Richard D'Ari

Escherichia coli Physiology in Luria-Bertani Broth

Luria-Bertani broth supports Escherichia coli growth to an optical density at 600 nm (OD(600)) of 7. Surprisingly, however, steady-state growth ceases at an OD(600) of 0.3, when the growth rate slows down and cell mass decreases. Growth stops for lack of a utilizable carbon source. The carbon sources for E. coli in Luria-Bertani broth are catabolizable amino acids, not sugars.

The leucine-Lrp regulon in E. coli : a global response in search of a raison d'être

Elaine B. Newman,’ Richard D’Ari,t and Rong Tuan Lin’ *Biology Department Concordia University Montreal, Quebec H3G lM8 Canada tlnstrtut Jacques Monod Centre National de la Recherche Scientifique Universite Paris 7 75251 Paris Cedex 05 France The concept of the regulon arose from studies showing that a single transcriptional regulator can control several distinct operons. The first regulons discovered were simi- lar to operons in that the member functions were involved in a specific physiological role, such as the uptake and utilization of maltose. The idea was generalized with the recognition of global responses, in which a number of func- tions are coordinately controlled in response to an environ- mental signal (e.g., temperature or pH). The factor mediat- ing transcriptional regulation in a global response can be a transcriptional repressor (LexA), a transcriptional activator (PhoB), a combined repressor-activator (Crp), an alterna- tive sigma factor (RpoH, NtrA), or an RNA polymerase effector (ppGpp). The sets of physiologically related functions that are grouped together in most global responses tend to reflect questions experimenters ask: How does the cell respond to starvation? How does the cell react to DNA damage? The leucine-Lrp regulon was discovered in aslightlydiffer- ent way. The leucine-responsive regulatory protein, Lrp, which governs expression of the leucine-Lrp regulon (Platko et al., 1990), was independently identified several times as a regulator of various members of the regulon (see table: Anderson et al., 1976; Andrews et al., 1986; Riccaetal., 1989; Linet al., 1990). Operonsofthe leucine- Lrp regulon can be controlled either positively or nega- tively, as in the Crp-CAMP regulon. Leucine is clearly a major effector of the regulon, in that it affects Lrp regulation in many, but not all, cases. However, we do not yet know what aspect of the environment leucine represents. This sets the leucine-Lrp regulon apart-a global response in search of a physiological rationale. The difference, how- ever, is probably more methodological than real. Lrp is a 19 kd DNA-binding protein, existing in solution as a dimer (Willins et al., 1991). Apart from its lack of tryptophan residues, Lrp has no unusual structural fea- tures compared with other DNA-binding proteins. The only E. coli DNA-binding protein with which it has sequence similarity is AsnC, activator of the asnA gene. In particular,

Quantitative evaluation of recA gene expression in Escherichia coli

SummaryA recA::lac operon fusion was constructed using the phage Mu d(Ap, lac) in Escherichia coli to obtain precise measurements of the level of recA gene expression in various genetic backgrounds. The RecA protein normally represents 0.02% of total protein. This value is known to increase dramatically after treatments interrupting DNA synthesis; kinetic experiments showed that the rate of recA expression increases 17-fold within 10 min after UV irradiation or thymine starvation. In mutants affected in SOS regulation or repair the following observations were made: (i) the tif-1 mutation in the recA gene does not alter the basal level of recA expression, suggesting that it improves the protease activity of RecA; (ii) the lexA3 mutation does not create a “super-repressor” of recA; (iii) the tsl-1 mutation in the lexA gene makes the LexA protein a poor repressor of recA at 30°C (2.5-fold derepression) and a poor substrate for RecA protease (3-fold stimulation of recA expression by UV); (iv) the spr-55 amber mutation in the lexA gene causes a 30-fold increase in recA expression, higher than all inducing treatments, and this level cannot be further increased by nalidixic acid; (v) the zab-53 mutation at the recA locus, known to abolish tsl-mediated induction of recA expression, is trans-recessive and thus probably affects a regulatory site on the DNA; (vi) uvrA, B and C, recB and recF mutations do not increase the basal level of recA expression, suggesting that there are not sufficient spontaneous lesions to cause induction even when any one of these three repair pathways is inoperative.

Unstable Escherichia coli L Forms Revisited: Growth Requires Peptidoglycan Synthesis

Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the beta-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the beta-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli-and possibly all L forms-have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.

Antagonistic regulation of Escherichia coli ribosomal RNA rrnB P1 promoter activity by GreA and DksA.

The Escherichia coli proteins DksA, GreA, and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription. DksA, with its co-factor ppGpp, inhibits rrnB P1 transcription initiation, whereas GreA and GreB activate RNAP to cleave back-tracked RNA during elongational pausing. Here, in vivo and in vitro evidence reveals antagonistic regulation of rrnB P1 transcription initiation by Gre factors (particularly GreA) and DksA; GreA activates and DksA inhibits. DksA inhibition is epistatic to GreA activation. Both modes of regulation are ppGpp-independent in vivo but DksA inhibition requires ppGpp in vitro. Kinetic experiments and studies of rrnB P1-RNA polymerase complexes suggest that GreA mediates conformational changes at an initiation step in the absence of NTP substrates, even before DksA acts. GreA effects on rrnB P1 open complex conformation reveal a new feature of GreA distinct from its general function in elongation. Our findings support the idea that a balance of the interactions between the three secondary channel-binding proteins and RNAP can provide a new mode for regulating transcription.

The SOS system.

In the bacterium Escherichia coli DNA damaging treatments such as ultraviolet or ionizing radiation induce a set of functions called collectively the SOS response, reviewed here. The regulation of the SOS response involves a repressor, the LexA protein, and an inducer, the RecA protein. After DNA damage an effector molecule is produced--possibly single stranded DNA--which activates the RecA protein to a form capable of catalysing proteolytic cleavage of LexA. The repressors of certain temperate prophages are cleaved under the same conditions, resulting in lysogenic induction. SOS functions are involved in DNA repair and mutagenesis, in cell division inhibition, in recovery of normal physiological conditions after the DNA damage is repaired, and possibly in cell death when DNA damage is too extensive. The SOS response also includes several chromosomal genes of unknown function, a number of plasmid encoded genes (bacteriocins, mutagenesis), and lysogenic induction of certain prophages. DNA damaging treatments seem to induce DNA repair and mutagenic activities and proviral development in many species, including mammalian cells. In general, substances which are genotoxic to higher eukaryotes induce the SOS response in bacteria. This correlation is the basis of the numerous bacterial tests for genotoxicity and carcinogenicity.

The leucine-responsive regulatory protein: more than a regulator?

The leucine-responsive regulatory protein (Lrp) is the regulator of the recently discovered leucine/Lrp regulon in Escherichia coli. Like other global regulators, it regulates the expression of 35 or more specific target operons. Studies of this global response have led to the suggestion that Lrp--and perhaps some other gene regulators--may also participate in the maintenance of chromosome structure and organization.

Escherichia coli metabolism in space

Cultures of the bacterium Escherichia coli were grown in the orbiting Biocosmos 2044 satellite in order to evaluate the effects of the space environment--weightlessness and heavy particle radiation--on growth parameters and energy metabolism, which have previously been reported to be affected, and on induction of the SOS response, which reflects DNA damage to the cell. We found no differences between the flight samples and control ground cultures in the growth yield per gram of carbon, in mean cell mass (from which we deduce that the growth rate was unaltered) or in the level of expression of the SOS response. These observations indicate that free-growing bacterial cells do not expend significant energy fighting gravity and that cosmic radiation within a space capsule does not produce significant levels of DNA damage.

The SOS chromotest: Direct assay of the expression of gene sfiA as a measure of genotoxicity of chemicals

We used a gene fusion, placing the lacZ gene encoding beta-galactosidase under the control of the sfiA promoter, to construct a new tester strain for genotoxic agents. The assay is performed in a few hours and involves simple enzymatic assays. The dose response curves contain a linear portion which enables to define the SOS Inducing Potency (SOSIP) of compounds. For the compounds tested SOSIPs extend over 7 decades and correlate generally well with the mutagenic potency assayed in the Salmonella/microsome assay (Mutatest) and in a phage induction assay (Inductest). Sensitivities (lowest amount detected) are comparable in the SOS Chromotest and Mutatest but lower in the Inductest. Our results suggest that at least part of the response in the Mutatest depends on the induction of an SOS function, and that most of the genotoxins are inducer of the SOS system -i.e. can lead to activation of the RecA protease.

Inducible sfi dependent division inhibition in Escherichia coli

SummaryBrief exposure of an Escherichia coli tif lon strain to 40° results in subsequent prolonged inhibition of cell division (part of the “SOS response”), which is completely and specifically suppressed by sfiA and sfiB mutations. This sfi dependent division inhibition requires protein synthesis during the 40° incubation period, implying the existence of a tif-inducible protein which results in cell division arrest. sfi dependent division inhibition is also induced early during thymine starvation in tif+ cells; at later times a sfi independent mechanism of division arrest is invoked as well.In lon mutants, known to lack a protease, the sfi dependent division inhibition is amplified, perhaps due to stabilization of the inducible protein involved in division arrest. In these strains the P1 lysogenization defect and the filamentation observed after a nutritional shift-up are sfi dependent, suggesting that P1 infection and nutritional shift-up may also induce the protein involved in division arrest. Bacteria are known to increase in size following a shift-up. Thus the latter observation suggests that the SOS response may be not only a last resort in time of distress but also a means permitting better adaptation of the cells to their environment.