Recombinant Bacillus thuringiensis subsp. konkukian MutS2 protein (mutS2), partial

What is MutS2 and how does it differ from MutS1 in Bacillus species?

MutS2 is part of the bacterial MutS protein family, which is subdivided into MutS1 and MutS2. While MutS1 family members function in mismatch repair (MMR) pathways recognizing DNA replication errors, MutS2 proteins have more diverse roles that vary between bacterial species .

The key structural difference is that MutS2 contains a C-terminal small MutS-related (Smr) domain with endonuclease activity. This domain is necessary but not sufficient for MutS2 functionality in many bacteria . In Bacillus thuringiensis subsp. konkukian, MutS2 shares structural similarities with other bacterial MutS2 proteins but has evolved specific functions that may differ from those in other species.

What is the taxonomic relationship between B. thuringiensis subsp. konkukian and other Bacillus species?

B. thuringiensis subsp. konkukian (including strain 97-27) is part of the broader "Bacillus cereus group." Genomic analysis reveals that this subspecies is more closely related to B. cereus and B. anthracis than to other B. thuringiensis serovars .

Recent phylogenetic studies using comparative sequence analysis divide the B. cereus group into two major clusters, with B. thuringiensis subsp. konkukian grouping in Cluster I alongside B. anthracis strains and certain B. cereus strains, rather than with typical insecticidal B. thuringiensis serovars in Cluster II .

B. thuringiensis subsp. konkukian strain 97-27 was initially isolated from necrotic tissue in a human patient and contains no identifiable insecticidal (cry) genes on its chromosome or its single plasmid pBT9727 . This differentiates it from typical insecticidal B. thuringiensis strains and supports its closer relationship to B. cereus and B. anthracis.

What role does MutS2 play in homologous recombination in Bacillus species?

The function of MutS2 in recombination varies dramatically between bacterial species. While MutS2 in Helicobacter pylori and Thermus thermophilus suppresses homologous recombination (antirecombination), B. subtilis MutS2 unexpectedly promotes recombination .

In B. subtilis, deletion of mutS2 (ΔmutS2) renders cells sensitive to mitomycin C (MMC), a DNA-damaging agent that requires homologous recombination for repair. The ΔmutS2 strains also show decreased transformation efficiency with both plasmid and chromosomal DNA, further supporting MutS2's role in promoting recombination .

Evidence suggests that B. subtilis MutS2 may function redundantly with the Holliday junction endonuclease RecU. Deletion of mutS2 in a strain lacking recU results in increased MMC sensitivity and decreased transformation efficiency compared to either single mutant . This data supports a model where B. subtilis MutS2 functions as a backup Holliday junction endonuclease.

How does the structure of MutS2 relate to its function in B. thuringiensis?

MutS2 contains several key domains that contribute to its function:

• N-terminal and ATPase domain: Essential for full functionality. The Walker A and Walker B motifs in the ATPase domain are critical for ATP binding and hydrolysis .

• C-terminal Smr domain: Contains the endonuclease activity. In B. subtilis, this domain is necessary but not sufficient for tolerance to DNA-damaging agents like MMC .

• KOW domain: Contributes to binding to collided ribosomes in B. subtilis .

Experimental evidence shows that the C-terminal Smr domain requires the N-terminal portion of MutS2 for proper function in vivo . Mutations in the Walker B motif of the ATPase domain completely abrogate MutS2 activity, while mutations in the KOW domain or Smr domain result in partial loss of activity .

What methodologies are used to study MutS2 function in Bacillus species?

Researchers employ several advanced techniques to investigate MutS2 function:

• CRISPR/Cas9 genome editing: A single plasmid-based CRISPR/Cas9 system has been developed specifically for B. subtilis to create markerless genetic manipulations . This system allows efficient plasmid removal after genome editing.

• Mitomycin C sensitivity assays: Used to assess homologous recombination capability in mutS2 deletion strains compared to wild-type .

• Transformation efficiency assays: Using both plasmid and chromosomal DNA to evaluate recombination function .

• Cryo-electron microscopy: Used to resolve high-resolution structures of MutS2 bound to stalled ribosomes .

• Reporter constructs: In vivo reporter systems using NanoLuc-BleR fusion proteins with stalling sequences to track the activity of MutS2 in ribosome rescue .

• Western blot analysis: Used to monitor protein production from reporter constructs in wild-type and mutant strains .

• Northern probes: Applied to analyze mRNA levels and decay products in translation stalling experiments .

How does MutS2 function in ribosome rescue mechanisms in B. subtilis?

Recent research reveals that B. subtilis MutS2 also plays a critical role in rescuing stalled ribosomes:

• Ribosome sensing: MutS2 detects collided ribosomes that occur when translation is stalled on mRNA .

• Ribosome splitting: Using its ABC ATPase activity, MutS2 splits stalled ribosomes into subunits. This requires ATP hydrolysis, as demonstrated in experiments using non-hydrolyzable ATP analogs (AMP-PNP) .

• Nascent peptide release: This splitting action releases truncated protein products from stalled ribosomes, directing them to the ribosome quality control pathway for degradation .

• No effect on mRNA levels: Unlike its E. coli counterpart SmrB, B. subtilis MutS2 does not appear to affect mRNA levels or induce mRNA decay when ribosomes stall .

Experimental evidence clearly demonstrates that the ATPase activity of MutS2 is critical for ribosome splitting, as Walker A and Walker B mutations completely abolish this function. In contrast, mutations in the SMR domain have only modest effects on ribosome rescue .

How can researchers reconcile the different functions of MutS2 across bacterial species?

MutS2 proteins have adapted diverse functions across bacterial species, requiring careful comparative analysis:

• Sequence divergence: Despite high coverage in sequence alignments, B. subtilis MutS2 shares only 35% identity with T. thermophilus MutS2 and 28% with H. pylori MutS2 . These sequence differences likely account for functional divergence.

• Domain-specific variations: The LDLK motif found in H. pylori MutS2 is absent in B. subtilis and T. thermophilus MutS2 . H. pylori MutS2 contains two nuclease sites (the Smr domain and an N-terminal LDLK motif), while B. subtilis MutS2 relies primarily on its Smr domain for function .

• Experimental approaches for cross-species comparison:

  • Complementation assays using MutS2 from different species

  • Domain-swapping experiments to identify functional regions

  • Comparative structural analysis of MutS2-DNA or MutS2-ribosome complexes

  • Holliday junction binding and resolution assays

• Dual functionality hypothesis: Some evidence suggests that MutS2 may have evolved dual roles in certain species, potentially functioning both in DNA recombination and ribosome rescue pathways . This requires experimental validation through separation-of-function mutations.

What are the optimal expression and purification strategies for recombinant B. thuringiensis subsp. konkukian MutS2?

Based on successful approaches with related proteins:

• Expression systems:

  • E. coli BL21(DE3) with pET-based vectors has been successfully used for MutS2 expression

  • Include a His-tag for purification (N-terminal or C-terminal depending on functional requirements)

  • Optimize codon usage for E. coli if expression levels are low

• Induction conditions:

  • IPTG induction at 0.5-1 mM when culture reaches OD600 of 0.6-0.8

  • Lower temperature (16-18°C) induction can improve protein solubility

  • Extended expression time (overnight) at lower temperatures

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Size exclusion chromatography to ensure homogeneity

  • Ion exchange chromatography for additional purity

• Activity considerations:

  • Ensure purified protein contains bound ATP or provide ATP in storage buffer

  • Test ATPase activity using colorimetric assays to confirm functionality

  • Verify endonuclease activity with Holliday junction substrates

What mutation strategies are most informative for studying MutS2 domain functions?

Based on published research, several key mutations provide insight into MutS2 function:

• ATPase domain mutations:

  • Walker A mutation (K44A): Prevents ATP binding

  • Walker B mutation (E122A): Prevents ATP hydrolysis while allowing binding

  • Both mutations completely abolish MutS2 function in ribosome rescue

• SMR domain mutations:

  • Deletion of entire SMR domain (ΔSMR): Tests necessity of domain

  • Point mutations in conserved residues (H743A): Tests specific catalytic functions

  • ALA mutation (changing DLR motif): Tests role of specific conserved residues

• KOW domain mutations:

  • Mutations in conserved residues affect ribosome binding

  • KOW-mut shows intermediate phenotypes in functional assays

• Truncation constructs:

  • N-terminal truncation (ΔN): Tests sufficiency of the Smr domain

  • C-terminal truncation (ΔC): Tests necessity of the Smr domain

  • Both constructs have been shown to fail to complement mutS2 deletion phenotypes

When designing mutations, researchers should consider:

• Creating stable, properly folded proteins

• Affecting only the intended function

• Including appropriate controls to verify expression levels

• Testing multiple functional outputs (e.g., MMC sensitivity, transformation efficiency, ribosome splitting)

How can CRISPR/Cas9 genome editing be optimized for studying MutS2 in Bacillus species?

A single plasmid-based CRISPR/Cas9 genome editing system has been developed specifically for markerless genetic manipulation in B. subtilis . This system can be adapted for B. thuringiensis with the following considerations:

• Plasmid design elements:

  • Temperature-sensitive origin of replication

  • Selectable marker appropriate for B. thuringiensis

  • Promoter for Cas9 expression optimized for Bacillus

  • sgRNA expression cassette with appropriate promoter

• Targeting efficiency factors:

  • Optimize guide RNA design for target specificity

  • Provide appropriate length homology arms (500-1000 bp)

  • Select PAM sites with high efficiency in Bacillus species

• Protocol optimization:

  • Transform at permissive temperature (30°C)

  • Transfer to non-permissive temperature (39°C) for integration

  • Screen for successful integrants using PCR verification

  • Remove plasmid by growth without selection at permissive temperature

• Applications for MutS2 research:

  • Generate clean deletion mutants without polar effects

  • Create point mutations in specific domains

  • Integrate reporter constructs at native loci

  • Tag endogenous MutS2 with fluorescent or affinity tags

This system allows efficient removal of the plasmid after genome editing, providing a significant advancement in markerless genetic manipulation of Bacillus species .

What experimental approaches can distinguish between MutS2's roles in recombination versus ribosome rescue?

Distinguishing between these functions requires carefully designed experiments:

• Separation-of-function mutations:

  • Identify mutations that specifically affect one function but not the other

  • Test each mutation for both recombination phenotypes (MMC sensitivity, transformation efficiency) and ribosome rescue phenotypes (reporter construct readout)

• Biochemical assays:

  • In vitro Holliday junction binding and resolution assays

  • In vitro ribosome splitting assays using purified components

  • Compare kinetic parameters for both activities

• Double mutant analysis:

  • Combine mutS2 deletion with deletions in known recombination genes (recU)

  • Combine mutS2 deletion with deletions in known ribosome rescue genes

  • Analyze epistatic relationships to place MutS2 in correct pathways

• Domain-specific complementation:

  • Express separate domains or chimeric proteins

  • Test which constructs can rescue specific defects

• Temporal regulation:

  • Create conditional mutS2 alleles to study function at different growth phases

  • Determine if MutS2 functions in distinct pathways during different cellular states

These approaches can help determine whether MutS2 has evolved dual functionality or if one function is primary and the other is secondary.