In vivo effects of pyrophosphate in Escherichia coli

Abstract Pyrophosphate (PP_i), a noncompetitive inhibitor of Rho poly(C)-dependent ATPase activity in vitro has been shown to relieve polarity in the lac operon. This indicates that PP_i could inhibit Rho activity in vivo too. An additional effect of PP_i on adenosine 3',5'-cyclic monophosphate (cAMP) synthesis during stationary phase of growth is also described.     

In vivo regulation of chromosomal beta-lactamase in Escherichia coli.

Chromosomal beta-lactamase, a periplasmic enzyme of Escherichia coli, was studied with respect to its regulation in vivo. Both the activity and the amount of beta-lactamase increased with growth rate. During a nutritional shift-down, chromosomal beta-lactamase activity followed stable ribonucleic acid accumulation. After a nutritional shift-up the differential rate of beta-lactamase synthesis did not increase immediately (like stable ribonucleic acid), but did increase after a lag period of 30 min. To determine whether beta-lactamase was under stringent control, strains carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and differing only in the allelic state of the relA gene were shifted from a permissive to a semipermissive temperature. No influence by the relA gene product was found on beta-lactamase synthesis. The regulation of this periplasmic enzyme is discussed in relation to that of some components of the translational apparatus.     

In vivo distribution of phosphothioredoxin and thioredoxin in Escherichia coli.

The phosphorylation state of thioredoxin was compared in intact cells and in crude extracts. In crude extracts, the extent of phosphorylation was 0.70 to 0.80 mol of phosphate per mol of thioredoxin, with approximately equal amounts of thioredoxin phosphorylated either on cysteinyl32 (formula: see text) or on cysteinyl35 (formula: see text). By comparison, the extent of thioredoxin phosphorylation in intact cells was nearly 1.0 with phosphate present almost exclusively on cysteine32. Nonphosphorylated thioredoxin was present as the reduced thiol form (formula: see text). These findings imply that (formula: see text) is the relevant in vivo species and that a mechanism is operative in crude extracts for transfer of phosphate from cysteine32 to cysteine35.     

In vivo regulation of the Escherichia coli araC promoter.

The ara pC promoter is known to be derepressed about fivefold for 20 to 30 min after the addition of arabinose. This transient derepression was studied by using araC::Mu lac insertions and araC-lacZ gene fusions. In strains containing increased levels of araC protein, the pC promoter became progressively less derepressible, but the ara pBAD promoter remained normally inducible. Repression of pC was reestablished 20 min after induction in araB mutants, but did not occur in arabinose-transport-deficient mutants. Finally, mutant araCc proteins which normally do not repress pC did so in the presence of arabinose.     

Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.     

In vivo studies of repair of 2-aminopurine in Escherichia coli.

The repair of the base analog 2-aminopurine has been studied in vivo by using a temperature-sensitive mutant of the cloned mutH gene of Escherichia coli. Our results suggest that the lethal event in killing of dam mutants by 2-aminopurine does not result simply from incorporation of 2-aminopurine into the DNA and its subsequent repair. Furthermore, a 10-fold increase in the level of 2-aminopurine incorporated into the DNA of a dam mutH double mutant has little effect on the mutation frequency of this strain. An alternative mechanism for the mutagenicity of 2-aminopurine in E. coli is proposed.     

In vivo methylation of elongation factor Tu of Escherichia coli.

A protein existing mainly in the supernatant fraction of Escherichia coli was found to be methylated by accepting the methyl moiety originating from methionine. The protein was identified as peptide synthesis elongation factor Tu (EF-Tu) by the following criteria. 1) The methylatable protein separated at the same position as purified EF-Tu on two-dimensional gel electrophoresis. 2) The methylatable protein interacted with antiserum specific for EF-Tu. Amino acid analysis of the methyl-labeled protein suggested that the site of methylation was an epsilon-amino group of lysine.     

In vivo microscopic assessment of cremasteric microcirculation during hindlimb allograft rejection in rats.

Experimental and clinical studies of vascular allogenic extremity transplantation have yielded disappointing results and have not been clinically useful. With recent advances in transplantation immunology, considerable interest has focused on the understanding of leukocyte-endothelial interaction at the microcirculatory level. The objective of this study was to characterize the alterations in leukocyte-endothelial interaction in the early stages of rat hindlimb allograft rejection. To study the changes at the microcirculatory level, a new microsurgical model was developed; the cremaster muscle was incorporated into the transplanted hindlimb. The purpose of this study was to report on the microcirculatory changes during rat hindlimb allograft rejection. A total of 24 transplantations were performed among the four experimental groups. In a control group, 12 rat hindlimb-cremaster grafts were transplanted between genetically identical animals, Lewis to Lewis. Microcirculatory measurements of graft survival were taken at 24 hours (group 1A, n = 6) and at 72 hours (group 1B, n = 6). In the rejection control group, 12 transplantations were performed across a major histocompatibility barrier between Lewis-Brown Norway and Lewis rats. Microcirculatory measurements were taken at 24 (group 2A, n = 6) and 72 hours (group 2A, n = 6) as above. The following parameters were evaluated to discover the leukocyte-endothelial interaction: endothelial edema index and the number of rolling, adherent, and transmigrating leukocytes and lymphocytes in the postcapillary venule. Physical signs of limb rejection, such as edema, erythema, scaling, plaque formation on the skin, hair loss, and skin surface temperature, were monitored. Microcirculatory signs of rejection included the following. There was a significant increase in the number of adherent leukocytes in allograft transplants at both 24 hours (205 percent; 2.05 +/- 0.38) and 72 hours (431 percent; 9.11 +/- 3.41) when compared with isograft controls (1.00 +/- 0.89 at 24 hours; 2.11 +/- 0.34 at 72 hours) (p < 0.05). The activation of leukocyte transmigration increased more than 7-fold in muscle allografts at 24 hours (0.55 +/- 0.25 versus 4.16 +/- 1.89) and more than 6-fold at 72 hours (0.72 +/- 0.38 versus 4.38 +/- 1.28) after transplantation (p < 0.05). Endothelial edema index, a measure of endothelial swelling and cellular deposit accumulation, increased more than 119 percent in the allograft group 72 hours after transplantation (1.23 +/- 0.07 versus 1.46 +/- 0.09) (p < 0.05). The first clinical signs of limb rejection were scaling of the skin or hair loss; they were observed between the seventh and ninth postoperative days. The composite rat hindlimb-cremaster model presented in this study introduces a new in vivo approach to monitor acute graft rejection using the intravital microscopy system. This is a valuable model for defining the timing, sequence, and correlation between immunologic events and clinical signs during the acute phase of allograft rejection.     

In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function

We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes-those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.     

In vivo and in vitro phosphorylation of DNA-binding proteins from Escherichia coli.

A deoxyribonucleoprotein (DNP) complex has been isolated from Escherichia coli cells by chromatography on Sephadex G-200. The DNP complex contains phosphoproteins and the content of phosphorus bound to the DNP protein is 3 times higher than in cytoplasmic proteins not bound to DNA. These results have been confirmed by in vivo (32-P-KH2PO4) and in vitro (32-P-ATP) phosphorylation of E. coli DNA-binding proteins isolated by chromatography on DNA--cellulose.     

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.     

In situ development of sublethal stress in Escherichia coli: effects on enumeration.

Development of sublethal stress in Escherichia coli exposed in situ to estuarine waters was examined during various seasons. An electrochemical detection technique was utilized to derive a stress index based upon the difference between a predicted electrochemical response time in Trypticase soy broth or EC medium at 44.5 degrees C estimated from a standard curve for unstressed cells and an observed response time for cells exposed to seawater. This stress index was related to recovery efficiencies of seawater-exposed cells, using a variety of standard and resuscitative enumeration procedures. Stress was further studied by determination of the adenylate energy charge. Sublethal stress as measured by the electrochemical detection method was an inverse function of water temperature, with maximum stress occurring after exposure to temperatures below 10 degrees C. Total adenylates and ATP decreased dramatically at low temperatures, although energy charge remained relatively constant under various environmental conditions. Decreases in E. coli ATP suggest that ATP may not be an adequate measure of biomass for in situ stressed cells. Discrepancies in enumeration efficiency were most pronounced at temperatures below 10 degrees C. Resuscitative procedures for solid-media techniques increased the recovery of stressed cells under cold water conditions but were not as effective as the standard most-probable-number procedure.     

In vivo analysis of integration of membrane proteins in Escherichia coli

The in vivo process of membrane protein integration was studied by pulse-labelling Escherichia coli cells, and assessing integral anchoring of labelled proteins to the lipid bilayer based on their resistance to alkali extraction. To conduct this experiment, conditions for extracting E. coli proteins with alkali were refined, and the immunoprecipitation procedures were improved to allow effective detection of integral membrane proteins. Examination of pulse-labelled, integral membrane proteins, including lactose permease (LacY), SecY, cytochrome omicron subunit II and leader peptidase revealed that all were in the alkali-insoluble fraction, indicating that membrane integration of these proteins takes place rapidly in wild-type cells. However, when LacY was synthesized in excess from a multicopy plasmid, significant proportions were found in the alkali-soluble fraction, indicating that the solubility in alkali is not an intrinsic property of the protein, and suggesting that LacY depends on some limited cellular factor for membrane integration. The unintegrated species of LacY sedimented slowly through an alkaline sucrose gradient. The secY24 mutant cells accumulated higher proportions of unintegrated LacY molecules at lower levels of overproduction than the sec+ cells. LacY overproduction in wild-type cells was found to inhibit processing (export) of beta-lactamase but not of OmpA and OmpF. These results are interpreted to mean that integration of LacY depends on multiple cellular components, one of which is also involved in export of beta-lactamase.     

In vivo transfer of the ti-plasmid of Agrobacterium tumefaciens to Escherichia coli.

Summary The Ti-plasmids are naturally self-transmissible from their normal host Agrobacterium to E. coli. They are however unable to stably establish themselves as a replicon in E. coli. It is nevertheless possible to study the Ti-plasmids in E. coli with the help of Ti::RP4 cointegrate plasmids that transfer and maintain themselves very efficiently in E. coli. An E. coli harbouring such a Ti::RP4 plasmid is unable to catabolize octopine and unable to induce crowngall tumours on plants.This work was supported by grants from the Kankerfonds van de A.S.L.K. and from the Fonds voor Kollektief Fundamenteel Onderzoek (n⁰ 10.316) to J.S. and M.V.M.     

In vivo incorporation of an alkyne into proteins in Escherichia coli

Using a genetic selection we identified mutants of the M. janaschii tyrosyl-tRNA synthetase that selectively charge an amber suppressor tRNA with para-propargyloxyphenylalanine in Escherichia coli. These evolved tRNA-synthetase pairs were used to site-specifically incorporate an alkynyl group into a protein, which was subsequently conjugated with fluorescent dyes by a [3+2]-cycloaddition reaction under mild reaction conditions.