|Melanie Berkmen with research assistants Kyle Swerdlow, Stephanie Laurer, Gianna Mancuso, and Azul Pinochet Barros]]
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'''Matt Broulidakis''' - B.S. Biochemistry, Honors Program, May 2014<br>
'''Matt Broulidakis''' - B.S. Biochemistry, Honors Program, May 2014<br>
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Matt joined the lab under the NSF grant in the summer of 2012 as a research assistant. He has explored the role of ''yddF'' in mating. Then, he examined the determinants of localization of ConE-GFP as well as analyzed the localization of other mating components through GFP and SNAP tags.  <br>
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Matt joined the lab under the NSF grant in the summer of 2012. He has explored the role of ''yddF'' in mating. Then, he examined the determinants of localization of ConE-GFP as well as analyzed the localization of other mating components through GFP and SNAP tags.  <br>
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'''Gianna Mancuso ''' - B.S. Biochemistry Forensic Science, May 2013<br>
'''Gianna Mancuso ''' - B.S. Biochemistry Forensic Science, May 2013<br>
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Gianna, along with Kyle Swerdlow, helped optimize the purification of His6-ConE. The research was presented at the 2012 ACS national conference and 2012 STEM conference at Suffolk University. She has also helped clone ConE fusions for use in bacterial two-hybrid.
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Gianna, along with Kyle Swerdlow, helped optimize the purification of His6-ConE. The research was presented at the 2012 ACS national conference and 2012 STEM conference at Suffolk University. She has also helped clone ConE fusions for use in bacterial two-hybrid.<br>
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'''Georgeanna Morton''' - B.S. Biochemistry, May 2013<br>
'''Georgeanna Morton''' - B.S. Biochemistry, May 2013<br>
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Geogeanna became a research assistant under the NSF grant in the summer of 2012. For her senior thesis, she purified wild-type and mutant His6-ConE tested the oligomerization of each using Blue Native-PAGE.  <br>
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Geogeanna became a research assistant under the NSF grant in the summer of 2012. For her senior thesis, she purified wild-type and mutant His6-ConE and tested the oligomerization of each using Blue Native-PAGE.  <br>
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'''Berkmen MB''', [[Laurer SJ, Giarusso BK, and Romero R]]. (2012)The Integrative and Conjugative Element ICE''Bs1'' of ''Bacillus subtilis''. Review chapter in the book Bacterial Integrative Mobile Genetic Elements, edited by Adam P. Roberts and Peter Mullany, Landes Bioscience.  
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'''Berkmen MB''', [[Murthy AC, Broulidakis MP]]. An Inquiry-Based Laboratory Module to Promote Understanding of the Scientific Method and Bacterial Conjugation. J Micriobiol & Biol Educ. ''In press''.
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'''Berkmen MB''', [[Laurer SJ, Giarusso BK, and Romero R]]. (2013) The Integrative and Conjugative Element ICE''Bs1'' of ''Bacillus subtilis''. Review chapter in the book Bacterial Integrative Mobile Genetic Elements, edited by Adam P. Roberts and Peter Mullany, Landes Bioscience.  
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Gourse RL, Gaal T, Aiyar SE, '''Barker MM''', Estrem ST, Hirvonen CA, Ross W. (1998) Strength and regulation without transcription factors: Lessons from bacterial rRNA promoters. Cold Spring Harb Sym 63: 131-139.
Gourse RL, Gaal T, Aiyar SE, '''Barker MM''', Estrem ST, Hirvonen CA, Ross W. (1998) Strength and regulation without transcription factors: Lessons from bacterial rRNA promoters. Cold Spring Harb Sym 63: 131-139.
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Singer SS, Henkels K, Deucher A, '''Barker MM''', Singer J, Trulzsch T. (1996) Growth hormone and aging change rat liver fatty acid binding protein levels. J Amer Coll Nutr 15: 169-174.<br>
 
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Current revision

Melanie Berkmen

Contents

Bio

Melanie Barker Berkmen
Associate Professor of Chemistry and Biochemistry at Suffolk University

How to contact me:
Email: mberkmen at suffolk.edu

Office Phone: 617-973-5321

Mailing address:
Suffolk University
Department of Chemistry and Biochemistry
41 Temple St.
Boston, MA 02114

Campus Address:
Office: Donahue Building, Room 513
Biochemistry Lab: Archer Building, Room 631

Courses I teach

FALL - CHEM 331 (Biochemistry I)
FALL - CHEM L331 (Biochemical Techniques Lab)

SPRING - CHEM L112 (General Chemistry Lab II)
SPRING - CHEM L432 (Advanced Biochemistry Research Lab)


Previously Taught Courses
FALL - CHEM L111 (General Chemistry Lab I)
SPRING - CHEM L333 (Advanced Biochemistry Research Lab)
FALL - CHEM 428 (Research & Seminar I)
SPRING - CHEM 429 (Research & Seminar II)
FALL 2005 (at MIT), course 7.341 (advanced seminar on bacterial molecular and cellular biology)

Education

(2002-2007) Jane Coffin Childs Postdoctoral Fellow
Massachusetts Insitute of Technology, Cambridge, MA
Laboratory of Alan D. Grossman


(2001) Ph.D., Cellular and Molecular Biology
University of Wisconsin-Madison, Madision, WI
Laboratory of Richard L. Gourse


(1995) B.S., Biochemistry
University of Dayton, Dayton, OH, summa cum laude

Research

My lab focuses on two broad questions in biology:

1. How do proteins come together to form a complex molecular machine, capable of such tasks like DNA transport through a membrane (e.g. in bacterial mating)?
2. How are the proteins that make up a complex molecular machine targeted to the correct location in the bacterial cell?

B. subtilis cells with ConE-GFP (green), membrane (red), and DNA (blue) shown.
B. subtilis cells with ConE-GFP (green), membrane (red), and DNA (blue) shown.


Bacterial mating or conjugation is the transfer of DNA from one bacterium to another via direct cell-to-cell contact through a mating pore. We use the genetically-tractable bacterium Bacillus subtilis as a model system to explore the function and subcellular localization of a putative component of the bacterial mating pore apparatus. We have been characterizing the protein ConE (formerly YddE) which is encoded on the B. subtilis conjugative element ICEBs1. ConE is related to proteins encoded on conjugative elements in numerous bacteria, including the Gram-positive pathogens S. aureus, C. difficile, and L. monocytogenes. ConE belongs to a large superfamily of ATP-dependent pumps, such as VirB4, FtsK, and SpoIIIE, involved in the extrusion of proteins and DNA through membrane pores. We have shown that ConE and its ATPase domain are essential for mating of ICEBs1. In addition, ConE-GFP localizes at the membrane, predominantly at the cell poles (see Figure). Given ConE’s localization, ATPase domain, and essentiality in conjugation, we propose that ConE and its homologs are the essential membrane-associated ATPase component of the Gram-positive mating pore apparatus. We are analyzing the role of ConE in conjugation, exploring its functional domains, and investigating its subcellular localization through a combination of bioinformatics, molecular, cellular, and biochemical techniques. Our research is funded through Suffolk University and an NSF-RUI grant from 2012-2015.





Current Members of the Berkmen Lab


Melanie Berkmen with research assistants Artemisa Bulku, Omar Pinkhasov, Matt Broulidakis, and Naira Aleksanyan.
Melanie Berkmen with research assistants Artemisa Bulku, Omar Pinkhasov, Matt Broulidakis, and Naira Aleksanyan.

Anastasia Murthy - Biochemistry Major, Honors Program, May 2015
Anastasia joined the lab under the NSF grant in the summer of 2013. Since then, she has explored the localization of several ConE mutants along with optimizing the purification of MBP-tagged ConE.

Omar Pinkhasov - Biochemistry Major, Honors Program, May 2015
Omar joined the lab under the NSF grant in the summer of 2014. He has purified full length ConB and a truncated version lacking its transmembrane segments, and explored the oligomerization of these proteins using Blue Native PAGE.

Artemisa Bulku - Biochemistry Major, Honors Program, May 2016
Artemisa joined the lab under the NSF grant in the summers of 2012 and 2013. She has cloned His10-ConE, along with several mutants of ConE. In addition, she has optimized the purification of His10-ConE, providing a better yield and purity.

Naira Aleksanyan - Biology Major, McNairs Scholars Program, May 2016
Naira joined the lab under the NSF grant in the summer of 2014. She has created several site-directed mutants of ConE and is exploring their effects on mating.

Former Members of the Berkmen Lab

Melanie Berkmen with research assistants Kyle Swerdlow, Stephanie Laurer, Gianna Mancuso, and Azul Pinochet Barros
Melanie Berkmen with research assistants Kyle Swerdlow, Stephanie Laurer, Gianna Mancuso, and Azul Pinochet Barros


Matt Broulidakis - B.S. Biochemistry, Honors Program, May 2014
Matt joined the lab under the NSF grant in the summer of 2012. He has explored the role of yddF in mating. Then, he examined the determinants of localization of ConE-GFP as well as analyzed the localization of other mating components through GFP and SNAP tags.

Minh Bui - B.S. Biochemistry, May 2014
Funded through the NSF-RUI grant, Minh constructed 4 different ConE mutations on the chromosome and analyzed their effects on mating for her senior thesis.

Kyle Swerdlow - B.S. Biochemistry, May 2014
Kyle worked very closely with Gianna Mancuso on the optimization of His6-ConE purification. They presented this research at the 2012 ACS national conference as well as the 2012 STEM conference at Suffolk University. Funded through the NSF-RUI grant during the summers of 2012 and 2013, Kyle used bacterial two-hybrid analysis to piece together key players in the ICEBs1 conjugation machinery for his senior thesis.

Gianna Mancuso - B.S. Biochemistry Forensic Science, May 2013
Gianna, along with Kyle Swerdlow, helped optimize the purification of His6-ConE. The research was presented at the 2012 ACS national conference and 2012 STEM conference at Suffolk University. She has also helped clone ConE fusions for use in bacterial two-hybrid.

Georgeanna Morton - B.S. Biochemistry, May 2013
Geogeanna became a research assistant under the NSF grant in the summer of 2012. For her senior thesis, she purified wild-type and mutant His6-ConE and tested the oligomerization of each using Blue Native-PAGE.

Azul Pinochet Barros - Biology/Philosophy, December 2012
Azul helped optimize our competent cells procedure. Afterwards, she cloned a Walker B mutant for our His6-ConE, but unfortunately this mutant did not express well in E. coli.

Stephanie Laurer - B.A. Biochemistry, Honors Program, May 2012
Stephanie used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. She presented this research with Bridget at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She also helped clone several ICEBs1 genes. On the side, Stephanie had an interest in genetically modified food. During her first year at Suffolk, she used a PCR-based assay to detect the Bt gene in corn. She found that at least half of the samples she tested (6 of 11) were genetically modified. She presented this work at the 2009 Suffolk science banquet where her poster won first place. She now acts as Clinical Research Assistant at Beth Israel Deaconess Medical Center.

Bridget Giarusso - B.S. Biochemistry, May 2011
As a research assistant, Bridget used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. She presented this research with Stephanie at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She helped clone several ICEBs1 genes.

Matt Hamada - B.S. Biochemistry, December 2010
For CHEM L333, Matt cloned the conC gene encoded on the ICEBs1 conjugative element. For CHEM L428/L429, Matt used fluorescence microscopy to determine whether conC and other ICEBs1 genes are required for ConE to localize at the cell poles. He presented his research with Cori at the 2009 Boston Bacterial Meeting. Currently, Matt is working as a laboratory research technician.

Cori Leonetti - B.S. Biochemistry, May 2010
For CHEM L333, Cori helped clone the conB gene encoded on the ICEBs1 conjugative element. Cori extended her CHEM L333 project for CHEM L428/L429. She tested whether conB and other ICEBs1 genes are required for mating. Cori presented her research with Matt at the 2009 Boston Bacterial Meeting. Cori is now a graduate student at Arizona State University studying microbiology.

Erin Cross - B.S. Biochemistry, May 2009
In CHEM L333, Erin and her class mates attempted to clone various C-terminal truncations of ConE fused to GFP. For CHEM L428/L429, she used fluorescence microscopy to analyze what parts of ConE are required for localization to the cell poles. She found that the C-terminal half of ConE is critical for localization. She presented this work at the national ACS meeting in March 2009 and at the 2009 Suffolk Science Banquet where her poster won 3rd place. Erin is now working as a laboratory research technologist at MMCRI.

Maria Muccioli (formerly Levicheva) - B.S. Biochemistry, Honors Program, May 2009
For CHEM L333, Maria constructed a his-tagged ConE. For CHEM L428/L429, she purified and characterized His6-ConE to enable future students to perform ATPase assays to determine whether ConE can hydrolyze ATP in vitro. She presented this work at the national ACS meeting in March 2009. Maria is now a graduate student in the molecular and cellular biology Ph.D. program at Ohio University.

Tamara Wong - B.S. Biochemistry Forensic Science, May 2009
Tamara helped cloned the His6-ConE construct so that we can test whether this protein can support mating. In addition, Tamara purified His6-ConE to enable future ATPase assays.

Emma-Kate Loveday - B.S. Biochemistry, May 2008
For her CHEM L428/L429 project, Emma-Kate constructed two variants of ConE and tested their effects on mating. She found that the Walker B (ATP hydrolysis domain) of ConE is essential for mating. She also found that the N-terminus of ConE does not contribute significantly to mating. She presented her work at the Boston Bacterial Meeting in June 2008 and the Cold Spring Harbor Molecular Genetics of Bacteria and Phages Meeting in August 2008. Emma is now a graduate student in the microbiology and immunology Ph.D. program at the University of British Columbia supported by an NSF Fellowship.


Publications

Suffolk undergraduate authors in red

Berkmen MB, Murthy AC, Broulidakis MP. An Inquiry-Based Laboratory Module to Promote Understanding of the Scientific Method and Bacterial Conjugation. J Micriobiol & Biol Educ. In press.

Berkmen MB, Laurer SJ, Giarusso BK, and Romero R. (2013) The Integrative and Conjugative Element ICEBs1 of Bacillus subtilis. Review chapter in the book Bacterial Integrative Mobile Genetic Elements, edited by Adam P. Roberts and Peter Mullany, Landes Bioscience.

Martinez KA 2nd, Kitko RD, Mershon JP, Adcox HE, Malek KA, Berkmen MB, Slonczewski JL. (2012) Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy. Appl Environ Microbiol., 78(10):3706-14.

Babic A, Berkmen MB, Lee CA, Grossman AD. (2011) Efficient gene transfer in bacterial cell chains. MBio, 2(2). pii: e00027-11. doi: 10.1128/mBio.00027-11.

Berkmen MB, Lee CA, Loveday EK, Grossman AD. (2010) Polar positioning of a conjugation protein from the integrative and conjugative element ICEBs1 of Bacillus subtilis. J Bacteriol, 192(1):38-45.

Kitko RD, Cleeton RL, Armentrout EI, Lee GE, Noguchi K, Berkmen MB, Jones BD, Slonczewski JL. (2009) Cytoplasmic acidification and the benzoate transcriptome in Bacillus subtilis. PLoS One, 4(12):e8255.

Vrentas CE, Gaal T, Berkmen MB, Rutherford ST, Haugen SP, Ross W, Gourse RL. (2008) Still looking for the magic spot: the crystallographically defined binding site for ppGpp on RNA polymerase is unlikely to be responsible for rRNA transcription regulation. J Mol Biol, 277(2): 551-64.

Wang JD, Berkmen MB, Grossman AD. (2007) Genome-wide co-orientation of replication and transcription reduces adverse effects on replication in Bacillus subtilis, PNAS, 104(13): 5608-5613.

Berkmen MB and Grossman AD. (2007) Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA. Mol Microbiol, 63(1): 150-165.

Berkmen MB, Grossman AD. (2006) Spatial and temporal organization of the Bacillus subtilis replication cycle. Mol. Microbiol, 62(1): 57-71.

Haugen SP, Berkmen MB, Ross W, Gaal T, Ward C, Gourse RL. (2006) rRNA promoter regulation by nonoptimal binding of σ region 1.2: An additional recognition element for RNA polymerase. Cell, 125(6): 1069-1082.

Paul BJ, Berkmen MB, Gourse RL (2005) DksA potentiates direct activation of amino acid promoters by ppGpp. PNAS, 102(22):7823-8.

Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL (2004) DksA: A critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell, 118(3): 311-322.

Wang JD, Rokop ME, Barker MM, Hanson NR, Grossman AD (2004) Multi-copy plasmids affect replisome positioning in Bacillus subtilis. J Bacteriol, 186(21):7084-90.

Barker MM, Gourse RL (2002) Control of stable RNA synthesis. In Translation Mechanisms. (Lapointe J, Brakier-Gingras L. ed.). Landes Biosciences, Austin, TX.

Barker MM, Gourse RL (2001) Regulation of rRNA transcription correlates with nucleoside triphosphate sensing. J Bacteriol, 183, 6315-6323.

Barker MM, Gaal T, Josaitis CA, Gourse RL. (2001) Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro. J Mol Biol 305(4): 673-688.

Barker MM, Gaal T, Gourse RL (2001) Mechanism of regulation of transcription initiation by ppGpp II. Models for positive control based on properties of RNAP mutants and competition for RNAP. J Mol Biol 305(4): 689-702.

Gourse RL, Gaal T, Aiyar SE, Barker MM, Estrem ST, Hirvonen CA, Ross W. (1998) Strength and regulation without transcription factors: Lessons from bacterial rRNA promoters. Cold Spring Harb Sym 63: 131-139.

Personal

In my free time I like to travel, snowboard, practice my Turkish, and eat delicious food.

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