Manuscript

Guanosine 5’,3’-bispyrophosphate coordinates global gene expression during glucose-lactose diauxie in Escherichia coli, Proc. Nat. Acad. Sci., 2006, 103:2374-2379

Microarray Data

• MG1655 wildtype glucose-lactose diauxie, 17 timepoints, triplicate microarrays
  
• rpoS diauxie, 13 timepoints, triplicate arrays
  
• crp "diauxie" 4 timepoints, quadruplicate arrays
  
• relA diauxie, 11 timepoints. triplicate arrays

Growth Data Sets

Wildtype Diauxie

RpoS Mutant Diauxie

Crp Mutant Diauxie

RelA Mutant Diauxie

Project summary. When cultured on a mixture of glucose and lactose, E. coli grows preferentially on glucose until it is exhausted, resulting in growth arrest while the cells adjust to growth on lactose, i.e., diauxie. K-means clustering of the transcriptome dataset of wildtype E. coli during glucose-lactose diauxie revealed three regulatory networks (RpoS, Crp, and RelA) that dominated the transcription profile. Therefore, we investigated the temporal changes in transcription during glucose-lactose diauxie in the wildtype and mutants lacking the key regulators, RpoS, Crp, and RelA (ppGpp synthetase).

Strains and growth conditions. E. coli MG1655 and isogenic mutants were cultured in a 2 l Biostat B fermentor (B. Braun Biotech International) containing 1 liter of Morpholinepropanesulfonic acid (MOPS) minimal medium with 0.5 g/l of glucose and 1.5 g/l of lactose, as described (3) . The temperature was maintained at 37 ºC and pH was kept constant at 7.2 by the addition of 2 M NaOH. The dissolved oxygen level was maintained above 20% of saturation by adjusting the agitation speeds in the range of 270-500 rpm with fixed 1 l/min air flow. Growth was monitored as absorbance at 600nm. E. coli D relA 251::kan R was a gift from M. Cashel, constructed as described (14) . The E. coli D crp ::kan R and D rpoS ::kan R strains were constructed by allelic replacement (15) of the entire genes. These mutant strains are isogenic with E. coli MG1655.

Microarray analysis. Microarray analysis was carried out essentially as described (16) . Total RNA was extracted from cells, diluted (1:1) in ice-cold RNAlater (Ambion) and purified using RNeasy columns (Qiagen), as described (3) . RNA was labeled by first strand cDNA synthesis using reverse transcriptase, random primers, and aminoallyl-dUTP incorporation; Cy-3 and Cy-5 dyes were chemically coupled in vitro to the aminoallyl-derivatized cDNA. The oligonucleotide microarrays used in this study were printed on GAPS II slides (Corning) with a probe set containing 70 base oligonucleotide probes for all E. coli MG1655 genes (Operon Biotechnologies) using a Molecular Dynamics Gen III Array Spotter (Amersham Biosciences). Slides were hydrated and flash-dried, UV-cross-linked, and blocked with succinic anhydride, then equal amounts of the Cy-3 and Cy-5 labeled samples were hybridized in triplicate to microarrays using a Discovery system and ChipMap reagents (Ventana Medical Systems). For all microarrays, the experimental sample was labeled with Cy-5 and the control, from early logarithmic growth of E. coli MG1655 wildtype on minimal glucose medium, was labeled with Cy-3. Hybridized slides were scanned on a GenePix4000 scanner (Axon), the data collected using GenePix (ver. 5.0) software, and uploaded to our database for analysis (http://www.ou.edu/microarray). The data were normalized by a local Lowess algorithm (17) implemented on our database and the replicate arrays averaged for analysis. Clustering algorithms were implemented in Spotfire DecisionSite for Functional Genomics software.

Wildtype Diauxie

.

Time (min) A600    Timepoint
 780           0.143   WT_tp1
 830           0.245   WT_tp2
 861           0.345   WT_tp3
 869           0.38    WT_tp4
 878           0.406   WT_tp5
 888           0.408   WT_tp6
 898           0.403   WT_tp7
 908           0.422   WT_tp8
 919           0.446   WT_tp9
 929           0.49    WT_10
 939           0.534   WT_tp11
 969           0.706   WT_tp12
 999           0.907   WT_tp13
1035           1.344   WT_tp14
1049           1.458   WT_tp15
1070           1.594   WT_tp16
1089           1.604   WT_tp17

Time (min) A600    Timepoint
120            0.257   rpoS tp1 
150            0.372   rpoS tp2
160            0.391   rpoS tp3
170            0.432   rpoS tp4
180            0.434   rpoS tp5
190            0.438   rpoS tp6
200            0.472   rpoS tp7
210            0.513   rpoS tp8
220            0.561   rpoS tp9
250            0.78    rpoS tp10
290            1.21    rpoS tp11
315            1.55    rpoS tp12
330            1.63    rpoS tp13

Time (min) A600    Timepoint
  0            0.054   none
 60            0.078   none
120            0.098   none
180            0.134   none
210            0.168   none
240            0.216   none
250            0.24    crp tp2
300            0.319   none
315            0.353   none
330            0.378   none
345            0.415   none
360            0.455   none
375            0.503   none
390            0.532   none
405            0.56    crp tp5
420            0.568   crp tp6
450            0.56    crp tp7

Time (min) A600    Timepoint
 832           0.198   tp1
 865           0.283   tp2
 896           0.437   tp4
 909           0.53    tp5
 916           0.526   tp6
 925           0.526   tp7
 935           0.532   tp8
 945           0.535   tp9
 955           0.535   tp10
 966           0.582   tp11
 976           0.626   tp12
1007           0.772   none
1036           0.959   none
1067           1.278   none
1095           1.479   none
1125           1.509   none
1140           1.544   none
1325           1.38    none