Beatrix Kuen

HETEROLOGOUS EXPRESSION AND SELF-ASSEMBLY OF THE S-LAYER PROTEIN SBSA OF BACILLUS STEAROTHERMOPHILUS IN ESCHERICHIA COLI

The cell surface of Bacillus stearothermophilus PV72 is covered by a regular surface layer (S‐layer) composed of a single species of protein, SbsA, with a molecular weight of 130 000. Recently, the sequence of the corresponding gene (sbsA) has been determined. The SbsA coding region including the signal sequence was cloned as a polymerase chain reaction (PCR) product into a low‐copy‐number vector under the transcriptional control of the λpL promoter. Expression of sbsA was shown to be thermally inducible from the resulting vector pBK4 in a strain of Escherichia coli expressing the λcI857 from the chromosome. As shown by ultrathin sectioning of whole cells and immunogold labelling using SbsA‐specific antibodies, expression of sbsA in E. coli led to accumulation of sheet‐like self‐assembling products of the protein in the cytoplasm. No SbsA protein was detected either in the periplasm or in the supernatant fractions. Long‐term expression of sbsA from pBK4, including in the late stationary phase, did not lead to degradation of SbsA.

Description of Bacillus thermoaerophilus sp. nov., To Include Sugar Beet Isolates and Bacillus brevis ATCC 12990†

Isolates of thermophilic bacteria obtained from an Austrian beet sugar factory were screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and freeze-fracture electron microscopy for the presence of glycosylated crystalline cell surface layers (S-layers). On the basis of similarities in the protein band patterns on SDS-PAGE gels and the lattice geometry of the S-layers as revealed by electron micrographs, the 31 isolates which we studied were clustered into five groups (groups I to V) and several strains which exhibited no common characteristics (group 0). We found that the organisms belonging to groups I to III had glycosylated S-layer proteins, but the highest carbohydrate contents were observed in group III organisms. Partial sequencing of the 16S ribosomal DNAs of selected representative strains of each group revealed that the group I, II, IV, and V isolates and the few group 0 strains were different from the group III strains. The results of DNA-DNA hybridization experiments, SDS-PAGE, and an analysis of polar lipids demonstrated that group III isolates L419-91, L420-91T (T = type strain), and L438-91 belong to the same species. We chose the group III organism Bacillus sp. strain L420-91T for further analysis because of the high carbohydrate content of its S-layer protein. The taxonomic position of this isolate was determined by using a polyphasic approach. Phenotypic, chemotaxonomic, and genomic analyses revealed that strains L420-91T, L419-91, and L438-91 represent a new Bacillus species. We observed high levels of similarity between these strains and Bacillus brevis ATCC 12990, which also had a glycosylated S-layer protein. Our results show that strains L420-91T, L419-91, and L438-91 and B. brevis ATCC 12990 belong to the same species and that this species is a new Bacillus species, which we name Bacillus thermoaerophilus. The type strain of this species is strain L420-91 (= DSM 10154).

S-Layer Variation in Bacillus stearothermophilus PV72 Is Based on DNA Rearrangements between the Chromosome and the Naturally Occurring Megaplasmids

Bacillus stearothermophilus PV72 expresses different S-layer genes (sbsA and sbsB) under different growth conditions. No stretches of significant sequence identity between sbsA and sbsB were detected. In order to investigate S-layer gene regulation in B. stearothermophilus PV72, we characterized the upstream regulatory region of sbsA and sbsB by sequencing and primer extension analysis. Both genes are transcribed from unique but different promoters, independently of the growth phase. Localization of sbsB in the sbsA-expressing strain PV72/p6 revealed that the coding region of the second S-layer gene sbsB is located not on the chromosome but on a natural megaplasmid of the strain, whereas the upstream regulatory region of sbsB was exclusively detected on the chromosome of PV72/p6. For sbsB expression, the coding region has to be integrated into the chromosomally located expression site. After the switch to sbsB expression, the sbsA coding region was removed from the chromosome but could still be detected on the plasmid of the sbsB-expressing strain PV72/p2. The sbsA upstream regulatory region, however, remained on the chromosome. This is the first report of S-layer variation not caused by intrachromosomal DNA rearrangements, but where variant formation depends on recombinational events between the plasmid and the chromosome.

Analysis of mutations to gyrA in quinolone-resistant clinical isolates of Enterobacter cloacae

The gyrA subgenes of a quinolone-resistant Enterobacter cloacae clinical isolate (ofloxacin MIC, 16 mg/L) and of a control, E. cloacae NCTC 10005 (ofloxacin MIC, 0.03 mg/L), were amplified by polymerase chain reaction (PCR) and sequenced. The resistant isolate had mutations at the codons for amino acids 83, 89 and 90. The first of these mutations led to replacement of serine-83 by tyrosine, whereas the other mutations were silent. Digestion of PCR-amplified DNA fragments with the restriction enzyme HinfI detected mutations at the same site in gyrA in six further quinolone resistant E. cloacae isolates.

Structural and Functional Analysis of the S-Layer Protein from Bacillus stearothermophilus

One feature common to many bacteria, regardless of their phylogenetic origin within the kingdoms Eucarya or Archaea, is the presence of a regularly ordered (glyco)protein border as the outermost macromolecular layer of the cell envelope. A recent list of organisms with such crystalline surface layers (S-layers) cites approximately 300 different prokaryotic species (Messner and Sleytr, 1992). However, most of the S-layers have only been described by electron microscopical or biochemical investigations and DNA sequence data of the corresponding genes are available for very few species (approximately 20). Here we report on the structural and functional properties of the S-layer of Bacillus stearothermophilus strain PV72 which have been deduced from computer analysis of DNA-sequence data (for sequencing details of the gene see the contribution by Kuen, Sara, Sleytr, and Lubitz in this book).