The gene coding for CelG, a family 9 cellulase from Clostridium cellulolyticum, was cloned and overexpressed in Escherichia coli. Four different forms of the protein were genetically engineered, purified, and studied: CelGL (the entire form of CelG), CelGcat1 (the catalytic domain of CelG alone), CelGcat2 (CelGcat1 plus 91 amino acids at the beginning of the cellulose binding domain [CBD]), and GST-CBD CelG (the CBD of CelG fused to glutathione S-transferase). The biochemical properties of CelG were compared with those of CelA, an endoglucanase from C. cellulolyticum which was previously studied. CelG, like CelA, was found to have an endo cutting mode of activity on carboxymethyl cellulose (CMC) but exhibited greater activity on crystalline substrates (bacterial microcrystalline cellulose and Avicel) than CelA. As observed with CelA, the presence of the nonhydrolytic miniscaffolding protein (miniCipC 1 ) enhanced the activity of CelG on phosphoric acid swollen cellulose (PASC), but to a lesser extent. The absence of the CBD led to the complete inactivation of the enzyme. The abilities of CelG and GST-CBD CelG to bind various substrates were also studied. Although the entire enzyme is able to bind to crystalline cellulose at a limited number of sites, the chimeric protein GST-CBD CelG does not bind to either of the tested substrates (Avicel and PASC). The lack of independence between the two domains and the weak binding to cellulose suggest that this CBD-like domain may play a special role and be either directly or indirectly involved in the catalytic reaction.

The bifunctional bacterial enzyme N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against ␤-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 Å, respectively. The S. pneumoniae GlmU molecule is organized in two separate domains connected via a long ␣-helical linker and associates as a trimer, with the 50-Å-long left-handed ␤-helix (L␤H) Cterminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the L␤H core and exchanged with the ␤-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent L␤H domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits.

Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases.

The small heat shock proteins (sHSP) are characterized by a chaperone activity to prevent irreversible protein denaturation. This study deals with the sHSP Lo18 induced by multiple stresses in Oenococcus oeni, a lactic acid bacterium. Using in situ immunocytochemistry and cellular fractionation experiments, we demonstrated the association of Lo18 with the membrane in O. oeni cells submitted to heat shock. The same result was obtained after exposure of cells to ethanol or benzyl alcohol, agents known to have an influence on membranes. For the different stresses, the protein was located on the periphery of the cell at membrane level and was also found within the cytoplasm. In order to determine if Lo18 could interact with the phospholipids, we used model membranes made of lipids extracted from O. oeni cells. Using fluorescence anisotropy of diphenylhexatriene (DPH) and generalized polarization of Laurdan, we showed that purified Lo18 interacts with these liposomes, and increases the molecular order of the lipid bilayer in these membranes when the temperature reaches 33.8 degrees C. All these data suggest that Lo18 could be involved in an adaptive response allowing the maintenance of membrane integrity during stress conditions in O. oeni cells.

To investigate if the primary function of the Agr system of Listeria monocytogenes is to monitor cell density, we followed Agr expression in batch cultures, in which the autoinducer concentration was uniform, and in biofilms. Expression was heterogeneous, suggesting that the primary function of Agr is not to monitor population density.Quorum sensing (QS) is the mechanism by which bacteria secrete signaling molecules called autoinducers that are sensed by neighboring cells in a population (30). The binding of these autoinducers to cognate receptors results in transcriptional regulation of gene expression. So far, for the species Listeria monocytogenes, one QS system, mediated by the agrBDCA operon, has been described (2, 7). Deletion of agrD or agrA results in impairment of major adaptive strategies, such as biofilm development (22, 23) and virulence (2, 21).Historically, the term QS was coined to illustrate that accumulation of autoinducers enables a coordinated control of gene expression resulting in a population-wide phenotype switch when the population reaches a threshold or quorum (6,8,18). However, recent reports indicate that adaptive functions of QS can be diverse and are not limited to population density sensing (20).For example, phenotypic heterogeneity of QS-regulated traits was reported in biofilms. Several subpopulations with distinct phenotypes organize Bacillus subtilis biofilms (13,14). Extracellular DNA release during the sessile growth of Enterococcus faecalis is directed by a fratricidal mechanism triggered by a quorum-responsive subpopulation (26). Heterogeneity was also observed in QS-regulated bioluminescence of Vibrio harveyi (1).Recent reports showed that confocal laser scanning microscopy (CLSM) associated with fluorescent reporter fusions may be used to trace the spatiotemporal expression of specific genes at a single-cell level within the overall biofilm structure (9, 12). When we traced Agr expression in biofilms, we detected green fluorescent protein (GFP) mainly in a network of elongated chains reminiscent of scaffoldings that surrounded densely populated microcolonies (22). This heterogeneous expression was surprising; indeed, maximum expression was expected within microcolonies, where the autoinducer concentration is maximum (19).Thus, the question of whether the function of this QS system was primarily to monitor population density arose. In order to test this hypothesis, a P agr -gfp fusion was integrated upstream of the agr locus of the L. monocytogenes EGD-e genetic background. This construct was designed to develop Agr expression reporters without affecting expression of the downstream agrBDCA operon (22).We followed GFP fluorescence by flow cytometry and microscopy during growth in batch homogenized liquid cultures, which represents environmental conditions prone to facilitate responses to cell density (confined cultures and no diffusion). Cells were collected by centrifugation (10 min at 8,000 ϫ g), washed, and diluted in 150 mM filtered NaCl solution before flow cytometry a...