In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.     

Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.     

Expression of Escherichia coli K-12 Arginine Genes in Pseudomonas fluorescens

Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 carrying the E. coli F110 plasmid.     

In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12.

Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene product by an enzyme or enzymes that reduce the protein to an active form.     

Catabolite repression in the D-serine deaminase system of Escherichia coli K-12

The induced synthesis of d-serine deaminase in Escherichia coli is subject to three catabolic effects: inhibition on inducer uptake, transient repression, and catabolite repression. Inhibition on d-serine uptake is not significant at the d-serine concentration normally used for induction. Transient repression and catabolite repression of d-serine deaminase synthesis are abolished by mutations in dsdCy, which appears to be an operator locus. The decline in the rate of constitutive synthesis observed in dsdCx mutants growing with glycerol as carbon source at temperatures above 37 C is due to catabolite repression. The low level of constitutivity at 37 C and the partial cis dominance of dsdCx mutants are not artifacts of catabolite repression. It is suggested that a product of one of the genes of the dsd operon may regulate the expression of the operon.     

Escherichia coli K-12 cell-cell interactions seen by time-lapse video.

The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.     

Reductive repression in Escherichia coli K-12 is mediated by oxygen radicals

Cyclic AMP (cAMP) content and the expression of cAMP-dependent phenotypes were positively correlated with respiration capacity in respiration-deficient mutants of Escherichia coli K-12 ("reductive repression," R. Hertz, and J. Bar-Tana, (1982) Arch. Biochem. Biophys. 213, 193-199). Reductive repression in respiration-deficient mutants could not be accounted for by respective changes in either the energy charge of adenine nucleotides or the redox state of pyridine nucleotides but could be ascribed to an increased formation of oxygen radicals under conditions of limited respiration. Scavengers of superoxide radicals eliminated reductive repression in respiration-deficient mutants with a concomitant increase in cAMP content. Such scavengers also effected a partial escape from permanent glucose catabolite repression, thus indicating a possible role played by oxygen radicals in both repression modes.     

Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction.

Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.     

Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.     

Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12.

We compare the nucleotide sequences of the regulatory regions of five genes or groups of genes of the arginine regulon of Escherichia coli K-12: argF, argI, argR, the bipolar argECBH operon and the carAB operon. All these regions harbour one or two copies of a conserved 18 bp sequence which appears to constitute the basic arginine operator sequence (ARG box). We discuss the influence of ARG box copy number, degree of dyad symmetry, base composition, and position relative to the cognate promoter site on the derepression-repression ratios of the genes of the regulon. A novel hypothesis, based on structural considerations, is also put forward to account for the absence ot attenuation control.     

Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12.

Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi. The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon. Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase.     

Reconstitution of binding protein-dependent ribose transport in spheroplasts of Escherichia coli K-12.

Purified Escherichia coli K-12 ribose binding protein was used to reconstitute the high affinity ribose transport system in spheroplasts derived from ribose-induced cells. It was not possible to reconstitute ribose transport in spheroplasts derived from uninduced cells or from transport-negative mutant strains, suggesting that one or more additional inducible components are required for binding protein-dependent ribose transport. It was possible to reconstitute transport in a ribokinase-deficient mutant which constitutively transports but does not utilize ribose.