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Jacqueline Plumbridge,2 Valley Stewart,3 Daniel Bertenthal,1,
Haidy Lee,1,
Gyaneshwar Prasad,1 Oleg Paliy,1 Parinya Charernnoppakul,1 and Sydney Kustu1*
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102,1 Institut de Biologie Physico-chimique, UPR9073, CNRS, 75005 Paris, France,2 Section of Microbiology, University of California, Davis, California 95616-86653
Received 28 April 2003/ Accepted 2 July 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using DNA microarrays, we observed apparent cross regulation of gene expression between galactose catabolism and that of lactose and N-acetylglucosamine, effects that would not have been readily detected by other means. However, when we explored the bases for these effects, we found that MG1655 (CGSC 6300 unless otherwise noted) grew slowly on galactose, and apparent cross-regulatory effects were due to the appearance of suppressor mutations that allow fast growth.
We here present a compendium of the growth defects and selected other properties of MG1655 (CGSC 6300), which we analyzed in different degrees of detail. We also document differences between the Stock Center isolate and other strains designated MG1655 which we obtained from other sources. In agreement with previous studies (2), we show that cDNAs from a variety of E. coli strains hybridize well to MG1655-based DNA microarrays and hence that these arrays can be used to perform genomic and physiological studies of any strain of interest. Given the problems with "MG1655," it may be advisable for laboratories to continue using the parental E. coli strains that they have characterized.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Repair of rph-1 mutation in MG1655 (CGSC 6300).
The rph-1 lesion in CGSC 6300 was repaired in several steps. The zib-207::Tn10 insertion (39) was introduced into VH1000 (rph+) (14) by P1vir-mediated transduction to yield NCM3414. The linkage between the insertion and rph is 
10%. P1vir phage grown on NCM3414 were used to introduce the rph+ allele into CGSC 6300 to yield NCM3416. Selection was done for tetracycline resistance, and screening was done for improved growth on N-C- medium with glucose as the carbon source. The presence of the rph+ allele in NCM3416 and the rph-1 allele in congenic strain NCM3415 was confirmed by sequencing.
Isolation of lac mutants from MG1655 (CGSC 6300) as galactose fast-growers. When MG1655 (CGSC 6300) was passed three times on minimal medium containing glucose or glycerol as the carbon source, its doubling time remained constant at 67 and 98 min, respectively. After being passed three times on galactose, its doubling time decreased to 86 min (see Results). At this point, 96 clones were purified on Luria-Bertani (LB) and screened on minimal medium with galactose or glucose as the carbon source and containing the indicator 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) to monitor ß-galactosidase activity. Like CGSC 6300, most clones were white to pale blue. However, 16 were blue on glucose, whereas CGSC 6300 was white, and were dark blue on galactose, whereas CGSC 6300 was pale blue. Two strains, NCM3559 and NCM3560, were studied further, as was a control strain, NCM3558, which was a galactose fast-grower but looked like CGSC 6300 on indicator plates. The population of galactose fast-growers was also screened on LB containing X-Gal and 0.5 mM IPTG to induce expression of the lactose operon. Three clones of the 300 screened were white, whereas CGSC 6300 was blue. One, NCM3556, was studied further. No white colonies were seen in a population of CGSC 6300 passed once on galactose, and none were seen in populations passed three times on glucose or glycerol. Lesions in strains NCM3556, NCM3559, and NCM3560 were tested for linkage to the lac operon by P1vir-mediated transduction. The strains were transduced to tetracycline resistance on LB plates containing X-Gal with phage grown on lac+ strain NCM3384 (mhpC281::Tn10), and transductants were screened for the appropriate change in color.
Isolation of a nagA null mutant of MG1655 (CGSC 6300) as a galactose fast-grower. MG1655 (CGSC 6300) was passed four times on minimal medium containing galactose as the carbon source. Then a portion of the culture was plated on rich medium (LB), and colonies were tested for growth on N-acetylglucosamine (GlcNAc) as the carbon source. One strain that failed to grow on GlcNAc, NCM3845, was chosen for further characterization. PCR amplification (nagA1 primer, 5'-GTTACCGTTAACGATGGTCTTGG; nagA2 primer, 5'-CCGGATCTTTACCGGCCACG) of the coding region of the nagA gene in NCM3845 yielded a fragment similar in size (1.1 kb) to that obtained from MG1655 (CGSC 6300), indicating that the lesion was not a large deletion. PCRs were performed with 1 µl of a 10-fold-diluted overnight LB culture of the strains tested in a 25-µl reaction volume (0.3 U of AmpliTaq enzyme, 1x reaction buffer, 200 µM deoxynucleoside triphosphate mix, and 300 nM each primer) at 94°C for 2 min, followed by 30 cycles of 30 s at 94°C, 30 s at 45°C, and 3 min at 72°C.
Isolation of Eut+ strains. Parental strains were grown on LB and then streaked on N-C- plates containing 0.4% glycerol, 10 mM ethanolamine hydrochloride, and 0.2 µM cyanocobalamin (CN-B12; Sigma). Plates were incubated at 37°C. No Eut+ colonies were observed on plates that lacked cyanocobalamin.
Construction of a lacA mutant.
Strain NCM3748 (lacA::Kanr) was obtained by insertion of a kanamycin resistance cassette 255 bp downstream of the translational start codon for the lacA gene of MG1655 (CGSC 6300), as described below. (i) The lacA gene with short flanking sequences of the lacY (24-bp) and cynX (52-bp) genes was amplified by PCR from genomic DNA isolated from MG1655 (CGSC 6300) with primers cynX1 (5'-CCCTGCGTTTTGCACCAG; position 155 bp downstream of the stop codon for lacA) and lacY3 (5'-CGTCAGGTGAATGAAGTCGC; position 90 bp upstream of lacA). PCRs were performed with 34 ng of genomic DNA in a 25-µl reaction volume (1 U of AmpliTaq enzyme, 1x reaction buffer, 200 µM deoxynucleoside triphosphate mix, and 320 nM each primer). They were initiated at 94°C for 5 min, followed by 30 cycles of 30 s at 94°C, 30 s at 50°C, and 2 min at 72°C. (ii) The 856-bp amplified DNA fragment was ligated according to the instructions of the manufacturer into the pNo Ta/T7 shuttle vector (Prime PCR Cloner; 5'
3', Inc.) to yield plasmid pJES1411. The identity of the PCR fragment was confirmed by sequence analysis. (iii) To eliminate the EcoRI site present in the polylinker, pJES1411 was digested with EcoRI and SacI, made blunt-ended with mung bean nuclease, and self-ligated to yield pJES1413. (iv) The 1.3-kb EcoRI fragment from plasmid pUC4K (Amersham Pharmacia Biotech), which carries a kanamycin resistance cassette, was ligated into pJES1413 which had been linearized with ApoI to yield pJES1415. (v) The 2.2-kb PmeI fragment of pJES1415, containing the lacA::Kanr construct, was moved into the SmaI site of the gene replacement vector pK03 (33), resulting in pJES1506. pK03 contains a temperature-sensitive origin of replication and the selectable markers sacB and cat. (vi) The lacA gene of MG1655 (CGSC 6300) was replaced as described previously (20, 33).
From the kanamycin-resistant transformants, one clone with the correct phenotype (Kanr, Cams, and sucrose resistant [growth on LB medium without NaCl and containing 6% sucrose (5)]) was chosen for further analysis. The presence of the lacA::Kanr insertion in this strain, NCM3748, was confirmed by PCR and by measuring transacetylase activity (not shown). That the insertion had indeed replaced the wild-type copy of lacA was confirmed by Southern analysis with full-length lacA and Kanr probes. Genomic DNAs were digested with BglI, HindIII, or both (data not shown).
Construction of a 
ycjT::Kanr deletion-disruption.
Strain NCM3467 (ycjT::Kanr) which carries a deletion of 686 bp of the ycjT gene (,2267 bp total; deletion starts 544 bp downstream of the translational start codon) and an insertion of a kanamycin resistance cassette, used for selection, was obtained from the recD strain NCM3426 (see Table 1) by the following steps. (i) The ycjT gene with flanking sequences of the ycjS and ycjU genes was amplified from strain MG1655 (CGSC 6300) with primers ycjT1 (5'-ACTAAGCTTAGATCCTGCCCAGGCGTACC) and ycjT2 (5'-AGTTCTAGAGCCAGAAGGCCGATAACCGC) and the Expand high-fidelity PCR system (Boehringer Mannheim-Roche). PCRs were performed with 34 ng of genomic DNA from MG1655 (CGSC 6300) in a 100-µl reaction volume (2.6 U of Expand enzyme mix, 1x reaction buffer, 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphate mix, and 300 nM each primer). They were initiated at 94°C for 2 min, followed by 30 cycles of 30 s at 94°C, 30 s at 50°C, and 5 min at 72°C. The primers introduced a HindIII restriction site at the 5' end and an XbaI site at the 3' end of the fragment. (ii) The 4-kb PCR product was digested with HindIII and XbaI and ligated into pBluescript-KS (Stratagene) which had also been digested with HindIII and XbaI to yield plasmid pJES1333. (iii) The identity of the insert was confirmed by sequence analysis. A difference from the published sequence, apparently due to a mutation introduced by PCR amplification, was detected downstream of the SacII restriction site in ycjU. (iv) The 1.3-kb EcoRI fragment from plasmid pUC4K was made blunt-ended with the Klenow fragment of DNA polymerase I and ligated into pJES1333 which had been digested with EcoRV and MscI (deleting 686 bp of ycjT) to yield pJES1336. (v) The 4.6-kb KpnI-SacII (upstream of the possible mutation noted above) fragment of pJES1366, carrying ycjS'-ycjT1366::Kanr-'ycjT was transformed into the recD strain NCM3426 to yield strain NCM3467 (kanamycin resistant). The presence of the ycjT1366::Kanr deletion-insertion was confirmed by PCR (data not shown) and moved by P1vir-mediated transduction into other strains (Table 1).
Determination of fnr phenotypes of various strains. Strains were tested anaerobically for growth on a variety of media containing appropriate electron donors and acceptors and for evolution of gas from glucose (see Results). The well-characterized fnr+ and fnr strains RK4353 and RK5279, respectively, were used as controls (55). Lesions in strains with phenotypic defects were tested for linkage to ycj-630::Tn10 by P1vir-mediated transduction. The ycj-630::Tn10 allele is 90% linked to fnr (55).
Analysis of fnr region of various strains by PCR.
To determine the basis of the Fnr- phenotype of MG1655 (CGSC 6300) (see Results), we attempted to amplify the fnr gene (b1344) and the chromosomal region surrounding it by PCR. PCR products were analyzed on 0.8% agarose gels and stained with ethidium bromide. PCR amplification of the fnr gene with the b1334 ORFmer primer set (Fnr-A/C) of Sigma Genosys produced a single PCR product of the expected size (0.75 kbp) for the Singer-Gross isolate of MG1655 (NCM3430), but a fragment of 3.0 kbp for the isolate of MG1655 from the CGSC (CGSC 6300). Control PCRs performed in the absence of one primer established that the 3' primer (Fnr-C) amplified a nonspecific fragment of the same size (3.0 kbp).
New primers were designed (fnr-5', 5'-CGCCATGAAGGTTATCTT-3'; fnr-3', 5'-CCTTCTGCCAGATCAATA-3'). PCRs were performed with 40 ng of genomic DNA in a 30-µl reaction volume (1 U of Taq polymerase [Perkin-Elmer], 1x reaction buffer, 125 µM deoxynucleoside triphosphate mix, and 350 nM each primer). They were initiated at 94°C for 4 min, followed by 25 cycles of 1 min at 94°C, 1 min at 50°C, and 80 s at 72°C. A single PCR product of the expected size (1.0 kbp) was amplified from Fnr+ strain NCM3430, and no product was obtained from Fnr- strain CGSC 6300. PCR amplification of fnr with our primer set and of every other gene from b1330 to b1346 with the ORFmer primer sets of Sigma-Genosys yielded a single product of the expected size for each gene from NCM3430. (The primer pair for b1336 was an exception: it failed). However, products were obtained only for b1330 and b1331 and for b1345 and b1346 from CGSC 6300. No products were detected for b1332 through b1344. The entire fnr region was amplified by long-range PCR according to the manufacturer's instructions (Expand PCR Long Range; Boehringer-Roche) with 40 ng of genomic DNA, 3.75 U of Expand polymerase, 1x reaction buffer 2, 500 µM deoxynucleoside triphosphate mix, and 300 nM each primer in a 50-µl reaction) with forward primer b1331 and reverse primer b1345 at 94°C for 120 s and then for 10 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 15 min, and 15 additional cycles with an increase of 20 s per cycle at 68°C (see Fig. 2).
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Determination of 
-amylase activity.
The -amylase activity assay was adapted from that of Freundlieb et al. (13) and modified. After centrifugation, cell pellets were washed and suspended in ice-cold sodium phosphate buffer (100 mM, pH 7.0). Cells were permeabilized with chloroform and sodium dodecyl sulfate (0.002% [wt/vol]) and warmed to 27°C for 5 min. Reactions were started by the addition of 4-nitrophenyl--D-hexa-(1 4)-glucopyranoside (PG6; Fluka Chemical Corp.), which was dissolved in the above buffer, to a final concentration of 1 mM. Reactions were stopped with Na2CO3, and cells were removed by centrifugation. The -amylase activity was calculated as 1,000 x A405/(t x v) (36). Activity reflects the sum of the activities of the MalT-inducible periplasmic -amylase MalS, the MalT-inducible cytoplasmic maltodextrin glucosidase MalZ (56), and the MalT-independent cytoplasmic -amylase AmyA (47).
Clinical isolates of E. coli. Twelve isolates obtained from the Tang Health Center at the University of California, Berkeley, were kindly provided by A. Manges and L. Riley in the School of Public Health. They had been identified as E. coli based on their ability to catabolize lactose (red colonies on MacConkey-lactose plates) and their ability to produce indole from tryptophan (35). Strains were frozen within three passages of the time of isolation. Strains NCM3601 (Tang identifier Seq404) and NCM3610 (Tang identifier Seq915) were isolated from patients with acute urinary tract infections (UTI), whereas NCM3611 (Tang identifier 10002-006) was an intestinal commensal strain isolated from the partner of a patient with an acute UTI. Each of these strains was isolated from a separate individual. Isolates from the Tang Center failed to grow on minimal medium, and many required nicotinic acid. Tang Center isolates readily yielded prototrophic derivatives, and derivatives of the strains listed above were chosen for microarray experiments because they grew well on minimal medium. Isolates from the Tang Center were resistant to phage P1vir.
Glass slide DNA microarray analysis. Cells were grown to mid-exponential phase, and growth was then quenched with 1/10 volume of 5% phenol in ethanol (57, 60). RNA extraction, synthesis of cDNAs containing fluorescent nucleotide analogues, hybridization to microarrays, scanning of fluorescence, and normalization were done essentially as described previously (60). Data are displayed in genome images (57, 60).
Operon organization is taken from the RegulonDB database (http://www.cifn.unam.mx/Computational_Genomics/regulondb/) with the modifications of Zimmer et al. (60). Genes are listed in the order transcribed, with the exception of those in the flagellar or chemotaxis regulon. For the latter, the direction of transcription of individual operons is indicated with arrows. For comparisons of CGSC 6300 and clinical isolates of E. coli grown on glucose with either glutamine or NH4Cl as the nitrogen source, data from three experiments with CGSC 6300 were averaged. Data from three experiments performed once each with prototrophic derivatives of clinical isolates NCM3601, NCM3610, and NCM3611 were also averaged.
Affymetrix DNA microarray analysis. E. coli K-12 strains NCM3722 and MG1655 (CGSC 6300) were grown in N-C- medium with either 0.4% glucose or 0.4% glycerol as the carbon source and either 10 mM NH4Cl or 2.5 mM arginine as the nitrogen source. Strains were adapted to growth in the appropriate medium by preculturing to the early stationary phase. Experimental cultures were incubated in a water bath at 37°C in 500- or 1,000-ml Erlenmeyer flasks containing 1/10 volume of medium. The starting OD600 was 0.025, and cells were harvested in mid-exponential phase (OD600 = 0.4 to 0.5) by adding 1/10 culture volume of 95% ethanol-5% phenol. Cell suspensions (25-ml portions) were then subjected to centrifugation at 6,000 rpm for 5 min, and pellets were frozen on dry ice. They were stored at -80°C prior to use.
Gene expression was assessed on Affymetrix E. coli Antisense GeneChip arrays (Affymetrix, Inc., Santa Clara, Calif.). The detailed protocol, which was slightly modified from that supplied to us by Affymetrix, Inc., is available at http://nature.berkeley.edu/~opaliy/papers/MG1655.html. Briefly, total RNA was prepared from E. coli cells by phenol-chloroform extraction, and 15 µg of total RNA was used to synthesize cDNA. After purification with a Qiagen cDNA purification kit (Qiagen, Inc.), the cDNA was fragmented with DNase I. The fragmented cDNA was end biotinylated by terminal transferase with biotinylated ddATP. Hybridization of the fragmented, biotinylated cDNA to a chip, washing, staining, and scanning of the chip were done on an Affymetrix workstation as described previously (Expression Analysis Technical Manual; Affymetrix, Inc., Santa Clara, Calif.). Raw data files were analyzed by the statistical algorithm in MAS 5.0 (Affymetrix, Inc.) with the default parameters. All experiments were scaled globally to the same target intensity of 1,500 with only the probe sets for E. coli genes and not intergenic regions. Data obtained from different strains grown on the same medium were compared to determine differences in mRNA levels. Results were exported into Microsoft Excel for further analysis.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When we attempted to grow MG1655 (CGSC 6300) in glycerol-limited continuous culture, growth defects due to internal uracil (pyrimidine) starvation became more problematic (Fig. 1). At a very low dilution rate of 0.34 h-1, the culture reached steady state at an OD600 of 0.6, approximately that expected, and exhausted glycerol from the medium (<0.02 mM remaining) (Fig. 1 and legend). However, at a faster dilution rate of 0.51 h-1, the OD600 decreased, that is, the culture started to wash out. Surprisingly, however, at dilution rates in this range (0.52 to 0.54 h-1), it established a new steady state (OD 0.36 to 0.4) in which large amounts of glycerol remained in the medium (1.3 to 1.6 mM of the 5 mM total that was provided in the reservoir).
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MG1655 (CGSC 6300) carries an fnr deletion. Strains carrying fnr null alleles exhibit specific defects in anaerobic respiration: they fail to use fumarate or nitrate as an electron acceptor for respiration. They also fail to respire with nitrite or dimethyl sulfoxide as the electron acceptor, and they synthesize little formate-hydrogen lyase (due to Fnr-dependent expression of nickel uptake). They cannot use glycerol as an electron donor for anaerobic growth but grow reasonably well on glucose (fermentation) and also show essentially normal aerobic respiratory metabolism.
We determined the Fnr phenotype of various strains by testing anaerobically for growth on glycerol-fumarate (growth on defined medium), for formate-nitrate respiration (phenotype on MacConkey-nitrate [MN] indicator medium), and for gas production from glucose (55). We confirmed genetic linkage to fnr by testing for transductional linkage to a Tn10 element near the fnr locus (55). In each case, experimental strains were compared to well-characterized controls (55) (see Materials and Methods). MG1655 (CGSC 6300) failed to grow on glycerol-fumarate plates, formed small dark red colonies on MacConkey-nitrate plates, and evolved little gas from glucose. Introduction of an fnr+ allele from strain RK4353 (55) by P1vir-mediated transduction restored growth on glycerol-fumarate and allowed the formation of large salmon-colored colonies on MN medium.
Both phenotypic and genetic analyses indicated that a number of additional strains held at the CGSC, some related to MG1655 and some not, were also Fnr-. These included BD792 (CGSC 6159), which, like MG1655, is W1485F- W1485F+ (CGSC 5024); the original wild-type E. coli K-12 strain EMG2 of Clowes and Hayes (CGSC 4401); and WG1 of Lederberg (CGSC 5073). However, an isolate of MG1655 obtained through Mitchell Singer from the laboratory of Carol Gross (NCM3430) was Fnr+, as were isolates obtained from the American Type Culture Collection (ATCC 47076) and the Blattner laboratory (both colony types; see below). An E. coli K-12 wild-type strain used in our laboratory (NCM3722) was also Fnr+. It appears that storage at or shipping from the CGSC may predispose strains to become Fnr- (37).
PCR-based amplification of the fnr region from MG1655 (CGSC 6300) and the Singer isolate (NCM3430) showed that CGSC 6300 carries a deletion of this region (Fig. 2). PCR amplification of individual genes in the region from CGSC 6300 indicated that the deletion extends from gene b1332 (included) to b1344 (included) and results in the loss of 13 genes (legend to Fig. 2) and about 14 kbp. Interestingly, the genes flanking the deletion code for a transposase (b1331) and a prophage integrase (b1345) (4). Amplification of the fnr region from the Blattner isolate of MG1655 (both colony types; see below) and ATCC 47076 indicated that both were fnr+ (not shown). This was also true of K-12 wild-type strain NCM3722.
MG1655 grows slowly on galactose. When E. coli MG1655 (CGSC 6300) was transferred from enriched medium to minimal medium with galactose as the carbon source or from minimal-glucose medium to minimal-galactose medium, it grew slowly (doubling time of 200 to 300 min; Table 2). As it was adapted to galactose by serial transfers, the culture grew more rapidly (doubling time of 70 to 80 min). The fast-growing population had unchanged doubling times on glucose or glycerol as the carbon source (data not shown). Clones isolated from the fast-growing population grew rapidly on galactose immediately upon transfer from other carbon sources or media (data not shown), indicating that they were genetically altered. Several were characterized further (see below).
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-galactosides, an -galactosidase, and a regulator.
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Cross-induction of mal regulon by growth on lactose.
The fact that induction of expression of the mal regulon did not occur with IPTG indicated that it was not mediated directly by the lactose repressor, LacI. The fact that it did not occur on glycerol or galactose as the carbon source (Table 3 and not shown) provided evidence that it was not due simply to relief of catabolite repression. To test its dependence on MalT, the activator of the maltose regulon, and maltose transport components, we first confirmed that induction could be detected at the protein level both for MalE, the periplasmic maltose binding protein (not shown), and for MalS plus MalZ, periplasmic and cytoplasmic -amylases, respectively (Table 4) (6, 12, 13). The -amylase activity, which was ninefold higher on glycerol plus maltose than on glycerol, was also 2.5 times higher on lactose than on glycerol. In a strain carrying a null allele in malT, the increases in -amylase activity on glycerol plus maltose and on lactose were eliminated, indicating that expression of malS plus malZ during growth on lactose is MalT dependent. Similarly, increased expression of the maltose binding protein on lactose was MalT dependent (not shown).
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-amylase activity on glycerol plus maltose, confirming that they prevented maltose transport, they caused no decrease in activity on lactose (Table 4), indicating that induction of malS on lactose was apparently not due to the presence of contaminating coinducer in the lactose. Rather, it appeared to be due to synthesis of the coinducer endogenously. These results were confirmed qualitatively for the maltose binding protein (not shown). Decker et al. (9) proposed that maltose and maltotriose may be synthesized endogenously from glucose and glucose 1-phosphate (Fig. 4) by a maltose phosphorylase. Hence, we inactivated ycjT (see Materials and Methods), which codes for a protein with 45% similarity over its entire length to MapA, a maltose phosphorylase from Lactobacillus sanfranciscensis (Blast website, http://www.ncbi.nlm.nih.gov/blast/) (10). Based on levels of the maltose binding protein (assessed by Western blotting; data not shown), the ycjT null allele did not affect induction of the maltose regulon on lactose, nor did it affect growth on maltose.
Apparent induction of lac operon by growth on galactose. Our microarray experiments were performed before we knew that MG1655 (CGSC 6300) was acquiring mutations to fast growth in galactose. In a population of cells growing rapidly on galactose, we observed four- to fivefold-higher levels of expression of the lac operon than in cells grown on glucose (Table 3). Higher expression of the lac operon was not observed in a culture grown on glycerol or in a population of cells growing slowly on galactose (not shown). Similar results were obtained by enzyme assays (Table 2) except that the degree of increase in ß-galactosidase activity in the population of galactose fast-growers (400 versus 10 U/ml/OD600) was at least 40-fold. We have noted such discrepancies between microarray data and enzyme assays previously (57).
To study the basis for higher expression of the lac operon in a culture of MG1655 "adapted to" galactose and growing on it rapidly, we screened 96 purified clones for a constitutive Lac phenotype (see Materials and Methods). Sixteen had this phenotype. Lesions in the two strains tested (NCM3559 and NCM3560) were linked by P1vir-mediated transduction to the lac operon (see Materials and Methods). These strains grew rapidly on galactose and had high ß-galactosidase (LacZ) and galactoside transacetylase (LacA) activities when grown on galactose, glycerol, or glucose as the carbon source (Table 2, but note the differences between the strains). The proportion of such strains (15%) accounted nicely for the ß-galactosidase activity observed in a population of galactose fast-growers derived from MG1655 (CGSC 6300) (3000 x 0.15 = 450).
In addition, we tested 300 clones from a population of galactose fast-growers for a LacZ- phenotype on LB agar plus IPTG, the inducer of the lac operon, and X-Gal. Three were white, indicating that they lacked ß-galactosidase activity. The fact that none of the clones from a culture grown on glucose or glycerol as the carbon source was white indicated that this phenotype was associated with fast growth on galactose. The lesion in one such strain (NCM3556) was shown to be linked to the lac operon. The strain grew rapidly on galactose, and although it had no detectable ß-galactosidase activity, it had high levels of transacetylase (Table 2). Such a phenotype can be accounted for by a small nonpolar deletion encompassing lacO and the beginning of lacZ (49). Together, the results indicated that high levels of expression of lacY and/or lacA resulted in fast growth on galactose in the MG1655 background. As expected, a galactose fast-grower that resembled its parental strain on both types of plates containing X-Gal (NCM3558) had very low levels of ß-galactosidase and galactoside transacetylase activity when grown on galactose. Thus, not all galactose fast-growers had increased lac expression.
To determine the roles of individual lac genes in fast growth of MG1655 (CGSC 6300) on galactose, we employed strains with well-characterized mutations in lacZ, lacY, or lacA (insertion constructed for this work; see Materials and Methods). These strains and one carrying both the lacZ and lacA mutations were grown on galactose plus IPTG or glycerol plus IPTG to induce expression of the lac operon (Table 5). The MG1655 parental strain (not adapted to galactose) grew rapidly on galactose plus IPTG, in agreement with the view that expression of one or more lac genes was sufficient to confer this ability. Only the strain with the (polar) lacY mutation grew slowly, indicating that high activity of the lactose permease probably accounted for improved growth on galactose. Enzyme assays (not shown) verified the genotypes of the strains, and a control experiment indicated that all mutant strains had the same doubling time as their parent on glycerol plus IPTG (Table 5).
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To study induction further, we introduced defined nag mutations into MG1655 and determined their effect on its doubling time on galactose (Table 6). Every lesion known to result in derepression of the nag operons (45) (nagC, nagA, and nagB insertion [due to polarity on nagA], but not a point mutation in nagB or an insertion in nagD or nagE) resulted in fast growth on galactose. nagC codes for the Nag repressor, and the absence of NagA activity results in accumulation of the coinducer N-acetylglucosamine 6-phosphate (45). To determine whether high levels of nagE or nagD were required for rapid growth, we tested the effect of deletion of both nag operons (nagEBACD). To our surprise, this also resulted in fast growth on galactose (NCM3820), indicating that no product of the nag operons, including the transporter, was required.
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nagEBACD derivative (NCM3820) because this strain accumulates coinducer internally (Table 6) (45). Comparison of strain MG1655 (CGSC 6300) to E. coli K-12 strain NCM3722 on glass slide DNA microarrays. Given the difficulties with MG1655 isolates, we tested whether MG1655-based DNA microarrays could be used to probe gene expression in other E. coli strains. We first compared mRNA levels in an E. coli K-12 strain with no known mutational lesions (NCM3722; see Materials and Methods) to those in strain MG1655 (CGSC 6300) for cells grown to the mid-exponential phase in LB (Fig. 5). To be sure there would be at least one difference between the strains, we induced expression of the lactose operon in NCM3722 by adding IPTG (to 100 µM) for 25 min before harvest.
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2-fold-higher levels of mRNA for most of the genes in this regulon (mean and median differences between the strains of 4.2 and 2.7, respectively, for 50 genes). By contrast, mRNA levels for the galactitol operon gatYZABCDR2 (b2096 to b2090) were lower in NCM3722 than in MG1655. Information and data on these and other differences between the two strains are available at http://nature.berkeley.edu/~opaliy/papers/MG1655.html. In most instances, differences in mRNA levels between the K-12 strain and MG1655 were probably due to differences in gene expression (see Discussion for, e.g., the flagellar and chemotaxis regulon and the galactitol operon). However, in some cases the presence of an insertion element may prevent expression in one of the strains or a gene(s) may be deleted (see Discussion). Like MG1655 (CGSC 6300), the K-12 strain NCM3722 showed higher levels of expression of the maltose regulon when grown on lactose than glucose (not shown).
Comparison of strain MG1655 (CGSC 6300) to E. coli K-12 strain NCM3722 on Affymetrix chips. Comparison was made for cells grown on N-C- medium with glucose and NH4Cl as the C and N sources, respectively, for cells grown with glycerol and NH4Cl as the C and N sources, respectively, and for cells grown with glycerol and arginine as the C and N sources, respectively. In all cases, mRNA levels for genes of the flagellar and chemotaxis regulons were much higher for the K-12 strain than for MG1655. After averaging over the media, the mean and median differences were 54- and 30-fold, respectively, for the 50 genes indicated above. Affymetrix arrays confirmed lower levels of mRNA in the K-12 strain for the galactitol operon. Information and data on these and other differences between the strains are available at http://nature.berkeley.edu/~opaliy/papers/MG1655.html.
Comparison of strain MG1655 (CGSC 6300) to clinical isolates of E. coli on glass slide DNA microarrays. To determine whether use of the MG1655 arrays could be extended to E. coli strains isolated recently from humans and whether such strains would have physiological responses similar to those of laboratory strains, we worked with two urinary tract isolates and an intestinal commensal strain (see Materials and Methods). These had been identified as E. coli based solely on the fact that they were Lac+ on indicator plates and produced indole from tryptophan. We compared mRNA levels for cells grown on glutamine as the sole nitrogen source, a nitrogen-limiting condition, to those for cells grown on ammonium. Differences in gene expression under these two conditions were compared to those for strain MG1655 (CGSC 6300) (Fig. 6).
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2). These genes extended to 13 operons containing 37 genes (see legend to Fig. 6) (60). Sixteen additional genes not under NtrC/Nac control were also more highly expressed on glutamine in all strains (R/G ratio, 2). Two that are readily rationalized were the gene for a putative glutaminase (b0485) and that for the glutamine-dependent asparagine synthetase (b0674). Differences in expression of the gadBC operon (b1493 to b1492) were very striking, as were those for operons in the hdeAB region (b3504 to b3517), about which we have commented previously (60). Expression of 14 genes in 11 operons was decreased on glutamine in all four strains (R/G ratio, 
0.5). These operons code for enzymes of the tricarboxylic acid cycle, carbohydrate transporters, the F1-F0 ATPase, and ribosomal proteins. Decreases in their expression may reflect global sensing of nitrogen-limited slow growth. Further analysis of expression differences between cells grown on glutamine and ammonium and between the urinary tract isolates and MG1655 is available at http://nature.berkeley.edu/~opaliy/papers/MG1655.html. Ethanolamine utilization (eut) operon. As expected from the sequence of MG1655 (4, 29), which indicated that a cryptic prophage was inserted between two genes whose products are both required to initiate ethanolamine degradation, eutA (b2451) and eutB (b2441), CGSC 6300 failed to grow on ethanolamine as the nitrogen source with glycerol as the carbon source. This was also true of E. coli K-12 strain NCM3722, but both strains readily gave rise to Eut+ derivatives (see Materials and Methods). Comparison of strain NCM3722 to itself when grown on ethanolamine plus ammonium versus ammonium alone showed about twofold increases in expression of several eut genes upstream of the site of the cryptic prophage insertion in MG1655 (eutS-eutN [b2462 to b2456]) but not of genes downstream (Fig. 7, rows 3 and 4) (http://nature.berkeley.edu/~opaliy/papers/MG1655.html). (Cyanocobalamin was present in both cultures, because utilization of ethanolamine depends on vitamin B12, which is not synthesized aerobically [29].)
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By contrast to the above regulatory differences, differences in mRNA levels for the rfb genes and genes in the adjacent glf-wbbHIJK-yefJ operon (higher in MG1655) may be due to the presence of different rfb insertions and deletions in the two strains (34). We have not tested this possibility further. Finally, detection of the fnr deletion on microarrays was problematic for two reasons. First, detection depends on gene expression. Most of the genes in the fnr region other than fnr itself have not been well studied, and hence it is not known whether they are expressed under the growth conditions we have used. Lack of expression would affect the results on both glass slides and Affymetrix arrays (which did, however, detect differences between NCM3722 and CGSC 6300). Second, since 1999 our PCR products for glass slides have been made by a West Coast consortium (laboratories of N. Cozzarelli, C. Gross, and S. Kustu) from the CGSC isolate of MG1655 (with Sigma-Genosys primers), and we have noted that genes in the fnr region (b1332 to b1344) gave "failed PCR products." Preliminary inspection indicated that other failed PCR products also tended to occur in clusters and affect genes that coded for open reading frames. We do not know whether any of the other clusters correspond to deletions in CGSC 6300. Oligonucleotides for Affymetrix arrays were designed from the published sequence of MG1655 (Affymetrix, Inc., Santa Clara, Calif.) and hence would include the fnr region.
The cDNAs from two urinary tract isolates and an intestinal commensal strain of E. coli recently isolated from humans hybridized well to MG1655 microarrays. Moreover, the responses of these recent isolates to nitrogen limitation were very similar to those of MG1655 (Fig. 6). The results indicated that studies of the central metabolism and physiology of laboratory strains are likely to be pertinent to pathogenic and commensal strains, too, and that the responses of such strains can also be studied directly on MG1655 microarrays.
One of the growth defects of MG1655 (CGSC 6300) not apparent from its genotype is slow growth on galactose (Table 2). Apparent cross regulation of gene expression between galactose and lactose metabolism—i.e., induction of the lac operon during growth on galactose (59) (Table 3)—and apparent cross-regulation between galactose and N-acetylglucosamine were actually due to the appearance of fast-growing mutants. Some (15%) of these galactose-fast-growing mutants had high levels of lac expression (Table 2). Studies with MG1655 derivatives carrying defined lac mutations showed that lacY, which codes for the lactose permease, is the only lac gene required (Table 5). LacY is known to transport galactose (15, 22, 50), and presumably increased galactose transport accounts for the fast growth.
MG1655 derivatives that lacked NagC, the repressor of the N-acetylglucosamine catabolic operons, or had high levels of N-acetylglucosamine 6-phosphate, the coinducer, also grew fast on galactose (Table 6). One such strain was isolated as a fast-growing suppressor. Unlike the case for lactose, none of the products of the nag operons was required for fast growth on galactose, and hence it appears that the absence of NagC or the absence of NagC function per se is what is necessary. NagC is also an activator of the glmUS operon, whose products are needed for biosynthesis of N-acetylglucosamine as a component of the cell envelope (Fig. 4) (41, 46). Decreased biosynthesis may result in increased availability of the precursor of N-acetylglucosamine, fructose 6-phosphate, and hence in increased flux through central carbon metabolism (Fig. 4). This may account for fast growth on galactose. The only other known effect of the absence of NagC is slightly increased expression (30%) of the manXYZ operon (42), which codes for components of the phosphotransferase system that have a wide substrate specificity for transport of hexoses. We have not investigated a possible role for ManXYZ in transport of galactose (Fig. 4).
By contrast to the cases above, cross regulation of gene expression between lactose and maltose (Table 3) does not seem to have a trivial explanation (Table 4) and occurred not only in MG1655 (CGSC 6300) but also in the unrelated E. coli K-12 strain NCM3722. Indeed, it has been observed previously (1, 9, 51). It is probably accounted for by endogenous generation of inducing maltosaccharides from glucose and glucose 1-phosphate, both products of lactose catabolism (Fig. 4A). Low-level induction of the maltose regulon in lactose-grown cells may be programmed to occur in E. coli, or at least not selected against, because lactose and maltosaccharides are found together in the mammalian intestine.
Finally, studies of MG1655 in glycerol-limited continuous culture (Fig. 1) provided a dramatic example of the principle that enteric bacteria do not perceive two unrelated nutrient limitations simultaneously (25, 30). As the availability of glycerol was increased by increasing the dilution rate, the cells became internally limited for uracil (pyrimidines) due to their defect in pyrE function. This was reflected in a marked decrease in cell yield and the appearance of suppressor mutations. For work with MG1655 in continuous culture, it is advisable to repair the rph-1 lesion or to supplement the medium with uracil.
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ACKNOWLEDGMENTS |
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We thank W. Boos, G. M. Church, O. Danot, A. Manges, H. Nikaido, L. Riley, and C. Turnbough for strains or plasmids and V. Wendisch and D. Zimmer for help with the initial phases of this project.
This work was supported by Public Health Service grant GM36877 from the National Institute of General Medical Sciences to V.S. and by National Institutes of Health grant GM38361 and a grant from the Torrey Mesa Research Institute, Syngenta Research and Technology, San Diego, Calif., to S.K.
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FOOTNOTES |
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Present address: Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1018 WV Amsterdam, The Netherlands.
Present address: Health Services Research Enhancement Award Program, San Francisco Veterans Affairs Medical Center, San Francisco, CA 94121.
Present address: UC Irvine College of Medicine, Irvine, CA 92697-4089.
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