Recombinant Bacillus licheniformis UPF0477 protein BLi01284/BL02661 (BLi01284, BL02661)

Which expression systems have been successfully used for recombinant production of BLi01284/BL02661?

Multiple expression systems have been successfully utilized for the recombinant production of BLi01284/BL02661, each with specific advantages depending on research objectives :

For studies focusing on basic biochemical characterization, E. coli systems typically provide sufficient quantity and quality of the recombinant protein. For more complex analyses requiring native-like post-translational modifications, the baculovirus or mammalian systems are recommended despite their higher cost and technical complexity .

What methodology is recommended for purification of recombinant BLi01284/BL02661?

Purification of recombinant BLi01284/BL02661 can be achieved through a multi-step process tailored to the expression system used. A methodological approach includes:

• Cell lysis: For E. coli systems, sonication or high-pressure homogenization in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 1 mM PMSF is recommended .

• Initial purification: Affinity chromatography using the appropriate tag (typically His-tag or Avi-tag as mentioned in the product descriptions) with imidazole gradient elution .

• Secondary purification: Size exclusion chromatography using Superdex 75 or 200 columns to remove aggregates and achieve >90% purity.

• Quality control: SDS-PAGE analysis to confirm >85% purity as indicated in product specifications , followed by Western blotting and mass spectrometry to verify protein identity.

For proteins expressed with the Avi-tag biotinylation system, streptavidin-based affinity purification can provide excellent purity in a single step. In cases where inclusion bodies form, particularly in E. coli systems, additional refolding steps using gradual dialysis against decreasing concentrations of urea or guanidine hydrochloride are necessary to obtain active protein .

How does the promoter selection affect expression efficiency of BLi01284/BL02661 in Bacillus licheniformis as a host system?

The selection of appropriate promoters significantly impacts the expression efficiency of BLi01284/BL02661 in Bacillus licheniformis host systems. Recent studies have characterized several promoter options with varying strengths and induction characteristics :

For optimal expression of BLi01284/BL02661 in B. licheniformis, the PbacA promoter has demonstrated superior performance for constitutive expression, while the Prha system offers excellent control for inducible expression . When using the rhamnose-inducible system, it's important to note that B. licheniformis requires approximately 36 hours to deplete 20 g/L of rhamnose, compared to just 9 hours for glucose .

Advanced promoter engineering approaches such as hybrid promoter construction and RBS (ribosome binding site) engineering can further enhance expression by 2-5 fold compared to standard promoters .

What are the methodological approaches to study the biochemical function of this putative phosphoesterase?

To elucidate the biochemical function of BLi01284/BL02661 as a putative phosphoesterase, a comprehensive methodological workflow should include:

• Substrate screening assay: Test activity against a panel of phosphorylated substrates including:

  • p-nitrophenyl phosphate (general phosphatase activity)

  • Various phosphorylated nucleotides (AMP, ADP, ATP)

  • Phosphorylated proteins/peptides

  • Phospholipids

• Enzyme kinetics characterization:

  • Determine optimal pH and temperature conditions (based on B. licheniformis native environment)

  • Measure Km, Vmax, and kcat values for identified substrates

  • Evaluate the effects of potential inhibitors and activators

• Metal ion dependency analysis: As many phosphoesterases require metal cofactors, systematically test activity in the presence of various metal ions (Mg²⁺, Mn²⁺, Zn²⁺, Ca²⁺) and with EDTA to establish cofactor requirements.

• Structural biology approaches:

  • X-ray crystallography or cryo-EM to determine three-dimensional structure

  • Site-directed mutagenesis of predicted catalytic residues based on structural data

  • Molecular docking of potential substrates

• In vivo functional validation:

  • Generate knockout strains of BLi01284 in B. licheniformis

  • Perform comparative metabolomics and phenotype analysis

  • Conduct complementation studies with wild-type and mutant versions

This multi-faceted approach has successfully elucidated the function of previously uncharacterized proteins in related Bacillus species and would be appropriate for determining the specific role of BLi01284/BL02661 .

How can multiple ribosomal binding sites enhance the expression of BLi01284/BL02661 in Bacillus licheniformis?

Recent breakthrough research has demonstrated that incorporating multiple ribosomal binding sites (RBSs) within a single mRNA leader sequence can dramatically enhance protein expression in Bacillus licheniformis . When applied to BLi01284/BL02661 expression, this methodology could provide significant advantages:

The mechanism involves the construction of expression plasmids carrying the BLi01284/BL02661 gene with varying numbers of RBSs within the mRNA leader region. Research has shown that protein expression levels increased proportionally with the number of RBSs, with dramatic improvements observed when increasing from one to four RBSs .

Quantitative data from similar experiments with GFP showed:

• Six RBSs increased fluorescence intensity 5-fold compared to a single RBS

• Protein with six RBSs constituted >50% of total intracellular protein

• The translation efficiency of six RBSs was approximately 100 times higher than previously strong promoters coupled with native 5'-UTRs

To implement this methodology for BLi01284/BL02661:

• Design and construction: Use the one-step method for ligation of tandem repeat sequences as described by researchers to create expression constructs with multiple RBSs (1, 3, 5, and 6 RBSs).

• Expression optimization: Clone these constructs into pHY300-PLK compatible plasmid with the P43 promoter.

• Quantification methodology: Measure protein production through SDS-PAGE densitometry, Western blotting, and phosphoesterase activity assays if functional assays are available.

This approach is particularly valuable for difficult-to-express proteins or when high yields are required for structural or biochemical studies. The technique has been proven effective for both intracellular and secreted proteins in B. licheniformis .

What are the challenges in resolving protein insolubility issues when expressing BLi01284/BL02661 in E. coli systems?

Expression of BLi01284/BL02661 in E. coli frequently results in inclusion body formation, creating significant challenges for obtaining soluble, functional protein . Addressing these insolubility issues requires a systematic approach:

Challenges and Solutions for BLi01284/BL02661 Insolubility:

• Expression conditions optimization:

  • Temperature reduction: Lowering expression temperature to 16-20°C can significantly improve folding

  • Induction modulation: Using lower IPTG concentrations (0.1-0.5 mM instead of 1 mM) and extending expression time

  • Media formulation: Supplementing with osmolytes like sorbitol (0.5 M) and betaine (2.5 mM) that act as chemical chaperones

• Fusion tag selection:

  • Solubility-enhancing tags: MBP (maltose-binding protein) and SUMO tags have shown superior performance over His-tags for similar Bacillus proteins

  • Tag position effects: N-terminal vs. C-terminal tag placement can significantly affect folding dynamics

• Co-expression strategies:

  • Chaperone co-expression: GroEL/GroES, DnaK/DnaJ/GrpE systems can assist proper folding

  • Rare codon supplementation: Co-expression of rare tRNAs for codons frequently used in Bacillus but rare in E. coli

• Refolding methodologies (when inclusion bodies are unavoidable):

  • Step-wise dialysis: Gradually reducing denaturant concentration over 24-48 hours

  • On-column refolding: Immobilizing denatured protein on affinity resin before refolding

  • Pulsatile refolding: Introducing protein into refolding buffer in pulses to prevent aggregation

• Buffer optimization:

  • Addition of low concentrations of non-ionic detergents (0.05% Triton X-100)

  • Inclusion of stabilizing agents like L-arginine (0.4-0.8 M)

  • Testing various pH conditions (pH 6.0-9.0) to identify optimal stability range

For phosphoesterases like BLi01284/BL02661, inclusion of potential cofactors (Mg²⁺, Mn²⁺) in the lysis and purification buffers often enhances stability and solubility by promoting proper folding through metal ion coordination .

How conserved is UPF0477 protein BLi01284/BL02661 across different Bacillus species, and what does this suggest about its function?

Genomic analysis of UPF0477 protein BLi01284/BL02661 across Bacillus species reveals important evolutionary patterns that provide insights into its potential function. Comparative genomic approaches have shown:

Orthologous genes to BLi01284/BL02661 are present in several closely related Bacillus species with varying degrees of sequence conservation :

The high conservation of this protein among soil-dwelling and industrially relevant Bacillus species suggests it may play a role in core metabolic functions rather than specialized adaptations. Amino acid residues predicted to be involved in catalytic activity show particularly high conservation (>95% identity), supporting the phosphoesterase functional annotation .

Genomic context analysis indicates that BLi01284/BL02661 is often located in proximity to genes involved in nucleotide metabolism and stress response pathways in most Bacillus species. This syntenic arrangement provides additional evidence that the protein may function in phosphate-related metabolic processes or stress signaling pathways .

Pan-genome analysis comparing B. licheniformis CBA7126 with closely related strains identified BLi01284/BL02661 as part of the core genome shared among all analyzed B. licheniformis strains, further supporting its importance in fundamental cellular processes .

What methodological approaches can be used to investigate the role of BLi01284/BL02661 in Bacillus licheniformis stress responses?

Investigation of BLi01284/BL02661's potential role in B. licheniformis stress responses requires a multi-faceted methodological approach:

• Transcriptomic analysis under stress conditions:

  • RNA-Seq analysis of B. licheniformis exposed to various stressors (osmotic stress, oxidative stress, heat shock)

  • qRT-PCR validation of BLi01284/BL02661 expression patterns during stress response

  • Promoter-reporter fusion constructs (using GFP or luciferase) to visualize temporal expression patterns

• Gene knockout and complementation studies:

  • CRISPR/Cas9 gene editing to create BLi01284 deletion mutants

  • Phenotypic characterization of mutants under various stress conditions

  • Complementation with wild-type and site-directed mutants to confirm phenotypes

• Protein-protein interaction network mapping:

  • Bacterial two-hybrid screening to identify interaction partners

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity-dependent biotin labeling (BioID) to identify transient interactions

• Phosphoproteome analysis:

  • Comparative phosphoproteomics between wild-type and ΔBLi01284 strains

  • Identification of differentially phosphorylated proteins during stress response

  • In vitro validation of potential substrate proteins

This methodological approach is supported by previous studies on B. licheniformis stress responses, which have shown that osmotic stress triggers complex transcriptional changes involving multiple systems, including SigB-controlled general stress response genes . The osmostress response in B. licheniformis involves synthesis and import of compatible solutes along with secondary oxidative stress responses , providing a framework for investigating potential roles of BLi01284/BL02661 in these pathways.

Based on research on osmotic stress in B. licheniformis, methodologies using 1M NaCl shock treatments followed by time-resolved transcriptional profiling would be particularly relevant for studying this protein's potential stress-related functions .

How can comparative genomics be leveraged to predict the biochemical function of UPF0477 protein BLi01284/BL02661?

Comparative genomics offers powerful approaches to predict the biochemical function of uncharacterized proteins like UPF0477 protein BLi01284/BL02661:

• Phylogenetic profiling methodology:

  • Construct a presence/absence matrix of BLi01284/BL02661 homologs across diverse bacterial species

  • Identify co-evolving genes that show similar phylogenetic distribution patterns

  • Apply statistical methods (mutual information, Pearson correlation) to quantify co-evolution strength

  • Functional prediction based on characterized co-evolving genes

• Gene neighborhood analysis:

  • Examine the genomic context of BLi01284/BL02661 across multiple Bacillus genomes

  • Identify conserved gene clusters and operonic structures

  • Apply the "guilt by association" principle to infer function based on neighboring genes with known functions

• Structural bioinformatics approaches:

  • Predict three-dimensional structure using homology modeling or AlphaFold2

  • Identify structural homologs using fold recognition algorithms

  • Analyze conserved domains and potential active sites

  • Predict binding pockets and potential substrates through molecular docking

• Integrated functional networks:

  • Combine multiple genomic features (co-expression, protein-protein interactions, genomic proximity)

  • Use machine learning algorithms to weight and integrate diverse evidence types

  • Validate predictions through targeted experimental approaches

Application of these methodologies to related Bacillus proteins has successfully predicted functions later confirmed experimentally. For example, OrthoANI (Orthologous Average Nucleotide Identity) analysis has been used to classify B. licheniformis strains and identify strain-specific functions . The genome structure comparison between B. licheniformis CBA7126 and related strains using MAUVE alignment has revealed important functional elements that could inform the analysis of BLi01284/BL02661 .

Published research indicates that B. licheniformis CBA7126 possesses 19 unique genes compared to closely related strains, with functions related to carbon metabolism and prophage elements . Similar comparative approaches could reveal the specialized role of BLi01284/BL02661 within B. licheniformis metabolic networks.

What experimental approaches can determine if BLi01284/BL02661 plays a role in ergothioneine biosynthesis in engineered B. licheniformis strains?

Recent research has established B. licheniformis as an effective platform for ergothioneine (EGT) production . To investigate whether BLi01284/BL02661 plays a role in this biosynthetic pathway, the following experimental approaches are recommended:

• Gene expression correlation analysis:

  • Quantify BLi01284/BL02661 expression levels during different phases of EGT production using RT-qPCR

  • Compare expression patterns with known EGT biosynthetic genes (EanA, EanB, EanAN, EanBN)

  • Analyze RNA-Seq data from EGT-producing strains to identify co-expressed gene clusters

• Gene disruption and overexpression studies:

  • Create a BLi01284 knockout strain using CRISPR/Cas9 gene editing

  • Measure EGT production in the knockout strain versus wild-type (using HPLC analysis as described in )

  • Overexpress BLi01284/BL02661 in EGT-producing strains and quantify impact on production

  • Perform complementation studies with mutant variants to identify essential domains

• Metabolomic analysis:

  • Compare metabolite profiles between wild-type and BLi01284-modified strains

  • Focus on intermediates in the EGT pathway (hercynine, hercynylcysteine sulfoxide)

  • Use LC-MS/MS to quantify changes in metabolic flux through the pathway

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation experiments with BLi01284/BL02661 and known EGT biosynthetic enzymes

  • Use bacterial two-hybrid assays to screen for interactions

  • Employ in vitro reconstitution of the EGT pathway with and without BLi01284/BL02661

Based on the methodologies used in successful EGT production studies, experiments should be conducted under optimal conditions for EGT synthesis: 37°C incubation, 250 rpm shaking, with appropriate substrate amino acids (particularly cysteine, which was shown to be rapidly utilized in EGT production) .

If BLi01284/BL02661 functions as a phosphoesterase in nucleotide metabolism, it might indirectly affect EGT biosynthesis through modulation of energy metabolism or regulation of biosynthetic gene expression, making these connections important to investigate.

How might BLi01284/BL02661 contribute to the antimicrobial properties of B. licheniformis?

B. licheniformis is known for producing various antimicrobial substances, including bacteriocins, bacitracins, and licheniformins with significant antimycobacterial activity . To investigate whether BLi01284/BL02661 contributes to these antimicrobial properties:

• Genetic manipulation and bioactivity testing:

  • Generate BLi01284 knockout and overexpression strains

  • Compare antimicrobial activity using agar diffusion assays against indicator strains (particularly Mycobacterium species)

  • Quantify production of known antimicrobials (bacitracin, licheniformins) using HPLC and bioassays

  • Perform complementation studies with site-directed mutants targeting predicted catalytic residues

• Secretome analysis:

  • Compare protein profiles in culture supernatants from wild-type and BLi01284-modified strains

  • Employ LC-MS/MS to identify differentially abundant antimicrobial peptides

  • Investigate post-translational modifications of secreted antimicrobials that might require phosphoester processing

• Transcriptional regulation studies:

  • Analyze expression of antimicrobial biosynthetic gene clusters in BLi01284 knockout versus wild-type

  • Investigate whether BLi01284/BL02661 affects quorum sensing systems that regulate antimicrobial production

  • Use chromatin immunoprecipitation sequencing (ChIP-seq) to identify potential regulatory interactions

• Phosphorylation state analysis:

  • If BLi01284/BL02661 functions as a phosphoesterase, investigate the phosphorylation status of regulatory proteins involved in antimicrobial synthesis

  • Employ phosphoproteomic analysis to identify potential substrates

  • Perform in vitro dephosphorylation assays with purified BLi01284/BL02661 and candidate substrates

Current research indicates that B. licheniformis has unique capabilities in synthesizing and producing a range of antibacterial compounds . If BLi01284/BL02661 is involved in regulatory phosphorylation/dephosphorylation events, it could potentially impact the expression or activation of these antimicrobial systems, particularly through interaction with transcriptional regulators like DegU, AbrB, or CcpA that are known to be involved in B. licheniformis' regulatory networks .

How can advanced promoter engineering strategies be applied to study BLi01284/BL02661 function through controlled expression?

Advanced promoter engineering strategies provide powerful tools for studying BLi01284/BL02661 function through precisely controlled expression. Recent developments in B. licheniformis promoter technology enable sophisticated experimental approaches:

• Inducible expression systems optimization:

  • Implement rhamnose-inducible (Prha) promoter systems for titratable expression of BLi01284/BL02661

  • Utilize mannose-inducible promoters (Pman) for gradual induction

  • Employ hybrid promoters combining the strength of constitutive promoters with regulatory elements from inducible systems

  • Optimize induction conditions based on documented response curves (e.g., rhamnose concentration 0-20 g/L)

• Transcription factor-based promoter engineering:

  • Incorporate recognition sites for key transcription factors (DegU, AbrB, CcpA, GlnR) to create synthetic promoters with custom regulation profiles

  • Design promoters responding to specific environmental conditions relevant to BLi01284/BL02661's hypothesized function

  • Use characterized transcription factor binding sites to build promoters with desired expression dynamics

• Multiple ribosome binding site (RBS) integration:

  • Apply the multi-RBS technology demonstrated in recent research to achieve ultra-high expression levels when needed

  • Construct expression cassettes with 1-6 RBSs to create an expression gradient for dose-response studies

  • Implement the one-step ligation method for tandem repeat sequences to efficiently generate multi-RBS constructs

  • Combine with secretion signals if export of BLi01284/BL02661 is desired

• Experimental application methodology:

  • Create a series of strains with BLi01284/BL02661 under different promoter controls

  • Monitor phenotypic effects across a spectrum of expression levels

  • Correlate expression level with specific cellular functions using transcriptomics and metabolomics

  • Identify minimum expression level required for function and toxic threshold

These strategies can be particularly powerful when combined with reporter systems (such as GFP fusion) to monitor expression levels in real-time. The documented 5-fold increase in protein production achieved with six RBSs compared to single RBS constructs provides a quantifiable range for expression tuning experiments.

For studying potential stress-response roles, combining stress-responsive promoters with BLi01284/BL02661 expression could reveal important functional relationships and regulatory mechanisms that would be difficult to elucidate with constitutive expression systems.

What mass spectrometry approaches are most effective for characterizing post-translational modifications of BLi01284/BL02661?

Characterizing potential post-translational modifications (PTMs) of BLi01284/BL02661 requires sophisticated mass spectrometry approaches tailored to phosphoesterase proteins:

• Sample preparation methodology:

  • Parallel purification from multiple expression systems (E. coli, B. licheniformis native)

  • Phosphatase inhibitor inclusion during extraction (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Dual proteolytic digestion approach (trypsin followed by Glu-C) to enhance sequence coverage

  • Enrichment strategies for phosphopeptides (TiO2, IMAC) if phosphorylation is suspected

• MS instrumentation and methodology:

  • High-resolution MS/MS using Orbitrap or Q-TOF systems for accurate mass determination

  • Electron transfer dissociation (ETD) or electron capture dissociation (ECD) for labile PTM preservation

  • Parallel reaction monitoring (PRM) for targeted analysis of predicted modification sites

  • Data-independent acquisition (DIA) for comprehensive PTM landscape analysis

• Data analysis workflow:

  • Open search algorithms to identify unexpected modifications

  • Site localization scoring (Ascore, ptmRS) for precise PTM position assignment

  • Quantitative analysis using label-free or TMT-based approaches to determine stoichiometry

  • Integration of MS data with structural models to evaluate functional implications

• Validation strategies:

  • Site-directed mutagenesis of identified PTM sites

  • Functional assays comparing wild-type and PTM-deficient variants

  • Temporal PTM profiling under different growth or stress conditions

  • In vitro enzymatic assays with purified proteins to confirm PTM effects on activity

For potential phosphoesterases like BLi01284/BL02661, particular attention should be paid to serine, threonine, and tyrosine phosphorylation, which could represent either regulatory modifications or enzyme-substrate intermediates. Additionally, given B. licheniformis' propensity for protein secretion , signal peptide processing and other N-terminal modifications should be carefully analyzed.

Cross-linking mass spectrometry (XL-MS) can also provide valuable insights into protein-protein interactions that might regulate BLi01284/BL02661 function or localize it within specific protein complexes, offering functional context beyond simple PTM identification.

How can structural biology approaches help elucidate the catalytic mechanism of BLi01284/BL02661?

Structural biology approaches provide essential insights into the catalytic mechanism of putative phosphoesterases like BLi01284/BL02661. A comprehensive structural biology workflow would include:

• Protein crystallography methodology:

  • Optimization of recombinant protein production with minimal flexible regions

  • High-throughput crystallization screening (sitting-drop vapor diffusion)

  • Co-crystallization with potential substrates, product analogs, and inhibitors

  • Heavy atom derivatization for phase determination if molecular replacement fails

  • High-resolution data collection at synchrotron radiation facilities

• Cryo-electron microscopy approach:

  • Sample preparation optimization for single-particle analysis

  • Collection of large datasets (>5000 micrographs) on high-end cryo-EM systems

  • 2D and 3D classification to identify conformational states

  • High-resolution refinement targeting sub-3Å resolution

  • Model building and refinement against the EM density

• NMR spectroscopy for dynamics:

  • ¹⁵N/¹³C-labeled protein production for backbone and sidechain assignment

  • Chemical shift perturbation experiments with ligands to identify binding sites

  • Relaxation dispersion experiments to characterize millisecond timescale dynamics

  • Hydrogen-deuterium exchange to identify protected regions

• Computational structure analysis:

  • Molecular dynamics simulations to sample conformational space

  • QM/MM calculations to model transition states during catalysis

  • Docking studies with potential substrates to predict binding modes

  • Evolutionary coupling analysis to identify co-evolving residue networks

• Structure-guided functional studies:

  • Alanine scanning of predicted catalytic and substrate-binding residues

  • Activity assays with structure-based mutants to validate mechanistic hypotheses

  • Engineering of substrate specificity based on structural insights

  • Design of specific inhibitors targeting the active site

The integration of these approaches allows for comprehensive characterization of the catalytic mechanism. For a putative phosphoesterase like BLi01284/BL02661, particular attention should be paid to identifying the catalytic triad or metal-coordinating residues typical of phosphoesterase enzymes, as well as substrate-binding pockets that determine specificity.

Recent advances in AlphaFold2 and RoseTTAFold can complement experimental approaches by providing initial structural models that can guide experimental design and interpretation, particularly useful if experimental structure determination proves challenging.

What systems biology approaches can integrate BLi01284/BL02661 into the broader metabolic and regulatory networks of B. licheniformis?

Systems biology approaches provide powerful frameworks for integrating BLi01284/BL02661 into the broader metabolic and regulatory networks of B. licheniformis:

• Multi-omics integration methodology:

  • Combine transcriptomics, proteomics, metabolomics, and fluxomics data from wild-type and BLi01284 knockout strains

  • Implement temporal profiling under various conditions (stress, different carbon sources)

  • Apply network inference algorithms to identify regulatory relationships

  • Develop mathematical models representing the integrated data

• Genome-scale metabolic modeling:

  • Update existing B. licheniformis metabolic models to include BLi01284/BL02661 function

  • Perform flux balance analysis (FBA) to predict metabolic consequences of BLi01284 perturbation

  • Conduct in silico gene knockout simulations to generate testable hypotheses

  • Validate model predictions with experimental measurements of growth and metabolite production

• Protein-protein interaction network mapping:

  • Perform affinity purification coupled with mass spectrometry (AP-MS) to identify interaction partners

  • Utilize bacterial two-hybrid or split-protein complementation assays for binary interaction detection

  • Construct temporal interaction networks under different conditions

  • Map interactions onto known regulatory pathways

• Regulatory network reconstruction:

  • Implement ChIP-seq to identify transcription factor binding sites genome-wide

  • Perform RNA-seq following BLi01284 perturbation to identify affected genes

  • Utilize clustered regularly interspaced short palindromic repeats interference (CRISPRi) for targeted gene repression

  • Construct causal network models from perturbation data

• Integration with industrial bioprocess data:

  • Correlate BLi01284/BL02661 expression with production metrics in industrial strains

  • Develop predictive models for optimizing expression in biomanufacturing contexts

  • Design synthetic regulatory circuits incorporating BLi01284/BL02661 for enhanced control

Recent research has highlighted B. licheniformis' complex stress response systems and sophisticated gene regulation mechanisms , providing context for integrating BLi01284/BL02661 into these networks. The established osmotic stress response pathways, which include SigB-controlled genes and compatible solute synthesis, offer a framework for investigating potential regulatory roles of this putative phosphoesterase .