Danièle Joseleau-Petit

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.

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.

ppGpp Concentration, growth without PBP2 activity, and growth-rate control in Escherichia coli

Escherichia coli strains partially induced for the stringent response are resistant to mecillinam, a β‐lactam antibiotic which specifically inactivates penicillin‐binding protein 2, the key enzyme determining cell shape. We present evidence that mecillinam resistance occurs whenever the intracellular concentration of the nucleotide ppGpp (guanosine 3’‐diphosphate 5’‐diphosphate), the effector of the stringent response, exceeds a threshold level. First, the ppGpp concentration was higher in a mecillinam‐resistant mutant than in closely related sensitive strains. Second, the ppGpp pool was controlled by means of a plasmid carrying a ptac‐relA′ gene coding for a hyperactive (p)ppGpp synthetase, ReiA′; increasing the ppGpp pool by varying the concentration of lac operon inducer IPTG resulted in a sharp threshold ppGpp concentration, above which cells were mecillinam resistant. Third, the ppGpp pool was increased by using poor media; again, at the lowest growth rate studied, the cells were mecillinam resistant, in all experiments, cells with a ppGpp concentration above 140pmoles/A600 were mecillinam resistant whereas those with lower concentrations were sensitive. We discuss a possible role for ppGpp as transcriptional activator of cell division genes whose products seem to become limiting in the presence of mecillinam, when cells form large spheres. We confirmed the well‐known inverse correlation between growth rate and ppGpp concentration but, surprisingly, for a given growth rate, the ppGpp concentration was lower in poor medium than in richer medium in which RelA is induced. We conclude that, for E. coli growing in poor media, the concentration of the nucleotide ppGpp is not the major growth rate determinant.

Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli

Escherichia coli loses its rod shape by inactivation of PBP2 (penicillin‐binding protein 2), target of the β‐lactam mecillinam. Under these conditions, cell division is blocked in rich medium. Division in the absence of PBP2 activity is restored (and resistance to mecillinam is conferred) when the three cell division proteins FtsQ, FtsA and FtsZ are overproduced, but not when only one or two of them are overproduced. Division in the absence of PBP2 activity is also restored by a doubling in the ppGpp pool, as in the argS201 mutant. However, the nucleotide ppGpp, a transcriptional regulator of many operons, does not govern any of the five promoters of the ftsQAZ operon, as shown by S1 mapping of ftsQAZ mRNA 5′ ends in exponentially growing wild‐type cells in the mecillinam‐resistant argS201 mutant (intermediate ppGpp level) or during the stringent response elicited by isoleucine starvation (high ppGpp level). Furthermore, the concentration of FtsZ protein is not increased in exponentially growing mecillinam‐resistant argS201 cells. These results show that the ftsQAZ operon is not the ppGpp target responsible for mecillinam resistance. We are currently trying to identify those targets that, at intermediate ppGpp levels, allow cells to divide as spheres in the absence of PBP2.

Global Stress Response in a Prokaryotic Model of DJ-1-Associated Parkinsonism

YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet-undefined function in the oxidative-stress response. YajL protects cells against oxidative-stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. Compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA repair proteins, RNA chaperones, and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative-stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.

The GemA protein of phage Mu and the GyrB gyrase subunitof Escherichia coli: The search for targets and interactions leading tothe reversion of Mu-induced mutations

The mutant bacteriophage Mugem2(Ts), known to synchronize the division of infected cells, to relax DNA supercoiling and, as prophage, to give rise to precisely excised revertants, has been thought to overexpress the gemA-mor operon, and genetic evidence suggests that the B subunit of DNA gyrase (GyrB) is the target of action of GemA. In two different double hybrid tests presented here, we find no evidence of GemA-GyrB protein-protein interaction. We do observe a GemA-GemA interaction, however, indicating that GemA can dimerize. In lacZ::Mu lysogens, overexpression of the gemA-mor operon from a plasmid, under control of the L-arabinose inducible p(araBAD) promoter, does not permit the recovery of Lac(+) revertants. These observations suggest that GyrB is not the direct target of GemA action and that the various phenotypes of Mugem2(Ts) are not caused by overexpression of the gemA-mor operon.

Does PBP2 Regulate Cell Division in E. coli

Although penicillin has not made war humane, it probably saved over a million lives during World War II. The action of penicillin and related β-lactam antibiotics was early recognized to be extrmely broad, affecting the vast majority of bacterial species, and at the same time highly specific, generally producing little effect on eukaryotic cells. As the complex structure of the bacterial cell wall became known, the mechanism of action of β-lactams was also revealed: they bind covalently to the PBPs (‘penicillin binding proteins’), a set of integral membrane proteins which catalyse the terminal steps in the synthesis of the rigid peptoglycan wall (for review see Waxman and Strominger, 1983; Ghuysen, 1991).

Le cycle cellulaire d'Escherichia coli

Resume Cette revue fait le point de nos connaissances actuelles a propos du cycle cellulaire des bacteries et d Escherichia coli en particulier. Nous traitons de trois types dapproche pour letude de la croissance et la division cellulaire: 1) La recherche devenements discrets qui se produisent une fois par cycle. Linitiation et la terminaison de la replication du DNA comme la septation cellulaire sont de tels evenements indiscutes aujourdhui. Il nen est pas de meme pour les doublements brusques de la vitesse dextension de la surface cellulaire ou de la biosynthese de la mureine, des proteines ou des phospholipides membranaires. Nous discutons les relations dans le temps entre les phenomenes cycliques reperes. 2) La recherche de sites de croissance localises dans le plan des enveloppes. Lextension localisee de la mureine est controversee aujourdhui. Linsertion de proteines a des sites privilegies de la membrane externe ou de la membrane interne est egalement discutee. 3) Lelucidation des mecanismes de regulation de linitiation de replication du DNA. Nous faisons le point sur le concept de « masse critique dinitiation . Nous passons en revue les donnees en faveur de lattachement du DNA aux enveloppes et du role de ces dernieres dans le declenchement de linitiation de replication.

Precise excision of bacteriophage Mu DNA

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.