The thermophilic strains HTA426 and HTA462 isolated from the Mariana Trench were identified as Geobacillus kaustophilus and G. stearothermophilus, respectively, based on physiologic and phylogenetic analyses using 16S rDNA sequences and DNA-DNA relatedness. The genome size of HTA426 and HTA462 was estimated at 3.23-3.49 Mb and 3.7-4.49 Mb, respectively. The nucleotide sequences of three independent lambda-phage inserts of G. stearothermophilus HTA462 have been determined. The organization of protein coding sequences (CDSs) in the two lambda-phage inserts was found to differ from that in the contigs corresponding to each lambda insert assembled by the shotgun clones of the G. kaustophilus HTA426 genome, although the CDS organization in another lambda insert is identical to that in the HTA426 genome.

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His6-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65 degrees C, respectively, and 50% of its activity remained after incubation at 60 degrees C for 32 min. The enzyme preferentially hydrolyzed L-leucine- p-nitroanilide (L-Leu- p-NA) followed by Cys derivative.

Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, recA and rpoB sequence analysis was evaluated as an alternative for distinguishing Geobacillus species. The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. The recA and rpoB sequences of 10 reference strains in clusters V, VIb and VIc were amplified and sequenced using consensus primers. Alignment of recA sequences in clusters V, VIb and VIc revealed three types of recA genes, consistent with the putative amino acid sequences and in vivo recA splicing analysis. The phylogram constructed from rpoB sequences showed more divergence than that constructed from 16S rRNA gene sequences. recA and rpoB sequence analysis differentiated closely-related Geobacillus species and provided direct evidence for reclassifying some species dubiously categorized as Geobacilli. Additionally, this study revealed three types of recA genes in the different Geobacillus species. This study highlights the advantage of recA and rpoB sequence analysis to supplement 16S rRNA gene sequence analysis for efficient and convenient determination of Geobacillus species.

In this work, the variability of spo0A gene in the genus Geobacillus and applicability of this gene for the taxonomy within this genus were evaluated. The protein Spo0A is the master regulator of the endospore-forming process in the all endospore-forming bacteria. Geobacillus genus-specific primers GEOSPO were designed based on the sequences of Geobacillus spo0A gene available through the public databases. Inter and intraspecific variability of Geobacillus spo0A gene was determined after sequencing of the GEOSPO-PCR products. Geobacillus spo0A sequence analysis showed that three species--Geobacillus thermodenitrificans, G. stearothermophilus, and G. jurassicus--could be easily identified. Similarity between the sequences of these species and the other species were in the range of 83.3%-92.0%. In contrast, intraspecific similarity of G. thermodenitrificans and G. stearothermophilus was high--above 99.0%. Similarity of spo0A sequences of G. subterraneus-G. uzenensis species cluster also matched this interval. Intercluster similarity between G. lituanicus-G. thermoleovorans-G. kaustophilus-G. vulcani and G. thermocatenulatus-G. gargensis-G. caldoxylosilyticus-G. toebii-G. thermoglucosidasius species clusters, as well as interspecific similarity within these two clusters was in the range of the intraspecific similarity determined for G. thermodenitrificans and G. stearothermophilus. It was also determined that spo0A cannot be used as the phylogenetic marker for the genus Geobacillus.

Beta-galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable beta-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. subtilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified beta-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this beta-galactosidase activity were 70 degrees C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70 degrees C were 50 and 9 h, respectively, and the K(m) and V(max) values were 2.96 mM and 6.62 micromol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe(2+), Zn(2+), Cu(2+), Pb(2+), and Sn(2+), inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.

To investigate the applicability of rpoB gene, which encodes the beta subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP- and BOX-polymerase chain reaction) were also used. rpoB DNA (458 bp) were amplified from 21 Geobacillus- and Bacillus type strains, producing different BOX- and REP-PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90-100% (16S rRNA) and 74-100% (rpoB) similarity among examined bacteria. BOX- and REP-PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.

Although efforts have been made the past few years, knowledge on genomic and phenotypic diversity and occurrence of the denitrification ability in Gram-positive bacteria are still fragmentary. Many environmental monitoring approaches have used nir, nor, and nos genes as marker genes for detection of denitrification or denitrifying bacteria. However, primers used in these methods often fail to detect the genes in specific bacterial taxa, such as Gram-positive denitrifiers. In this study, novel primer sets specifically targeting nirK, qnorB, and nosZ genes of the Firmicute genus Geobacillus were developed by genomic mining and tested in parallel with commonly used primers on a set of phylogenetically closely related denitrifying geobacilli. Novel nirK and qnorB sequences were recovered from all strains tested, whereas nosZ was detected in part of the strain set, which was in agreement with observed phenotypes. Interspecies and modest intraspecies variations in amplified fragment length polymorphism (AFLP) patterns were observed, verifying presence of genomic variation within the strain set. Our study shows that closely related Gram-positive denitrifiers may differ in denitrification phenotype and genotype. But foremost, novel primers targeting very divergent nirK, qnorB, and nosZ gene sequences of Gram-positive denitrifiers, are now available for cultivation-independent environmental surveys.

A thermophilic, arsenate resistant bacterial strain was isolated from a geothermal field located in the area surrounding Monterotondo (Tuscany, Italy). Based on 16S rRNA gene analysis and recN comparisons the strain was identified as Geobacillus kaustophilus. Cells of the strain, designated A1, were rod-shaped, 2-3 �gm long and reacted negatively to Gram staining, despite its taxonomic classification as a Gram positive microorganism. Strain A1 is a thermophilic spore-forming bacterium, and grows optimally at pH 6.5 and 55 �XC. An arsenate MIC of 80 mM was determined for strain A1, and the close relative G. kaustophilus DSM 7263(T) showed similar levels of arsenate resistance. These observations were consistent with the presence of arsenic detoxification genes in the genome of G. kaustophilus HTA426. Furthermore, strain A1 growth was not inhibited by 5 mM antimonite and 15 mM arsenite, the highest tested concentrations. This is the first description of arsenic resistance in a Geobacillus strain and supports the hypothesis that members of the genus may have a role in the biogeochemical cycling of arsenic.

Bacillus stearothermophilus IAM11001 produced three beta-galactosidases, beta-galactosidase I, II, and III (beta-gal I, II, and III), which are detectable by polyacrylamide (nondenatured) gel electrophoresis. By connecting restriction fragments of the chromosomal DNA to plasmid vectors, followed by transformation of Escherichia coli, two beta-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were isolated. Identification of the gene products and Southern hybridization analyses with a 2.7-kilobase-pair EcoRI fragment containing the bgaA gene as probe revealed that a single bgaA gene exists on the genome and that beta-gal II and beta-gal III consist of a common subunit (the bgaA gene product; molecular weight, 120,000), but differ in their assembly (beta-gal II is a dimer, and beta-gal III is a tetramer). The bgaB gene product (molecular weight, 70,000) in Bacillus subtilis harboring pHG5 (a hybrid plasmid consisting of pUB110 and a 2.9-kilobase-pair EcoRI fragment) was estimated to be the beta-gal I protein from its heat stability. Southern hybridization and immunological testing indicated that the two genes have no homology.

The nucleotide sequence of the bgaB gene, which encodes the thermostable beta-galactosidase I of Bacillus stearothermophilus, and its flanking region was determined. A 2,016-base-pair open reading frame observed was concluded to be for beta-galactosidase I (Mr 78,051) from observations that the amino acid composition of the enzyme and the sequence of 14 amino acids from the amino-terminus of the enzyme coincided with those deduced from this open frame. A 107-base-pair HaeIII-AluI fragment just upstream of the estimated Shine-Dalgarno sequence of the bgaB gene had promoter activity toward cat-86 (chloramphenicol acetyltransferase gene) and produced the enzyme at a level equivalent to 7% of the total cellular protein of B. subtilis. From the base sequence of this DNA region and the transcriptional start site determined by S1 nuclease mapping, the -35 and -10 sequences are estimated to be TTGACA and TAATTT, respectively, which are similar to the consensus sequence of B. subtilis sigma 43 RNA polymerase.

Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.