The role of magnesium and calcium ions in the glucose dehydrogenase activity of <i>Klebsiella pneumoniae</i> NCTC 418

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 μM CaCl2 in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl2 was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg2+ and Ca2+ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca2+ ions could replace Mg2+ ions. The high GDH activity which has been found in cells grown under Mg2−-limited conditions in the presence of higher concentrations of Ca2+ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Dissipation of Proton Motive Force is not Sufficient to Induce the Phage Shock Protein Response in Escherichia coli

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H+-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response

Quantitative Assessment of Oxygen Availability: Perceived Aerobiosis and Its Effect on Flux Distribution in the Respiratory Chain of Escherichia coli

Despite a large number of studies on the role of oxygen in cellular processes, there is no consensus as to how oxygen availability to the cell should be defined, let alone how it should be quantified. Here, a quantitative definition for oxygen availability (perceived aerobiosis) is presented; the definition is based on a calibration with reference to the minimal oxygen supply rate needed for fully oxidative catabolism (i.e., complete conversion of the energy source to CO(2) and water for glucose-limited conditions). This quantitative method is used to show how steady-state electron fluxes through the alternative cytochrome oxidases of Escherichia coli are distributed as a function of the extent of aerobiosis of glucose-limited chemostat cultures. At low oxygen availability the electron flux is mainly via the high-affinity cytochrome bd oxidase, and, at higher oxygen availability, a similar phenomenon occurs but now via the low-affinity cytochrome bo oxidase. The main finding is that the catabolic activities of E. coli (and specifically its respiratory activity) are affected by the actual oxygen availability per unit of biomass rather than by the residual dissolved oxygen concentration of the culture

Requirement of ArcA for Redox Regulation in Escherichia coli under Microaerobic but Not Anaerobic or Aerobic Conditions

In Escherichia coli, the two-component regulatory ArcAB system functions as a major control system for the regulation of expression of genes encoding enzymes involved in both aerobic and anaerobic catabolic pathways. Previously, we have described the physiological response of wild-type E. coli to changes in oxygen availability through the complete range from anaerobiosis to full aerobiosis (S. Alexeeva, B. de Kort, G. Sawers, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 182:4934-4940, 2000, and S. Alexeeva, K. J. Hellingwerf, and M. J. Teixeira de Mattos, J. Bacteriol. 184:1402-1406, 2002). Here, we address the question of the contribution of the ArcAB-dependent transcriptional regulation to this response. Wild-type E. coli and a mutant lacking the ArcA regulator were grown in glucose-limited chemostat cultures at controlled levels of oxygen availability ranging from full aerobiosis to complete anaerobiosis. A flux analysis of the distribution of catabolic fluxes over parallel pathways was carried out, and the intracellular redox state (as reflected by the NADH/NAD ratio) was monitored for all steady states. Deletion of ArcA neither significantly altered the in vivo activity of the pyruvate dehydrogenase complex and pyruvate formate lyase nor significantly affected catabolism under fully aerobic and fully anaerobic conditions. In contrast, profound effects of the absence of ArcA were seen under conditions of oxygen-restricted growth: increased respiration, an altered electron flux distribution over the cytochrome o- and d-terminal oxidases, and a significant change in the intracellular redox state were observed. Thus, the ArcA regulator was found to exert major control on flux distribution, and it is concluded that the ArcAB system should be considered a microaerobic redox regulator

Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r

The C and D cell cycle periods of seven Escherichia coli K-12 strains and three E. coli B/r strains were determined by computer simulation of DNA histograms obtained by flow cytometry of batch cultures grown at several different generation times. To obtain longer generation times two of the K-12 strains were cultivated at several different dilution rates in glucose-limited chemostats. The replication period (C period) was found to be similar in K-12 and B/r strains grown at similar generation times. At generation times below 60 min the C period was constant; above 60 min it increased linearly with increasing generation time. The period from termination of replication to cell division (D period) was more variable. It was much shorter in B/r than in K-12 strains. Like the C period it was relatively constant at generation times below 60 min and it increased with increasing generation times at longer generation times. In glucose-limited chemostats good correlation was found between D periods and generation times, whereas batch cultures exhibited carbon-source-dependent variations. Chemostat cultures showed cell cycle variations very similar to those obtained in batch cultures. These flow cytometric determinations of cell cycle periods confirm earlier determinations of the C period and establish that the D period also varies with generation time in slowly growing cultures. In addition they extend the range of growth rates at which cell cycle periods have been determined in E. coli K-12

Oxygen Relieves the CO₂ and Acetate Dependency of <em>Lactobacillus johnsonii</em> NCC 533

<div><p>Oxygen relieves the CO₂ and acetate dependency of <i>Lactobacillus johnsonii</i> NCC 533. The probiotic <i>Lactobacillus johnsonii</i> NCC 533 is relatively sensitive to oxidative stress; the presence of oxygen causes a lower biomass yield due to early growth stagnation. We show however that oxygen can also be beneficial to this organism as it relieves the requirement for acetate and CO₂ during growth. Both on agar- and liquid-media, anaerobic growth of <i>L. johnsonii</i> NCC 533 requires CO₂ supplementation of the gas phase. Switching off the CO₂ supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO₂ dependency. Analogously, <i>L. johnsonii</i> NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth medium for lactobacilli already suffices for normal growth. Analogous to the CO₂ requirement, oxygen supply relieves this acetate-dependency for growth. The <i>L. johnsonii</i> NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO₂ and acetyl-CoA producing systems. Therefore, C1- and C2- compound production is predicted to largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/C1-generation is corroborated by the observation that in a POX deficient mutant of <i>L. johnsonii</i> NCC 533, oxygen is not able to overcome acetate dependency nor does it relieve the CO₂ dependency.</p> </div

Current topics in signal transduction in bacteria

Among the signal transfer systems in bacteria two types predominate: two-component regulatory systems and quorum sensing systems. Both types of system can mediate signal transfer across the bacterial cell envelope; however, the signalling molecule typically is not taken up into the cells in the former type of system, whereas it usually is in the latter. The Two-component systems include the recently described (eukaryotic) phosphorelay systems; quorum sensing systems can be based upon autoinducers of the N-acylated homoserine lactones, and on autoinducers of a peptidic nature. A single bacterial cell contains many signalling modules that primarily operate in parallel. This may give rise to neural-network behaviour. Recently, however, for both types of basic signal transfer modules, it has been demonstrated that they also can be organised in series (i.e. in a hierarchical order). Besides their hierarchical position in the signal transduction network of the cell, the spatial distribution of individual signalling modules may also be an important factor in their efficiency in signal transfer. Many challenges lie hidden in future work to understand these signal transfer processes in more detail. These are discussed here, with emphasis on the mutual interactions between different signal transfer processes. Successful contributions to this work will require rigorous mathematical modelling of the performance of signal transduction components, and -networks, as well as studies on light-sensing signal transduction systems, because of the unsurpassed time resolution obtainable in those latter systems, the opportunity to apply repeated reproducible stimuli, etc. The increased understanding of bacterial behaviour that already has resulted--and may further result--from these studies, can be used to fine-tune the beneficial activities of bacteria and/or more efficiently inhibit their deleterious ones

Effect of acetate depletion on aerobic and anaerobic growth.

<p>Growth of <i>L. johnsonii</i> NCC 533 in a chemically defined medium with 12 mM Na-acetate (square symbols) and without 12 mM Na-acetate (round symbols) in stirred pH controlled cultures sparged with N₂+5% CO₂ (closed symbols) or N₂+20% O₂+5% CO₂ (open symbols) at a rate of 500 ml/min. Data are average of independent triplicate experiments ± standard deviation.</p

Aerobic CO₂ requirement of a NCC 9333 mutant.

<p>Growth of the NCC 533 (closed symbols) and NCC 9333 (open symbols) as measured at OD₆₀₀ in stirred batch cultures sparged with N₂+20% O₂+5% CO₂. The gas regime was switched after 3 hours of exponential growth to N₂+20% O₂. Data are the average of quadruple independent experiments ± standard deviation.</p