Recombinant Buchnera aphidicola subsp. Baizongia pistaciae Lysine--tRNA ligase (lysS)

What is Buchnera aphidicola subsp. Baizongia pistaciae Lysine--tRNA ligase and how does it function?

Buchnera aphidicola subsp. Baizongia pistaciae Lysine--tRNA ligase (lysS) is an aminoacyl-tRNA synthetase responsible for catalyzing the attachment of lysine to its cognate tRNA (tRNA^Lys) during protein synthesis. This enzyme belongs to one of two evolutionary unrelated forms of lysyl-tRNA synthetase - either LysRS1 or LysRS2. LysRS1 is predominantly found in most archaea and some bacteria, while LysRS2 occurs in eukarya, most bacteria, and a few archaea . The enzyme functions by first activating lysine with ATP to form lysyl-adenylate, then transferring the lysyl group to the 3' end of tRNA^Lys, producing charged lysyl-tRNA^Lys which can be used by ribosomes during protein synthesis.

The proper functioning of lysS is essential for accurate translation of the genetic code, as incorrect charging of tRNA could lead to amino acid misincorporation and potentially defective proteins. In the specialized context of Buchnera as an obligate endosymbiont with a reduced genome, maintaining accurate protein synthesis is crucial both for the bacterium's survival and for fulfilling its symbiotic role in providing essential amino acids to its aphid host.

How does lysS in Buchnera compare to lysyl-tRNA synthetases in free-living bacteria?

Buchnera aphidicola has undergone extensive genome reduction during its evolution as an obligate endosymbiont, retaining only genes essential for survival and symbiotic function. This evolutionary trajectory has important implications for lysS:

• Reduced genetic redundancy: Unlike many free-living bacteria that may possess multiple aminoacyl-tRNA synthetase genes or redundant systems, Buchnera typically maintains only the minimal necessary enzymatic machinery for protein synthesis.

• Evolutionary rate: As part of an endosymbiotic system, Buchnera's lysS likely experiences different selective pressures compared to free-living bacteria. The genomic variations in Buchnera often track lineage-specific changes in their aphid hosts through genetic drift .

• Substrate specificity: The search results indicate significant differences in substrate specificity between LysRS1 and LysRS2, particularly in their interaction with lysine analogs. For example, S-(2-aminoethyl)-l-cysteine inhibits LysRS1 200-fold less effectively than it inhibits LysRS2 . This difference in substrate recognition impacts bacterial growth in the presence of such analogs, potentially influencing which form of LysRS is found in different organisms.

• Charging efficiency: Experimental data shows that the level of tRNA^Lys charging can vary significantly between strains expressing different LysRS enzymes. For instance, approximately 62% of tRNA^Lys is acylated in exponentially growing wild-type B. subtilis cells, while strains expressing B. burgdorferi LysRS1 showed reduced charging levels of approximately 44% .

What evolutionary pressures shape lysS in endosymbiotic bacteria?

The evolution of lysS in Buchnera aphidicola is shaped by several key pressures:

• Genome streamlining: As an obligate endosymbiont, Buchnera has undergone substantial genome reduction, retaining only essential genes. The preservation of lysS underscores its critical function in protein synthesis.

• Host-symbiont coevolution: Patterns of genomic covariation between Buchnera and its aphid host can arise from near-neutral processes of mutation accumulation and genomic decay, causing lineage-specific genomic changes in Buchnera to track the lineage of their aphid host via drift . This coevolutionary relationship likely extends to lysS.

• Metabolic integration: Buchnera provides essential amino acids to its aphid host that are lacking in the phloem sap diet . The efficient functioning of lysS is crucial for synthesizing the proteins involved in these amino acid biosynthetic pathways.

• Selective constraints on accuracy: Despite genome reduction and accelerated evolution in many genes, aminoacyl-tRNA synthetases like lysS must maintain sufficient accuracy to prevent mistranslation, which could be detrimental to both the endosymbiont and its host.

• Adaptation to the intracellular environment: The intracellular environment of the aphid bacteriocyte, where Buchnera resides, may influence the optimal activity parameters of lysS compared to homologs in free-living bacteria.

What expression systems are most effective for producing recombinant Buchnera aphidicola lysS?

Producing functional recombinant lysS from Buchnera aphidicola presents several challenges due to the unique characteristics of this endosymbiont. Based on approaches used for similar systems, the following strategies are recommended:

• Expression host selection:

  • Escherichia coli remains the preferred expression host due to its well-established genetic tools and high protein yields

  • BL21(DE3) and its derivatives are particularly suitable for recombinant protein expression

  • Rosetta strains may be beneficial as they supply tRNAs for rare codons that might be present in Buchnera genes

• Vector design considerations:

  • Codon optimization is crucial given Buchnera's AT-rich genome (typically >70% AT content)

  • Fusion tags can enhance solubility; common options include:

    • His6-tag for purification convenience

    • MBP (maltose-binding protein) for enhanced solubility

    • SUMO for proper folding and native N-terminus after cleavage

• Expression conditions:

  • Lower induction temperatures (16-20°C) often improve folding of recombinant proteins

  • Extended expression periods (overnight) at lower temperatures may increase yield of correctly folded protein

  • IPTG concentration should be optimized (typically 0.1-0.5 mM)

• Special considerations:

  • Co-expression with molecular chaperones (GroEL/ES, DnaK/J) may improve folding

  • Addition of rare tRNAs can overcome codon bias issues

  • If toxicity occurs, consider using tightly controlled inducible promoters

The effectiveness of heterologous expression systems for aminoacyl-tRNA synthetases is evidenced in the search results, which mention successful preparation of B. burgdorferi LysRS1 for experimental use .

What purification strategies yield active recombinant lysS enzyme?

Purifying active recombinant lysS requires careful attention to maintaining protein stability and function throughout the process. A comprehensive purification strategy typically includes:

• Initial extraction conditions:

  • Lysis buffer composition is critical:

    • Buffer: 50 mM Tris-HCl or HEPES, pH 7.5-8.0

    • Salt: 100-300 mM NaCl to maintain solubility

    • Glycerol: 10-20% to enhance stability

    • Reducing agent: 1-5 mM DTT or β-mercaptoethanol to protect cysteine residues

    • Magnesium: 5-10 mM MgCl₂ (critical for aminoacyl-tRNA synthetase activity)

    • Protease inhibitors: PMSF or commercial cocktails to prevent degradation

• Multi-step purification approach:

  • Affinity chromatography: Typically the first step, using His-tag or other fusion partners

  • Ion-exchange chromatography: Further purification based on charge properties

  • Size-exclusion chromatography: Final polishing step that also allows buffer exchange

• Activity preservation considerations:

  • Maintain temperature at 4°C throughout purification

  • Monitor enzyme activity after each purification step

  • Minimize exposure to freezing/thawing cycles

  • Consider adding stabilizing agents like ATP (0.1-1 mM) during purification

• Quality assessment:

  • SDS-PAGE to verify purity

  • Mass spectrometry to confirm identity

  • Dynamic light scattering to assess homogeneity

  • Aminoacylation assays to confirm activity

The search results indicate that specific aminoacylation conditions have been established for both LysRS1 and LysRS2 , suggesting that the activity of purified recombinant lysS can be reliably assessed using appropriate assay conditions.

How can the activity and kinetic properties of recombinant lysS be accurately measured?

Accurate measurement of lysS activity is essential for characterizing the recombinant enzyme. Multiple complementary approaches can be employed:

• Radioactive aminoacylation assays:

  • Traditional gold standard method using [³H]-labeled lysine or ³²P-labeled tRNA

  • Reaction components typically include:

    • Purified recombinant lysS (1-10 μg/ml)

    • tRNA^Lys (either total tRNA or in vitro transcribed specific tRNA^Lys)

    • ATP (2-5 mM)

    • Lysine (10-100 μM) including radioactive tracer

    • Magnesium chloride (5-15 mM)

    • Buffer (typically HEPES or Tris, pH 7.5-8.0)

  • The direct attachment of amino acids to tRNA can be monitored by ³²P labeling of the tRNA using E. coli CCA-adding enzyme, followed by aminoacylation and product visualization through gel electrophoresis

• Non-radioactive alternative methods:

  • Pyrophosphate release assays using enzymatic coupling with malachite green detection

  • HPLC-based measurement of AMP formation

  • Mass spectrometry to detect charged versus uncharged tRNA

• Kinetic parameter determination:

  • K_m determination for lysine (typically 10-100 μM range)

  • K_m determination for tRNA^Lys (typically in the 0.1-5 μM range)

  • k_cat calculation to determine turnover number

  • Catalytic efficiency (k_cat/K_m) comparison with other LysRS enzymes

• Inhibition studies:

  • Determination of K_i values for lysine analogs such as S-(2-aminoethyl)-l-cysteine

  • Comparative inhibition between different LysRS forms, as the search results show that S-(2-aminoethyl)-l-cysteine inhibits LysRS1 200-fold less effectively than it inhibits LysRS2

Table 1: Comparative kinetic parameters for LysRS1 and LysRS2, with typical ranges based on experimental data from various bacterial sources including data inferred from the search results .

How does lysS contribute to understanding the Buchnera-aphid symbiotic relationship?

The study of lysS provides several unique insights into the Buchnera-aphid symbiotic relationship:

How can recombinant lysS be used to investigate endosymbiont genome evolution?

Recombinant lysS serves as an excellent model for investigating broader patterns in endosymbiont genome evolution:

• Molecular signatures of genome reduction:

  • Comparison of Buchnera lysS with homologs from free-living relatives can reveal how enzyme function is maintained (or altered) in the context of genome streamlining.

  • Analysis of synonymous vs. non-synonymous substitution rates (dN/dS) in lysS across different Buchnera strains can identify selective pressures unique to the endosymbiotic lifestyle.

• Functional constraints in reduced genomes:

  • Kinetic characterization of recombinant lysS can determine whether endosymbionts maintain optimal enzyme function or tolerate suboptimal activity.

  • The search results show that when heterologous LysRS1 from B. burgdorferi was expressed in B. subtilis, the level of charged tRNA^Lys was reduced to approximately 44% compared to 62% in wild-type cells , suggesting functional differences between enzymes from different sources.

• Co-evolutionary patterns:

  • Studies of Buchnera-aphid systems reveal evidence of genomic covariation, where "genomic (co)variation is segregating among different clonal strains" .

  • By comparing lysS sequences across Buchnera strains from different aphid species or host plants, researchers can determine whether this gene follows broader patterns of host-symbiont coevolution.

• Neutral vs. adaptive evolution:

  • Experimental characterization of recombinant lysS variants can help distinguish between neutral drift and adaptive evolution.

  • The search results discuss testing "for the potential contributions of adaptive versus neutral processes in shaping the distribution of variation within the Buchnera genome" , an approach that could be applied specifically to lysS.

• Polymorphism within endosymbiont populations:

  • Interestingly, the search results note that "we observed polymorphism across haploid Buchnera genomes within clonal lineages suggests that there are different Buchnera strains segregating in aphid hosts from the same strain" .

  • This within-host diversity could be explored by examining lysS sequence variants within single aphid individuals.

What does lysS reveal about adaptation to endosymbiotic lifestyles across bacterial lineages?

The study of lysS across different bacterial endosymbionts reveals important patterns of convergent and divergent evolution in adaptation to endosymbiotic lifestyles:

• Substrate specificity adaptations:

  • The search results highlight significant differences in substrate specificity between LysRS1 and LysRS2, particularly in their interaction with lysine analogs like S-(2-aminoethyl)-l-cysteine .

  • This raises interesting questions about whether endosymbionts show consistent patterns in lysS substrate specificity that might represent adaptations to the intracellular environment.

  • Comparison of structures of LysRS1 and LysRS2 complexed with lysine revealed significant differences in their potential to bind lysine analogues with backbone replacements , suggesting different evolutionary trajectories.

• Enzyme efficiency trade-offs:

  • The reduced genetic capacity of endosymbionts may lead to trade-offs between enzyme efficiency and metabolic cost.

  • Characterization of lysS from diverse endosymbionts can reveal whether there are consistent patterns in kinetic parameters that might represent adaptation to endosymbiotic lifestyle.

• Multimodal regulatory responses:

  • The search results describe how B. aphidicola responds to variations in leucine demand through "an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number" .

  • Similar regulatory mechanisms might exist for lysS and other aminoacyl-tRNA synthetases, representing adaptations to fluctuating metabolic demands in the endosymbiotic environment.

• Evolutionary distribution patterns:

  • The search results note that LysRS1 and LysRS2 "are almost never found together in a single organism" .

  • The distribution of these two unrelated forms across different endosymbiont lineages might reveal whether certain forms are better suited to endosymbiotic lifestyles.

• Robustness to environmental fluctuations:

  • Endosymbionts must function within the changing intracellular environment of their hosts.

  • Characterization of lysS stability and activity across different conditions can indicate adaptations for robustness in the face of environmental fluctuations.

Table 2: Predicted LysRS types across different insect endosymbionts and their potential adaptive significance (inferred from broader knowledge of these systems, as specific LysRS characterization data is limited in the search results).

What factors influence the specificity of lysS for lysine versus lysine analogs?

The specificity of lysS for lysine versus structural analogs is critical for accurate protein synthesis and has important implications for enzyme evolution and potential antimicrobial development. Several factors influence this specificity:

• Active site architecture:

  • The search results highlight significant structural differences between LysRS1 and LysRS2 in their lysine-binding pockets, particularly in their ability to accommodate lysine analogs with backbone replacements .

  • These structural differences explain the observed 200-fold difference in inhibition by S-(2-aminoethyl)-l-cysteine between the two enzyme forms .

• Key recognition elements:

  • The α-amino group of lysine is typically recognized through hydrogen bonding with conserved acidic residues

  • The ε-amino group forms salt bridges with acidic residues in the binding pocket

  • The carboxyl group interacts with positively charged residues or hydrogen bond donors

  • Any modifications to the backbone or side chain of lysine can disrupt these specific interactions

• Conformational changes upon binding:

  • Aminoacyl-tRNA synthetases often undergo conformational changes upon substrate binding

  • These induced-fit mechanisms can contribute to specificity by optimizing interactions with the correct substrate

• Pre-transfer and post-transfer editing:

  • Some aminoacyl-tRNA synthetases possess editing domains that hydrolyze misactivated amino acids

  • The presence or absence of such editing capabilities can significantly impact specificity

• Experimental evidence from analog studies:

  • The search results describe how S-(2-aminoethyl)-l-cysteine (thialysine) interacts differently with LysRS1 versus LysRS2

  • In vitro tests showed that S-(2-aminoethyl)-l-cysteine is a poor substrate for LysRS1 and inhibits LysRS1 200-fold less effectively than it inhibits LysRS2

  • These differences have biological consequences, as B. subtilis strains producing LysRS1 alone were relatively insensitive to growth inhibition by S-(2-aminoethyl)-l-cysteine, whereas strains producing LysRS2 showed significant growth inhibition

This differential sensitivity to lysine analogs has important evolutionary implications, as noted in the search results: "These growth effects arising from differences in amino acid recognition could contribute to the distribution of LysRS1 and LysRS2 in different organisms" .

How do mutations in lysS affect enzyme function and bacterial fitness?

The impact of mutations in lysS on enzyme function and bacterial fitness can be substantial, affecting multiple aspects of cellular physiology:

Understanding the relationship between lysS mutations and fitness is particularly important in the context of endosymbionts like Buchnera with their reduced genomes and intimate host associations.

What technical challenges arise when studying recombinant proteins from unculturable endosymbionts?

Studying recombinant proteins from unculturable endosymbionts like Buchnera aphidicola presents unique technical challenges:

• Sequence verification limitations:

  • Without the ability to directly culture the organism, obtaining accurate gene sequences can be challenging

  • Genomic DNA must be extracted from insect tissues containing the endosymbiont

  • Potential PCR bias or sequencing errors can complicate accurate gene amplification

  • The search results note the challenge of within-host diversity, as "there are different Buchnera strains segregating in aphid hosts from the same strain"

• Expression system incompatibilities:

  • Codon usage in Buchnera differs significantly from common expression hosts like E. coli

  • Buchnera genes are typically AT-rich (>70% AT content), requiring codon optimization

  • Post-translational modifications present in the native environment may be absent in heterologous systems

  • The search results show that heterologous expression can affect enzyme function, as seen with B. burgdorferi LysRS1 in B. subtilis

• Protein folding and stability issues:

  • Recombinant proteins from endosymbionts may misfold in heterologous expression systems

  • The intracellular environment of the aphid bacteriocyte differs from standard expression hosts

  • Long-term evolutionary adaptation to the endosymbiotic lifestyle may have resulted in proteins optimized for specific intracellular conditions

• Functional assay development:

  • Natural substrates (e.g., Buchnera tRNA^Lys) may not be readily available

  • The search results highlight that tRNA charging efficiency can vary significantly between homologous and heterologous systems

  • Reconstructing physiologically relevant assay conditions is challenging without detailed knowledge of the bacteriocyte environment

• Validation limitations:

  • Without the ability to perform genetic manipulation in Buchnera, validating in vitro findings in the native context is virtually impossible

  • Comparative approaches using related culturable organisms provide only approximate validation

These technical challenges necessitate creative experimental approaches, often combining recombinant protein studies with genomic analyses, metabolomic profiling, and detailed characterization of the host-symbiont interface.

How might lysS function be harnessed for developing new antimicrobial strategies?

The essential nature of lysS and its divergent forms across bacterial lineages present intriguing opportunities for antimicrobial development:

• Exploiting structural differences between LysRS forms:

  • The search results highlight significant differences in the lysine-binding pockets of LysRS1 and LysRS2, particularly in their ability to accommodate lysine analogs

  • These structural differences result in a 200-fold difference in inhibition by S-(2-aminoethyl)-l-cysteine

  • This differential sensitivity could be exploited to develop selective inhibitors targeting specific bacterial groups based on their LysRS type

• Targeting bacterial-specific features:

  • Most pathogenic bacteria possess LysRS2, which differs from human LysRS

  • Structural differences between bacterial and human LysRS2 could be leveraged for selective inhibition

  • The search results demonstrate that LysRS2 is more susceptible to inhibition by certain lysine analogs , providing a starting point for drug development

• Resistance considerations:

  • The search results note that "diversity of the aminoacyl-tRNA synthetases prevents infiltration of the genetic code by noncanonical amino acids, thereby providing a natural reservoir of potential antibiotic resistance"

  • Understanding these resistance mechanisms is crucial for developing robust antimicrobial strategies

  • Multi-target approaches might help overcome potential resistance mechanisms

• Delivery challenges for intracellular pathogens:

  • Many intracellular pathogens share characteristics with endosymbionts like Buchnera

  • Insights from Buchnera lysS could inform approaches to targeting aminoacyl-tRNA synthetases in intracellular pathogens

  • Drug delivery systems might need to penetrate host cell membranes to reach these targets

• Combination therapy potential:

  • Inhibitors targeting lysS could be combined with other antibiotics for synergistic effects

  • Such combinations might help prevent resistance development while allowing lower doses of each compound

The search results provide direct evidence that differential sensitivity to amino acid analogs can significantly impact bacterial growth, as "B. subtilis strains producing LysRS1 alone were relatively insensitive to growth inhibition by S-(2-aminoethyl)-l-cysteine, whereas a WT strain or merodiploid strains producing both LysRS1 and LysRS2 showed significant growth inhibition under the same conditions" .

What comparative genomic approaches could reveal new insights about lysS evolution in endosymbionts?

Advanced comparative genomic approaches offer powerful tools for understanding lysS evolution in endosymbionts:

• Pan-symbiont analysis:

  • Comparing lysS sequences across diverse insect endosymbionts could reveal convergent adaptation patterns

  • The search results describe approaches for analyzing "patterns of genomic covariation between the Buchnera and aphid host genome" that could be applied specifically to lysS

  • This could help distinguish between neutral drift and adaptive evolution in endosymbiont genes

• Molecular evolution rate analysis:

  • Calculating synonymous (dS) and non-synonymous (dN) substitution rates for lysS across different Buchnera strains

  • Comparing these rates with those of other essential genes to determine if lysS is under unusual selective pressure

  • The search results discuss "drifting Buchnera genomes" that "track the microevolutionary trajectories" of their hosts , providing context for such analyses

• Population genomics of within-host variation:

  • The search results note evidence of within-host diversity, as "we observed polymorphism across haploid Buchnera genomes within clonal lineages suggests that there are different Buchnera strains segregating in aphid hosts from the same strain"

  • Deep sequencing of lysS from individual aphids could reveal the extent and patterns of this within-host diversity

  • This approach could help understand the evolutionary dynamics of lysS at the microevolutionary scale

• Structural bioinformatics:

  • Homology modeling of Buchnera lysS based on crystal structures of related enzymes

  • Identifying structurally conserved regions versus variable regions

  • The search results mention "comparison of structures of LysRS1 and LysRS2 complexed with lysine" , which provides a framework for such analyses

• Host-symbiont coevolution mapping:

  • Correlating patterns of lysS evolution with changes in host genomes

  • Testing whether lysS evolution tracks host phylogeny or ecological factors

  • The search results discuss testing "genetic versus geographic distance, covariation between Buchnera and M. periscae hosts, and plant host specialization" as factors influencing genetic differentiation

These comparative approaches could reveal whether lysS evolution in Buchnera follows patterns seen in other endosymbiont genes, or whether its essential role in protein synthesis subjects it to unique evolutionary constraints.

How might CRISPR-based approaches advance research on lysS in the Buchnera-aphid system?

Despite the challenges of working with unculturable endosymbionts, CRISPR-based technologies offer innovative approaches for studying lysS in the Buchnera-aphid system:

• Host-directed symbiont modification:

  • While direct genetic manipulation of Buchnera remains challenging, CRISPR systems delivered to aphid hosts could potentially target symbiont genes

  • dCas9-based approaches could allow modulation of lysS expression without disrupting this essential gene

  • This would require careful design of guide RNAs specific to Buchnera lysS that don't cross-react with host sequences

• In situ visualization approaches:

  • CRISPR-based imaging systems using catalytically inactive Cas9 fused to fluorescent proteins could allow visualization of lysS transcripts or genomic loci within intact bacteriocytes

  • This could provide insights into the spatial organization of protein synthesis machinery within the symbiont cells

• Transcriptome modulation:

  • CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) systems delivered to aphids could potentially modulate lysS expression in Buchnera

  • This would allow investigation of how lysS expression levels affect symbiont function and host fitness

  • The search results describe how Buchnera can respond to metabolic requirements through transcriptional regulation , providing context for such studies

• Host gene editing to study interaction:

  • CRISPR-mediated editing of aphid genes involved in bacteriocyte formation or function

  • This could indirectly affect the environment in which Buchnera lysS operates

  • The search results discuss patterns of genomic covariation between Buchnera and its aphid host , suggesting intimate functional interactions that could be probed through this approach

• CRISPR-based sensors:

  • Development of CRISPR-Cas-based sensors that respond to specific metabolites relevant to the symbiotic relationship

  • This could help understand how lysS activity relates to broader metabolic integration between host and symbiont

  • The search results describe how "symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid" , suggesting metabolic sensing systems that might be detectable with appropriate tools

While these approaches remain technically challenging, they represent frontier technologies that could overcome the traditional barriers to studying unculturable endosymbionts and their essential genes like lysS.