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Study of the Subunits and Glycine Be...

Yang, Rui.

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Study of the Subunits and Glycine Betaine Uptake of the Gbu Transporter of Listeria monocytogenes.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Study of the Subunits and Glycine Betaine Uptake of the Gbu Transporter of Listeria monocytogenes./
Author:	Yang, Rui.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	148 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-03, Section: B.
Contained By:	Dissertations Abstracts International81-03B.
Subject:	Agriculture. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13808579
ISBN:	9781085732765

Study of the Subunits and Glycine Betaine Uptake of the Gbu Transporter of Listeria monocytogenes.
Yang, Rui.

Study of the Subunits and Glycine Betaine Uptake of the Gbu Transporter of Listeria monocytogenes. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 148 p.

Source: Dissertations Abstracts International, Volume: 81-03, Section: B.

Thesis (Ph.D.)--University of California, Davis, 2019.

This item is not available from ProQuest Dissertations & Theses.

Listeria monocytogenes is a food-borne pathogenic bacterium that can grow in salt solutions and in the refrigerator. One major strategy for hyperosmotic and chill stress resistance is the uptake the glycine betaine by the ATP-binding cassette (ABC) transporter Gbu. The Gbu transporter consists of subunits GbuA, GbuB and GbuC. GbuA is an ATPase transducing the energy needed for the uptake of glycine betaine; GbuB is a transmembrane protein translocating glycine betaine; GbuC is a solute-binding protein (SBP), which binds glycine betaine for future transport.First, this study analyzed the GbuC and GbuAB (GbuA and GbuB complex) subunits. The importance of the SBP in an ABC transporter is unknown, as some ABC transporters lack an SBP. The importance of GbuC, the SBP of Gbu, was investigated by creating a mutant derivative of L. monocytogenes that possesses GbuAB but lacks GbuC. Glycine betaine uptake assay and growth rate experiments indicated that GbuC is critical for activity of the Gbu transporter under hyperosmotic and cold stress. GbuC is anchored to the membrane via an N-terminal diacylglycerol lipid tether. This study measured the sizes of the tether in the GbuC proteins expressed at different temperatures and concluded the size of the tether was unaffected by changing the growth temperature from 30 ºC to 4 ºC, which profoundly affects the fatty acid composition of the membrane. The mass of the tether is consistent with that of unsaturated fatty acids, probably two 16:1, which is a minor component of the membrane lipid complement. Tagging proteins with histidine is a common technique for detection and purification of proteins, such as GbuC. This study also tested whether a recombinant GbuC carrying a C-terminal histidine tag is still functional. Since the modified Gbu transporter, carrying the histidine-tagged GbuC, showed a normal glycine betaine uptake rate, the histidine tag probably minimally affected the activity of the GbuC. The GbuA and GbuB subunits (GbuAB) are encoded by two genes, and the integrity of the GbuAB is required in some studies. However, the GbuAB complex was unstable during protein purification.Second, this study investigated the interactions among hyperosmolality, membrane and the activity of the Gbu transporter of L. monocytogenes. Fatty acid analysis of L. monocytogenes cells revealed that hyperosmolarity increased the C17:C15 fatty acids ratio and the average fatty acid chain length. The effects of hyperosmolarity on membrane fluidity were analyzed in L. monocytogenes. An increase in membrane fluidity was observed when KCl was added to raise osmolality. As a membrane protein, the Gbu transporter was also observed to be affected by the addition of a cationic amphipath (tetracaine) and the alteration of fatty acid composition. The addition of tetracaine decreased the activity of the Gbu transporter under hyperosmotic stress. The glycine betaine transport of the Gbu transporter under hyperosmolality was slightly stimulated by the shortened average fatty acid chain length induced by the addition of cerulenin, mimicking growth at low temperature.

ISBN: 9781085732765Subjects--Topical Terms:

518588
Agriculture.
Subjects--Index Terms:

ABC transporter

Study of the Subunits and Glycine Betaine Uptake of the Gbu Transporter of Listeria monocytogenes.
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020 $a 9781085732765
035 $a (MiAaPQ)AAI13808579
035 $a AAI13808579
040 $a MiAaPQ $c MiAaPQ
100 1 $a Yang, Rui. $3 610549
245 1 0 $a Study of the Subunits and Glycine Betaine Uptake of the Gbu Transporter of Listeria monocytogenes.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2019
300 $a 148 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-03, Section: B.
500 $a Advisor: Smith, Gary M.
502 $a Thesis (Ph.D.)--University of California, Davis, 2019.
506 $a This item is not available from ProQuest Dissertations & Theses.
506 $a This item must not be sold to any third party vendors.
520 $a Listeria monocytogenes is a food-borne pathogenic bacterium that can grow in salt solutions and in the refrigerator. One major strategy for hyperosmotic and chill stress resistance is the uptake the glycine betaine by the ATP-binding cassette (ABC) transporter Gbu. The Gbu transporter consists of subunits GbuA, GbuB and GbuC. GbuA is an ATPase transducing the energy needed for the uptake of glycine betaine; GbuB is a transmembrane protein translocating glycine betaine; GbuC is a solute-binding protein (SBP), which binds glycine betaine for future transport.First, this study analyzed the GbuC and GbuAB (GbuA and GbuB complex) subunits. The importance of the SBP in an ABC transporter is unknown, as some ABC transporters lack an SBP. The importance of GbuC, the SBP of Gbu, was investigated by creating a mutant derivative of L. monocytogenes that possesses GbuAB but lacks GbuC. Glycine betaine uptake assay and growth rate experiments indicated that GbuC is critical for activity of the Gbu transporter under hyperosmotic and cold stress. GbuC is anchored to the membrane via an N-terminal diacylglycerol lipid tether. This study measured the sizes of the tether in the GbuC proteins expressed at different temperatures and concluded the size of the tether was unaffected by changing the growth temperature from 30 ºC to 4 ºC, which profoundly affects the fatty acid composition of the membrane. The mass of the tether is consistent with that of unsaturated fatty acids, probably two 16:1, which is a minor component of the membrane lipid complement. Tagging proteins with histidine is a common technique for detection and purification of proteins, such as GbuC. This study also tested whether a recombinant GbuC carrying a C-terminal histidine tag is still functional. Since the modified Gbu transporter, carrying the histidine-tagged GbuC, showed a normal glycine betaine uptake rate, the histidine tag probably minimally affected the activity of the GbuC. The GbuA and GbuB subunits (GbuAB) are encoded by two genes, and the integrity of the GbuAB is required in some studies. However, the GbuAB complex was unstable during protein purification.Second, this study investigated the interactions among hyperosmolality, membrane and the activity of the Gbu transporter of L. monocytogenes. Fatty acid analysis of L. monocytogenes cells revealed that hyperosmolarity increased the C17:C15 fatty acids ratio and the average fatty acid chain length. The effects of hyperosmolarity on membrane fluidity were analyzed in L. monocytogenes. An increase in membrane fluidity was observed when KCl was added to raise osmolality. As a membrane protein, the Gbu transporter was also observed to be affected by the addition of a cationic amphipath (tetracaine) and the alteration of fatty acid composition. The addition of tetracaine decreased the activity of the Gbu transporter under hyperosmotic stress. The glycine betaine transport of the Gbu transporter under hyperosmolality was slightly stimulated by the shortened average fatty acid chain length induced by the addition of cerulenin, mimicking growth at low temperature.
590 $a School code: 0029.
650 4 $a Agriculture. $3 518588
650 4 $a Microbiology. $3 536250
653 $a ABC transporter
653 $a Cold stress
653 $a Gbu
653 $a Hyperosmotic stress
653 $a Listeria monocytogenes
690 $a 0473
690 $a 0410
710 2 $a University of California, Davis. $b Food Science. $3 3193559
773 0 $t Dissertations Abstracts International $g 81-03B.
790 $a 0029
791 $a Ph.D.
792 $a 2019
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13808579