Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression

Abstract

Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumarate-nitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.

FIG. 1.

MG1655 becomes internally limited for uracil in a glycerol-limited chemostat. Cell yield (OD₆₀₀; dotted lines) was plotted as a function of the dilution rate, which was varied from slowest (left) to fastest (right). At the dilution rates indicated on the line at the top, the medium in the reservoir was supplemented with uracil at the concentrations indicated. Symbols: black squares, unsupplemented; red circles, 20 mg/liter; green triangles, 60 mg/liter; blue crosses, 100 mg/liter. The directions of changes in OD₆₀₀ at fixed dilution rates are indicated by arrows. When uracil was removed from the medium in the reservoir at the end of the experiment, the OD₆₀₀ remained high (solid inverted triangle).

FIG. 2.

PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica), uspE (ydaA; UspA paralog) (17), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt (O-6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT (ydaH; p-aminobenzoyl-glutamate transport; oxidized folate recycling) (23), abgB (ydaI; p-aminobenzoyl-glutamate; oxidized folate recycling) (23), abgA (ydaJ; p-aminobenzoyl-glutamate; oxidized folate recycling) (23), abgR (ydaK; p-aminobenzoyl-glutamate regulator, LysR-type) (23), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

FIG. 3.

Appearance of different isolates of MG1655 on several media. MG1655 was obtained from the CGSC (CGSC 6300), the American Type Culture Collection (ATCC 47076), Mitchell Singer (from the laboratory of Carol Gross) (NCM3430), and the Blattner laboratory (NCM3629). Upon receipt, the isolate from the Blattner laboratory gave a mixture of large and small colonies on both LB and minimal glucose agar (see text). The properties of both are shown. Media were LB (A); N⁻C⁻ medium with glucose (0.4%) and NH₄Cl (10 mM) (B); and N⁻C⁻ medium with glycerol (0.2%) and NH₄Cl (10 mM) (C). Cells were streaked on LB before being transferred to the medium indicated and were incubated aerobically at 37°C for 1 to 2 days.

FIG. 4.

Diagram of the utilization of lactose, galactose, and N-acetylglucosamine (GlcNAc) as sole carbon sources and related pathways discussed in this work. The gene products indicated are LacY, lactose transporter; LacZ, β-galactosidase; GalP, low-affinity galactose transporter; MglABC, high-affinity galactose transporter; GalK, galactokinase; GalT, galactose 1-phosphate uridylyltransferase; GalE, UDP-galactose-4-epimerase; Pgm, phosphoglucomutase; Pgi, phosphoglucoisomerase; NagE, GlcNAc transporter; NagA, GlcNAc-6-phosphate deacetylase; NagB, GlcN-6-phosphate deaminase; ManXYZ, glucosamine transporter; GlmS, GlcN-6-phosphate synthase; GlmM, phosphoglucosamine mutase; GlmU, GlcN 1-phosphate acetylase; and UDP-GlcNAc synthase. As indicated by a bent arrow, LacY is known to transport galactose (15, 50), and elevated LacY expression apparently allows fast growth of MG1655 (CGSC 6300) on galactose as the carbon source (see text). Cells grown on lactose as the carbon source apparently synthesize inducers of the maltose regulon endogenously. Such synthesis from glucose and glucose 1-phosphate has been proposed previously (1, 9, 51), although the enzymes involved have not been defined. The product of gene ycjT does not appear to be solely responsible for endogenous synthesis of inducer (see text). NagC (not shown) regulates both the synthesis and degradation of GlcNAc (see Discussion). Inactivation of NagC allows fast growth of MG1655 on galactose, apparently by decreasing endogenous synthesis of GlcN 6-phosphate and thus increasing the flux of fructose 6-phosphate through central metabolism (see Discussion).

FIG. 5.

Genome image of a DNA microarray comparison of RNA levels in prototrophic E. coli K-12 strain NCM3722 and MG1655 (CGSC 6300). Strains were grown in LB medium, and expression of the lactose operon was induced in NCM3722 by addition of 100 μM IPTG 25 min before harvest. cDNAs from NCM3722 and MG1655 were labeled with Cy5 (red) or Cy3 (green), respectively, and spots in fluorescence scanning images obtained after hybridization were rearranged in genome order (57, 60). The b number centuries are indicated to the left, and decades are indicated on top. Blanks represent b numbers that do not correspond to open reading frames or no longer exist.

FIG. 6.

Aligned genome images of DNA microarrays of MG1655 (CGSC 6300) and prototrophic derivatives of clinical isolates of E. coli. The strains were MG1655 (CGSC 6300), rows 1 and 5; derivative of NCM3601 (a urinary tract isolate), row 2; derivative of NCM3610 (a urinary tract isolate), row 3; derivative of NCM3611 (an intestinal commensal isolate), row 4. They were grown in N⁻C⁻ medium with 0.4% glucose as the carbon source and 5 mM glutamine or 10 mM NH₄Cl as the nitrogen source. The cDNAs from cells grown with glutamine or NH₄Cl were labeled with Cy5 (red) or Cy3 (green), respectively. Operons thought to be in the extended NtrC/Nac regulon (60) that are more highly expressed on glutamine in all strains include glnK-amtB (b0450 to b0451), glnHPQ (b0809 to b0811), potFGHI (b0854 to b0857), ycdGHIJKLM (b1006 to b1012), ydcSTUVW (b1440 to b1444), ddpXABCDE (b1483 to b1488), yeaGH (b1783 to b1784), yedL (b1932), cbl (b1987), nac (b1988), argT (b2310), ygjG (b3073), and glnALG (b3868 to b3870). See text for discussion of other differences in expression. A different print was used for the experiment in row 5 than for the others.

FIG. 7.

Portions of genome images showing expression of the eut operon under two conditions. The borders of the eut operon are indicated (lines), along with the direction of transcription (arrow) and the site of insertion of the cryptic prophage in MG1655 (4). For the experiment in rows 1 and 2, NCM3954, a Eut⁺ derivative of E. coli K-12 strain NCM3722, was grown aerobically on N⁻C⁻ medium with 0.4% glycerol as the carbon source and 10 mM ethanolamine (Cy5 label) or NH₄Cl (Cy3 label) as the nitrogen source. Cultures were supplemented with cyanocobalamin (0.2 μM) in both cases. The images are from two prints present on the same glass slide. Rows 3 and 4, E. coli K-12 strain NCM3722 was grown aerobically on N⁻C⁻ medium with 5 mM ethanolamine plus 5 mM NH₄Cl (indocarbocyanine) or with 10 mM NH₄Cl alone (indodicarbocyanine). Cyanocobalamin (0.2 μM) was present in both cases. See text for discussion of individual genes.