TABLE 1

Identity of protein spots that differ between the ancestor and evolved clones and two protein spots shown as internal standards for visualization

Protein spot no.aProtein (gene)bFunctional groupcExpression lower in:dQuantitatione
M1AhpC (ahpC)Cell processes, protection1.060 ± 0.065
0.991 ± 0.034
M2GapA (gapA)Metabolism, energy metabolism0.999 ± 0.011
0.993 ± 0.079
1CirA (cirA)*Transport, outer membraneEvol0.656 ± 0.011
0.678 ± 0.052
2FadL (fadL)*Transport, outer membraneAnc1.956 ± 0.080
2.800 ± 0.195
3LamB (lamB)*Transport, outer membraneEvol(0)OFF
OFF
4ArtJ (artJ)Transport, periplasmic spaceEvol0.504 ± 0.038
0.536 ± 0.037
5GlnH (glnH)Transport, periplasmic spaceEvol(0)OFF
OFF
6–7MalE (malE)*Transport, periplasmic spaceEvol(0)OFF
OFF
8–9MetQ (metQ)Transport, periplasmic spaceEvol(0)OFF
OFF
10MglB (mglB)*Transport, periplasmic spaceEvol0.736 ± 0.007
0.554 ± 0.007
11MglB (mglB)*Transport, periplasmic spaceEvol0.599 ± 0.036
0.451 ± 0.010
12ModA (modA)*Transport, periplasmic spaceEvol0.433 ± 0.008
0.408 ± 0.034
13RbsB (rbsB)*Transport, periplasmic spaceEvol(0)OFF
OFF
14TolB (tolB)Transport, periplasmic spaceEvol0.165 ± 0.020
0.139 ± 0.031
15TolB (tolB)Transport, periplasmic spaceEvol0.206 ± 0.008
0.258 ± 0.044
16ZnuA (znuA)Transport, periplasmic spaceAnc(0)ON
ON
17AroG (aroG)Metabolism, amino-acid biosynthesisEvol0.127 ± 0.008
0.203 ± 0.014
18IlvB (ilvB)**Metabolism, amino-acid biosynthesisAnc(0)ON
ON
19LeuA (leuA)*Metabolism, amino-acid biosynthesisAnc(0)ON
ON
20LeuA (leuA)*Metabolism, amino-acid biosynthesisAnc(0)ON
ON
21SerC (serC)*Metabolism, amino-acid biosynthesisEvol0.320 ± 0.008
0.380 ± 0.012
22AceB (aceB)Metabolism, central intermediary metabolismAnc4.959 ± 0.186
6.017 ± 0.285
23GpmA (gpmA)Metabolism, central intermediary metabolismEvol0.403 ± 0.071
0.344 ± 0.013
24NfnB (nfnB)Metabolism, central intermediary metabolismAnc(0)ON
ON
25ATPF (atpF)Metabolism, energy metabolismEvol0.461 ± 0.002
0.524 ± 0.025
26ATPF (atpF)Metabolism, energy metabolismEvol0.409 ± 0.064
0.492 ± 0.014
27Mdh (mdh)*Metabolism, energy metabolismEvol0.532 ± 0.047
0.545 ± 0.024
28SucC (sucC)*Metabolism, energy metabolismEvol0.631 ± 0.005
0.742 ± 0.022
29SucD (sucD)*Metabolism, energy metabolismEvol0.463 ± 0.014
0.484 ± 0.029
30–31PflB (pflB)*Metabolism, energy metabolismEvol(0)OFF
OFF
32FabI (fabI)Metabolism, fatty acid biosynthesisEvol0.497 ± 0.012
0.424 ± 0.026
33FklB (fklB)Information transfer, chaperone, foldingEvol0.374 ± 0.017
0.333 ± 0.012
34GroEL (groL)**Information transfer, chaperone, foldingEvol0.300 ± 0.037
0.399 ± 0.040
35HtpG (htpG)Information transfer, chaperone, foldingEvol0.447 ± 0.026
0.454 ± 0.044
36Aat (aat)Information transfer, protein degradationEvol0.555 ± 0.038
0.564 ± 0.026
37HslU (hslU)Information transfer, protein degradationEvol0.468 ± 0.063
0.414 ± 0.020
38Fur (fur)*Information transfer, transcriptionEvol(0)OFF
OFF
39AsnS (asnS)**Information transfer, translationEvol0.556 ± 0.043
0.653 ± 0.019
40AsnS (asnS)**Information transfer, translationEvol0.505 ± 0.032
0.379 ± 0.029
41GlyS (glyS)Information transfer, translationEvol0.216 ± 0.039
0.238 ± 0.024
42GlyS (glyS)Information transfer, translationEvol0.200 ± 0.019
0.157 ± 0.021
43Mannosyl-transferasefCell processesAnc(0)ON
ON
44MinD (minD)Cell processes, cell divisionEvol0.218 ± 0.029
0.292 ± 0.017
45MinD (minD)Cell processes, cell divisionEvol0.272 ± 0.028
0.255 ± 0.019
46GTP-dependent nucleic acid- binding proteinfGTP-binding proteinEvol0.739 ± 0.043
0.731 ± 0.061
47BipA (bipA)GTP-binding protein, elongation factorEvol0.543 ± 0.029
0.442 ± 0.031
48BipA (bipA)GTP-binding protein, elongation factorEvol0.542 ± 0.044
0.583 ± 0.040
49YcdO (ycdO)Hypothetical, unknownEvol0.559 ± 0.068
0.633 ± 0.027
50OppA (oppA)Transport, periplasmic spaceAra + 10.315 ± 0.006
0.975 ± 0.090
51ArgT (argT)Metabolism, amino-acid biosynthesisAncg2.204 ± 0.322
1.054 ± 0.037
52MetE (metE)Metabolism, amino-acid biosynthesisAra + 10.544 ± 0.048
0.983 ± 0.055
53MetE (metE)Metabolism, amino-acid biosynthesisAra + 10.409 ± 0.040
0.950 ± 0.039
54GlpK (glpK)*Metabolism, carbon compound utilizationAra − 11.066 ± 0.137
0.256 ± 0.027
55TktA (tktA)Metabolism, central intermediary metabolismAra + 10.559 ± 0.049
0.996 ± 0.045
56PykF (pykF)Metabolism, energy metabolismAra − 1(0)1.112 ± 0.041
OFF
57GuaA (guaA)*Metabolism, nucleotide biosynthesisAra − 10.996 ± 0.096
0.274 ± 0.015
58PurH (purH)Metabolism, nucleotide biosynthesisAra + 10.615 ± 0.060
0.964 ± 0.120
59EF-Tu (tufAB)**Information transfer, translationAncg2.476 ± 0.163
h
60SodB (sodB)*Cell processes, adaptation to stressAra + 10.370 ± 0.030
1.000 ± 0.039
  • a The protein spots M1 and M2 show similar intensities across all genotypes and serve as internal standards for visualization only (Figure 1). Protein spots 1–49 show significant and parallel changes in the two independently evolved clones compared to their ancestor (○ in Figure 1). Protein spots 50–60 show significant changes in only one of the two evolved clones (▵ and ▿ in Figure 1). Some proteins were identified in two different spots; this pattern corresponds to post-translational modifications or to degradation products (in each case, both protein spots showed the same changes in the evolved clones).

  • b Regulation by cAMP–CRP and ppGpp is indicated by “*” and “**,” respectively, for the functionally characterized genes (Cashel et al. 1996 and Salgado et al. 2004 for the table; Man et al. 1997 for serC; Zhang et al. 2005 for sodB; Zheng et al. 2004 for modA, kbl, and leuA; Traxler et al. 2006 for asnS).

  • c Functional groupings of the proteins are shown according to the data from GenProtEC (http://genprotec.mbl.edu).

  • d Anc, expression lower in the ancestor clone; Anc(0), proteins absent from the ancestor but appearing in both evolved clones; Evol, expression lower in both evolved clones; Evol(0), proteins disappearing in both evolved clones; Ara + 1, expression lower than in the ancestor only in the Ara + 1 evolved clone; Ara − 1, expression lower than in the ancestor only in the Ara − 1 evolved clone; Ara − 1(0), protein spot disappearing in the Ara-1 evolved clone.

  • e Quantitative gel analysis was performed using the Melanie II software (Genebio). For every spot, the protein expression of each evolved clone was standardized using the ancestral strain as a reference, after first standardizing for total protein volume over all spots (see materials and methods). For example, a value of 2.0 indicates that the relative abundance of a particular protein spot is twice as high in an evolved clone as in the ancestor, whereas a value of 0.5 indicates that the relative abundance of a protein in the evolved clone is only half that measured in the ancestor. The upper and lower entries for each protein are relative abundances measured in the Ara + 1 and Ara − 1 evolved clones, respectively. Mean values are shown along with standard deviations based on the three independent sets of gels. OFF, protein spots absent in the evolved clones, but present in the ancestor. ON, protein spots absent in the ancestor, but present in the evolved clones.

  • f Based on protein sequence comparisons. A gene name could not be assigned.

  • g ArgT and EF-Tu are significantly higher only in the Ara + 1 evolved clone.

  • h The Melanie II software was unable to quantify the EF-Tu protein spot in the Ara − 1 evolved clone because the spot was fuzzy and precise delineation was impossible for two of the three replicate gels.