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 (fumaratenitrate 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.

Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA

“…First, our E ␣ p ϭ 0.5% measured in glucose (Table 3, entry 5) is lower than previously measured ␣ p values in glucose (Table 3, ␣ p ϭ 1-1.4%, entries 3,4,7,8). Because E expression varies with growth rate (21,23,24), the discrepancy in ␣ p values may be due to a much slower doubling time of MG1655 (133 min), a partial purine auxotroph (32), than the other strains (40-79 min). Second, slower-growing cells have a 1.4-fold increase in the ratio of 70 to E (Tables 1 and 4), which might alter competition.…”

“…Quantifying Intracellular Levels of E , 32 , 70 , and E. Our data for the levels of 70 , E , 32 , and E in cells growing exponentially in M9 glucose (M9 minimal) and M9 glucose supplemented with amino acids (M9 complete) are summarized in Table 1. Determinations of cell number and total protein mass per cell, taken at the same OD 450 as sampling for intracellular protein concentrations ( Table 2, which is published as supporting information on the PNAS web site), were used to convert our measurements to molecules per cell and fmol͞ g total cellular protein (Table 1).…”

“…His-tagged 70 , E , 32 , and the periplasmic domain of RseA, as well as E and GST-tagged E , were overproduced and purified as previously described (36)(37)(38)(39)(40) (43). Cells were grown at 30°C in M9 glucose medium, prepared as described (44) and supplemented with 0.2% glucose, 1 mM MgSO 4 , and 2 g͞ml thiamine (M9 minimal) or in M9 glucose plus amino acids (M9 complete), which contained the previous components and all amino acids (40 g͞ml).…”

“…In fact, discrepancies in these numbers have led to the common perceptions that (i) as most RNAP is elongating, only a minor fraction could be bound to DNA nonspecifically (27,28), and (ii) cellular levels of 70 are much less than that of RNAP (29); thus, s may compete because of their excess over free RNAP. We have remeasured the levels of E, 70 , 32 , and E in E. coli K12 MG1655. Analysis of our data, which are consistent with most of the primary data in the literature, suggests that in vivo (i) only a minor fraction of RNAP (Ͻ20%) is involved in elongation and (ii) 70 is in excess of total E. Using an equilibrium model based on our new values and relevant data from the literature, we explored the possible partitioning of E and E between promoters, nonspecific DNA sites, and the cytoplasm.…”

Bacillus subtilis, the model Gram‐positive bacterium: 20 years of annotation refinement

“…The genome was therefore entirely re‐sequenced using NGS methods ten years later (Barbe et al ., 2009). It can now be expected that, barring the inevitable mutations that appear during propagation in laboratories [see the situation for E. coli (Soupene et al ., 2003)], this final sequence corresponds to an exact sequence [International Nucleotide Sequence Database Collaboration (INSDC) AccNum AL009126.2], that does not need to be re‐sequenced. In contrast, sequence annotations inevitably keep changing as the identification of gene function improves almost on a daily basis.…”