ALBERT

Notes: Abstract: 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 activitiy. 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 α/β-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 β-ε-Ser-α motif. The active site is covered by a lid-like a-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. This movable lid-like α-helix explains at a molecular level the lipase-specific phenomenon of interfacial activation. At least some of the pathogenic bacterial species produce a lipase which has been studied with respect to its role as a virulence factor. Lipases of Propionibacterium acnes and Staphylococcus epidermidis may be involved in colonization and persistence of these bacteria on the human skin. Lipases of S. aureus and P. aeruginosa are produced during the bacterial infection process and, at least in vitro, considerably impair the function of different cell types involved in the human immune response like macrophages or platelets. The present state of knowledge suggests to classify the lipases as important bacterial virulence factors which exert their harmful effects in combination with other bacterial enzymes, in particular the phospholipases C. Most of the steadily increasing interest in bacterial lipases is based on their biotechnological applications which are partly based on their potential to catalyze not only hydrolysis but also synthesis of a variety of industrially valuable products. Optically active compounds, various esters and lactones are among the substances synthesized using bacterial lipases. Recently, an important application emerged with the addition of bacterial lipases to household detergents in order to reduce or even replace synthetic detergent chemicals which pose considerable environmental problems. As a main conclusion, [ipases represent an extremely versatile group of bacterial extracellular enzymes that are capable of performing a variety of important reactions, thereby presenting a fascinating field tot future research.

Notes: Abstract Using a broad host-range, mobilizable plasmid vector, the endoglucanase gene eglX from Pseudomonas fluorescens var. cellulosa was cloned and expressed in the bacterial ethanologen Zymomonas mobilis. The enzyme was intracellular in this new host. It was produced throughout the growth phase and was not repressed by glucose. We postulate that transcription of the eglX gene was initiated from a promoter of the vector in Z. mobilis.

Notes: Summary Several thousand mutagenized clones of Escherichia coli were screened for methyl group incorporation into protein in crude extracts, in order to isolate mutants lacking the full complement of methyl groups in ribosomal proteins. One mutant isolated by this method and designated prm-1 incorporated 6–7 methyl groups per ribosome upon incubation of its ribosomes with a partially purified enzyme preparation from E. coli wild-type. The methyl groups were located exclusively in the 50S particle and for the most part (85%) in protein L11. Three methylated amino acids were detected: ε-N-trimethyllysine, ε-N-monomethyllysine, and an uncharacterized amino acid. These accounted respectively for 4.6, 1.3 and 0.9 methyl groups per ribosome. These results indicate that protein L11 in wild-type contains a stoichiometric amount of these methylated amino acids which are absent in mutant prm-1. Since this mutant is fully viable, its methylation deficiency does not result in a major defect in ribosome assembly or functioning.

Notes: Summary Two genes governing ribosomal protein methylation have been located on the map of Escherichia coli by conjugation and transduction crosses between wild-type and prm (protein methylation) mutants. The Prm phenotype of recombinants was determined by an in vitro assay of methylgroups incorporation into protein. Gene prmA, governing methylation of protein L11 is situated at minute 71 on the map and is cotransduced with aroE (30%) and with rpsL (5%). Gene prmB, governing methylation of protein L3 is at minute 50, very close to aroC (98.5% co-transduction). A cold-sensitive phenotype was found associated with mutation prmB and was used to score a large number of recombinants in a three factor cross. The results of this cross suggest the order aroC-prmB-purF. The striking symmetrical clustering of aro, prm and rim (ribosome maturation) genes is discussed.

Notes: Summary Spontaneous mutants of Escherichia coli K12 displaying an increased level of the kanamycin resistance conferred by plasmid pGR71 were selected. Several mutants obtained in this way apparently carry large chromosomal deletions extending into galU and/or bglY (27 min). This positive selection of deletions allowed detection of a new locus located between galU and bglY. Deletions of this locus are responsible for increased resistance to kanamycin (Irk), decreased resistance to l-serine in minimal medium (Drs) and decreased resistance to chloramphenicol (Drc) when a cat gene is present in the bacteria.

Notes: Summary The ribosomes of an Escherichia coli mutant, designated prm-2, can be methylated in vitro by an enzymatic fraction from wild-type. This enzyme is inactive on the ribosomes from another mutant, prm-1, reported previously to be methyl group-defidient in protein L11. In vitro methylation of prm-2 ribosomes resulted in the incorporation of about one methyl group per molecule of protein L3. After acid hydrolysis, all the methyl groups were found in a very basic compound which was identified as methylamine. This compound could have been generated by acid hydrolysis of N-methylated amide-groups from glutamine or asparagine. Therefore, chemically-synthesized N4-methyl-asparagine and N5-methylglutamine were chromatographed together with an enzymatic hydrolysate of methylated prm-2 proteins. In all the chromatographic systems studied the methylated amino acid was found in the same position as N5-methylglutamine. These results indicated that mutant prm-2 lacks one residue of N5-methylglutamine present in ribosomal protein L3 of wild type E. coli.

Notes: Summary Haploid hybrids between Salmonella typhimurium Hfr and Escherichia coli Fexercise two additive types of restriction and modification (SA and SB) on phage γ. System SA had been detected previously in S. typhimurium with phage L. Independent mutants in the SA and SB systems were isolated. P22- and P1-mediated transductions in S. typhimurium and in hybrids established that the genes governing these systems are independent but linked and situated counter-clockwise of serB on the map, in the order: pyrB-hsd SA-hsd SB-serB.

Notes: Summary bglY mutants ofEscherichia coli K12 which show higher levels of kanamycin resistance (Kmr) in the presence of plasmid pGR71 have been previously described. In this work, we show that this increased resistance to an aminoglycoside antibiotic is not due either to low drug uptake or to alteration of its target, the ribosome. The copy number of plasmid pGR71 is not modified. The fact that increased antibiotic resistance is observed with only some of the Kmr determinants used in this study suggests a specific role for thebglY gene product. Moreover, for one such determinant, a higher level of resistance was observed when it was inserted in the chromosome but not when harbored by a plasmid. This discrepancy can be explained by the twin transcriptional-loop model, which proposes that transcription can lead to local variation in topology. Akan-lacZ fusion was constructed from the Kmr gene of plasmid pGR71 and inserted into a low copy number vector. Assay of β-galactosidase in wild-type and mutant strains showed that expression of the antibiotic resistance gene was directly affected by H1 protein, thebglY gene product.

Notes: Summary E. coli x S.typhimurium partially diploid hybrids were constructed to investigate the possibility of genetic complementation between the SA and the SB restriction and modification systems of S. typhimurium and the K and B systems of E. coli. An hsdR K - mutation was complemented in a stable hybrid in which the hsd SA + -hsd SB + -serB + portion of the S. typhimurium chromosome was integrated at a non-homologous locus. By isolating hsd - mutants in that hybrid, it was shown that complementation occured between K and SB, but not between K and SA. Similarly, in a set of F-prime merodiploids bearing the SA, SB and B systems, complementation was observed between B and SB, but not between B and SA.