Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of beta-(2-->1)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated Mr of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (kcat/Km) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.

The rrs (16S rDNA) gene sequences of nitrogen-fixing endospore-forming bacilli isolated from the rhizosphere of wheat and maize were determined in order to infer their phylogenetic position in the Bacillaceae. These rhizosphere strains form a monophyletic cluster with Paenibacillus azotofixans, Paenibacillus polymyxa and Paenibacillus macerans. Two of them (RSA19 and TOD45) had previously been identified as Bacillus circulans (group 2) by phenotypic characterization (API 50CH). Evidence for nitrogen fixation by P. azotofixans, P. polymyxa, P. macerans and putative B. circulans strains RSA19 and TOD45 was provided by acetylene-reduction activity, and confirmed by amplifying and sequencing a nifH fragment (370 nt). The phylogenetic tree of nifH-derived amino acid sequences was compared to the phylogenetic tree of rrs sequences. All Paenibacillus nifH sequences formed a coherent cluster distinct from that of related nitrogen-fixing anaerobic clostridia and Gram-positive high-G+C-content frankiae. The nifH gene was neither detected in the B. circulans type strain (ATCC 4513T) nor in the type strains of Bacillus subtilis, Bacillus cereus, Bacillus alcalophilus, Bacillus simplex, Brevibacillus brevis and Paenibacillus validus. Accordingly, nitrogen fixation among aerobic endospore-forming Firmicutes seems to be restricted to a subset of species in the genus Paenibacillus.

Clusters of genes encoding enzymes for tetrapyrrole biosynthesis were cloned from Bacillus sphaericus, Bacillus stearothermophilus, Brevibacillus brevis and Paenibacillus macerans. The sequences of all hemX genes found, and of a 6.3 kbp hem gene cluster from P. macerans, were determined. The structure of the hem gene clusters was compared to that of other Gram-positive bacteria. The Bacillus and Brevibacillus species have a conserved organization of the genes hemAXCDBL, required for biosynthesis of uroporphyrinogen III (UroIII) from glutamyl-tRNA. In P. macerans, the hem genes for UroIII synthesis are also closely linked but their organization is different: there is no hemX gene and the gene cluster also contains genes, cysG8 and cysG(A)-hemD, encoding the enzymes required for synthesis of sirohaem from UroIII. Bacillus subtilis contains genes for three proteins, NasF, YInD and YInF, with sequence similarity to Escherichia coli CysG, which is a multi-functional protein catalysing sirohaem synthesis from UroIII. It is shown that YInF is required for sirohaem synthesis and probably catalyses the precorrin-2 to sirohaem conversion. YInD probably catalyses precorrin-2 synthesis from UroIII and NasF seems to be specific for nitrite reduction.

To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is produced mainly by Bacillus strains. CGTase from Bacillus macerans IFO3490 produces alpha-cyclodextrin as the major hydrolysis product from starch, whereas thermostable CGTase from Bacillus stearothermophilus NO2 produces alpha- and beta-cyclodextrins. To analyze the cyclization characteristics of CGTase, we cloned different types of CGTase genes and constructed chimeric genes. CGTase genes from these two strains were cloned in Bacillus subtilis NA-1 by using pTB523 as a vector plasmid, and their nucleotide sequences were determined. Three CGTase genes (cgt-1, cgt-5, and cgt-232) were isolated from B. stearothermophilus NO2. Nucleotide sequence analysis revealed that the three CGTase genes have different nucleotide sequences encoding the same amino acid sequence. Base substitutions were found at the third letter of five codons among the three genes. Each open reading frame was composed of 2,133 bases, encoding 711 amino acids containing 31 amino acids as a signal sequence. The molecular weight of the mature enzyme was estimated to be 75,374. The CGTase gene (cgtM) of B. macerans IFO3490 was composed of 2,142 bases, encoding 714 amino acids containing 27 residues as a signal sequence. The molecular weight of the mature enzyme was estimated to be 74,008. The sequence determined in this work was quite different from that reported previously by other workers. From data on the three-dimensional structure of a CGTase, seven kinds of chimeric CGTase genes were constructed by using cgt-1 from B. stearothermophilus NO2 and cgtM from B. macerans IFO3490. We examined the characteristics of these chimeric enzymes on cyclodextrin production and thermostability. It was found that the cyclization reaction was conferred by the NH2-terminal region of CGTase and that the thermostability of some chimeric enzymes was lower than that of the parental CGTases.

tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.

�\-Cyclodextrin glycosyltransferase (�\-CGTase) can convert starch into �\-cyclodextrin with various proportions of �]-cyclodextrin and/or �^-cyclodextrin in the products. To improve the �\-cyclodextrin-forming specificity, directed evolution on the wild-type �\-CGTase was performed by constructing mutant library with error-prone PCR method. The positive mutant strains were selected in combination of starch plate screening with HPLC detection of the products. An �\-CGTase from the mutant strain (assigned No. 95) was found to be able to increase the �\:�] ratio in product mixture from 3.4 to 7.8 in comparison with the wild-type �\-CGTase. Sequence alignment indicated that two mutations occurred in the No. 95 mutant �\-CGTase, which were Y167H and A536V. Reverse mutation revealed that Y167H was responsible for this change. A series of 167 site-substituted mutants could improve the �\:�] ratio to different extents as indicated by saturated mutagenesis, with Y167H as the best substitution. In conclusion, Y167 was confirmed to be one of the main subsites in the -6 domain of �\-CGTase that is responsible for the �\:�] ratio in the product mixture. Y167H is most preferable among all types of mutant enzymes tested at this site. The reconstructed Y167H (i.e., No. 95) �\-CGTase showed better potential for �\-cyclodextrin production on industrial scale.

The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta-1,3-1,4-glucanase of Bacillus macerans has been determined. The bglM gene comprises an open reading frame (ORF) of 711 bp (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta-1,3-1,4-glucanases from B. subtilis and B. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilic Bacillus endo-beta-glucanases. The B. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm in E. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 of B. macerans endo-beta-glucanase could be identified.

The three-dimensional structure of the hybrid Bacillus 1,3-1,4-beta-glucanase (beta-glucanase; 1,3-1,4-beta-D-glucan 4-glucanohydrolase, lichenase, EC 3.2.1.73) designated H(A16-M) was determined by x-ray crystallography at a resolution of 2.0 A and refined to an R value of 16.4% using stereochemical restraints. The protein molecule consists mainly of two seven-stranded antiparallel beta-pleated sheets arranged atop each other to form a compact, sandwich-like structure. A channel crossing one side of the protein molecule accommodates an inhibitor, 3,4-epoxybutyl beta-D-cellobioside, which binds covalently to the side chain of Glu-105, as seen in a crystal structure analysis at 2.8-A resolution of the protein-inhibitor complex (R = 16.8%). That Glu-105 may be indispensible for enzyme catalysis by H(A16-M) is suggested by site-directed mutagenesis of this residue, which inevitably leads to an inactive enzyme.

H(A16-M) is a hybrid endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus. Its crystal structure was refined using synchrotron X-ray diffraction data up to a maximal resolution of 0.16 nm. The R value of the resulting model is 14.3% against 21,032 reflections > 2 sigma. 93% of the amino acid residues are in the most favorable regions of the Ramachandran diagram, and geometrical parameters are in accordance with other proteins solved at high resolution. As shown earlier [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291], the protein folds into a compact jellyroll-type beta-sheet structure. A systematic analysis of the secondary structure reveals the presence of two major antiparallel beta-sheets and a three-stranded minor mixed sheet. Amino acid residues involved in catalysis and substrate binding are located inside a deep channel spanning the surface of the protein. To investigate the stereochemical cause of the observed specificity of endo-1,3-1,4-beta-D-glucan 4-glucanohydrolases towards beta-1,4 glycosyl bonds adjacent to beta-1,3 bonds, the high-resolution crystal structure has been used to model an enzyme-substrate complex. It is proposed that productive substrate binding to the subsites p1, p2 and p3 of H(A16-M) requires a beta-1,3 linkage between glucose units bound to p1 and p2.

The gene for cyclodextrin glucanotransferase from Bacillus macerans was cloned in an Escherichia coli bacteriophage, lambda D69, and was recloned in a Bacillus subtilis plasmid, pUB110. Starting from an ATG initiation codon, a unique reading frame was shown to extend for 2,142 base pairs (714 amino acids). The nucleotide sequence revealed that the enzyme is composed of two identical subunits.

The diversity of dinitrogenase reductase gene (nifH) fragments in Paenibacillus azotofixans strains was investigated by using molecular methods. The partial nifH gene sequences of eight P. azotofixans strains, as well as one strain each of the close relatives Paenibacillus durum, Paenibacillus polymyxa, and Paenibacillus macerans, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are two nifH sequence clusters, designated clusters I and II, in P. azotofixans. The data further indicated that there was sequence divergence among the nifH genes of P. azotofixans strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all nifH cluster II sequences were similar to the alternative (anf) nitrogenase sequence. A nested PCR assay for the detection of nifH (cluster I) of P. azotofixans was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the P. azotofixans nifH gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of nifH in P. azotofixans strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among different nifH genes. Moreover, nifH is probably a multicopy gene in P. azotofixans. Both similarities and differences were detected in the P. azotofixans nifH DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples.

Paenibacillus (formerly Bacillus) macerans is capable of succinate oxidation under oxic conditions and fumarate reduction under anoxic conditions. The reactions are catalyzed by different enzymes, succinate dehydrogenase (Sdh) and fumarate reductase (Frd). The genes encoding Sdh (sdhCAB) were analyzed. The gene products of sdhA and sdhB were similar to the subunits of known Sdh and Frd enzymes. The hydrophobic subunit SdhC showed close sequence similarity to the class of Sdh/Frd enzymes containing diheme cytochrome b. From the sdhCAB gene cluster two transcripts were produced, one comprising sdhCAB, the other sdhAB. The transcripts were found only during aerobic growth, and the amount was directly proportional to Sdh activity, but inversely proportional to Frd activity.