Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the alpha/beta-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved beta-epsilon Ser-alpha motif. The active site is covered by a lid-like alpha-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

The extracellular lipase of Bacillus subtilis 168 was purified from the growth medium of an overproducing strain by ammonium sulfate precipitation followed by phenyl-Sepharose and hydroxyapatite column chromatography. The purified lipase had a strong tendency to aggregate. It exhibited a molecular mass of 19000 Da by SDS/PAGE and a p l of 9.9 by chromatofocusing. The enzyme showed maximum stability at pH 12 and maximum activity at pH 10. The lipase was active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C, acyl groups. Using trioleyl glycerol as substrate, the enzyme preferantially cleaved the 1 (3)-position ester bond. No interfacial activation effect was observed with triacetyl glycerol as substrate.Lipases are a class of enzymes which catalyse the hydrolysis of long-chain triacylglycerides. Microbial lipases are currently receiving much attention because of their potential use in industrial processes. In Bacillus subtilis, the presence of an extracellular lipase has been reported [I], but the enzyme has never been purified. Recently, Dartois et al. [2] have cloned and sequenced the lip gene responsible for the extracellular lipolytic activity in B. subtilis 168. They observed that, in contrast to other lipases, the B. subtilis enzyme lacks the conserved pentapeptide Gly-Xaa-Ser-Xaa-Gly reported to contain the nucleophilic serine residue essential for catalysis [3, 41. In B. subtilis, the first glycine residue of the pentapeptide is replaced by an alanine (residue 106) [2]. The same feature was reported more recently for the lipase of Bacillus pumilus [5] which shows 74% amino acid identity with the B. subtilis enzyme. In the present work, we have purified and performed a preliminary characterization of the B. szibtilis lipase, our final aim being to determine its threedimensional structure in the framework of an inter-laboratory collaboration.

The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. In this paper, we show that motile behavior was affected in Escherichia coli hns mutants. The loss of motility resulted from a complete lack of flagella. A decrease in the level of transcription of theflhD andfliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation. Furthermore, the Fla-phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex. These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway.In order to survive and develop in highly varied environments, microorganisms have to constantly monitor external conditions and respond to changes in pH, osmolarity, temperature, or chemicals. One of these adaptative behaviors causes motile bacteria to swim toward attractants (e.g., amino acids or carbohydrates) and away from repellents (e.g., alcohols and other toxic substances) in response to environmental cues.Bacterial motility is dependent on the presence of flagella. In Eschenichia coli, the biosynthesis of these multicomponent structures requires the expression of about 40 genes clustered at several regions on the chromosome. The transcription of the flagellar operons forms an ordered cascade in which the expression of genes located at a given level requires the transcription of another gene at an upper level. At the top of the hierarchy, the flhC and flhD genes constitute the master operon which controls the expression of all other flagellar genes (15,23).The expression of the master operon is sensitive to catabolite repression and is positively regulated by the cyclic AMPcatabolite activator protein (cAMP-CAP) complex (2, 4). Furthermore, the transcription of flagellar genes requires the DnaK, DnaJ, and GrpE heat shock proteins (29). The organization of the bacterial membrane is also known to affect swarming properties of E. coli. For example, flagellum formation is impaired in lipopolysaccharide (LPS)-deficient strains (17,26). Similarly, the pss mutation results in a drastic reduction in the membrane of phosphatidylethanolamine and in loss of flagellation (27) (20) and is under negative autoregulation in the exponential growth phase (7,8,30). In E. coli, mutations at the hns locus are highly pleiotropic and are known to affect the expression of several apparently unrelated genes (6, 10, 33).We have previously described the isolation of pleiotropic hns mutants of E. coli. These strains showed an increased resistance to kanamycin in the presence of plasmid pGR71 (5, 21). In contrast, an increased susceptibility to chloramphenicol was observed in the hns mutants, in spite of similar chloramphenicol acetyltransferase activities in wild-type and mutant strains (21). Th...

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A derepressed R factor, RYzdrdz, was transferred at a frequency of 5 x I O -~ between two strains of Yersinia enterocolitica mated on a membrane. Under the same conditions transfer of this R factor from Escherichia coli to Y. enterocolitica was observed at a frequency of only 7.7 x IO-~. This frequency was greatly increased when the recipient strain was heat-treated before mating. Heat exposure for optimum fertility was 50 to 52 "C for a period of 2 to 3 min. Mutants of Y. enterocolitica were isolated which were infected by RYzdrd;! from E. coli or from Y. enterocolitica at the same frequency. These observations strongly suggest that a DNA restriction and modification system in Y. enterocolitica causes its low recipient ability for plasmids from other species.