Structural and functional characterization of the bacterial biofilm activator RemA.

Bacillus subtilis can form structurally complex biofilms on solid or liquid surfaces, which requires expression of genes for matrix production. The transcription of these genes is activated by regulatory protein RemA, which binds to poorly conserved, repetitive DNA regions but lacks obvious DNA-binding motifs or domains. Here, we present the structure of the RemA homologue from Geobacillus thermodenitrificans, showing a unique octameric ring with the potential to form a 16-meric superstructure. These results, together with further biochemical and in vivo characterization of B. subtilis RemA, suggests that the protein can wrap DNA around its ring-like structure through a LytTR-related domain.

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.

Directed Computational Evolution of Quorum-Quenching Lactonases from the Amidohydrolase Superfamily.

In this work, we present a generalizable directed computational evolution protocol to effectively reduce the sequence space to be explored in rational enzyme design. The protocol involves in silico mutation modeling and substrate docking to rapidly identify mutagenesis hotspots that may enhance an enzyme's substrate binding and overall catalysis. By applying this protocol to a quorum-quenching Geobacillus kaustophilus lactonase, GKL, we generated 1,881 single mutants and docked high-energy intermediates of nine acyl homoserine lactones onto them. We found that Phe28 and Tyr99 were two hotspots that produced most of the predicted top 20 mutants. Of the 180 enzyme-substrate combinations (top 20 mutants × 9 substrates), 51 (28%) exhibited enhanced substrate binding and 22 (12%) had better overall activity when compared with wild-type GKL. X-ray crystallographic studies of Y99C and Y99P provided rationalized explanations for the enhancement in enzyme function and corroborated the utility of the protocol.

Anoxybacillus and Geobacillus biofilms in the dairy industry: effects of surface material, incubation temperature and milk type.

Anoxybacillus (A. flavithermus, A. kamchatkensis subsp. asachharedens, A. caldiproteolyticus and A. tepidamans) and Geobacillus (two strains of G. thermodenitrificans, G. thermoglucosidans and G. vulcanii) isolates and reference strains in whole milk were evaluated for their biofilm production on six different abiotic surfaces. G. thermodenitrificans DSM 465T had the highest cell counts (>4 log10 CFU cm-2) on glass and stainless steel (SS) at 55 and 65 °C, respectively. G. thermodenitrificans D195 had the highest counts on SS at 55 °C (>5 log10 CFU cm-2) and polyvinyl chloride (PVC) at 65 °C (>4 log10 CFU cm-2), indicating the existence of strain variation. The ideal surfaces for all strains were SS and glass at 55 °C, but their preferences were polystyrene and SS at 65 °C. Moreover, Anoxybacillus members were more prone to form biofilms in skim milk than in semi-skim and whole milk, whereas the results were the opposite for Geobacillus. Both the attachment and sporulation of Geobacillus in whole milk was higher than in semi-skim or skim milk. This study proposes that the surface material, temperature and milk type had a cumulative effect on biofilm formation.

Comparative transcriptomic analysis revealed the key pathways responsible for organic sulfur removal by thermophilic bacterium Geobacillus thermoglucosidasius W-2.

Biodesulfurization is a promising method to desulfurize sulfur-containing compounds in oil with its unique advantages, such as environment-friendly treatments and moderate reaction conditions. In this study, a thermophilic bacterium Geobacillus thermoglucosidasius W-2 was reported to show nearly 40% and 55% desulfurization rates on heavy oil with 2.81% and 0.46% initial total sulfur content, respectively. Subsequently, comparative transcriptome analysis indicated that several possible key desulfurization-related genes of this strain were found to be differentially up-regulated induced by benzothiophene and dibenzothiophene, respectively. These desulfurization-related genes were considered to conduct key step to convert organic sulfur to inorganic sulfur. Moreover, the characterization of thermophilic alkanesulfonate monooxygenase systems SsuD1/SsuE1 and SsuD2/SsuE2 revealed that the enzymes exhibit considerable thermal and pH stability and wide substrates applicability. These enzymes probably endowed the strain W-2 with the ability to desulfurize oil and eliminate the sulfur-containing surfactants. Thus, this study provides novel alkanesulfonate monooxygenase systems that have the application potential for heavy oil biodesulfurization, oil demulsification and other biocatalytic processes.

Genome analysis of a thermophilic exopolysaccharide-producing bacterium - Geobacillus sp. WSUCF1.

Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.

Low frequency ultrasound inactivation of thermophilic bacilli (Geobacillus spp. and Anoxybacillus flavithermus) in the presence of sodium hydroxide and hydrogen peroxide.

The vegetative cells and spores of Geobacillus spp. and Anoxybacillus flavithermus were subjected to 20 kHz ultrasound with a power ∼8 W. Ultrasonication had considerable effect on vegetative cells (5-log reduction in Geobacillus spp. and 1.6-log reduction in A.flavithermus). TEM imaging of the ultrasonicated vegetative cells showed an extensive damage both internally and externally. However, spores showed high resistance towards ultrasound treatment in the absence of NaOH and H2O2, although the outer layers such as the exosporium and the outer coat layer were disrupted, resulting in the reduced resistance of spores towards sonication. The combination of 0.12 M NaOH and 10 min ultrasonication inactivated 6 log spores of Geobacillus spp. A 7 log spore reduction of A.flavithermus was achieved by combining 0.17 M NaOH with 10 min ultrasonication. Ultrasonication combined with 1% H2O2 inactivated ∼7 log Geobacillus spp. spores in 6 min and ∼7 log A.flavithermus spores in 3 min. These ultrasound treatments in the presence of NaOH and H2O2 are synergistic as they showed a greater spore reduction when compared to NaOH combined with high temperature (85 °C), where only 1 and 3 log reduction was achieved in Geobacillus spp. and A.flavithermus spores, respectively.

Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species.

The genus Geobacillus comprises thermophilic bacilli capable of endospore formation. The members of this genus provide thermostable proteins and can be used in whole cell applications at elevated temperatures; therefore, these organisms are of biotechnological importance. While these applications have been described in previous reviews, the present paper highlights the environmental adaptations and genome diversifications of Geobacillus spp. and their applications in evolutionary-protein engineering. Despite their obligate thermophilic properties, Geobacillus spp. are widely distributed in nature. Because several isolates demonstrate remarkable properties for cell reproduction in their respective niches, they seem to exist not only as endospores but also as vegetative cells in diverse environments. This suggests their excellence in environmental adaptation via genome diversification; in fact, evidence suggests that Geobacillus spp. were derived from Bacillus spp. while diversifying their genomes via horizontal gene transfer. Moreover, when subjected to an environmental stressor, Geobacillus spp. diversify their genomes using inductive mutations and transposable elements to produce derivative cells that are adaptive to the stressor. Notably, inductive mutations in Geobacillus spp. occur more rapidly and frequently than the stress-induced mutagenesis observed in other microorganisms. Owing to this, Geobacillus spp. can efficiently generate mutant genes coding for thermostable enzyme variants from the thermolabile enzyme genes under appropriate selection pressures. This phenomenon provides a new approach to generate thermostable enzymes, termed as thermoadaptation-directed enzyme evolution, thereby expanding the biotechnological potentials of Geobacillus spp. In this review, we have discussed this approach using successful examples and major challenges yet to be addressed.

Survival and Adaptation of the Thermophilic Species Geobacillus thermantarcticus in Simulated Spatial Conditions.

Astrobiology studies the origin and evolution of life on Earth and in the universe. According to the panspermia theory, life on Earth could have emerged from bacterial species transported by meteorites, that were able to adapt and proliferate on our planet. Therefore, the study of extremophiles, i.e. bacterial species able to live in extreme terrestrial environments, can be relevant to Astrobiology studies. In this work we described the ability of the thermophilic species Geobacillus thermantarcticus to survive after exposition to simulated spatial conditions including temperature's variation, desiccation, X-rays and UVC irradiation. The response to the exposition to the space conditions was assessed at a molecular level by studying the changes in the morphology, the lipid and protein patterns, the nucleic acids. G. thermantarcticus survived to the exposition to all the stressing conditions examined, since it was able to restart cellular growth in comparable levels to control experiments carried out in the optimal growth conditions. Survival was elicited by changing proteins and lipids distribution, and by protecting the DNA's integrity.

Effect of moisture equilibration time and medium on contact angles of bacterial spores.

Contact angle measurement of microorganisms is often described in literature, either to investigate their hydrophobic characteristic or the adhesion behavior of cells. However, in some key aspects the preparation methods differ. Thus, it is difficult to compare results and to choose a procedure for repetition of measurements. The aim of this paper is to point out some critical points during microorganism film preparation that can alter the resulting contact angles. Depending on the moisturizing medium and equilibration time, contact angles differ significantly.

Evidence-based validation of quorum quenching from Lysinibacillus sphaericus and Geobacillus sp. in bioremediation of oil sludge.

Many studies on quorum quenching focus on the discovery and characterization of novel acyl-homoserine lactonases (AHL-lactonases) because these enzymes could be used in the control of diseases caused by Gram-negative bacteria. The effects of quorum quenching are also remarkable in the performance of bacterial consortia in applications such as bioremediation. In the current work, we demonstrated the presence of a potential novel AHL-lactonase-encoding locus (Bsph_3377) from Lysinibacillus sphaericus and Geobacillus sp. The deduced amino acid sequences for this enzyme possess the characteristic domains and motifs involved in Zn-binding from AHL lactonases and were grouped into 1 clade within the phylogeny of the lactonases from firmicutes, showing 70% of identity with the lactonase AhlS from Solibacillus silvestris. We demonstrated the locus transcription by RT-qPCR and its relationship with the suppression of the pathogenicity of Pectobacterium carotovorum. Additionally, we analyzed the interaction of these bacilli with a commercial consortium in the bioremediation of a hydrocarbon-contaminated soil, showing inhibitory effects on its establishment. These results represent a new contribution in the understanding of the potential biotechnological applications of L. sphaericus and Geobacillus sp. as well as in the research on antibacterial techniques based on quorum-sensing disruption.

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.

Changes in Sodium, Calcium, and Magnesium Ion Concentrations That Inhibit Geobacillus Biofilms Have No Effect on Anoxybacillus flavithermus Biofilms.

This study investigated the effects of varied sodium, calcium, and magnesium concentrations in specialty milk formulations on biofilm formation by Geobacillus spp. and Anoxybacillus flavithermus. The numbers of attached viable cells (log CFU per square centimeter) after 6 to 18 h of biofilm formation by three dairy-derived strains of Geobacillus and three dairy-derived strains of A. flavithermus were compared in two commercial milk formulations. Milk formulation B had relatively high sodium and low calcium and magnesium concentrations compared with those of milk formulation A, but the two formulations had comparable fat, protein, and lactose concentrations. Biofilm formation by the three Geobacillus isolates was up to 4 log CFU cm(-2) lower in milk formulation B than in milk formulation A after 6 to 18 h, and the difference was often significant (P ≤ 0.05). However, no significant differences (P ≤ 0.05) were found when biofilm formations by the three A. flavithermus isolates were compared in milk formulations A and B. Supplementation of milk formulation A with 100 mM NaCl significantly decreased (P ≤ 0.05) Geobacillus biofilm formation after 6 to 10 h. Furthermore, supplementation of milk formulation B with 2 mM CaCl2 or 2 mM MgCl2 significantly increased (P ≤ 0.05) Geobacillus biofilm formation after 10 to 18 h. It was concluded that relatively high free Na(+) and low free Ca(2+) and Mg(2+) concentrations in milk formulations are collectively required to inhibit biofilm formation by Geobacillus spp., whereas biofilm formation by A. flavithermus is not impacted by typical cation concentration differences of milk formulations.

Secreted protease mediates interspecies interaction and promotes cell aggregation of the photosynthetic bacterium Chloroflexus aggregans.

Interspecies interactions were studied in hot spring microbial mats where diverse species of bacterial cells are densely packed. The anoxygenic photosynthetic bacterium, Chloroflexus aggregans, has been widely found in the microbial mats as a major component in terrestrial hot springs in Japan at the temperature from 50 to 70°C. C. aggregans shows cellular motility to form a microbial mat-like dense cell aggregate. The aggregating ability of C. aggregans was affected by another bacterial species, strain BL55a (related to Bacillus licheniformis) isolated from the microbial mats containing C. aggregans. Cell aggregation rate of C. aggregans was promoted by the addition of culture supernatants of strain BL55a. Similar effects were also detected from other bacterial isolates, specifically Geobacillus sp. and Aeribacillus sp. Protease activity was detected from the culture supernatants from all of these isolates. The promoting effect of strain BL55a was suppressed by a serine protease inhibitor, phenylmethylsulfonyl fluoride. A purified serine protease, subtilisin obtained from B. licheniformis, showed a promoting effect on the cell aggregation. These results suggest that an extracellular protease, secreted from co-existing bacterial species promoted the aggregating motility of C. aggregans. This is the first report that exogenous protease affects bacterial cellular motility.

Inactivation of chemical and heat-resistant spores of Bacillus and Geobacillus by nitrogen cold atmospheric plasma evokes distinct changes in morphology and integrity of spores.

Bacterial spores are resistant to severe conditions and form a challenge to eradicate from food or food packaging material. Cold atmospheric plasma (CAP) treatment is receiving more attention as potential sterilization method at relatively mild conditions but the exact mechanism of inactivation is still not fully understood. In this study, the biocidal effect by nitrogen CAP was determined for chemical (hypochlorite and hydrogen peroxide), physical (UV) and heat-resistant spores. The three different sporeformers used are Bacillus cereus a food-borne pathogen, and Bacillus atrophaeus and Geobacillus stearothermophilus that are used as biological indicators for validation of chemical sterilization and thermal processes, respectively. The different spores showed variation in their degree of inactivation by applied heat, hypochlorite, hydrogen peroxide, and UV treatments, whereas similar inactivation results were obtained with the different spores treated with nitrogen CAP. G. stearothermophilus spores displayed high resistance to heat, hypochlorite, hydrogen peroxide, while for UV treatment B. atrophaeus spores are most tolerant. Scanning electron microscopy analysis revealed distinct morphological changes for nitrogen CAP-treated B. cereus spores including etching effects and the appearance of rough spore surfaces, whereas morphology of spores treated with heat or disinfectants showed no such changes. Moreover, microscopy analysis revealed CAP-exposed B. cereus spores to turn phase grey conceivably because of water influx indicating damage of the spores, a phenomenon that was not observed for non-treated spores. In addition, data are supplied that exclude UV radiation as determinant of antimicrobial activity of nitrogen CAP. Overall, this study shows that nitrogen CAP treatment has a biocidal effect on selected Bacillus and Geobacillus spores associated with alterations in spore surface morphology and loss of spore integrity.

Optimized culture condition for enhancing lytic performance of waste activated sludge by Geobacillus sp. G1.

Hydrolysis is known as the rate-limiting step during waste activated sludge (WAS) digestion. The optimization of the culture conditions of Geobacillus sp. G1 for enhancing WAS hydrolysis was conducted in this study with uniform design and response surface methodology. Taking the lysis rate of Escherichia coli as the response, the Plackett-Burman design was used to screen the most important variables. Experimental results showed that the maximum predicted lysis rate of E. coli was 50.9% for 4 h treatment time with concentrations of skim milk, NaCl and NH4SO4 at 10.78, 4.36 and 11.28 g/L, respectively. The optimized dosage ratio of Geobacillus sp. G1 to WAS was 35%:65% (VG1:VWAS). Under this condition, soluble protein was increased to 695 mg chemical oxygen demand (COD)/L, which was 5.0 times higher than that obtained in the control (140 mg COD/L). The corresponding protease activity reached 1.1 Eu/mL. Scanning electron microscopy showed that abundant cells were apparently lysed with treatment of Geobacillus sp. G1.

The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet?

The genus Geobacillus comprises endospore-forming obligate thermophiles. These bacteria have been isolated from cool soils and even cold ocean sediments in anomalously high numbers, given that the ambient temperatures are significantly below their minimum requirement for growth. Geobacilli are active in environments such as hot plant composts, however, and examination of their genome sequences reveals that they are endowed with a battery of sensors, transporters and enzymes dedicated to hydrolysing plant polysaccharides. Although they appear to be relatively minor members of the plant biomass-degrading microbial community, Geobacillus bacteria have achieved a significant population with a worldwide distribution, probably in large part due to adaptive features of their spores. First, their morphology and resistance properties enable them to be mobilized in the atmosphere and transported long distances. Second, their longevity, which in theory may be extreme, enables them to lie quiescent but viable for long periods of time, accumulating gradually over time to achieve surprisingly high population densities.

Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis†KBAB4.

Temperate phages can integrate their genomes into a specific region of a host chromosome to produce lysogens (prophage). During genome insertion, prophages may interrupt the gene coding sequence. In Bacillus subtilis, the sigma factor gene sigK is interrupted by a 48 kb prophage-like element. sigK is a composite coding sequence from two partial genes during sporulation. For over two decades, however, no further examples of DNA element-mediated gene reconstitution other than sigK have been identified in spore formers. Here we report that the gene for dipicolinic acid (DPA) synthetase ß subunit spoVFB in B. weihenstephanensis KBAB4 is interrupted by a prophage-like element named vfbin. DPA is synthesized in the mother cell and required for maintaining spore dormancy. We found that spoVFB was a composite coding sequence generated in the mother cell via chromosomal rearrangement that excised vfbin. Furthermore, vfbin caused excision after phage-inducer treatment, but vfbin appeared to be defective as a prophage. We also found various spore-forming bacteria in which sporulation-related genes were disrupted by prophage-like DNA elements. These results demonstrate the first example of a similar mechanism that affects a sporulation gene other than sigK and suggest that this prophage-mediated DNA rearrangement is a common phenomenon in spore-forming bacteria.

Preconditioning with cations increases the attachment of Anoxybacillus flavithermus and Geobacillus species to stainless steel.

Preconditioning of Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 with cations prior to attachment often significantly increased (P ≤ 0.05) the number of viable cells that attached to stainless steel (by up to 1.5 log CFU/cm(2)) compared with unconditioned bacteria. It is proposed that the transition of A. flavithermus and Geobacillus spp. from milk formulations to stainless steel product contact surfaces in milk powder manufacturing plants is mediated predominantly by bacterial physiological factors (e.g., surface-exposed adhesins) rather than the concentrations of cations in milk formulations surrounding bacteria.

Geobacillus thermoglucosidasius endospores function as nuclei for the formation of single calcite crystals.

Geobacillus thermoglucosidasius colonies were placed on an agar hydrogel containing acetate, calcium ions, and magnesium ions, resulting in the formation of single calcite crystals (calcites) within and peripheral to the plating area or parent colony. Microscopic observation of purified calcites placed on the surface of soybean casein digest (SCD) nutrient medium revealed interior crevices from which bacterial colonies originated. Calcites formed on the gel contained [1-(13)C]- and [2-(13)C]acetate, demonstrating that G. thermoglucosidasius utilizes carbon derived from acetate for calcite formation. During calcite formation, vegetative cells swam away from the parent colony in the hydrogel. Hard-agar hydrogel inhibited the formation of calcites peripheral to the parent colony. The calcite dissolved completely in 1 M HCl, with production of bubbles, and the remaining endospore-like particles were easily stained with Brilliant green dye. The presence of DNA and protein in calcites was demonstrated by electrophoresis. We propose that endospores initiate the nucleation of calcites. Endospores of G. thermoglucosidasius remain alive and encapsulated in calcites.