oubcf
Growth Transitions

July, 2002
Chang, et al., 2002, Molec Microbiol 45: 289-306 (PDF)

Data Sets

Diauxic Growth Experimental Design and Data

H2O2 Treatment Experimental Design and Data

Minimal Glucose Medium Aerobic and Anaerobic Growth Curve Experimental Design and Data

Project summary. When conditions cause bacterial growth to stop, extensive reprogramming of physiology and gene expression allows for the cellís survival.† We used whole-genome DNA arrays to determine the system response in E. coli cells experiencing transient growth arrest caused by glucose-lactose diauxie and H2O2-treatment, and also entry into stationary phase.† The results show that growth arrested cells induce stringent control of several gene systems.† The vast majority of genes encoding the transcription and translation apparatus immediately down-regulate, followed by a global return to steady-state when growth resumes. Approximately one-half of the amino acid biosynthesis genes down-regulate during growth arrest, with the notable exception of the his operon which transiently up-regulates in the diauxie experiment.† Nucleotide biosynthesis down-regulates, a result that is again consistent with the stringent response.† Likewise, aerobic metabolism down-regulates during growth arrest, and the results led us to suggest a model for stringent control of the ArcA regulon.† The stationary phase stress response fully induces during growth arrest, whether transient or permanent, in a manner consistent with known mechanisms related to stringent control.† Cells similarly induce the addiction module antitoxin and toxin genes during growth arrest; the latter are known to inhibit translation and DNA replication.† The results indicate that, in all aspects of the response, cells do not distinguish between transient and potentially permanent growth arrest (stationary phase).† We introduce an expanded model for the stringent response that integrates induction of stationary phase survival genes and inhibition of transcription, translation, and DNA replication.† Central to the model is the reprogramming of transcription by ppGpp, which provides for the cellís rapid response to growth arrest and, by virtue of its brief half-life, the ability to quickly resume growth as changing conditions allow.

Diauxie Experimental Design and Data

Time (min)†††††††† A600††††††††††† Timepoint
90.0000†††††††††† 0.1500 ††††††††††† logD1
135.0000†††††††† 0.2700 ††††††††††† logD2
165.0000†††††††† 0.3500 ††††††††††† logD3
180.0000†††††††† 0.4020 ††††††††††† logD4
185.0000†††††††† 0.4400 ††††††††††† logD5
190.0000†††††††† 0.4400 ††††††††††† logD
195.0000†††††††† 0.4720 ††††††††††† logD7
205.0000†††††††† 0.4560 ††††††††††† logD8
215.0000†††††††† 0.4560 ††††††††††† logD9
225.0000†††††††† 0.4800 ††††††††††† logD10
235.0000†††††††† 0.5400 ††††††††††† logD11
273.0000†††††††† 0.7920 ††††††††††† logD12
345.0000†††††††† 1.4080 ††††††††††† logD13
360.0000†††††††† 1.6800 ††††††††††† logD14
375.0000†††††††† 1.6480 ††††††††††† logD15
390.0000†††††††† 1.6400 ††††††††††† logD16
420.0000†††††††† 1.6480 ††††††††††† logD17

Culture conditions. E. coli MG1655 (CGSC #6300) was cultured in 1 liter of morpholinepropanesulfonic acid (MOPS) minimal medium containing 0.5 g/liter of glucose and 1.5 g/liter of lactose (Neidhardt et al., 1974) in a 2 liter Biostat B fermentor. The temperature was maintained at 37 degrees C and pH was kept constant at 7.2 by the addition of 2 M NaOH. The dissolved oxygen level was maintained above 20% of its saturation level by adjusting the agitation speeds in the range of 400-600 rpm with fixed 1 liter/min air flow rate. Growth was monitored by measuring the optical density (OD) at 600nm. These growth conditions resulted in a diauxic lag lasting 36 min. The culture continued growing until entering stationary phase upon exhaustion of carbon and nitrogen. RNA samples were isolated at 5 and 10 min intervals following the diauxic lag, and at appropriate intervals during the experiment, by pipeting into ice-cold RNAlater™ (Ambion, Austin, TX) followed by purification using an RNeasy™ Mini Kit (Qiagen, Valencia, CA). The RNA samples were labeled by first strand cDNA synthesis. Labeled targets were hybridized to DNA arrays (Panorama E. coli Gene Arrays, Sigma Genosys Biotechnologies, Inc., The Woodlands, TX). The hybridized arrays were scanned by phosphorimaging at a pixel density of 100 microns (10,000 dots/cm2) with a STORM 820 PhosphoImager (Molecular Dynamics, Sunnyvale, CA) following exposure to a Kodak Storage Phosphor Screen GP (Eastman Kodak Co., Rochester, NY) for 24 hrs. The array membranes were consecutively hybridized, stripped, and rehybridized.

Spot-finding and quantitation. Image analysis software (ArrayVision, Imaging Research, Inc.) was used for spot-finding and quantitation of the E. coli Panorama arrays. The raw spot intensities were represented in a row-column format and exported into Microsoft Excel spreadsheets for further analysis, or as comma-delimited files (.csv) for upload to the database. Raw data from each experimental replicate were analyzed in Excel workbooks containing manually executed macros written in Visual Basic, or the data were processed in the database. The first step in the analysis associates the array coordinate for each spot with a unique spot number, the gene name, and related gene annotation. On the membrane arrays there are two spots for each gene, and these were treated as separate determinations. The raw data were normalized by expressing spot intensities as a percentage of the sum of all of the gene-specific spot intensities. The second step in the analysis applies the student t-test to determine the probability that the average of the experimental replicates is significantly different from the average of the control replicates . The P values (derived from the student t-test) for the normalized and natural log transformed data were calculated. The third step calculates relative gene expression between conditions by introducing a threshold value, chosen to be representative of the limit of detection of expressed genes (usually the 500th lowest expressed gene), and then calculating the ratio of the experimental/control expression levels such that genes that are more highly expressed in the experimental condition are given a positive value, and genes that are more highly expressed in the control condition are given a negative value.

Control for ratio calculation. The early log phase control values used for the diauxie experiment represent the average of 12 hybridizations: two each from time points 1 and 2 of the H2O2-treatment and time points 1 and 3 of the glucose-lactose diauxie and 4 from time point 2 of the glucose-lactose diauxie. This approach is possible because gene expression is essentially steady-state in early-logarithmic phase and because there is excellent correlation between these time points. The use of these control values allowed us to directly compare the H2O2-treatment and glucose-lactose diauxie.

E. coli Gene Expression Database (Oracle) Interface

H2O2 Treatment Experimental Design

Time (min)†††††††† A600††††††††††† Timepoint
100.0000†††††††† 0.2480†††††††††††† logH1
140.0000†††††††† 0.3840†††††††††††† logH2
145.0000†††††††† 0.3760†††††††††††† logH3
150.0000†††††††† 0.3800†††††††††††† logH4
160.0000†††††††† 0.3960†††††††††††† logH5
170.0000†††††††† 0.4080†††††††††††† logH6
180.0000†††††††† 0.4160†††††††††††† logH7
200.0000†††††††† 0.5040†††††††††††† logH8
255.0000†††††††† 0.9000†††††††††††† logH9
330.0000†††††††† 1.7800†††††††††††† logH10
345.0000†††††††† 1.7840†††††††††††† logH11
390.0000†††††††† 1.7840†††††††††††† logH12

Culture conditions. E. coli MG1655 (CGSC #6300) was cultured in 1 liter of morpholinepropanesulfonic acid (MOPS) minimal medium containing 2 g/liter of glucose (Neidhardt et al., 1974) in a 2 liter Biostat B fermentor. The temperature was maintained at 37 degrees C and pH was kept constant at 7.2 by the addition of 2 M NaOH. The dissolved oxygen level was maintained above 20% of its saturation level by adjusting the agitation speeds in the range of 400-600 rpm with fixed 1 liter/min air flow rate. Growth was monitored by measuring the optical density (OD) at 600nm. Immediately following timepoint 2 (H2), a sublethal concentration of H2O2 (50 ug/ml) was added to cause an immediate inhibition of growth, followed by a 40 min lag prior to resuming growth. The culture continued growing until entering stationary phase upon exhaustion of carbon and nitrogen.

E. coli Gene Expression Database (Oracle) Interface

Aerobic and Anaerobic Growth Experimental Design

Time (min)

Aerobic

Timepoint

Time (min)

Anaerobic

0

0.051

1

0

0.065

97

0.148

2

30

0.0755

143

0.252

3

90

0.129

180

0.388

4

180

0.247

240

0.775

5

300

0.4905

280

1.285

6

330

0.568

300

1.61

7

360

0.613

315

1.7

8

375

0.6105

330

1.765

9

390

0.61

360

1.765

10

405

0.605

Experimental Design

Culture conditions. E. coli MG1655 (CGSC #6300) was cultured in 1 liter of morpholinepropanesulfonic acid (MOPS) minimal medium containing 2.0 g/liter of glucose (Neidhardt et al., 1974) in a 2 liter Biostat B fermentor. The temperature was maintained at 37 degrees C and pH was kept constant at 7.2 by the addition of 2 M NaOH. For the aerobic culture, the dissolved oxygen level was maintained above 20% of its saturation level by adjusting the agitation speeds in the range of 400-600 rpm with fixed 1 liter/min air flow rate. For the anaerobic culture, the medium contained 35 mM Na H2CO3 and was sparged with a mixture of nitrogen and carbon dioxide gases (90:10) at a fixed 1 liter/min flow rate. Growth was monitored by measuring the optical density (OD) at 600nm.

Data Legend

Aerobic Growth Data Set (Timepoint 2 control; Excel: 3.1 Mb)
TGC_2_conpct: average normalized spot intensity for control timepoint 2; values expressed as a percentage of the sum of all of the gene-specific spot intensities
TGC_10_tstpct: average normalized spot intensity for test timepoint 10
TGC_10_V_2_logratio: log10 ratio of timepoint 10 vs. timepoint 2
TGC_10_V_2_pln: P value for the corresponding log ratio calculated from the normalized, natural log transformed data

Aerobic Growth Data Set (Timepoint 1-4 averaged control; Excel: 3.4 Mb)
TGC_1-4_conpct : average normalized spot intensity for control timepoints 1-4; values expressed as a percentage of the sum of all of the gene-specific spot intensities
TGC_1_tstpct: average normalized spot intensity for test timepoint 1
TGC_1_logratio: log10 ratio of timepoint 1 vs. control average timepoints 1-4
TGC_1_pln: P value for the corresponding log ratio calculated from the normalized, natural log transformed data

Anaerobic Growth Data Set (Timepoint 2 control; Excel: 3.1 Mb)
AGTC_2_conpct: average normalized spot intensity for control timepoint 2; values expressed as a percentage of the sum of all of the gene-specific spot intensities
AGTC_10_tstpct: average normalized spot intensity for test timepoint 10
AGTC_10_V_2_logratio: log10 ratio of timepoint 10 vs. timepoint 2
AGTC_10_V_2_pln: P value for the corresponding log ratio calculated from the normalized, natural log transformed data

Anaerobic Growth Data Set (Timepoint 1-4 averaged control; Excel: 3.4 Mb)
AGTC_1-4_conpct : average normalized spot intensity for control timepoints 1-4; values expressed as a percentage of the sum of all of the gene-specific spot intensities
AGTC_1_tstpct: average normalized spot intensity for test timepoint 1
AGTC_1_logratio: log10 ratio of timepoint 1 vs. control average timepoints 1-4
AGTC_1_pln: P value for the corresponding log ratio calculated from the normalized, natural log transformed data

Aerobic vs. Anaerobic Comparison (Timepoints 1-6 averaged Excel: 1.5Mb)
Aer_pct: average normalized spot intensity for aerobic timepoints 1-6; values expressed as a percentage of the sum of all of the gene-specific spot intensities
Anaer_pct: average normalized spot intensity for anaerobic timepoints 1-6
Anaer/Aer_LOGRATIO: log10 ratio of Anaerobic average timepoints 1-6 vs. Aerobic average timepoints 1-6
Anaer/Aer_PLN: P value for the corresponding log ratio calculated from the normalized, natural log transformed data
Aer_STDDEV: Standard deviation of Aerobic timepoints 1-6
Anaer_STDDEV: Standard deviation of Anaerobic timepoints 1-6

E. coli Gene Expression Database (Oracle) Interface

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