Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.     

Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014.

Summary The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or DNA fragments in vectors derived from p353-2 or p8014-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be efficiently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.This paper is dedicated with great appreciation to Dr. Frits Berends on the occasion of his retirement as Head of the Biochemistry Department of the TNO Medical Biological Laboratory     

A mutant of Escherichia coli K12 deficient in the 5''-3'' exonucleolytic activity of DNA polymerase I. II. Purification and properties of the mutant enzyme

Summary The enzymatic properties of purified DNA polymerase I from a strain of Escherichia coli K12 with a mutation in the polA gene have been studied. The polymerizing activity of the mutant enzyme is similar to that of the enzyme from isogenic wild-type cells, when the activity is measured on exonuclease III treated calf-thymus DNA. Also the 3–5 exonucleolytic activity is not significantly different for both enzyme preparations. The 5–3 exonucleolytic activity of DNA polymerase I isolated from the mutant strain, however, is much lower than that of wild-type DNA polymerase I. The products formed by the action of the wild-type and the mutant enzyme on nicked circular double-stranded DNA of phage X174 (RFII DNA) were analysed by sucrose-gradient sedimentation and electron-microscopy. When RFII DNA was incubated with wild-type enzyme 80% of the molecules were converted into linear molecules. All linear molecules were shorter than one phage genome. Only 25% of the molecules were branched. After incubation of RFII DNA with the mutant enzyme 62% of the molecules have become linear. More than 90% of these linear molecules were branched and the majority of them was longer than one phage genome.     

Degradation of lignin and lignin related compounds by protoplasts isolated from Fomes annosus

Protoplasts from a lignolytic fungus Fomes annosus were prepared through enzymatic hydrolysis of mycelium utilizing Novozym, a wall lytic enzyme preparation. Isolated protoplasts and living mycelium were compared in their ability to degrade ¹⁴C-labelled lignin related phenols and dehydropolymers of labelled coniferyl alcohol (synthetic lignin). The amounts of ¹⁴CO₂ released from O¹⁴CH₃-groups, ¹⁴C-2-side chains and ¹⁴C-rings by protoplasts was in the same range as those for intact mycelium. The methoxyl groups of synthetic lignin were more rapidly metabolized by protoplasts than by mycelium. When calculated in dpm of released ¹⁴CO₂ per mg protein the decomposition of ¹⁴C-labelled synthetic lignin and lignin-related monomers in a hyphae-free system of protoplasts was considerable higher than that obtained by the intact mycelium. The presence of intact hyphae is thus not necessary for lignin degradation to occur.Non-common-abbreviations used DHP Dehydropolymer of coniferyl alcohol - LS lignosulfonates prepared from DHP     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

The effect of the ribosomal protein S1 from Escherichia coli on the synthesis in vitro of bacterial-, DNA phage- and RNA phage proteins.

Summary Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 fromEscherichia coli on the synthesis of the coat protein of the RNA-containing phages Q and MS2, on that of an early and a ate enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicated that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.     

Cholesterol absorption inhibitor Ezetimibe blocks uptake of oxidized LDL in human macrophages

Ezetimibe belongs to a group of selective and very effective 2-azetidione cholesterol absorption inhibitors which act on the level of cholesterol entry into enterocytes. Recent data indicated that the drug prevents the formation of a heterocomplex consisting of annexin-2 and caveolin-l and leads to specific inhibition of an NPCILI-dependent cholesterol uptake pathway required for uptake of micellar cholesterol into enterocytes. Earlier studies have shown that caveolin-l and annexin-2 are also expressed in human macro-phages and we show in this study that human macrophages express NPC1L1. Moreover in human macrophages, Ezetimibe(SCH58235) and its analogue, SCH354909, are bound to specific cell surface receptors followed by endocytosis via the classical endocytic pathway. SCH58235 had no effect on uptake and/or processing of acetylated LDL (Ac-LDL). In contrast, the compound inhibited uptake of oxidized LDL (Ox-LDL) by -50% in a dose-dependent manner. SCH58235 blocked the lipid-induced induction of LXR/RXR target genes ABCAI, ABCGI, and apolipoprotein E distinctively more effectively in macrophages loaded with Ox-LDL than in those loaded with Ac-LDL. Based on these findings, we presume that the caveolin-l-, annexin-2-, and NPClLI-dependent cholesterol uptake system that is operating in enterocytes may also contribute to class B scavenger receptor-dependent uptake of Ox-LDL in human monocyte-derived macrophages.