Reclassification of Geobacillus galactosidasius and Geobacillus yumthangensis as Parageobacillus galactosidasius comb. nov. and Parageobacillus yumthangensis comb. nov., respectively.

Members of the genus Geobacillus within the phylum Firmicutes are Gram-stain-positive, aerobic, endospore-forming, obligate thermophiles. In 2016, the genus Geobacillus was subdivided into two genera based on whole-genome approaches. The new genus, Parageobacillus, comprises five genomospecies. In this study, we recommend the reclassification of two Geobacillus species, Geobacillus galactosidasius and Geobacillus yumthangensis, into the genus Parageobacillus. We have applied whole genome approaches to estimate the phylogenetic relatedness among the 18 Geobacillus and Parageobacillus type strains for which genome sequences are currently publicly available. The phylogenomic metrics AAI (average amino acid identity), ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization) denoted that the type strains of G. galactosidasius and G. yumthangensis belong to the genus Parageobacillus. Furthermore, a phylogeny based on comparison of the 16S rRNA gene sequences, recN gene sequences and core genes identified from the whole-genome analyses designated that the type strains of G. galactosidasius and G. yumthangensis belong in the genus Parageobacillus. With these findings, we consequently propose that G. galactosidasius and G. yumthangensis should be reclassified as Parageobacillus galactosidasius comb. nov. and Parageobacillus yumthangensis comb. nov.

A systematic review of the genera Geobacillus and Parageobacillus: their evolution, current taxonomic status and major applications.

The genus Geobacillus, belonging to the phylum Firmicutes, is one of the most important genera and comprises thermophilic bacteria. The genus Geobacillus was erected with the taxonomic reclassification of various Bacillus species. Taxonomic studies of Geobacillus remain in progress. However, there is no comprehensive review of the characteristic features, taxonomic status and study of various applications of this interesting genus. The main aim of this review is to give a comprehensive account of the genus Geobacillus. At present the genus acomprises 25 taxa, 14 validly published (with correct name), nine validly published (with synonyms) and two not validly published species. We describe only validly published species of the genera Geobacillus and Parageobacillus. Vegetative cells of Geobacillus species are Gram-strain-positive or -variable, rod-shaped, motile, endospore-forming, aerobic or facultatively anaerobic, obligately thermophilic and chemo-organotrophic. Growth occurs in the pH range 6.08.5 and a temperature of 37-75 °C. The major cellular fatty acids are iso-C15:o, iso-C16:0 and iso-C17:o. The main menaquinone type is MK-7. The G-+C content of the DNA ranges between 48.2 and 58 mol%. The genus Geobacillus is widely distributed in nature, being mostly found in many extreme locations such as hot springs, hydrothermal vents, marine trenches, hay composts, etc. Geobacillus species have been widely exploited in various industrial and biotechnological applications, and thus are promising candidates for further studies in the future.

Pan-Genome Analyses of Geobacillus spp. Reveal Genetic Characteristics and Composting Potential.

The genus Geobacillus is abundant in ecological diversity and is also well-known as an authoritative source for producing various thermostable enzymes. Although it is clear now that Geobacillus evolved from Bacillus, relatively little knowledge has been obtained regarding its evolutionary mechanism, which might also contribute to its ecological diversity and biotechnology potential. Here, a statistical comparison of thirty-two Geobacillus genomes was performed with a specific focus on pan- and core genomes. The pan-genome of this set of Geobacillus strains contained 14,913 genes, and the core genome contained 940 genes. The Clusters of Orthologous Groups (COG) and Carbohydrate-Active Enzymes (CAZymes) analysis revealed that the Geobacillus strains had huge potential industrial application in composting for agricultural waste management. Detailed comparative analyses showed that basic functional classes and housekeeping genes were conserved in the core genome, while genes associated with environmental interaction or energy metabolism were more enriched in the pan-genome. Therefore, the evolution of Geobacillus seems to be guided by environmental parameters. In addition, horizontal gene transfer (HGT) events among different Geobacillus species were detected. Altogether, pan-genome analysis was a useful method for detecting the evolutionary mechanism, and Geobacillus' evolution was directed by the environment and HGT events.

Geobacillin 26 - high molecular weight bacteriocin from a thermophilic bacterium.

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine.

Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China.

A rod-shaped, spore-forming, thermophilic, chemoorganotrophic, aerobic or facultatively anaerobic bacterial strain, 1017T, was isolated from production water sampled at the Dagang oilfield (PR China), and was characterized by using a polyphasic approach. The strain is capable of anaerobic glucose fermentation. Nitrate is reduced to nitrite. Optimal growth was observed at 60-65 °C, at pH between pH 7.0 and 7.5, and with 1-2 % (w/v) NaCl. The major cellular fatty acids were iso-C17 : 0, anteiso-C17 : 0, iso-C15 : 0, iso-C16 : 0 and C16 : 0. The predominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on the 16S rRNA, gyrB and parE gene sequences indicated that the isolate belonged to the genus Geobacillus and was most closely related to Geobacillus thermoleovorans KCTC 3570T (99.5, 96.1 and 97.9 % sequence similarity, respectively). Genome sequencing revealed a genome size of 3.57495 Mb and a DNA G+C content of 51.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 1017T and G. thermoleovorans KCTC 3570T were 95.9 and 64.9 %, respectively. Results of phylogenomic metrics analysis of the genome and 1172 core genes of strain 1017T and its physiological and biochemical characteristics confirmed that strain 1017T represented a novel species of the genus Geobacillus, for which the name Geobacillusproteiniphilus sp. nov. is proposed. The type strain is 1017T (=VKM B-3132T=KCTC 33986T).

Geobacillus yumthangensis sp. nov., a thermophilic bacterium isolated from a north-east Indian hot spring.

A thermophilic, spore-forming, rod-shaped bacterium isolated from the Yumthang hot spring in North Sikkim, India was subjected to taxonomic studies. The thermophilic bacterial isolate was designated as strain AYN2T. Cells were Gram-stain-positive, aerobic, motile, rod-shaped, catalase-positive and methyl red-negative. Strain AYN2T was able to grow in the pH range from 6 to 10 (optimum, pH 7.5-8.0), at 40-70 °C (60 °C) and in NaCl concentrations of 0-4 % (1 %). The major cellular fatty acids were iso-C15 : 0 (12.8 %), iso-C16 : 0 (13.9 %) and iso-C17 : 0 (13.8 %). No matches were found in the rtsba6 Sherlock libraries. The G+C content of the genomic DNA was 42.11 mol%. Based on phylogenetic analysis of the 16S rRNA gene sequences, strain AYNT showed highest sequence similarity to the type strain of Geobacillus toebii (96 %). However, the phenotypic properties of strain AYN2T were clearly distinct from those of G. toebii and related species. On the basis of polyphasic analysis, strain AYN2T represents a novel species in the genus Geobacillus, for which the name Geobacillus yumthangensis sp. nov. is proposed. The type strain is AYN2T(MTCC=12749=KCTC=33950= JCM 32596).

New xylanolytic enzyme from Geobacillus galactosidasius BS61 from a geothermal resource in Turkey.

In this study, isolation, conventional and molecular characterizations of ten thermophilic bacteria from Rize/Ayder were carried out. Xylanase from Geobacillus galactosidasius BS61 (GenBank number: KX447660) was purified by acetone precipitation, Diethylaminoethyl-cellulose and Sephadex G-100 chromatographies. The xylanase of G. galactosidasius BS61 in clarifying fruit juice was also investigated. Enzyme was purified 29.80-fold with 75.18% yield; and molecular weight was determined as 78.15 kDa. The optimum temperature of xylanase was 60 °C. The enzyme activity was maintained fully after 24 h and over 50% after 168 h at pH 4.0-10.0, while optimum pH was 7.0. Km and Vmax for beech wood xylan were measured as 3.18 mg mL-1, 123 U mg protein-1. In addition, Ca2+, Na+, Al3+, Zn2+, Cd2+, Mg2+, Ni2+, Cu2+ had decreasing effect on enzyme activity, while enzyme activity had been protected against anions, especially HSO3- and HPO42- stimulated enzyme activity. Xylanase applications (with 15 U/mL enzyme activity) in orange and pomegranate juices were increased; and the sugar and turbidity amounts were reduced 17.36% ±â€¯1.18 and 30.52 ±â€¯1.23, respectively. These results indicated that the xylanase of G. galactosidasius BS61 has biotechnological potential in juice clarification due to its stability against metal ions, chemicals and high pH-values.

Pervasiveness of UVC254-resistant Geobacillus strains in extreme environments.

We have characterized a broad collection of extremophilic bacterial isolates from a deep subsurface mine, compost dumping sites, and several hot spring ecosystems. Spore-forming strains isolated from these environments comprised both obligate thermophiles/thermotolerant species (growing at > 55 °C; 240 strains) and mesophiles (growing at 15 to 40 °C; 12 strains). An overwhelming abundance of Geobacillus (81.3%) and Bacillus (18.3%) species was observed among the tested isolates. 16S rRNA sequence analysis documented the presence of 24 species among these isolates, but the 16S rRNA gene was shown to possess insufficient resolution to reliably discern Geobacillus phylogeny. gyrB-based phylogenetic analyses of nine strains revealed the presence of six known Geobacillus and one novel species. Multilocus sequence typing analyses based on seven different housekeeping genes deduced from whole genome sequencing of nine strains revealed the presence of three novel Geobacillus species. The vegetative cells of 41 Geobacillus strains were exposed to UVC254, and most (34 strains) survived 120 J/m2, while seven strains survived 300 J/m2, and cells of only one Geobacillus strain isolated from a compost facility survived 600 J/m2. Additionally, the UVC254 inactivation kinetics of spores from four Geobacillus strains isolated from three distinct geographical regions were evaluated and compared to that of a spacecraft assembly facility (SAF) clean room Geobacillus strain. The purified spores of the thermophilic SAF strain exhibited resistance to 2000 J/m2, whereas spores of two environmental Geobacillus strains showed resistance to 1000 J/m2. This study is the first to investigate UV resistance of environmental, obligately thermophilic Geobacillus strains, and also lays the foundation for advanced understanding of necessary sterilization protocols practiced in food, medical, pharmaceutical, and aerospace industries.

Biodegradation of sulfamethazine by an isolated thermophile-Geobacillus sp. S-07.

Sulfamethazine (SM2) is an antimicrobial drug that is frequently detected in manure compost, is difficult to degrade at high temperatures and is potentially threatening to the environment. In this study, a thermophilic bacterium was isolated from the activated sludge of an antibiotics pharmaceutical factory; this bacterium has the ability to degrade SM2 at 70 °C, which is higher than the traditional manure composting temperature. The strain S-07 is closely related to Geobacillus thermoleovorans based on its 16S rRNA gene sequence. The optimal conditions for the degradation of SM2 are 70 °C, pH 6.0, 50 rpm rotation speed and 50 mL of culture volume. More than 95% of the SM2 contained in media was removed via co-metabolism within 24 h, which was a much higher percentage than that of the type strain of G. thermoleovorans. The supernatant from the S-07 culture grown in SM2-containing media showed slightly attenuated antibacterial activity. In addition, strain S-07 was able to degrade other sulfonamides, including sulfadiazine, sulfamethoxazole and sulfamerazine. These results imply that strain S-07 might be a new auxiliary bacterial resource for the biodegradation of sulfonamide residue in manure composting.

Comparative genomic analysis of the flagellin glycosylation island of the Gram-positive thermophile Geobacillus.

BACKGROUND: Protein glycosylation involves the post-translational attachment of sugar chains to target proteins and has been observed in all three domains of life. Post-translational glycosylation of flagellin, the main structural protein of the flagellum, is a common characteristic among many Gram-negative bacteria and Archaea. Several distinct functions have been ascribed to flagellin glycosylation, including stabilisation and maintenance of the flagellar filament, motility, surface recognition, adhesion, and virulence. However, little is known about this trait among Gram-positive bacteria. RESULTS: Using comparative genomic approaches the flagellin glycosylation loci of multiple strains of the Gram-positive thermophilic genus Geobacillus were identified and characterized. Eighteen of thirty-six compared strains of the genus carry these loci, which show evidence of horizontal acquisition. The Geobacillus flagellin glycosylation islands (FGIs) can be clustered into five distinct types, which are predicted to encode highly variable glycans decorated with distinct and heavily modified sugars. CONCLUSIONS: Our comparative genomic analyses showed that, while not universal, flagellin glycosylation islands are relatively common among members of the genus Geobacillus and that the encoded flagellin glycans are highly variable. This suggests that flagellin glycosylation plays an important role in the lifestyles of members of this thermophilic genus.

Phylogenomic re-assessment of the thermophilic genus Geobacillus.

Geobacillus is a genus of Gram-positive, aerobic, spore-forming obligate thermophiles. The descriptions and subsequent affiliations of the species in the genus have mostly been based on polyphasic taxonomy rules that include traditional sequence-based methods such as DNA-DNA hybridization and comparison of 16S rRNA gene sequences. Currently, there are fifteen validly described species within the genus. The availability of whole genome sequences has provided an opportunity to validate and/or re-assess these conventional estimates of genome relatedness. We have applied whole genome approaches to estimate the phylogenetic relatedness among the sixty-three Geobacillus strains for which genome sequences are currently publicly available, including the type strains of eleven validly described species. The phylogenomic metrics AAI (Average Amino acid Identity), ANI (Average Nucleotide Identity) and dDDH (digital DNA-DNA hybridization) indicated that the current genus Geobacillus is comprised of sixteen distinct genomospecies, including several potentially novel species. Furthermore, a phylogeny constructed on the basis of the core genes identified from the whole genome analyses indicated that the genus clusters into two monophyletic clades that clearly differ in terms of nucleotide base composition. The G+C content ranges for clade I and II were 48.8-53.1% and 42.1-44.4%, respectively. We therefore suggest that the Geobacillus species currently residing within clade II be considered as a new genus.

Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein.

Regulation of translation is critical for maintaining cellular protein levels, and thus protein homeostasis. The conserved RNA-binding protein CsrA (also called RsmA; for carbon storage regulator and regulator of secondary metabolism, respectively; hereafter called CsrA) represents a well-characterized example of regulation at the level of translation initiation in bacteria. Binding of a CsrA homodimer to the 5'UTR of an mRNA occludes the Shine-Dalgarno sequence, blocking ribosome access for translation. Small noncoding RNAs (sRNAs) can competitively antagonize CsrA activity by a well-understood mechanism. However, the regulation of CsrA by the protein FliW is just emerging. FliW antagonizes the CsrA-dependent repression of translation of the flagellar filament protein, flagellin. Crystal structures of the FliW monomer reveal a novel, minimal ß-barrel-like fold. Structural analysis of the CsrA/FliW heterotetramer shows that FliW interacts with a C-terminal extension of CsrA. In contrast to the competitive regulation of CsrA by sRNAs, FliW allosterically antagonizes CsrA in a noncompetitive manner by excluding the 5'UTR from the CsrA-RNA binding site. Our phylogenetic analysis shows that the FliW-mediated regulation of CsrA regulation is the ancestral state in flagellated bacteria. We thus demonstrate fundamental mechanistic differences in the regulation of CsrA by sRNA in comparison with an ancient regulatory protein.

Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production.

The ability to grow at high temperatures makes thermophiles attractive for many fermentation processes. In this work, we used evolutionary engineering to increase ethanol production in the thermophile Geobacillus thermoglucosidasius. This bacterium is a facultative anaerobe, grows at an optimal temperature of 60°C, and can ferment diverse carbohydrates. However, it natively performs mixed-acid fermentation. To improve ethanol productivity, we first eliminated lactate and formate production in two strains of G. thermoglucosidasius, 95A1 and C56-YS93. These deletion strains were generated by selection on spectinomycin, which represents, to the best of our knowledge, the first time this antibiotic has been shown to work with thermophiles. Both knockout strains, however, were unable to grow under microaerobic conditions. We were able to recover growth in G. thermoglucosidasius 95A1 by serial adaptation in the presence of acetic acid. The evolved 95A1 strain was able to efficiently produce ethanol during growth on glucose or cellobiose. Genome sequencing identified loss-of-function mutations in adenine phosphoribosyltransferase (aprt) and the stage III sporulation protein AA (spoIIIAA). Disruption of both genes improved ethanol production in the unadapted strains: however, the increase was significant only when aprt was deleted. In conclusion, we were able to engineer a strain of G. thermoglucosidasius to efficiently produce ethanol from glucose and cellobiose using a combination of metabolic engineering and evolutionary strategies. This work further establishes this thermophile as a platform organism for fuel and chemical production. Biotechnol. Bioeng. 2016;113: 2156-2167. © 2016 Wiley Periodicals, Inc.

(13)C metabolic flux analysis of the extremely thermophilic, fast growing, xylose-utilizing Geobacillus strain LC300.

Thermophiles are increasingly used as versatile hosts in the biotechnology industry. One of the key advantages of thermophiles is the potential to achieve high rates of feedstock conversion at elevated temperatures. The recently isolated Geobacillus strain LC300 grows extremely fast on xylose, with a doubling time of less than 30 min. In the accompanying paper, the genome of Geobacillus LC300 was sequenced and annotated. In this work, we have experimentally validated the metabolic network model using parallel (13)C-labeling experiments and applied (13)C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], and [5-(13)C]xylose, was used for the first time. Isotopic labeling of biomass amino acids was measured by gas chromatography mass spectrometry (GC-MS). Isotopic labeling of carbon dioxide in the off-gas was also measured by an on-line mass spectrometer. The (13)C-labeling data was then rigorously integrated for flux elucidation using the COMPLETE-MFA approach. The results provided important new insights into the metabolism of Geobacillus LC300, its efficient xylose utilization pathways, and the balance between carbon, redox and energy fluxes. The pentose phosphate pathway, glycolysis and TCA cycle were found to be highly active in Geobacillus LC300. The oxidative pentose phosphate pathway was also active and contributed significantly to NADPH production. No transhydrogenase activity was detected. Results from this work provide a solid foundation for future studies of this strain and its metabolic engineering and biotechnological applications.

Characteristics of Newly Isolated Geobacillus sp. ZY-10 Degrading Hydrocarbons in Crude Oil.

An obligately thermophilic strain ZY-10 was isolated from the crude oil in a high-temperature oilfield, which was capable of degrading heavy crude oil. Phenotypic and phylogenetic analysis demonstrated that the isolate should be grouped in the genus Geobacillus, which shared thd highest similarity (99%) of the 16S rDNA sequence to Geobacillus stearothermophilus. However, the major cellular fatty acid iso-15:0 (28.55%), iso-16:0 (24.93%), iso-17:0 (23.53%) and the characteristics including indole production, tolerance to NaN3 and carbohydrate fermentation showed some difference from the recognized species in the genus Geobacillus. The isolate could use tridecane, hexadecane, octacosane and hexatridecane as sole carbon source for cell growth, and the digesting rate of long-chain alkane was lower than that of short-chain alkane. When the isolate was cultured in the heavy crude oil supplement with inorganic salts and trace yeast extract, the concentration of short-chain alkane was significantly increased and the content of long-chain alkane was decreased, suggesting that the larger hydrocarbon components in crude oil were degraded into shorter-chain alkane. Strain ZY-10 would be useful for improving the mobility of crude oil and upgrading heavy crude oil in situ.

The genus Geobacillus and their biotechnological potential.

The genus Geobacillus comprises a group of Gram-positive thermophilic bacteria, including obligate aerobes, denitrifiers, and facultative anaerobes that can grow over a range of 45-75°C. Originally classified as group five Bacillus spp., strains of Bacillus stearothermophilus came to prominence as contaminants of canned food and soon became the organism of choice for comparative studies of metabolism and enzymology between mesophiles and thermophiles. More recently, their catabolic versatility, particularly in the degradation of hemicellulose and starch, and rapid growth rates have raised their profile as organisms with potential for second-generation (lignocellulosic) biorefineries for biofuel or chemical production. The continued development of genetic tools to facilitate both fundamental investigation and metabolic engineering is now helping to realize this potential, for both metabolite production and optimized catabolism. In addition, this catabolic versatility provides a range of useful thermostable enzymes for industrial application. A number of genome-sequencing projects have been completed or are underway allowing comparative studies. These reveal a significant amount of genome rearrangement within the genus, the presence of large genomic islands encompassing all the hemicellulose utilization genes and a genomic island incorporating a set of long chain alkane monooxygenase genes. With G+C contents of 45-55%, thermostability appears to derive in part from the ability to synthesize protamine and spermine, which can condense DNA and raise its Tm.

Effects of ultrasonic-assisted thermophilic bacteria pretreatment on hydrolysis, acidification, and microbial communities in waste-activated sludge fermentation process.

A novel pretreatment method combining ultrasonic with thermophilic bacteria (Geobacillus sp. G1) was employed to pretreat waste-activated sludge (WAS) for enhancing the WAS hydrolysis and subsequent volatile fatty acids (VFAs) production. The soluble protein and carbohydrate were mostly released from intracellular ultrasonic-assisted Geobacillus sp. G1 pretreatment, and accumulated to 917 ± 70 and 772 ± 89 mg COD/L, respectively, which were 2.53- and 2.62-fold higher than that obtained in control test. Excitation emission matrix (EEM) fluorescence spectroscopy revealed the highest fluorescence intensity (FI) of protein-like substances, indicating the synergistic effect of ultrasonic and Geobacillus sp. G1 pretreatments on WAS hydrolysis. The maximum VFAs accumulation was 4437 ± 15 mg COD/L obtained in ultrasonic-assisted Geobacillus sp. G1 pretreatment test. High-throughput pyrosequencing analysis investigated that the microbial communities were substantial determined by the pretreatment used. The hydrolysis enhancement was caused by an increase in extracellular enzymes, which was produced by one of dominant species Caloramator sp. The positive effect was well explained to the enhancement of WAS hydrolysis and final VFAs accumulation.

Geobacillus icigianus sp. nov., a thermophilic bacterium isolated from a hot spring.

A Gram-reaction-positive, motile, thermophilic spore-forming strain, G1w1(T), was isolated from a hot spring of the Valley of Geysers, Kamchatka (Russia). Based on data from the present polyphasic taxonomic study, including phylogenetic analysis of 16S rRNA and spo0A gene sequences, the strain is considered to represent a novel species of the genus Geobacillus, for which the name Geobacillus icigianus sp. nov. is proposed. The type strain is G1w1(T) ( = VKM B-2853(T) = DSM 28325(T)).

Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping.