Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system.

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min(-1) mg of dry weight(-1). In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity.     

Chemotaxis inhibitory protein of Staphylococcus aureus binds specifically to the C5a and formylated peptide receptor

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is an exoprotein produced by several strains of S. aureus, and a potent inhibitor of neutrophil and monocyte chemotaxis toward C5a and formylated peptides like fMLP. These chemoattractants act on their target cells by binding and activating the C5aR and formylated peptide receptor (FPR), respectively. In the present report, we examined the mechanism by which CHIPS affects both of these receptors. We showed that CHIPS blocked binding of anti-C5aR mAb and formylated peptide to human neutrophils as efficiently at temperatures of 0 and 37 degrees C, implying that it is independent of signal transducing systems. This was confirmed by showing that CHIPS acts completely independently of ATP. Additionally, CHIPS was not internalized upon binding to neutrophils. Furthermore, we showed that CHIPS binds specifically to the C5aR and FPR expressed on U937 cells. This binding was functional in blocking C5a- and fMLP-induced calcium mobilization in these cell lines. These results suggest that CHIPS binds directly to the C5aR and FPR, thereby preventing the natural ligands from activating these receptors. The apparent K(d) values of CHIPS for the C5aR and FPR were 1.1 +/- 0.2 nM and 35.4 +/- 7.7 nM, respectively. Moreover, after screening a wide variety of other G protein-coupled receptors, CHIPS was found to affect exclusively the C5aR and FPR. This selectivity and high-affinity binding with potent antagonistic effects makes CHIPS a promising lead for the development of new anti-inflammatory compounds for diseases in which damage by neutrophils plays a key role.     

Understanding glucose transport by the bacterial phosphoenolpyruvate:glycose phosphotransferase system on the basis of kinetic measurements in vitro

The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed. Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor. Literature data on the K(m) values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously. No kinetic parameters were fitted. As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant. Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration. For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values. The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller.     

Distribution of Microcystins in a Lake Foodweb: No Evidence for Biomagnification

Microcystins, toxins produced by cyanobacteria, may play a role in fish kills, although their specific contribution remains unclear. A better understanding of the eco-toxicological effects of microcystins is hampered by a lack of analyses at different trophic levels in lake foodwebs. We present 3 years of monitoring data, and directly compare the transfer of microcystin in the foodweb starting with the uptake of (toxic) cyanobacteria by two different filter feeders: the cladoceran Daphnia galeata and the zebra mussel Dreissena polymorpha. Furthermore foodwebs are compared in years in which the colonial cyanobacterium Microcystis aeruginosa or the filamentous cyanobacterium Planktothrix agardhii dominated; there are implications in terms of the types and amount of microcystins produced and in the ingestion of cyanobacteria. Microcystin concentrations in the seston commonly reached levels where harmful effects on zooplankton are to be expected. Likewise, concentrations in zooplankton reached levels where intoxication of fish is likely. The food chain starting with Dreissena (consumed by roach and diving ducks) remained relatively free from microcystins. Liver damage, typical for exposure to microcystins, was observed in a large fraction of the populations of different fish species, although no relation with the amount of microcystin could be established. Microcystin levels were especially high in the livers of planktivorous fish, mainly smelt. This puts piscivorous birds at risk. We found no evidence for biomagnification of microcystins. Concentrations in filter feeders were always much below those in the seston, and yet vectorial transport to higher trophic levels took place. Concentrations of microcystin in smelt liver exceeded those in the diet of these fish, but it is incorrect to compare levels in a selected organ to those in a whole organism (zooplankton). The discussion focuses on the implications of detoxication and covalent binding of microcystin for the transfer of the toxin in the foodweb. It seems likely that microcystins are one, but not the sole, factor involved in fish kills during blooms of cyanobacteria.     

Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells

Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.