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Proteome Analysis To Assess Physiological Changes In Escherichia coli Grown Under Glucose Limited Fed-Batch Conditions

Babu Raman, M. P. Nandakumar, Vignesh Muthuvijayan, and Mark R. Marten
Department of Chemical and Biochemical Engineering
University of Maryland, Baltimore County

MATERIALS AND METHODS (Detailed Version)

Strain and Reagents: Wild type E.coli K12 strain W3110 [F^–, IN(rrnD-rrnE) rph-1, λ^–] (Hill and Harnish 1981) obtained from Coli Genetic Stock Center (Yale University, CT) was used for all studies. Most chemicals used for fermentations were of ACS grade or higher, while for 2-D gel protocols electrophoresis or higher grade chemicals were used. Vendor information is indicated as follows: ^aFisher Scientific (Pittsburgh, PA), ^bSigma-Aldrich Corp. (St. Louis, MO), ^cBio-Rad Laboratories (Hercules, CA).  

Preculture: 1ml of frozen E.coli cells was used to inoculate 50ml of Luria-Bertani media (^aYeast extract 5g/l, ^aBacto tryptone 10g/l, ^aNaCl 10g/l) in 250ml Erlenmeyer flask and grown at 37^oC, 250 revolutions per minute (rpm) in shaker/incubator (New Brunswick Scientific Co., Inc., Edison, NJ). To acclimatize the cells to fermentor conditions, 100μl of this culture at an optical density at 600nm (OD₆₀₀) ~ 1.0 was used to inoculate 100ml of minimal media containing 5g/l ^aGlucose (same as batch medium; see below) in 500ml Erlenmeyer flask and grown at 37^oC and 250rpm. 50ml of this culture at mid-exponential phase (OD₆₀₀ ~ 1.0) was used to inoculate the fermentor (0.5% v/v inoculum). 

Fermentation: Fermentations were conducted in a 20L BioFlow IV fermentor (New Brunswick Scientific Co., Inc., Edison, NJ) with 10L working volume. Media was adapted from DeLisa et al. (DeLisa et al. 1999) and prepared according to Riesenberg et al. (Riesenberg et al. 1991) . All the components were added for a working volume of 12l, except glucose (added for 10l). ^aKH₂PO₄ 13.3g/l, ^a(NH₄)₂HPO₄ 4g/l, ^aCitric acid 1.7g/l, ^bEDTA 8.4mg/l, and trace metal solution (^aCoCl₂·6H₂O 2.5mg/l, ^aMnCl₂·4H₂O 15mg/l, ^aCuCl₂·4H₂O 1.5mg/l, ^aH₃BO₃ 3mg/l, ^bNa₂MoO₄·2H₂O 2.5mg/l, ^bZn(CH₃COO)₂·2H₂O 13mg/l, ^bFe(III)Citrate 100mg/l)] were dissolved in ~10l of deionized (DI) water and sterilized in the fermentor for 20mins at 121^oC. ^aMgSO₄·7H₂O 1.2g/l, and glucose 5g/l were autoclaved separately for 20mins at 121^oC and added to the cooled fermentor, along with filter sterilized ^bThiamine·HCl 4.5mg/l. Pluronic (Novozymes North America Inc., Franklinton, NC) was added as needed during the fermentation to control foaming. pH was initially adjusted to and controlled during the fermentation at 6.8 using 3N NaOH. Fermentor was operated at 37^oC, 1 bar head pressure, 600rpm agitation and 1vvm air supply. NBS Biocommand Interface and AFS Biocommand software (New Brunswick Scientific Co., Inc., Edison, NJ) were used to acquire data on dissolved oxygen (DO), base pump and other process variables. Immediately after the initial glucose (5g/l) was consumed (~12hrs), seen as a sudden DO increase, glucose was fed continuously at ~30g/hr (400g/l at 1.25ml/min). The feed rate was calculated based on specific glucose consumption rate at the end of batch phase.

Analytical Methods: Samples were harvested at regular intervals for optical density and glucose measurements. OD₆₀₀ was measured using UV-VIS scanning spectrophotometer (UV-2101 PC, Shimadzu Scientific Instrument Inc., Columbia, MD). For glucose measurements, 1ml of fermentation sample was centrifuged at 16000g, 4^oC for 15mins and the supernatant was stored at –20^oC. Glucose concentration was estimated using a glucose assay kit (Sigma-Aldrich Corp., St. Louis, MO).

Proteome Analysis:

Sample Preparation: 2-D PAGE sample preparation protocol was adapted from http://ca.expasy.org. 20-100ml of culture broth (depending on culture OD₆₀₀) was harvested and centrifuged at 1500g, 4^oC for 30mins in Avanti J-25 I Centrifuge (Bechman Coulter, Inc., Fullerton, CA). Supernatant was discarded and cells were resuspended in 15ml of ice-cold wash buffer (^aKCl 3.0mM, ^aKH₂PO₄ 1.5mM, ^aNaCl 68.0mM, ^aNaH₂PO₄ 9.0mM), followed by centrifugation at 2500g, 4^oC for 15mins. The above wash step was repeated four times. After the last wash step, cells were resuspended in an appropriate volume (see below) of sonication/lysis buffer (Tris·HCl buffer pH 8.0 10mM, ^aMgCl₂ 1.5mM, KCl 10mM, ^bDithiothreitol (DTT) 0.5mM, ^bPMSF 1.5mM, ^cSodium dodecyl sulfate (SDS) 0.1% w/v). Cell solution was sonicated on ice at 40% power in repeated 30s ON/30s OFF cycles for a total of 6mins (3mins effective sonication) using a micro tip in Sonic dismembrator (Model 550, Fisher Scientific, Pittsburgh, PA). Sonicated samples were centrifuged at 16000 g, 4^oC for 30mins in a micro-centrifuge (Biofuge-pico, Heraeus Instruments Inc., Newtown, CT) to remove cell debris. An appropriate volume (see below) of Dnase/Rnase stock solution (Tris·HCl buffer pH 8.0 0.5M, MgCl₂ 50mM, Dnase I 1.0mg/ml, Rnase A 0.25 mg/ml; Dnase I and Rnase A from Worthington Biochemical Corporation, Lakewood, NJ) was added to the supernatant and solution was incubated on ice for 30mins. Protein samples were then aliquot and stored at –80^oC until further use. Protein concentration was estimated using BCA protein assay kit (Pierce Biotechnology Inc., Rockford, IL). Note, cells from 100ml of OD₆₀₀ 1.0 culture, resuspended in 2ml of sonication buffer and digested with 20μl of Dnase/Rnase stock added per ml buffer yielded ~2ml of 5mg/ml protein sample.

Isoelectric Focusing (IEF): Immobiline drystrip reswelling tray, Immobiline drystrip kit, Multiphor II IEF apparatus, Electrophoresis Power Supply EPS-3501 XL, Multitemp II Thermostatic Circulator, 18cm pH 3-10 non-linear (NL) immobilized pH gradient (IPG) strips, and IPG buffer 3-10 NL, were all purchased from Amersham Biosciences (Piscataway, NJ). Frozen protein samples (~ 4-5mg/ml; 100μg protein load per strip) were mixed with solubilization buffer [Tris·HCl buffer pH 8.0 67mM, ^cUrea 7M, ^bThiourea 2M, ^bCHAPS 4% w/v, DTT 1% w/v, IPG buffer 3-10 NL 2% v/v, ^aBromophenol Blue (BPB) trace] in 1:9 volume ratio and solubilized for 12hrs at room temperature (RT) on orbital shaker. Samples were then diluted with rehydration buffer (Urea 7M, Thiourea 2M, CHAPS 4% w/v, DTT 1% w/v, IPG buffer 3-10 NL 2% v/v, ^bGlycerol 10% v/v, BPB trace) such that 350μl of the final solution contained 100μg of protein. (Note, in both buffers DTT and IPG buffer were added just prior to use). IPG strips were in-gel rehydrated with protein sample solution (350μl per strip) for 16hrs at RT in Immobiline drystrip reswelling tray according to manufacturer’s instructions. Rehydrated strips were focused in Multiphor II apparatus according to manufacturer’s instructions with following modifications. Cathode and anode electrode strips were presoaked in 0.5mM ^aNaOH and 6mM ^aH₃PO₄, respectively. ^aKerosene was used as heat transfer fluid between the cooling plate and immobiline dry strip tray and ^asilicon oil between the tray and aligner. Prior to IEF, strips were completely covered with ^alight paraffin oil. Strips were focused for a total of ~35kVh by ramping up the voltage in six phases: I 0-150V in 0.01hr, II retained at 150V for 1hr; III 150-300V in 0.01hr; IV retained at 300V for 3hrs; V 300-3500V in 6hrs; VI retained at 3500V for 6-7hrs, using EPS-3501 XL power supply. During focusing, temperature was maintained at 20^oC using Multitemp II Thermostatic Circulator. Strips were then stored at –80^oC in culture tubes (Fisher Scientific, Pittsburgh, PA) until further use.

Slab Gel Casting: 1.5mm thick large-format (18.5cm x 19cm) 12% T/ 2.67% C, continuous Tris·HCl linear gradient gels (10-100 kDa separation) were cast using the Multi-gel casting chamber (Bio-Rad Laboratories, Hercules, CA), according to manufacturer’s instructions. For casting ten gels, 70.08g  ^cacrylamide and 1.92g ^cpiperazine diacrylamide were dissolved in 150ml 1.5M Tris·HCl buffer pH 8.8, made to 598ml total with DI water and degassed for 1hr. 2ml of freshly prepared 10% w/v ^aammonium persulfate and 200μl ^aTEMED were added and monomer solution was immediately poured into the casting chamber from the top, up to a height of 19cm. Solution in each sandwich was simultaneously overlaid with 1ml of water saturated ^an-butanol using 1ml syringes and gels were polymerized for 2hrs at RT. After polymerization, gel sandwiches were thoroughly rinsed with DI water to remove traces of butanol, overlaid with gel storage solution (Tris·HCl buffer pH 8.8 0.375M, SDS 0.1% w/v) and stored in sample bags at 4^oC until further use (stored for up to two weeks).

Equilibration and SDS PAGE: Prior to SDS PAGE, focused strips were equilibrated in SDS equilibration buffer (Tris·HCl buffer pH 8.8 50mM, Urea 6M, Glycerol 30% v/v, SDS 2% w/v, BPB trace) containing 1% w/v DTT for 20mins, followed by another 20mins in ^bIodoacetamide (IOAC) (2.5% w/v) containing equilibration solution on orbital shaker. Equilibrated strips were trimmed ~3mm at either end, loaded on top of slab gels and overlaid with 2.5ml of preheated 0.5%w/v ^bagarose (low-melting) solution in SDS running buffer (^cTris base 25mM, ^cglycine 192mM, SDS 0.1% w/v, pH 8.3). SDS PAGE protein separation was performed using PROTEAN II XL electrophoresis cell (Bio-Rad laboratories, Hercules, CA) according to manufacturer’s instructions. Gels were run at constant current, 12mA/gel for 45mins and then at 30mA/gel until bromophenol blue dye migrated to the gel end (~4hrs), using EC-135 Power Supply (E-C Apparatus Corporation, Holbrook, NY). During gel run, temperature was maintained at 12^oC using a re-circulating water bath (LAUDA RM6-Brinkmann, Westbury, NY).

Staining: All staining steps were done at RT using high quality (18 megaohm) DI water. Gels were stained in 9”x 9” Nalgene staining boxes, 250ml of solution / gel / box, with constant shaking on orbital shaker (Lab-Line MAXI-Rotator, Barnstead International, Dubuque, IO). Staining protocol was adapted from Blum et al. (Blum et al. 1987) . Briefly, gels were fixed in 50% v/v ^aCH₃OH/ 10% v/v ^bCH₃COOH for 1hr followed by overnight incubation in 5% v/v CH₃OH/ 1% v/v CH₃COOH. The following day, gels were washed with water four times, 5mins per wash step. Gels were then sensitized by incubation in 0.02% w/v ^bNa₂S₂O₃·5H₂O for 90s followed by three water washes of 30s each. After which, gels were incubated in 0.2% w/v ^aAgNO₃ for 30mins in dark. Followed by a quick water rinse, gels were developed in a solution containing 6% w/v ^aNa₂CO₃, 4mg/l Na₂S₂O₃·5H₂O, and 500μl/l ^aformalin (37% w/v formaldehyde) for ~10mins until desired intensity was reached. Developing was stopped with 6% v/v acetic acid for 10mins, followed by water washes. Gels were stored at 4^oC in sample bags with little DI water until further use.

Image Analysis: Stained gels were digitalized at 400 DPI resolution using GS-800 imaging densitometer (Bio-Rad Laboratories, Hercules, CA). Gel images (8-bit TIFF) were analyzed using Melanie 3.0 2-D PAGE image analysis software package (Genebio S.A., Geneva, Switzerland) as follows. Spot features were detected using default and altered settings. Since some real spots were missed and many extraneous spots were detected, spots were manually edited. After spot detection, 20 spots common to all gels and scattered across the area of the gel image were selected as landmarks for gel-to-gel alignment. Aligned gels were matched on a spot-to-spot basis without allowing multiple pairing. Spot pairing was manually checked for missed pairs and mismatched spots and was edited. In this study, for each condition three gels were used for image analysis. Gels were pair-wise matched to each other and composite gels containing spots common to all three gels were created for each sample. Composite gels were then compared to each other to determine differences in protein expression between the different conditions. Spot quantity was expressed in vol% (volume of a spot / total volume of all the spots in a gel) since it minimizes differences due to staining.

Reproducibility Studies: Multiple gels run from the same sample were compared to estimate variability in protein expression due to gel running procedures. We also compared gels of the same sample harvested from replicate fermentations to estimate variations resulting from experimental differences. In both cases, more than 90% of spots matched between gels, were within a two-fold differential expression (DE) ratio. Spots showing higher variability were either extraneous random spots, or spots in crowded and streaked regions of the gel where spot detection was ambiguous, and such regions were excluded from our analysis. Based on these studies, spots that were differentially expressed by more than 2-fold between gels run from different experimental conditions were considered as significantly up- or down-regulated.

Protein Identification

Preparative gels: Protein samples (conc. ~ 4-5mg/ml; 400μg protein load per strip) were mixed with solubilization buffer in 1:2 volume ratio and solubilized for 24hrs at RT. Rehydration buffer was added such that 350μl of the final solution contained ~400μg of protein. IPG strips were rehydrated (350μl per strip) in protein solution for 16hrs at RT in Immobiline drystrip reswelling tray and focused in IPGphor IEF system (Amersham Biosciences, Piscataway, NJ) according to manufacturer’s instructions. Light paraffin oil was used to cover the strips. Prior to IEF, 20μl of rehydration buffer (without protein) was added in the sample well close to acidic end of strips to minimize electroendosmosis. Strips were focused at 20^oC for a total of ~30 kVh by ramping up the voltage, as described for analytical gels. SDS PAGE and silver staining were done, as described earlier.

In-gel digestion: HPLC water (Fisher Scientific, Pittsburgh, PA) was used for in-gel digestion. Candidate spots were cored from six preparative gels, cut into 1mm³ cubes and pooled together in acetonitrile (ACN)-washed 1.5ml Eppendorf tubes. Gel pieces were washed four times with water, 5mins per wash, and destained in 100μl of a freshly prepared solution containing 30mM ^bK₃FeCN₆ and 100mM Na₂S₂O₃ in 1:1 ratio, for 10mins at RT, according to Gharahdaghi et al. (Gharahdaghi et al. 1999) . In-gel digestion protocol was adapted from Shevchenko et al. (Shevchenko et al. 1996) . After destaining, gel pieces were thoroughly washed with water 4-5 times, 15mins per wash, until they turned colorless and transparent. This was followed by incubation in 100μl of 100mM ^bAmmonium bicarbonate (ABC) for 20mins at RT. Pieces were then rinsed with water and shrunk twice in 100% ^bACN, each time for 10mins. ACN was removed and the white sticky gel pieces were dried in Speed-Vac Plus SC110A (Thermo Savant, Holbrook, NY) for 20mins at low setting. 100μl of 10mM DTT in 100mM ABC was added to the dried gel pieces and incubated at 56^oC for 1hr in a water bath (Fisher Scientific, Pittsburgh, PA). Samples were cooled for 15mins, DTT solution was removed and gel pieces were incubated in 100μl of 55mM IOAC in 100mM ABC at RT for 45mins in dark. IOAC solution was removed and gel pieces were washed with 100μl of 100mM ABC for 10mins and dehydrated by adding an equal volume of 100% ACN (final solution 50mM ABC, 50% ACN) for 15mins. Liquid phase was removed and gel pieces were rehydrated in ABC, shrunk again by addition of ACN as described above and dried in the speed-vac for 20mins. Dried gel pieces were then rehydrated in 20-30μl of digestion buffer (enough to cover) - 50mM ABC, 2.5mM ^bCaCl₂ and 8-12.5 ng/μl (depending on the darkness of the spot) trypsin (sequencing grade modified trypsin, Promega Corporation, Madison, WI) - for 1hr at 4^oC. Excess trypsin solution was removed and gel pieces were covered with the above digestion solution (without trypsin) and proteins were digested for 16hrs at 37^oC in a water bath. Note a blank piece of gel with no protein and SDS PAGE separated ^bcarbonic anhydrase was processed in parallel with the unknown samples to identify contaminant peaks and to monitor the quality of digestion, respectively.

Peptide extraction: After overnight digestion, supernatant was collected and gel pieces were covered with freshly prepared 50mM ABC and incubated at RT for 45mins. An equal volume of 100% ACN was added (final solution 25mM ABC, 50% ACN) and peptides were extracted for 2.5hrs. Above steps were repeated with 5% ^bformic acid instead of 50mM ABC. Peptides were further extracted overnight in 5% formic acid / 50% ACN (enough to cover gel pieces). Supernatant pooled from all the steps was completely dried in speed-vac at low setting. Dried peptides were dissolved in 10μl 0.1% ^bTrifluoroacetic acid (TFA), and desalted using zip-tips^TM (Millipore Corporation, Billerica, MA), according to manufacturer’s instructions. Peptides were eluted in 2-3μl of 60% ACN / 0.1% TFA solution.

MALDI-TOF MS: 0.5μl of peptide sample was mixed with an equal volume of a saturated (25mg/ml) solution of ^bα-cyano-4-hydroxycinnamic acid in 70% ACN/0.1% TFA on the target plate and air dried by dried droplet method. Peptide mass spectra were acquired in linear, positive mode, using Autoflex series MALDI-TOF and analyzed using XMASS/XTOF NT 5.1.1 software (Bruker Daltonics Inc. Billerica, MA). Peptide masses were externally calibrated using a mixture of ^bLeu-5-enkephalin ([MH+] m/z 556.64) and ^bInsulin B oxidized chain ([MH+] m/z 3496.97) and internally using a trypsin autolytic peak ([MH+] m/z 2212.42).

Database search: Isoelectric point (pI) and molecular weight (MW) calibration of 2-D gels was done using 2-D SDS PAGE standards (Bio-Rad laboratories, Hercules, CA) and mid-range MW markers (Promega Corporation, Madison, WI), respectively. Average peptide masses obtained from MALDI-TOF MS in the 1000-3000 Da range were used. Proteins were identified through peptide mass fingerprinting in the NCBI non-redundant E.coli database using ProFound database search engine (Genomic Solutions, Ann Arbor, MI). Following parameters were used in database searches: MW 0-100 kDa, pI 3-10 (a more narrow MW and pI window was used in some searches), one missed cleavage (in some cases, digestion was lesser efficient), cysteine modified by IOAC, peptides singly protonated [MH+], and average peptide mass tolerance in %. ProFound calculates “expectation value” to rate search results, which in simple terms is the score that the sequence matched was a random hit. Hence, smaller the expectation value, more likely that a particular match is not a random one. The software highlights protein identifications thus considered as confident.

Criteria for confident identification were that the protein should (a) be highlighted by ProFound, (b) match within 0.05% peptide mass tolerance, (c) have at least 20% sequence coverage and (d) match at least 4 peptides. Some (5) high MW proteins (45-85 kDa) had < 20% sequence coverage, but matched > 5 peptides, while few (2) low MW proteins (10-15 kDa) matched < 4 peptides but had > 20% sequence coverage. In such specific cases, identification was considered confident even if only 3 out of 4 criteria were met. In this study, out of 57 spots identified, 89% were identified within 0.03% peptide mass tolerance, 91% had more than 20% sequence coverage (all with > 15% coverage), and 98% of identifications matched more than 4 peptides.

Figure 1: Reproducibility studies: (a) Optical density at 600nm (OD₆₀₀) and Glucose concentration (g/l) data from four different batch fermentations is plotted against fermentation time (hrs). (b) Vol% of spots matched between two gels run from the same sample is plotted on a log-log plot. (c) Vol% of spots matched between exponential phase gels from two different fermentations is plotted on a log-log plot. Dotted lines in (b) and (c) indicate the two-fold differential expression (DE) boundary.

  
Figure 2: Differential comparison of protein expression in Escherichia coli under exponential phase and fed-batch phase in glucose limited fed-batch fermentation. 30 protein spots (10 identified) had significantly higher expression in exponential phase, while 60 spots (47 identified) showed higher expression under fed-batch conditions. Spots with circled gene names were not present in exponential phase gels.

Table 1.0: Catabolite repressed genes and operons upregulated under glucose-limited conditions in fed-batch phase.  

REFERENCES

Bell AW, Buckel SD, Groarke JM, Hope JN, Kingsley DH, Hermodson MA. 1986. The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12. J. Biol. Chem. 261(17):7652-8.

Black PN, DiRusso CC. 1994. Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli. Biochim. Biophys. Acta. 1210(2):123-45.

Blum H, Beier H, Gross HJ. 1987. Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels. Electrophoresis 8:93-99.

Busby S, Kolb A. 1996. The CAP modulon. In: Lin ECC, Lynch AS, editors. Regulation of gene expression in Escherichia coli. Austin, Texas, USA: R. G. Landes company. p 255-279.

DeLisa MP, Li J, Rao G, Weigand WA, Bentley WE. 1999. Monitoring GFP-operon fusion protein expression during high cell density cultivation of Escherichia coli using an on-line optical sensor. Biotechnol. Bioeng. 65(1):54-64.

Gavigan SA, Nguyen T, Nguyen N, Senear DF. 1999. Role of multiple CytR binding sites on co-operativity, competition, and induction at the Escherichia coli udp promoter. J. Biol. Chem. 274(23):16010-9.

Gharahdaghi F, Weinberg CR, Meagher DA, Imal BS, Mische SM. 1999. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis 20:601-605.

Hill CW, Harnish BW. 1981. Inversions between ribosomal RNA genes of Escherichia coli. Proc. Natl. Acad. Sci., U. S. A. 78:7069-7072.

Isaacs Jr. H, Chao D, Yanofsky C, Saier Jr. MH. 1994. Mechanism of catabolite repression of tryptophanase synthesis in Escherichia coli. Microbiology 140(8):2125-34.

Karp PD, Arnaud M, Collado-Vides J, Ingraham J, Paulsen IT, M. H. SJ. 2004. The E. coli EcoCyc Database: No Longer Just a Metabolic Pathway Database. ASM News 70(1):25-30.

Kumari S, Beatty CM, Browning DF, Busby SJW, Simel EJ, Hovel-Miner G, Wolfe AJ. 2000. Regulation of Acetyl-Coenzyme A synthetase in Escherichia coli. J. Bacteriol. 182(15):4173-9.

Liu J, Beacham IR. 1990. Transcription and regulation of cpdB gene in Escherichia coli K12 and Salmonella typhimurium LT2: Evidence for modulation of constitutive promoters by cyclic AMP-CRP complex. Mol. Gen. Genet. 222(1):161-5.

Nystrom T, Larsson C, Gustafsson L. 1996. Bacterial defense against aging: role of the Escherichia coli ArcA regulator in gene expression, readjusted energy flux and survival during stasis. EMBO J. 15(13):3219-28.

Ozbudak EM, Thattai M, Lim HN, Shraiman BI, van Oudenaarden A. 2004. Multistability in the lactose utilization network of Escherichia coli. Nature 427:737-740.

Park SJ, McCabe J, Truna J, Gunsalus RP. 1994. Regulation of citrate synthase (gltA) gene of Escherichia coli in response to anaerobiosis and carbon supply: Role of arcA gene product. J. Bacteriol. 176(16):5086-92.

Park SJ, Gunsalus RP. 1995. Oxygen, iron, carbon and superoxide control of the fumarase fumA and fumC genes of Escherichia coli: Role of the arcA, fnr and soxR gene products. J. Bacteriol. 177(21):6255-62.

Park SJ, Tseng CP, Gunsalus RP. 1995. Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: Role of ArcA and Fnr. Mol. Microbiol. 15(3):473-82.

Plumbridge J. 1998. Control of the expression of the manXYZ operon in Escherichia coli: Mlc is a negative regulator of the mannose PTS. Mol. Microbiol. 27(2):369-80.

Riesenberg D, Schulz V, Knorre WA, Pohl H-D, Korz D, Sanders EA, Rob A, Deckwer W-D. 1991. High cell density cultivation of Escherichia coli at controlled specific growth rate. J. Biotechnol. 20:17-28.

Saier Jr. MH. 1996. Cyclic AMP-independent catabolite repression in bacteria. FEMS Microbiol. Lett. 138:97-103.

Shevchenko A, Wilm M, Vorm O, Mann M. 1996. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68:850-858.

Stern MJ, Higgins CF, Ames GF. 1984. Isolation and characterization of lac fusions to two nitrogen-regulated proteins. Mol. Gen. Genet. 195(1-2):219-27.

Takeda S, Matsushika A, Mizuno T. 1999. Repression of the gene encoding succinate dehydrogenase in response to glucose is mediated by the EIICB(Glc) protein in Escherichia coli. J. Biochem. (Tokyo) 126(2):354-60.

Zhi J, Mathew E, Freundlich M. 1998. In vitro and in vivo characterization of three major dadAX promoters in Escherichia coli that are regulated by cyclic AMP-CRP and Lrp. Mol. Gen. Genet. 258(4):442-7.

Zhu Y, Lin ECC. 1988. A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli. J. Bacteriol. 170(5):2352-8.