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Chapter 7 : Control of Gene Expression by Compartmentalization: the put Operon
Author:
Stanley Maloy1
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Affiliations:
1: Department of Biology, San Diego State University, San Diego, CA 92182-4614
Content Type: Monograph
Publication Year:
2011
Category: History of Science; Microbial Genetics and Molecular Biology
Book DOI:
10.1128/9781555816810
Chapter DOI:
10.1128/9781555816810.ch7
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Abstract:
In this chapter, the author tells a story about the characterization of the operon from Salmonella. The author commenced his research trying to figure out why high phosphate concentrations inhibit cell division in Microcyclus flavus. In the final analysis, the most obvious change was in the distribution of phospholipids; however, it was not clear if this was the reason why cells stopped dividing or a secondary response to the inhibition of cell division. This led to his lifelong interest in membranes and convinced him that if he wanted to understand cause and effect, he will need to do genetics. His research in John’s lab focused on the regulation of proline utilization by the PutA protein, a bifunctional protein that could function as either a membrane-associated enzyme or a repressor. The proline utilization (put) operon allows cells to use proline as a sole source of carbon, nitrogen, and energy. In vitro studies confirmed that PutA binds to specific operator sites in the put control region. Instead of simply responding to the presence or absence of proline, induction of the put operon requires several physiological conditions needed for its catabolism: high concentrations of proline, an appropriate terminal electron acceptor, and the absence of other substrates that could compete for functional membrane binding sites.
Citation:
Maloy S.
2011.
Control of Gene Expression by Compartmentalization: the put Operon,
p 55-63.
In
Maloy S, Hughes K, Casadesús J (ed),
The Lure of Bacterial Genetics.
ASM Press, Washington, DC.
doi: 10.1128/9781555816810.ch7
Figures
Control of Gene Expression by Compartmentalization: the put Operon
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Citation:
Maloy S.
2011.
Control of Gene Expression by Compartmentalization: the put Operon,
p 55-63.
In
Maloy S, Hughes K, Casadesús J (ed),
The Lure of Bacterial Genetics.
ASM Press, Washington, DC.
doi: 10.1128/9781555816810.ch7
/content/10.1128/9781555816810.ch07.ch07fig01
FIGURE 1
Proline utilization pathway. Proline is transported by the putP gene product, a sodium-proline sym-porter. Once inside the cell, proline is degraded in two enzymatic steps by the putA gene product, a multifunctional protein with proline-FAD dehydrogenase (i) and P5C-NAD dehydrogenase (ii) activities.
10.1128/9781555816810/f0057-01_thmb.gif
10.1128/9781555816810/f0057-01.gif
FIGURE 1
Proline utilization pathway. Proline is transported by the putP gene product, a sodium-proline sym-porter. Once inside the cell, proline is degraded in two enzymatic steps by the putA gene product, a multifunctional protein with proline-FAD dehydrogenase (i) and P5C-NAD dehydrogenase (ii) activities.
Citation:
Maloy S.
2011.
Control of Gene Expression by Compartmentalization: the put Operon,
p 55-63.
In
Maloy S, Hughes K, Casadesús J (ed),
The Lure of Bacterial Genetics.
ASM Press, Washington, DC.
doi: 10.1128/9781555816810.ch7
/content/10.1128/9781555816810.ch07.ch07fig02
FIGURE 2
put control region. The putA and putP genes are divergently transcribed from promoters within the 420-bp region located between the putP and putA translation start sites. Gray arrows represent the putP and putA promoters. Rectangles labeled O
1 to O
5 represent PutA binding sites (“operator sites”). Dark rectangles indicate IHF binding sites. The squiggle to the left of O
1 represents a strong intrinsic bend.
10.1128/9781555816810/f0058-01_thmb.gif
10.1128/9781555816810/f0058-01.gif
FIGURE 2
put control region. The putA and putP genes are divergently transcribed from promoters within the 420-bp region located between the putP and putA translation start sites. Gray arrows represent the putP and putA promoters. Rectangles labeled O 1 to O 5 represent PutA binding sites (“operator sites”). Dark rectangles indicate IHF binding sites. The squiggle to the left of O 1 represents a strong intrinsic bend.
Citation:
Maloy S.
2011.
Control of Gene Expression by Compartmentalization: the put Operon,
p 55-63.
In
Maloy S, Hughes K, Casadesús J (ed),
The Lure of Bacterial Genetics.
ASM Press, Washington, DC.
doi: 10.1128/9781555816810.ch7
/content/10.1128/9781555816810.ch07.ch07fig03
FIGURE 3
Model for the physiological regulation of proline utilization. At low intracellular proline, PutA is cytoplasmic and functions as a transcriptional repressor of the put operon. As the concentration of proline increases, the FAD cofactor in PutA is reduced and proline is converted to P5C. Reduction of FAD alters the conformation of PutA and increases its hydrophobicity. The increased hydrophobicity drives PutA to the inner leaflet of the cytoplasmic membrane (CM) where it cannot bind DNA but functions as an enzyme. As proline levels decrease due to degradation, the FADH2 is reoxidized and PutA regains its dimeric, hydrophilic structure. Consequently, PutA accumulates in the cytoplasm and represses transcription of the put operon. (Note that the higher order structure of the repressing protein-DNA complex and phosphorylation of PutA are not shown in this figure.)
10.1128/9781555816810/f0060-01_thmb.gif
10.1128/9781555816810/f0060-01.gif
FIGURE 3
Model for the physiological regulation of proline utilization. At low intracellular proline, PutA is cytoplasmic and functions as a transcriptional repressor of the put operon. As the concentration of proline increases, the FAD cofactor in PutA is reduced and proline is converted to P5C. Reduction of FAD alters the conformation of PutA and increases its hydrophobicity. The increased hydrophobicity drives PutA to the inner leaflet of the cytoplasmic membrane (CM) where it cannot bind DNA but functions as an enzyme. As proline levels decrease due to degradation, the FADH2 is reoxidized and PutA regains its dimeric, hydrophilic structure. Consequently, PutA accumulates in the cytoplasm and represses transcription of the put operon. (Note that the higher order structure of the repressing protein-DNA complex and phosphorylation of PutA are not shown in this figure.)
Citation:
Maloy S.
2011.
Control of Gene Expression by Compartmentalization: the put Operon,
p 55-63.
In
Maloy S, Hughes K, Casadesús J (ed),
The Lure of Bacterial Genetics.
ASM Press, Washington, DC.
doi: 10.1128/9781555816810.ch7
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