Recombinant Bacillus weihenstephanensis Elongation factor Tu (tuf)

What is Elongation factor Tu (tuf) and what is its significance in Bacillus weihenstephanensis?

Elongation factor Tu (EF-Tu), encoded by the tuf gene, is a critical component of the bacterial protein synthesis machinery that functions during the elongation phase of translation. In this process, EF-Tu forms a ternary complex with GTP and aminoacyl-tRNA, delivering the latter to the ribosome A-site during protein synthesis. The specific EF-Tu from Bacillus weihenstephanensis is of particular interest due to B. weihenstephanensis's psychrotolerant (cold-tolerant) nature, suggesting potential adaptations in the protein synthesis machinery that facilitate function at lower temperatures. The full sequence of B. weihenstephanensis EF-Tu consists of 396 amino acids, which forms the complete functional protein . Research interest in this protein stems from its potential role in cold adaptation mechanisms, which may provide insights into bacterial survival in refrigerated environments and applications in biotechnology requiring low-temperature enzymatic activity.

The significance of studying B. weihenstephanensis EF-Tu extends to food safety applications, as this psychrotolerant bacterium belongs to the Bacillus cereus group and can grow at temperatures as low as 7°C, potentially contaminating refrigerated foods . Understanding the functional properties of EF-Tu in this organism may contribute to developing better detection methods and control strategies for food safety. Additionally, comparative studies between psychrotolerant and mesophilic bacterial EF-Tu proteins can reveal evolutionary adaptations to different temperature niches, providing fundamental insights into protein structure-function relationships and environmental adaptation mechanisms in bacteria.

What are the optimal storage and handling conditions for recombinant B. weihenstephanensis EF-Tu?

Proper storage and handling of recombinant B. weihenstephanensis Elongation factor Tu is essential for maintaining its structural integrity and functional activity. According to product specifications, the recombinant protein should be stored at -20°C for regular use, while extended storage requires conservation at either -20°C or -80°C to prevent degradation and maintain stability . Repeated freezing and thawing cycles should be strictly avoided as they can significantly compromise protein quality through denaturation and aggregation. For researchers working with this protein over short periods, working aliquots can be maintained at 4°C for up to one week to minimize freeze-thaw damage while ensuring convenient access for experiments . Prior to opening any vial containing the lyophilized or reconstituted protein, brief centrifugation is recommended to bring the contents to the bottom and prevent loss of material.

For reconstitution, the recommended protocol involves using deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL . The addition of glycerol at 5-50% final concentration is strongly advised for aliquots intended for long-term storage, with 50% being the standard recommendation to prevent freezing damage. The shelf life of the reconstituted protein in liquid form is approximately 6 months when stored at -20°C or -80°C, while the lyophilized form maintains stability for up to 12 months under the same storage conditions . These careful handling procedures ensure that experiments utilizing this recombinant protein yield reliable and reproducible results by maintaining the structural and functional integrity of the EF-Tu protein throughout the research period.

How can researchers confirm the identity and taxonomic classification of B. weihenstephanensis strains?

Molecular identification of B. weihenstephanensis involves analyzing specific signature sequences in several genetic regions. The most reliable molecular markers include specific signature sequences in the 16S rRNA gene, the cold shock protein gene (cspA), and several housekeeping genes including glycerol uptake facilitator protein (glpF), guanylate kinase (gmK), phosphoribosylaminoimidazolecarboxamide formyltransferase (purH), and triosephosphate isomerase (tpi) . These signature sequences serve as reliable molecular identifiers that distinguish B. weihenstephanensis from other closely related species in the B. cereus group. Multilocus sequence analysis (MLSA) based on these housekeeping genes provides a robust method for classifying strains, with psychrotolerant B. cereus and B. mycoides strains containing B. weihenstephanensis-specific signature sequences clustering together in phylogenetic analyses .

For comprehensive strain identification, researchers should implement both phenotypic testing and molecular characterization. A recommended workflow begins with testing growth at 7°C and 43°C, followed by PCR amplification and sequencing of the signature regions in 16S rRNA, cspA, and the housekeeping genes mentioned above. Sequence analysis and comparison against reference strains will provide confirmation of taxonomic identity. Additionally, multiplex quantitative PCR (mqPCR) assays have been developed that can detect B. weihenstephanensis with high sensitivity (detection limit of 10 copy numbers) in various matrices including food samples , offering rapid identification capabilities for research and food safety applications.

What are the optimal growth conditions for B. weihenstephanensis and how do they influence protein expression?

Bacillus weihenstephanensis exhibits specific growth parameters that distinguish it from other members of the Bacillus cereus group, particularly in relation to temperature tolerance. The optimal growth temperature (Topt) for B. weihenstephanensis KBAB4 has been determined to be 31.91°C (with confidence intervals of 30.93°C to 32.60°C), which is substantially lower than the optimal growth temperatures of thermotrophic Bacillus species . The minimum growth temperature (Tmin) is approximately 2.72°C, confirming its psychrotolerant nature and ability to grow at refrigeration temperatures, while its maximum growth temperature (Tmax) is around 40.91°C, explaining its inability to grow at 43°C, which serves as a diagnostic criterion . Regarding pH tolerance, B. weihenstephanensis KBAB4 demonstrates optimal growth at pH 7.71 (pHopt), with a minimum pH (pHmin) of 4.35, indicating moderate acid tolerance . These growth parameters are critical considerations when designing experiments involving this organism, particularly for protein expression studies.

What methodologies are most effective for purifying recombinant B. weihenstephanensis EF-Tu?

Effective purification of recombinant Bacillus weihenstephanensis Elongation factor Tu requires a carefully designed protocol that preserves the protein's native structure and activity. Based on the available product information, commercially produced recombinant B. weihenstephanensis EF-Tu demonstrates a purity of >85% as verified by SDS-PAGE analysis . To achieve such purity levels in research settings, affinity chromatography represents the most efficient initial purification step, typically utilizing a histidine tag system if the recombinant protein is engineered with such a tag. The specific tag type will be determined during the manufacturing process and should be considered when designing the purification strategy . For histidine-tagged proteins, immobilized metal affinity chromatography (IMAC) using Ni-NTA or cobalt-based resins allows for specific binding of the target protein while permitting non-tagged proteins to flow through the column.

Following the initial affinity purification step, size exclusion chromatography (SEC) provides further purification while simultaneously allowing buffer exchange into conditions optimal for downstream applications or storage. For B. weihenstephanensis EF-Tu, which functions in a psychrotolerant organism, it is advisable to perform all purification steps at reduced temperatures (4-15°C) to maintain protein stability and native conformation. Ion exchange chromatography may be employed as an intermediate step between affinity and size exclusion chromatography, particularly if higher purity than 85% is required for specific applications. The theoretical isoelectric point of the protein, derived from its amino acid sequence, should guide the selection of anion or cation exchange chromatography and operational pH conditions.

Quality control of the purified protein should include SDS-PAGE analysis to confirm molecular weight and purity, Western blotting for identity confirmation if antibodies are available, and activity assays to verify functional integrity. Activity assays might include GTP binding assays, as EF-Tu is a GTPase, or tRNA binding studies to confirm functional capability. Mass spectrometry analysis can provide definitive confirmation of protein identity and detect any post-translational modifications. For B. weihenstephanensis EF-Tu specifically, functional assays at various temperatures (4°C, 20°C, 30°C, and 37°C) would be informative to characterize temperature-dependent activity profiles, reflecting the psychrotolerant nature of the source organism. Proper storage of the purified protein in appropriate buffer conditions with glycerol as a cryoprotectant is essential for maintaining stability during short-term and long-term storage periods.

How can researchers assess the impact of environmental stressors on EF-Tu function in B. weihenstephanensis?

Assessing the impact of environmental stressors on Elongation factor Tu function in Bacillus weihenstephanensis requires a multifaceted experimental approach that combines biochemical, molecular, and physiological techniques. Temperature represents a primary stressor of interest given B. weihenstephanensis's psychrotolerant nature, with experiments typically examining EF-Tu function across a temperature range from 4°C to 40°C to span the organism's growth boundaries . In vitro translation assays using purified B. weihenstephanensis EF-Tu can directly measure protein synthesis rates at different temperatures, potentially revealing adaptations that maintain functionality in cold conditions. These assays should include comparative analyses with EF-Tu from mesophilic Bacillus species to highlight functional differences that may contribute to psychrotolerance. GTPase activity assays measuring the rate of GTP hydrolysis by EF-Tu at different temperatures provide another direct measure of functional impact, as this activity is essential for the protein's role in translation.

pH stress represents another important variable to examine, particularly considering B. weihenstephanensis's pH growth range of 4.35 to above 7.71 . Researchers can assess EF-Tu stability and activity across this pH range using circular dichroism spectroscopy to detect structural changes and in vitro functional assays to measure activity changes. The impact of combined stressors, such as simultaneous exposure to suboptimal temperature and pH conditions, may reveal synergistic effects that are particularly relevant to understanding bacterial survival in complex natural environments. Oxidative stress, another common environmental challenge, can be simulated by exposing purified EF-Tu or B. weihenstephanensis cultures to hydrogen peroxide or other oxidizing agents, followed by activity assessments to determine functional resilience.

What techniques are available for studying the structural characteristics of B. weihenstephanensis EF-Tu?

Investigating the structural characteristics of Bacillus weihenstephanensis Elongation factor Tu requires the application of diverse biophysical and computational techniques. X-ray crystallography represents the gold standard for determining high-resolution protein structures, providing detailed atomic coordinates that reveal the three-dimensional arrangement of the protein. For B. weihenstephanensis EF-Tu, crystallization conditions would need to be optimized specifically for this psychrotolerant protein, potentially testing crystallization at different temperatures (4°C, 15°C, and 20°C) to identify conditions that yield well-diffracting crystals. Nuclear Magnetic Resonance (NMR) spectroscopy offers an alternative approach for structural determination that can also provide information about protein dynamics in solution, which may be particularly relevant for understanding cold adaptation mechanisms. While NMR is typically limited to smaller proteins, specific domains of EF-Tu could be analyzed using this method if the full-length protein proves challenging.

Computational approaches provide valuable complementary insights, particularly given the availability of the complete amino acid sequence of B. weihenstephanensis EF-Tu . Homology modeling using solved structures of EF-Tu from other Bacillus species as templates can generate preliminary structural models that highlight potential regions of interest. The amino acid sequence (MAKAKFERSK PHVNIGTIGH VDHGKTTLTA AITTVLAKAG GAEARGYDQI DAAPEERERG ITISTAHVEY ETETRHYAHV DCPGHADYVK NMITGAAQMD GGILVVSAAD GPMPQTREHI LLSRQVGVPY IVVFLNKCDM VDDEELLELV EMEVRDLLSE YGFPGDDIPV IKGSALKALQ GEAEWEEKII ELMTEVDAYI PTPERETDKP FLMPIEDVFS ITGRGTVATG RVERGIVKVG DVVEIIGLAE ENATTTVTGV EMFRKLLDQA QAGDNIGALL RGVAREDIQR GQVLAKTGSV KAHAKFKAEV FVLSKEEGGR HTPFFANYRP QFYFRTTDVT GIIQLPEGTE MVMPGDNIEM TIELIAPIAI EEGTKFSIRE GGRTVGYGVV ATIVAE) can be analyzed for unique features that might contribute to cold adaptation . Molecular dynamics simulations can further explore the dynamic behavior of the protein at different temperatures, potentially revealing flexibility characteristics that contribute to function in cold environments.

How does B. weihenstephanensis EF-Tu contribute to cold adaptation mechanisms?

The contribution of Elongation factor Tu to cold adaptation in Bacillus weihenstephanensis likely involves specific structural and functional modifications that maintain translation efficiency at low temperatures. Psychrotolerant bacteria must overcome challenges including decreased enzyme activity, reduced membrane fluidity, and stabilized RNA secondary structures at low temperatures, with the protein synthesis machinery being particularly vulnerable to cold-induced inhibition. EF-Tu, as a central component of the translational apparatus, requires specific adaptations to maintain function in cold environments where B. weihenstephanensis can grow (as low as 7°C) . These adaptations may include increased structural flexibility in specific regions that facilitate continued function at lower temperatures, where proteins from mesophilic organisms would become too rigid and functionally compromised. The amino acid composition of B. weihenstephanensis EF-Tu may show characteristics typical of cold-adapted proteins, such as reduced proline content, increased glycine content, reduced arginine/lysine ratio, or other substitutions that promote flexibility while maintaining stability at lower temperatures.

The GTPase activity of EF-Tu, which is essential for its function in translation, may exhibit a shifted temperature optimum in B. weihenstephanensis compared to mesophilic counterparts. While mesophilic EF-Tu typically shows optimal activity around 37°C, B. weihenstephanensis EF-Tu likely maintains significant activity at temperatures closer to its optimal growth temperature of approximately 32°C and retains substantial activity at even lower temperatures . This temperature-activity relationship would align with the organism's ability to grow at refrigeration temperatures. The interaction of EF-Tu with other components of the translation machinery, particularly aminoacyl-tRNAs and the ribosome, may also show temperature-dependent adaptations that maintain efficient protein synthesis in cold conditions. These interaction dynamics can significantly impact translation efficiency and fidelity at different temperatures.

Gene expression patterns also likely contribute to cold adaptation mechanisms involving EF-Tu. In many bacteria, cold shock induces increased expression of translation factors to compensate for reduced efficiency at low temperatures. B. weihenstephanensis may constitutively express higher levels of EF-Tu than mesophilic relatives when growing at low temperatures, or it may maintain robust expression across a broader temperature range. Research investigating comparative expression levels of the tuf gene across different Bacillus species at various temperatures would provide valuable insights into this regulatory aspect of cold adaptation. Additionally, post-translational modifications specific to B. weihenstephanensis EF-Tu might play a role in fine-tuning its activity according to environmental temperature, representing another potential adaptation mechanism that warrants investigation using proteomic approaches.

What role does EF-Tu play in the environmental distribution and food safety implications of B. weihenstephanensis?

The functional properties of Elongation factor Tu in Bacillus weihenstephanensis likely influence the organism's ecological distribution and food safety significance by supporting growth and survival in refrigerated environments. B. weihenstephanensis's psychrotolerant nature, demonstrated by its ability to grow at temperatures as low as 7°C, enables this bacterium to proliferate in cold environments where mesophilic competitors cannot grow . This ecological advantage in refrigerated settings has significant food safety implications, as conventional cold storage methods used to inhibit bacterial growth may not effectively control B. weihenstephanensis populations. The maintained functionality of the protein synthesis machinery at low temperatures, in which EF-Tu plays a critical role, directly contributes to this adaptation by allowing continued protein production and cellular maintenance under refrigeration conditions. Consequently, research into B. weihenstephanensis EF-Tu function at low temperatures has direct relevance to understanding the organism's ability to contaminate and grow in refrigerated foods.

Detection methods for B. weihenstephanensis in food matrices further highlight the practical significance of understanding this organism's distinctive characteristics. Multiplex quantitative PCR (mqPCR) assays have been developed that can detect B. weihenstephanensis with high sensitivity in food samples such as potato salad and milk . These detection methods have established detection dynamic ranges for B. weihenstephanensis of 10^5 CFU/mL to 10^1 CFU/mL in potato salad and 10^5 CFU/mL to 10^2 CFU/mL in milk . While these assays typically target conserved or species-specific genomic regions rather than the tuf gene specifically, understanding the molecular biology and cold adaptation mechanisms of B. weihenstephanensis, including the role of EF-Tu, provides context for interpreting detection results and assessing potential growth and toxin production risks in refrigerated foods.

The relationship between EF-Tu function and B. weihenstephanensis virulence potential requires further investigation. As a member of the Bacillus cereus group, B. weihenstephanensis may possess genes encoding enterotoxins similar to those produced by B. cereus, though their expression patterns and temperature dependence may differ. The efficiency of protein synthesis at refrigeration temperatures, facilitated by cold-adapted EF-Tu, could potentially support toxin production in contaminated foods even during cold storage. Research comparing the temperature dependence of toxin gene expression and translation efficiency between B. weihenstephanensis and mesophilic B. cereus strains would provide valuable insights into this food safety concern. Additionally, comparing EF-Tu sequences across different B. weihenstephanensis isolates from diverse environmental sources may reveal correlations between specific EF-Tu variants and ecological distribution patterns, potentially identifying signature adaptations associated with particular environmental niches or geographic regions.

How can comparative studies of EF-Tu across Bacillus species advance our understanding of bacterial evolution and adaptation?

Comparative analyses of Elongation factor Tu across Bacillus species offer a powerful approach for exploring evolutionary adaptations to diverse environmental niches, particularly temperature adaptation. The Bacillus genus encompasses remarkable ecological diversity, including psychrotolerant species like B. weihenstephanensis (optimal growth at ~32°C), mesophilic species like B. cereus (optimal growth at ~37°C), and thermophilic species like B. licheniformis (optimal growth at ~49°C) . This temperature adaptation spectrum provides an excellent model system for studying how highly conserved proteins like EF-Tu evolve to function optimally across different thermal environments while maintaining their essential cellular functions. Sequence alignments and phylogenetic analyses of EF-Tu proteins from these diverse Bacillus species can reveal specific amino acid substitutions that correlate with optimal growth temperature, identifying potential adaptive mutations. These analyses might reveal distinct patterns of conservation and variation in different functional domains of EF-Tu, such as the GTP-binding domain, the tRNA-binding domain, and regions involved in ribosome interaction.

Structural comparisons between EF-Tu proteins from psychrotolerant, mesophilic, and thermophilic Bacillus species can provide deeper insights into adaptation mechanisms. Cold-adapted proteins typically exhibit increased flexibility in specific regions compared to their mesophilic counterparts, while thermophilic proteins generally show increased rigidity and thermal stability. Biophysical characterization of recombinant EF-Tu proteins from different Bacillus species, including thermal stability measurements, conformational flexibility assessments, and kinetic analyses of GTPase activity across temperature ranges, can reveal how structural properties correlate with function at different temperatures. These comparative functional studies could demonstrate whether cold adaptation in B. weihenstephanensis EF-Tu involves a shift in temperature optima for activity, maintenance of activity over a broader temperature range, or altered interaction dynamics with other components of the translation machinery.

Evolutionary analyses examining selection pressures on the tuf gene across Bacillus species can identify whether specific regions of EF-Tu have experienced positive selection during adaptation to different thermal environments. These analyses may reveal whether thermal adaptation has occurred through similar or different molecular mechanisms across independent lineages that have adapted to similar environments, addressing fundamental questions about the predictability and constraints of adaptive evolution. The highly conserved nature of EF-Tu across bacterial species makes it particularly valuable for such studies, as it allows researchers to distinguish between general constraints on protein structure and function versus specific adaptations to environmental conditions. Additionally, examining the genomic context of the tuf gene across Bacillus species may reveal differences in gene regulation, copy number, or operon structure that contribute to thermal adaptation through altered expression patterns rather than protein sequence changes alone. These comparative genomic approaches complement protein-level analyses to provide a comprehensive understanding of how this essential component of the bacterial translation machinery has evolved to support growth across diverse thermal environments.

What methodological approaches are most effective for detecting B. weihenstephanensis in complex food matrices?

Effective detection of Bacillus weihenstephanensis in complex food matrices requires specialized methodological approaches that account for both its psychrotolerant nature and its genetic similarity to other members of the Bacillus cereus group. Multiplex quantitative PCR (mqPCR) assays have demonstrated high sensitivity and specificity for B. weihenstephanensis detection in food samples such as potato salad and milk, with detection limits as low as 10^1 CFU/mL in potato salad and 10^2 CFU/mL in milk . These molecular detection methods typically target signature sequences in the 16S rRNA gene, cspA gene, or specific housekeeping genes (glpF, gmK, purH, and tpi) that distinguish B. weihenstephanensis from closely related Bacillus species . For developing effective detection protocols, researchers should include appropriate DNA extraction procedures optimized for different food matrices, as food components can inhibit PCR reactions and affect assay sensitivity. Internal amplification controls should be incorporated to identify potential false negatives due to PCR inhibition, and appropriate positive and negative controls are essential for assay validation.

Cultural methods complementing molecular detection provide important physiological information about detected strains. The defining growth characteristic of B. weihenstephanensis—ability to grow at 7°C but not at 43°C—serves as a key confirmatory test . A recommended workflow combines initial selective enrichment at 7°C in appropriate media to favor psychrotolerant Bacillus species, followed by plating on selective media such as Mannitol Egg Yolk Polymyxin (MYP) agar or chromogenic Bacillus cereus selective media. Suspected colonies can then be subjected to confirmatory PCR testing and growth temperature profiling. For quantitative assessment in food samples, most probable number (MPN) methods combined with PCR confirmation can provide estimates of viable cell concentrations, while direct quantitative PCR on food samples offers rapid but less discriminatory quantification that includes both viable and non-viable cells.

Advanced techniques employing next-generation sequencing approaches offer powerful tools for complex sample analysis. Metagenomic sequencing can detect B. weihenstephanensis in the context of the entire microbial community of a food sample, revealing potential interactions with other microorganisms that might influence growth and toxin production. For highly sensitive detection, digital PCR methods can detect extremely low concentrations of target DNA, potentially identifying contamination below the threshold of conventional PCR methods. Immunological methods using antibodies specific to B. weihenstephanensis cell surface antigens represent another approach, though cross-reactivity with other Bacillus cereus group members remains a challenge. Each detection method has specific advantages and limitations, making a polyphasic approach combining cultural, molecular, and potentially immunological methods the most comprehensive strategy for reliable detection and characterization of B. weihenstephanensis in complex food matrices, particularly for research applications requiring detailed strain characterization beyond simple presence/absence determination.

What are the key physiological and molecular parameters of B. weihenstephanensis compared to other Bacillus species?

Bacillus weihenstephanensis exhibits distinct physiological and molecular characteristics that differentiate it from other members of the Bacillus cereus group and broader Bacillus genus. These differences are evident in growth parameters, genetic markers, and ecological distribution patterns. The table below summarizes key comparative parameters between B. weihenstephanensis and selected Bacillus species, providing reference data for researchers studying these organisms:

This comparative data highlights the psychrotolerant nature of B. weihenstephanensis, particularly its ability to grow at refrigeration temperatures while being unable to grow at higher temperatures typical of mesophilic and thermophilic Bacillus species. The phylogenetic placement of B. weihenstephanensis in Group II distinguishes it from mesophilic B. cereus strains in Group I, despite their close relatedness in the B. cereus group . These physiological differences reflect underlying genetic adaptations, including modifications to essential cellular components like Elongation factor Tu that must maintain functionality across the organism's growth temperature range.

The molecular characteristics distinguishing B. weihenstephanensis include specific signature sequences in the 16S rRNA gene, cspA gene, and several housekeeping genes (glpF, gmK, purH, and tpi) . These molecular markers serve as reliable identifiers for taxonomic classification and detection purposes. The distinctive temperature-dependent growth profile of B. weihenstephanensis has significant implications for food safety, as it enables this organism to grow in refrigerated foods where mesophilic competitors are inhibited. Understanding these physiological and molecular parameters is essential for researchers designing experiments involving B. weihenstephanensis and its proteins, including the Elongation factor Tu, as they provide the environmental context in which these proteins have evolved to function.

What are the recommended experimental conditions for working with recombinant B. weihenstephanensis EF-Tu?

Working with recombinant Bacillus weihenstephanensis Elongation factor Tu requires careful attention to experimental conditions to maintain protein stability and functionality. The table below provides detailed recommendations for various experimental procedures involving this protein:

These recommended conditions are derived from the physiological parameters of B. weihenstephanensis and standard protocols for working with recombinant proteins, particularly those from psychrotolerant organisms. The optimal growth temperature of B. weihenstephanensis (approximately 32°C) provides a reference point for protein activity studies, though researchers should test activity across a range of temperatures to characterize temperature-dependent functionality. Similarly, the optimal pH for growth (approximately 7.7) informs buffer selection for functional studies.

For researchers conducting comparative studies between B. weihenstephanensis EF-Tu and homologous proteins from mesophilic or thermophilic species, it is essential to maintain consistent experimental conditions across all proteins being compared. Temperature-dependent properties should be characterized using identical methodologies and buffer compositions, with temperature as the only variable. When reporting experimental results, researchers should provide detailed descriptions of all experimental conditions, including buffer compositions, protein concentrations, assay temperatures, and incubation times, to ensure reproducibility. Additionally, researchers should validate the functional activity of purified recombinant B. weihenstephanensis EF-Tu using appropriate assays, such as GTP binding or hydrolysis assays, to confirm that the protein retains its native activity before proceeding with more specialized experimental applications.