Intracellular crowding defines the mode and sequence of substrate uptake by Escherichia coli and constrains its metabolic activity

Beg et al. 10.1073/pnas.0609845104.

Supporting Information

Files in this Data Supplement:

SI Data Set 1
SI Data Set 2
SI Data Set 3
SI Text
SI Figure 5
SI Figure 6
SI Figure 7
SI Figure 8
SI Table 1
SI Figure 9
SI Figure 10
SI Figure 11
SI Figure 12
SI Figure 13
SI Figure 14
SI Figure 15
SI Figure 16
SI Figure 17
SI Figure 18
SI Figure 19
SI Figure 20
SI Figure 21
SI Figure 22
SI Figure 23
SI Figure 24
SI Figure 25
SI Figure 26
SI Data Set 4
SI Data Set 5
SI Figure 27




SI Figure 5

Fig. 5. Predicted and measured maximum growth rate comparison. This figure is a reproduction of Fig. 1 (main text) after replacing the gamma distribution by a uniform distribution for the crowding coefficients. (a) Comparison between the predicted (y axis) and measured (x axis) growth rates m of E. coli MG1655 grown in M9 minimal medium with various carbon sources. For a perfect match between experiments and theory the symbols should be on the black line. The symbols indicate the carbon substrate identified in the legend. The predicted growth rates were obtained by using a uniform distribution for the crowding coefficients ai in the interval [0, 2<a>] with <a> = 0.0040 h•g/mmol (see Materials and Methods). The error bars represent the standard deviation over 1,000 sets of specific ai parameters. (b) Same plot for single gene deletion E. coli mutants growing in glucose, the deleted genes being indicated in the key. The mutant growth rates m- are given relative to the predicted and measured maximum growth rate m of wild-type E. coli cells growing in glucose-limited medium.





SI Figure 6

Fig. 6. Predicted vs. measured hierarchy of substrate utilization. This figure is a reproduction of Fig. 2c, after replacing the gamma distribution by a uniform distribution for the crowding coefficients. (a) The measured concentration of the indicated carbon sources in the growth medium. The experiments were performed in triplicate and averages and standard deviations are shown. Three substrate utilization phases, phase 1 (exclusive glucose), phase 2 (mixed substrate), and phase 3 (glycerol and acetate) are indicated in light blue, purple, and while backgrounds, respectively. (b) Predicted substrate uptakes from the growth medium based on the FBAwMC model, using a uniform distribution for the crowding coefficients ai in the interval [0, 2<a>] with <a> = 0.0040 h•g/mmol (see Materials and Methods). The color coding for substrate uptake curves is identical in a and b.





SI Figure 7

Fig. 7. Distribution of the ai coefficients as obtained from an independent estimate for about 100 E. coli enzymes. The arrow indicates the value of <a> obtained from the fit to the growth rate data.





SI Figure 8

Fig. 8. Real-time growth profile data collected from the Labfors bioreactor (showing changes in oxygen concentrations as blue curves, and pH as green curves) of E. coli MG1655 growth in M9 minimal medium containing five substrates (glucose, maltose, galactose, glycerol, and lactate) in equal proportions from three individual experiments. The optical density (black curves) was measured separately on a spectrophotometer and fed into the software. Samples for substrate analysis were collected from all three experiments and the substrate concentration plots (between zero to 10 h of growth) in Fig. 2b are the average of analysis results from three experiments. The OD600, pO2, and pH plots in Fig. 2a are also average of three plots from the above three figures. The samples for microarray analysis were collected only once from Experiment 3 (Bottom) between 2 and 8 h of growth.





SI Figure 9

Fig. 9. Principal component analysis of the microarray data from time-series experiment on mixed-substrate medium.





SI Figure 10

Fig. 10. Glucose. The green curve denotes ptsG.





SI Figure 11

Fig. 11. Lactate. The green curve denotes lldP.





SI Figure 12

Fig. 12. Maltose. The green curve denotes malE.





SI Figure 13

Fig. 13. Galactose. The green curve denotes mglA.





SI Figure 14

Fig. 14. Glycerol. The green curve denotes glpQ.





SI Figure 15

Fig. 15. Acetate. The green curve denotes acs.





SI Figure 16

Fig. 16. GO analysis and P values for cluster A.





SI Figure 17

Fig. 17. GO analysis and P values for cluster B.





SI Figure 18

Fig. 18. GO analysis and P values for cluster C.





SI Figure 19

Fig. 19. Expression pattern for cluster 1.





SI Figure 20

Fig. 20. GO enrichment table for cluster 1.





SI Figure 21

Fig. 21. Expression patterns for cluster 2.





SI Figure 22

Fig. 22. GO enrichment table for cluster 2.





SI Figure 23

Fig. 23. Expression patterns for cluster 3.





SI Figure 24

Fig. 24. GO enrichment table for cluster 3.





SI Figure 25

Fig. 25. Expression patterns for cluster 4.





SI Figure 26

Fig. 26. GO enrichment table for cluster 4.





SI Figure 27

Fig. 27. Gene expression profile of various genes known to be up-regulated during stationary phase stress response.





Table 1. Major functions and reactions catalyzed by the transporters/enzymes encoded by the genes involved in substrate uptake, as shown in Fig. 3

Carbon source and gene

Functions and reactions catalyzed by enzymes/proteins

Glucose

  

ptsG

Functions
 

The product of ptsG gene (glucose-specific PTS permease) is responsible for uptake of exogenous glucose from the medium, releasing the phosphate ester into the cell cytoplasm in preparation for metabolism, primarily via glycolysis (1, 2). PtsG/Crr, the glucose-specific PTS permease, belongs to the functional superfamily of the phosphoenolpyruvate (PEP)-dependent sugar-transporting phosphotransferase system (PTS). The PTS transports and simultaneously phosphorylates its sugar substrates in a process called group translocation.

 

Reaction
 

phosphoenolpyruvate + β-D-glucose[periplasmic space] → β-D-glucose 6-phosphate + pyruvate

Maltose

  

malEFGK

Functions
 

malKFGE operon plays the major role in the maltose transport system and it belongs to the ATP-binding cassette (ABC) superfamily of transporters (3). malE is the periplasmic maltose-binding protein, malF and malG are the integral membrane components of the ABC transporter, and malK is the ATP-binding component of the ABC transporter (MalFGK2) (4, 5)

 

Reactions
 

maltose[extracellular space] « maltose[cytosol]
 

ATP + maltose[periplasmic space] + H2O « ADP + phosphate + maltose[cytosol]

malQ

Functions
 

malQ codes for amylomaltase, which is responsible for degrading maltose after transport into the cell (6). The glucose liberated in the degradation reaction is then used in glycolysis. Amylomaltase also recognizes maltotriose and larger maltodextrins (donors), cleaving off the reducing glucose residue and transferring the remaining dextrinyl residue onto the nonreducing end of maltodextrin (acceptors), including maltose and glucose. Amylomaltase thus produces glucose and longer maltodextrins from maltotriose, the smallest donor substrate, as well as from longer linear maltodextrins (4).

 

Reaction
 

H2O + maltose «2 β-D-glucose

 

maltotriose + maltose « maltotetraose + β-D-glucose

glk

Functions
 

Under normal conditions, glucokinase plays a minor role in E. coli glucose metabolism. Under anabolic stress the enzyme is required to supplement the levels of glucose 6-phosphate.

 

Reaction
 

β-D-glucose + ATP « β-D-glucose 6-phosphate + ADP

Galactose

  

mglABC

Functions
 

MglABC is a β-methyl galactoside transport system that is a member of the ABC superfamily of transporters (3). The mglB gene codes for a galactose-binding protein that serves both as the galactose chemoreceptor as well as the recognition component of the b-methyl galactoside transport system, which utilizes the galactose-binding protein; mglC encodes the integral membrane component; and mglA encodes the ATP-binding component of the ABC transporter (7).

 

Reaction
 

ATP + β-D-galactose[periplasmic space] + H2O « ADP + phosphate + β-D-galactose[cytosol]

galE

Functions
 

galE codes for UDP-galactose 4-epimerase, which catalyzes a hydride transfer and the interconversion of UDP-galactose and UDP-glucose as part of galactose catabolism (8).

 

Reaction
 

UDP-D-glucose « UDP-galactose

galK

Functions
 

Galactokinase, coded by galK, catalyzes the first step in galactose metabolism.

 

Reaction
 

D-galactose + ATP → α-D-galactose 1-phosphate + ADP

galT

Functions
 

galT codes for galactose-1-phosphate uridylyltransferase, which catalyzes an interconversion reaction in galactose catabolism.

 

Reactions
 

UDP-D-glucose + α-D-galactose 1-phosphate « α-D-glucose 1-phosphate + UDP-galactose

 

α-D-galactose 1-phosphate + UTP « UDP-galactose + diphosphate

galP

Functions
 

GalP is one of two, along with MglABC, major routes for galactose transport into E. coli. 2-Deoxy-D-galactose is a specific substrate for GalP but not for MglABC, and GalP operates by a sugar-proton symport mechanism whereas MglABC does not.

 

Reaction
 

H+[periplasmic space] + β-D-galactose[periplasmic space] « H+[cytosol] + β-D-galactose[cytosol]

pgm

Functions
 

pgm codes for phosphoglucose mutase, which catalyzes conversion of glucose 1-phosphate to glucose 6-phosphate. Maximum activity is obtained only in the presence of α-D-glucose 1,6-bisphosphate. This bisphosphate is an intermediate in the reaction, being formed by transfer of a phosphate residue from the enzyme to the substrate, but the dissociation of bisphosphate from the enzyme complex is much slower than the overall isomerization.

 

Reaction
 

α-D-glucose 1-phosphate → α-D-glucose 6-phosphate

Glycerol

  

glpK

Function
 

glpK codes for glycerol kinase, which catalyzes the MgATP-dependent phosphorylation of glycerol to yield sn-glycerol 3-phosphate (9). This is also the rate-limiting step in glycerol utilization in E. coli.

 

Reaction
 

glycerol + ATP → sn-glycerol 3-phosphate + ADP

glpF

Function
 

The glycerol facilitator, GlpF, allows the facilitated diffusion of glycerol into the cell (10).
 

Reaction
 

glycerol[periplasmic space] « glycerol[cytosol]

gpsA

Functions
 

gpsA codes for glycerol-3-phosphate dehydrogenase [NAD(P)+], which catalyzes the NAD(P)H-dependent reduction of the glycolytic intermediate dihydroxyacetone phosphate to produce glycerol 3-phosphate (11)

 

Reaction
 

sn-glycerol 3-phosphate + NAD(P)+ « dihydroxyacetone phosphate + NAD(P)H + H+

Lactate

  

lldP

Function
 

LldP (or LctP) is a lactate/proton symporter responsible for the uptake of L-lactate. The lldP/lctP gene is located in a lactate-inducible operon with the lctD and lctR genes encoding a lactate dehydrogenase and a regulatory protein, respectively (12).

 

Reaction
 

H+[periplasmic space] + lactate[periplasmic space] « H+[cytosol] + lactate[cytosol]

dld

Function
 

dld codes for D-lactate dehydrogenase. There are three lactate dehydrogenase enzymes in E. coli that interconvert pyruvate and lactate. One is an NAD-linked fermentative dehydrogenase. The other two are membrane-bound flavoproteins, each specific for the D- or L-isomer, and are involved in the aerobic respiratory chain of E. coli. The D-lactate dehydrogenase is coded for by the dld gene, and it is the primary source of energy to drive the active transport of certain sugars and amino acids into the cell (13).

 

Reaction
 

ubiquinone-8 + D-lactate « ubiquinol-8 + pyruvate

Acetate

  

ackA

Function
 

The ackA gene product has propionate kinase activity as well as acetate kinase activity. It is unclear whether the two ack genes, ackA and ackB, code for two distinct acetate kinase enzymes or control a single enzyme. Helps in conversion of acetate to acetyl phosphate. The ackA-encoded propionate kinase 2 has an important role in propionyl-CoA metabolism (14). Acetate kinase can also catalyze acetylation of CheY, increasing signal strength for flagellar rotation (15).

 

Reactions
 

ATP + propionate « ADP + propionyl-P

acetate + ATP « acetylphosphate + ADP

pta

Function
 

pta gene codes for phosphate acetyltransferase, which can utilize both acetyl-CoA and propionyl-CoA.

 

Reactions
 

phosphate + acetyl-CoA « acetylphosphate + CoA

propionyl-CoA + phosphate « propionyl-P + CoA

acs

Function
 

acs gene codes for acetyl-CoA synthetase (ACS). There are two distinct pathways by which E. coli converts acetate to acetyl-CoA. ACS catalyzes one of them. It is thought that this ACS pathway functions in a mainly anabolic role, scavenging acetate present in the extracellular medium (16). ACS also can catalyze acetylation of CheY, increasing signal strength for flagellar rotation (17).

 

Reactions
 

CoA + 4-coumarate + ATP → coumaroyl-CoA + diphosphate + AMP
CoA + propionate + ATP « propionyl-CoA + diphosphate + AMP
CoA + acetate + ATP « acetyl-CoA + diphosphate + AMP

1. Buhr A, Erni B (1993) J Biol Chem 268:11599-11603.

2. Postma PW, Lengeler JW, Jacobson GR (1993) Microbiol Rev 57:543-594.

3. Wu LF, Mandrand-Berthelot MA (1995) Biochimie 77:744-750.

4. Boos W, Shuman H (1998) Microbiol Mol Biol Rev 62:204-229.

5. Joly N, Bohm A, Boos W, Richet E (2004) J Biol Chem 279:33123-33130.

6. Neidhardt FC, Curtiss R, III, Ingraham JL, Lin ECC, Low KB, Jr, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology (Am Soc Microbiol, Washington, DC), 2nd Ed.

7. Harayama S, Bollinger J, Iino T, Hazelbauer GL (1983) J Bacteriol 153:408-415.

8. Swanson BA, Frey PA (1993) Biochemistry 32:13231-13236.

9. Pettigrew DW, Ma DP, Conrad CA, Johnson JR (1988) J Biol Chem 263:135-139.

10. Stroud RM, Miercke LJ, O'Connell J, Khademi S, Lee JK, Remis J, Harries W, Robles Y, Akhavan D (2003) Curr Opin Struct Biol 13:424-431.

11. Lin EC (1976) Annu Rev Microbiol 30:535-578.

12. Dong JM, Taylor JS, Latour DJ, Iuchi S, Lin EC (1993) J Bacteriol 175:6671-6678.

13. Matsushita K, Kaback HR (1986) Biochemistry 25:2321-2327.

14. Hesslinger C, Fairhurst SA, Sawers G (1998) Mol Microbiol 27:477-492.

15. Barak R, Abouhamad WN, Eisenbach M (1998) J Bacteriol 180:985-988.

16. Kumari S, Tishel R, Eisenbach M, Wolfe AJ (1995) J Bacteriol 177:2878-2886.

17. Ramakrishnan R, Schuster M, Bourett RB (1998) Proc Natl Acad Sci USA 95:4918-4923.

This Article

  1. Published online before print July 24, 2007, doi: 10.1073/pnas.0609845104 PNAS July 31, 2007 vol. 104 no. 31 12663-12668
  1. AbstractFree
  2. Figures Only
  3. Full Text
  4. Full Text (PDF)
  5. » Supporting Information
About Our New Site Design >>
Link: Advanced Search

This Week's Issue

  1. July 22, 2008, 105 (29)
  1. Current Issue
  1. Alert me to new issues of PNAS

Most