Ole Michelsen

Excess capacity of H(+)-ATPase and inverse respiratory control in Escherichia coli.

With succinate as free‐energy source, Escherichia coli generating virtually all ATP by oxidative phosphorylation might be expected heavily to tax its ATP generating capacity. To examine this the H(+)‐ATPase (ATP synthase) was modulated over a 30‐fold range. Decreasing the amount of H(+)‐ATPase reduced the growth rate much less than proportionally; the H(+)‐ATPase controlled growth rate by < 10%. This lack of control reflected excess capacity: the rate of ATP synthesis per H(+)‐ATPase (the turnover number) increased by 60% when the number of enzymes was decreased by 40%. At 15% H(+)‐ATPase, the enzyme became limiting and its turnover was increased even further, due to an increased driving force caused by a reduction in the total flux through the enzymes. At smaller reductions of [H(+)‐ATPase] the total flux was not reduced, revealing a second cause for increased turnover number through increased membrane potential: respiration was increased, showing that in E.coli, respiration and ATP synthesis are, in part, inversely coupled. Indeed, growth yield per O2 decreased, suggesting significant leakage or slip at the high respiration rates and membrane potential found at low H(+)‐ATPase concentrations, and explaining that growth yield may be increased by activating the H(+)‐ATPase.

Bacillus subtilis strain deficient for the protein‐tyrosine kinase PtkA exhibits impaired DNA replication

Bacillus subtilis has recently come into the focus of research on bacterial protein‐tyrosine phosphorylation, with several proteins kinases, phosphatases and their substrates identified in this Gram‐positive model organism. B. subtilis protein‐tyrosine phosphorylation system PtkA/PtpZ was previously shown to regulate the phosphorylation state of UDP‐glucose dehydrogenases and single‐stranded DNA‐binding proteins. This promiscuity towards substrates is reminiscent of eukaryal kinases and has prompted us to investigate possible physiological effects of ptkA and ptpZ gene inactivations in this study. We were unable to identify any striking phenotypes related to control of UDP‐glucose dehydrogenases, natural competence and DNA lesion repair; however, a very strong phenotype of ΔptkA emerged with respect to DNA replication and cell cycle control, as revealed by flow cytometry and fluorescent microscopy. B. subtilis cells lacking the kinase PtkA accumulated extra chromosome equivalents, exhibited aberrant initiation mass for DNA replication and an unusually long D period.

Hemin Reconstitutes Proton Extrusion in an H+-ATPase-Negative Mutant of Lactococcus lactis

H(+)-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H(+)-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells are capable of coupling NADH oxidation to proton translocation.

The Extent to Which ATP Demand Controls the Glycolytic Flux Depends Strongly on the Organism and Conditions for Growth

Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells.

The transmembrane topology of the α subunit from the ATPase in Escherichia coli analyzed by PhoA protein fusions

The atpB gene encodes the α subunit of the H+‐ATPase of E. coli. The topology of this membrane protein has been analyzed by PhoA fusions. The results support an eight transmembrane segment model that is consistent with the hydropathic profile.

The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid.

Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.

Detection of Bacteriophage-Infected Cells of Lactococcus lactis by Using Flow Cytometry

ABSTRACT Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret this change as a consequence of a cease in cell growth, while the ongoing cell divisions leave the cells as single cells. Late in the infection cycle, cells with low-density cell walls appear, and these cells can be detected on cytograms of light scatter versus, for instance, fluorescence of stained DNA. We describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture. The method can be performed in real time and therefore increases the chance of successful intervention in the fermentation process.

Experimental determination of control by the H+-ATPase inEscherichia coli

Strains carrying deletions in theatp genes, encoding the H+-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of anatp deletion mutant was surprisingly high (some 75–80% of wild-type growth rate). The rate of glucose and oxygen consumption of these mutants was increased compared to the wild-type rates. In order to analyze the importance of the H+-ATPase at its physiological level, the cellular concentration of H+-ATPase was modulated around the wild-type level, using genetically manipulated strains. The control coefficient by the H+-ATPase with respect to growth rate and catabolic fluxes was measured. Control on growth rate was absent at the wild-type concentration of H+-ATPase, independent of whether the substrate for growth was glucose or succinate. Control by the H+-ATPase on the catabolic fluxes, including respiration, was negative at the wild-type H+-ATPase level. Moreover, the turnover number of the individual H+-ATPase enzymes increased as the H+-ATPase concentration was lowered. The negative control by the H+-ATPase on catabolism may thus be involved in a homeostatic control of ATP synthesis and, to some extent, explain the zero control by the H+-ATPase onE. coli growth rate.

Modulation of cellular energy state and DNA supercoiling in E. coli

Changes in DNA supercoiling have been shown to affect the expression of various genes in procaryotes. One of the factors that may control the level of DNA supercoiling in E. coli is the enzyme DNA gyrase, which utilizes ATP to introduce negative supercoils in topologically constrained DNA. Furthermore, the activity of DNA gyrase has been demonstrated to be sensitive to the ratio between [ATP] and [ADP] around the enzyme, in vitro 1. Consequently, cellular free energy metabolism may regulate transcription.

Uncoupler resistance in E. coli Tuv and Cuv is due to the exclusion of uncoupler by the outer membrane

The uncoupler resistant bacterial strains E. coli Tuv and Cuv share the high deoxycholate sensitivity of the parent strain, Doc S. However, both Tuv and Cuv show greater resistance than Doc S to other detergents. Measurement of the periplasmic volume indicates that the outer membrane of Doc S is freely permeable to both TPP+ and hydroxymethylinulin. Tuv and Cuv are able to exclude these compounds. EDTA treatment was necessary prior to measuring membrane potential in Tuv and Cuv. Under conditions where delta phi could be measured, uncouplers acted to dissipate delta phi with equal potency in all strains. Uncoupler resistant proline uptake in Tuv and Cuv was abolished by EDTA treatment. Transduction experiments with phage P1 showed that uncoupler resistance could be transferred from Tuv to Doc S. Such transductants were no longer sensitive to novabiocin. The gene for uncoupler resistance cotransduced with the gene pyrE (82 min). Plating efficiency experiments with P1 suggests that detergent sensitivity in Doc S arises from an rfa (81 min) mutation. This mutation is no longer present in Tuv.