Andrea Scheberl

Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively Alkaliphilic Bacillus pseudofirmus OF4 Lead to Characterization of an S-Layer Protein with a Role in Alkaliphily

The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na(+)-replete or suboptimal Na(+) concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na(+). By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7. 5 under Na(+)-replete and suboptimal Na(+) conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na(+)-dependent transport of alpha-aminoisobutyric acid, or H(+)-dependent synthesis of ATP. However, the capacity for Na(+)-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8. 5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.

Homologs of the Rml Enzymes from Salmonella enterica Are Responsible for dTDP-β-l-Rhamnose Biosynthesis in the Gram-Positive Thermophile Aneurinibacillus thermoaerophilus DSM 10155

ABSTRACT The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of l-rhamnose- and d-glycero-d-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-l-rhamnose (dTDP-l-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-l-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-d-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91T produces d-rhamnose- and 3-acetamido-3,6-dideoxy-d-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91T for l-rhamnose and d-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91T is not able to synthesize dTDP-l-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.

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).

Taxonomic Comparison of Different Thermophilic Sugar Beet Isolates with Glycosylated Surface Layer (S-Layer) Proteins and their Affiliation to Bacillus smithii

Summary During the beet sugar campaign 1991/1992 we have characterized thermophilic bacteria from the extraction plant of an Austrian beet sugar factory in a polyphasic approach. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and freeze-fracture electron microscopy revealed the presence of weakly glycosylated, oblique crystalline cell surface layers (S-layers) on all sugar factory isolates. By partial 16S rDNA sequencing and DNA-DNA hybridisation it was demonstrated that the strains belong to the species Bacillus smithii. In the course of the campaign, however, different strains of B. smithii became dominant, which was demonstrated by fingerprinting using SDS-PAGE, polar lipid analysis, random amplified polymorphic DNA (RAPD) assays, fluorophore-assisted carbohydrate electrophoresis (FACE). Possible explanations for this divergence in strain development are discussed from the technological point of view.

Isolation of Glucocardiolipins from Geobacillus stearothermophilus NRS 2004/3a

Glucose-substituted cardiolipins account for about 4 mol% of total phospholipid extracted from exponentially grown cells of Geobacillus stearothermophilus NRS 2004/3a. Individual glucocardiolipin species exhibited differences in fatty acid substitution, with iso-C(15:0) and anteiso-C(17:0) prevailing. The compounds were purified to homogeneity by a novel protocol and precharacterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.