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In culture supematants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as Iisteriolysin 0 (LLO). In the case of L. ivanovii a second major supematant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supematants of L. ivanovii a sphingomyelinase and a Iecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-tenninal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.
A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular Ievel. Nucleotide sequence determination revealed the presence of two open reading frames. 8oth open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.
A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the grain-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DHSa as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately SO times that of the combined E. coli catalases. The nucleutide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of tbe L. seeligeri catalase gene.
During an investigation into the substrate specificity and processing of subtilisin Carlsberg from Bacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97-101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, M<sub>r</sub>=42 kDa) was not processed to the mature form (M<sub>r</sub> = 30 kDa) and was not released into the medium by a proteasedeficient B. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg. (ii) In culture supernatants from B. subtilis harbouring the cloned wild-type subtilisin Carlsberg gene the transient appearance (at 0-3 h after onset of stationary phase) of a processing intermediate (P38c, M<sub>r</sub> = 38 kDa) oftbis protease could be demonstrated. P38c very probably represents a genuine proform of subtilisin Carlsberg.
A gene (Imsod) encoding superoxide dismutase (SOD; EC 1.15.1.1) of the facultative intracellular pathogen, Listeria monocytogenes, was cloned by functional complementation of an SOD-deficient Escherichia coli mutant. The nucleotide sequence was determined and the deduced amino acid (aa) sequence (202 aa) showed close similarity to manganese-containing SOD's from other organisms. Subunits of the recombinant L. monocytogenes SOD (re-SOD) and of both E. coli SODs formed enzymatically active hybrid enzymes in vivo. DNA/DNA-hybridization experiments showed that this type of recombinant re-sod gene is conserved within the genus Listeria.
The completc DNA scqucnccs coding for thc thiol-activated cytolysins from Listeria ivanovii, ivanolysin 0 (ILO) and for sccligerolysin 0 (LSO) from Listeria seeligeri have been dctermined. Thc deduced amino acid scquences revealed that: (i) the primary translation products comprise 528 (ILO) and 530 (LSO) amino acids. respectively. (ii) ILO contains two cysteines. LSO has a substitution in the conserved cysteine motif.
Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNAseq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semiquantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.
The internal transcribed spacer 2 (ITS2) is a widely used phylogenetic marker. In the past, it has mainly been used for species level classifications. Nowadays, a wider applicability becomes apparent. Here, the conserved structure of the RNA molecule plays a vital role. We have developed the ITS2 Database (http://its2.bioapps .biozentrum.uni-wuerzburg.de) which holds information about sequence, structure and taxonomic classification of all ITS2 in GenBank. In the new version, we use Hidden Markov models (HMMs) for the identification and delineation of the ITS2 resulting in a major redesign of the annotation pipeline. This allowed the identification of more than 160 000 correct full ength and more than 50 000 partial structures. In the web interface, these can now be searched with a modified BLAST considering both sequence and structure, enabling rapid taxon sampling. Novel sequences can be annotated using the HMM based approach and modelled according to multiple template structures. Sequences can be searched for known and newly identified motifs. Together, the database and the web server build an exhaustive resource for ITS2 based phylogenetic analyses.
Mammalian Sun1 belongs to an evolutionarily conserved family of inner nuclear membrane proteins, which are known as SUN domain proteins. SUN domain proteins interact with KASH domain partners to form bridging complexes, so-called LINC complexes, that physically connect the nuclear interior to the cytoskeleton. LINC complexes are critical for nuclear integrity and play fundamental roles in nuclear positioning, shaping and movement. The mammalian genome codes for at least five different SUN domain proteins used for the formation of a number of different LINC complexes. Recently, we reported on the identification of everal Sun1 isoforms, which tremendously enlarges the alternatives to form functional LINC complexes. We now confirmed that Sun1 actually exists in at least seven distinct splice variants. Besides that, we observed that expression of individual Sun1 isoforms remarkably depends on the cell type, suggesting a cell type-specific adaption of Sun1 dependent LINC complexes to specific cellular and physiological requirements.