Insights into the Geobacillus stearothermophilus species based on phylogenomic principles.

BACKGROUND: The genus Geobacillus comprises bacteria that are Gram positive, thermophilic spore-formers, which are found in a variety of environments from hot-springs, cool soils, to food manufacturing plants, including dairy manufacturing plants. Despite considerable interest in the use of Geobacillus spp. for biotechnological applications, the taxonomy of this genus is unclear, in part because of differences in DNA-DNA hybridization (DDH) similarity values between studies. In addition, it is also difficult to use phenotypic characteristics to define a bacterial species. For example, G. stearothermophilus was traditionally defined as a species that does not utilise lactose, but the ability of dairy strains of G. stearothermophilus to use lactose has now been well established. RESULTS: This study compared the genome sequences of 63 Geobacillus isolates and showed that based on two different genomic approaches (core genome comparisons and average nucleotide identity) the Geobacillus genus could be divided into sixteen taxa for those Geobacillus strains that have genome sequences available thus far. In addition, using Geobacillus stearothermophilus as an example, we show that inclusion of the accessory genome, as well as phenotypic characteristics, is not suitable for defining this species. For example, this is the first study to provide evidence of dairy adaptation in G. stearothermophilus - a phenotypic feature not typically considered standard in this species - by identifying the presence of a putative lac operon in four dairy strains. CONCLUSIONS: The traditional polyphasic approach of combining both genotypic and phenotypic characteristics to define a bacterial species could not be used for G. stearothermophilus where many phenotypic characteristics vary within this taxon. Further evidence of this discordant use of phenotypic traits was provided by analysis of the accessory genome, where the dairy strains contained a putative lac operon. Based on the findings from this study, we recommend that novel bacterial species should be defined using a core genome approach.

Genotypic and phenotypic characterization of foodborne Geobacillus stearothermophilus.

Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.

Thermophilic hydrogen production from sludge pretreated by thermophilic bacteria: analysis of the advantages of microbial community and metabolism.

In this study, the effects of thermophilic bacteria pretreatment and elevated fermentation temperature on hydrogen production from sludge were examined. The highest hydrogen yield of 19.9mlH2g(-1) VSS was achieved at 55°C by using pretreated sludge, which was 48.6% higher than raw sludge without pretreatment, and 28.39% higher than when fermented at 35°C. To explore the internal factors of this superior hydrogen production performance, the microbial community and the metabolism analysis were performed by using high-throughput sequencing and excitation-emission matrix. The pretreated sludge showed better utilization of dissolved organic matter and less inhibition of metabolism, especially at thermophilic condition. The 454 sequencing data indicated that microbial abundance was distinctly reduced and extremely high proportion of hydrogen-producing bacteria was found in the thermophilic community (Thermoanaerobacterium accounted for 93.75%). Thus, the pretreated sludge and thermophilic condition showed significant advantages in the hydrogen production using waste sludge as substrate.

Characterization of thermophilic bacilli from a milk powder processing plant.

AIMS: To determine whether strains of Geobacillus stearothermophilus isolated from a milk powder manufacturing plant were different in their ability to form biofilms and produce spores. In addition, this study evaluated whether there were other physiological characteristics that could differentiate these strains. METHODS AND RESULTS: Ten G. stearothermophilus strains and one Anoxybacillus species were isolated from a milk powder manufacturing plant. A microtitre plate assay was used to show that these strains differed in their abilities to form biofilms and produce spores. Scanning electron microscopy showed differences in the biofilm morphologies of three of the G. stearothermophilus strains. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty acid profiling further showed that they had distinct characteristics. CONCLUSIONS: These G. stearothermophilus strains, isolated from the same environment, showed differences in their ability to form biofilms and produce endospores. Based on the multiple characterization methods used in this study, these strains of G. stearothermophilus isolated from one manufacturing plant are diverse. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in the ability of G. stearothermophilus to form biofilms and produce spores may influence the cleaning method used to control the growth of thermophilic bacilli in a dairy processing environment.

Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain.

The implications of Asn315 and Val450 in the atypical starch hydrolysis profile of Bacillus stearothermophilus Amy (a-amylase) US100 have been suggested previously [Ben Ali, Mhiri, Mezghani and Bejar (2001) Enzyme Microb. Tech. 28, 537-542]. In order to confirm this hypothesis, three mutants were generated. Of these two have a single mutation, N315D or V450G, whereas the third contains both mutations. Analysis of the starch breakdown-profile of these three mutants, as well as of the wild-type, allowed us to conclude that each single mutation induces a small variation in the hydrolysis product. However, the major end product produced by the double mutant shifts from maltopentaose/maltohexaose to maltose/maltotriose, confirming the involvement of these two residues in starch hydrolysis. The superimposition of AmyUS100 model with that of Bacillus licheniformis shows in AmyUS100 an additional loop containing residues Ile214 and Gly215. Remarkably, the deletion of these two residues increases the half-life at 100 degrees C from 15 min to approx. 70 min. Moreover, this engineered amylase requires less calcium, 25 p.p.m. instead of 100 p.p.m., to reach maximal thermostability.

Near-infrared surface-enhanced-Raman-scattering-mediated detection of single optically trapped bacterial spores.

A novel methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants. This technique may be useful in many applications; most notably, development of novel detection schemes toward potentially harmful bacteria. This method would also be useful as an ancillary environmental monitoring system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally invasive medical applications). This unique detection scheme is based on the near-infrared (NIR) surface-enhanced Raman scattering (SERS) from single, optically trapped, bacterial spores. The SERS spectra of bacterial spores in aqueous media have been measured using SERS substrates based on approximately 60-nm-diameter gold colloids bound to 3-aminopropyltriethoxysilane derivatized glass. The light from a 787-nm laser diode was used to trap and manipulate as well as simultaneously excite the SERS of an individual bacterial spore. The collected SERS spectra were examined for uniqueness and the applicability of this technique for the strain discrimination of Bacillus stearothermophilus spores. Comparison of normal Raman and SERS spectra reveals not only an enhancement of the normal Raman spectral features but also the appearance of spectral features absent in the normal Raman spectrum.

Molecular analysis of maleate cis-trans isomerase from thermophilic bacteria.

Several strains of thermophilic bacteria containing maleate cis-trans isomerase were isolated from soil samples and identified as Bacillus stearothermophilus, Bacillus circulans, Bacillus brevis, and Deleya halophila. The maleate cis-trans isomerase was purified and characterized from one of the isolated strains, B. stearothermophilus MI-102. The purified enzyme of strain MI-102 showed higher thermal stability than the enzyme of a mesophile, Alcaligenes faecalis IFO13111. The seven maleate cis-trans isomerase genes (maiA) of thermophile were cloned and sequenced. B. stearothemophilus MI-102 MaiA has 67% amino acid identity with A. faecalis MaiA. All eight amino acid sequences of maiA gene products had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. To probe the catalytic mechanism, three cysteine residues in the conserved regions of A. faecalis MaiA were replaced with serine by site-directed mutagenesis. The results suggest that Cys80 and Cys198 play important roles in the enzyme activity.

Crystallization and preliminary X-ray analysis of an intracellular xylanase from Bacillus stearothermophilus T-6.

Xylanases (1,4-beta-D-xylan xylanhydrolases; E.C. 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans. The structural characterization of xylanase active sites is of great interest, since it can lead to a better understanding of their catalytic mechanism and contribute significant knowledge to the rational design of specific oligosaccharide-binding sites via protein engineering. An intracellular xylanase gene (xynA2) from Bacillus stearothermophilus T-6 has recently been cloned and sequenced. The xynA2 gene encodes for an intracellular enzyme (IXT6) of 331 amino acids, with a calculated molecular weight of 38 639 Da and a pI of 5.72. Based on sequence homology, the enzyme belongs to family 10 of the glycosyl hydrolases. The xynA2 gene product (IXT6) was overproduced in Escherichia coli and purified to homogeneity. Crystallographic studies of IXT6 were initiated in order to study the specificity and mechanism of catalysis of this unique xylanase, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. The M1 crystal form was found to be the most suitable for detailed crystal structure analysis. These crystals belong to a C--centered monoclinic crystal system (space group C2) with unit-cell parameters a = 170.6, b = 82.5, c = 80.0 A, beta = 91.43 degrees. They are mechanically strong, are fairly stable in the X-ray beam and diffract X--rays to better than 2.5 A resolution. A full 2.9 A resolution diffraction data set (97.9% completeness, R(merge) = 8.4%) has recently been collected from one crystal at room temperature using X-ray synchrotron radiation (lambda = 1.125 A) and a MAR300 imaging-plate area detector. A comparable 2.5 A data set was measured at 90 K using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate area detector (97.2% completeness, R(merge) = 6.9%). Molecular-replacement studies and multiple anomalous dispersion (MAD) experiments are currently in progress in order to determine the detailed three-dimensional structure of IXT6.

Sequencing and phylogenetic analysis of the spoIIA operon from diverse Bacillus and Paenibacillus species.

In order to clone the spoIIA operon from three different Bacillus and Paenibacillus species, we designed two sets of PCR primers based on three previously published Bacillus spoIIA sequences. One set of primers corresponded to the C-terminal region of SpoIIAB and a region near the middle of SpoIIAC. These primers were used to amplify the corresponding region of spoIIA from Bacillus stearothermophilus and Paenibacillus polymyxa (previously called Bacillus polymyxa [see Ash, C., Priest, F.G., Collins, M.D., 1993. Molecular identification of ribosomal-RNA group 3 bacilli using a PCR probe test - proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 64, 253-260]. The other set of primers, corresponding to an N-terminal and a C-terminal region of SpoIIAC, was used for B. sphaericus. The PCR products were used as probes for Southern blotting of homologous chromosomal DNA. DNA corresponding to spoIIA from the three organisms was identified by screening chromosomal DNA libraries, and cloned. Sequence analysis showed that all spoIIA sequences were conserved, but conservation was strongest in SpoIIAC and least strong in SpoIIAA. In the promoter the -35 region was conserved well but the -10 region rather poorly. Within the proteins, certain regions were particularly strongly conserved, suggesting that they are essential to the function of the protein. Phylogenetic analysis of spoIIA suggested that B. stearothermophilus is close to B. subtilis and B. licheniformis, but that P. polymyxa and B. sphaericus are remote from B. subtilis.

Cloning and nucleotide sequence of Bacillus stearothermophilus pyruvate carboxylase.

A gene for prokaryotic pyruvate carboxylase (PC) was cloned from Bacillus stearothermophilus. It has an open reading frame of 3441 base pairs which can code for a protein of 128,353 Da. Not only the molecular size and domain organization but also the deduced amino acid sequence of B. stearothermophilus PC are similar to those of eukaryotic PCs.

[Isolation and identification of extrem thermophilic bacteria from hot springs of Sichuan and Tibet].

Eleven strains of extrem thermophilic bacteria belonging to the Bacillus Stearothermophiles were isolated from the hot springs of Sichuan and Tibet. The cells were gram-positive, sporulating and motile. The optimum temperature for growth was between 65 degrees C to 70 degrees C; the maximum 94 degrees C, and minimum 40 degrees C. The colour of colony was yellow to bright orange. The G+C content of the DNA in strains has been found to be in the range 48.4-53.15 mol%.

Aerobic thermophilic treatment of sewage sludge at pilot plant scale. 1. Operating conditions.

The aerobic thermophilic treatment process of sewage sludge was studied at different bioreactor scales in a pilot plant installation. Since, for a satisfactory sludge disinfection, the Swiss legislation requires minimal incubation times of all volume elements, the bioreactors were operated in repetitive batch mode (draw and fill). Different retention times and frequencies of the volume changes were applied in order to prove the capability of the particular operation modes in assuring high degradative potential. The main enzymatic activity involved during the aerobic treatment was proteolysis: the RQ values ranged between 0.8 and 0.9 depending on the applied operating conditions. Although not in a linear manner, the efficiency of the microflora decreased as the bioreactor scale increased, when this increase corresponded with a reduction of the specific power input. The sludge oxidation rates can be tuned by some process operating conditions such as the volume change frequency, the changed volume quantities and the retention times. It was possible to improve the microbial degradative efficiency by an increased frequency of the changes, while the mean retention time influenced in particular the ultimate product quality, described as residual organic matter content of the sludge. The microflora present was also satisfactorily active at mean hydraulic retention times of less than 10 h. The organic matter concentration of the inlet sewage sludge plays an important role: it influences the aerobic degradation process positively.

Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies.

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

Evidence for movement of the alpha-amylase gene into two phylogenetically distant Bacillus stearothermophilus strains.

The gene for an alpha-amylase cloned from strain DY-5 of Bacillus stearothermophilus was used to examine to what extent the corresponding genes are structurally similar in other B. stearothermophilus strains. The structure of the gene itself was almost identical in DY-5 and a group of strains represented by strain 799. The gene was not detected at all in strain DSM2334, which was phenotypically amylase deficient. Comparison of the structure of 5S rRNA and electrophoretic pattern of the ribosomal proteins indicates that strains DY-5 and DSM2334 are closely related to each other, whereas strain 799 is phylogenetically very distant from the two. We estimate that strain 799 separated from DY-5 and DSM2334 some 420 million years ago. Nucleotide sequencing of the region containing the amylase gene from strains DY-5 and 799 revealed the presence of a 3.4-kilobase stretch that was highly similar in the two strains. Furthermore, comparison of the restriction map surrounding the amylase gene of DY-5 with that of a corresponding region in DSM2334 indicated that the former strain contained an extra segment 5.5 kilobases in length, which included the 3.4-kilobase stretch mentioned above. This segment was missing in DSM2334. It thus appears that the alpha-amylase gene was brought into strains DY-5 and 799 from outside despite a large phylogenetic distance.

Comparative 16S rRNA oligonucleotide analyses and murein types of round-spore-forming bacilli and non-spore-forming relatives.

The phylogenetic incoherency of the genus Bacillus as presently described is demonstrated by analysis of both published and new data from comparative 16S rRNA cataloguing of nine Bacillus species and a number of related non-Bacillus taxa, i.e. Caryophanon latum, Filibacter limicola and Planococcus citreus. While the ellipsoidal-spore-forming bacilli, e.g. B. subtilis and allied species, formed a coherent cluster, the round-spore-forming bacilli showed a higher degree of relationship to the non-spore-forming organisms than these bacilli show among each other. Thus B. sphaericus clustered with C. latum, B. globisporus grouped with F. limicola, B. pasteurii with Sporosarcina ureae, and 'B. aminovorans' with P. citreus, respectively. These organisms formed two related subclusters which, in their phylogenetic depth, are comparable to that of the B. subtilis subline. With the exception of 'B. aminovorans', the 16S rRNA phylogeny was entirely consistent with the distribution of murein types. Even more distantly related to and grouping outside the main Bacillus cluster was B. stearothermophilus, which displayed a moderate relationship to Thermoactinomyces vulgaris. Taxonomic problems arising from the new insights into the intrageneric relationships of Bacillus are discussed.

The isolation and characterization of bacteriophages infecting obligately thermophilic strains of Bacillus.

Twenty-four thermophilic bacteriophages have been isolated from diverse sources such as compost, soil, silage and rotting straw. Although considerable individual host specificity was observed, the phages were able to infect most of the major taxonomic groups of Bacillus thermophiles. The phages varied considerably in morphology and size; the phage heads were either cylindrical or polyhedral with tails varying in length between 15 and 500 nm. Most of the phages were stable at 50 degrees C for 4-5 h but at 70 degrees C the plaque-forming units decreased by between 10(2)- and 10(7)-fold in 2 h. The DNA of morphologically similar phages was examined by restriction enzyme analysis, and some differences in the DNA fragment patterns were found. Efficiency of plating data indicated that 'B. caldotenax' has a restriction and modification system. These phages may be valuable for the study of the genetics of thermophilic bacilli: transduction of 'B. caldotenax' and 'B. caldovelox' by phage JS017 has been observed.

Identification of Bacillus strains using the API system.

A system is described for the rapid and accurate identification of Bacillus isolates using a matrix of results from tests in the API 20E and API 50CHB strips and from supplementary tests. API System tests have been shown to be more reproducible than the classical tests. A taxonomy based upon API tests is in good agreement with those obtained by other methods. The results matrix can also be used in computer assisted identification.

[Numerical taxonomy of a thermophilic "Bacillus" species isolated from West African rice soils (author's transl)]. / Taxonomie numérique de Bacillus thermophiles isolés de sols de rizère de l'Afrique de l'Ouest.

Fifty-seven strains of endospore-forming thermophilic bacteria, 37 of which were capable of denitrification, were isolated from rice soils of West Africa. They were compared with 17 strains of similar bacteria from culture collections, utilizing a total of 123 morphological, physiological and biochemical characteristics. A numerical analysis was performed using the complete linkage-clustering method and the Khi2 test. Seventy-five percent (55 strains) could be included in 12 groups at a taxonomic distance of 0.015. Wild strains of denitrifiers issued in phenons 8 to 12 and strains of phenon 4 (not denitrifying) were related to the named strains of phenons 1 and 7 (Bacillus stearothermophilus). Twenty-two wild strains, and 5 strains from culture collections, were only thermotolerating without growth at 65 degrees C. The strains of phenon 3 were related to the 3 named strains of B. coagulans. Phenons 5 and 6 were composed of strains related to B. circulans. The strains of phenon 2 denitrified and showed a swollen central endospore; they were closely related to B. brevis. The denitrifying thermophilic strains isolated from rice soils (phenons 8 to 12) were related to the first group (B. kaustophilus) of Walker and Wolf but their base compositions of DNA were significantly different from those found for the reference strains.

Phenotypic and genotypic characterization of some thermophilic species of Bacillus.

Some thermophilic species of Bacillus were characterized using biochemical tests, antibiotic sensitivity, bacteriocin and bacteriophage sensitivity, esterase patterns, DNA hybridization and % G+C content. The three caldo-active strains of bacilli isolated by Heinen (1971) were compared with strains of B. stearothermophilus. Eight of the strains examined showed characteristics which enabled them to be placed in the three main taxonomic groups suggested by Walker & Wolf (1971). Two strains showed characteristics of both groups 1 and 3. Examination of genotype data showed taxonomic groupings which differed from those based on phenotypic characterization.