Recombinant Uncharacterized protein Rv1498c/MT1546 (Rv1498c, MT1546)

What is the basic characterization of Mycobacterium tuberculosis Rv1498c protein?

Rv1498c is a protein encoded in the Mycobacterium tuberculosis genome that functions as a probable methyltransferase. The protein is 205 amino acids in length and is located at coordinates 1689303-1689920 on the negative strand of the M. tuberculosis H37Rv genome . Structurally, it contains the ATP/GTP-binding site motif A (PS00017) and is categorized under the functional category of "Intermediary metabolism and respiration" . The protein has been identified through proteomics at the Statens Serum Institute (Denmark) and through mass spectrometry in whole cell lysates of M. tuberculosis H37Rv, but not in culture filtrate or membrane protein fractions .

What is the predicted function of Rv1498c in Mycobacterium tuberculosis?

Rv1498c is annotated as a probable methyltransferase involved in methylation processes within the bacterium . Methyltransferases typically catalyze the transfer of methyl groups from a donor (often S-adenosylmethionine, SAM) to an acceptor molecule. Bioinformatic analysis suggests similarity to a methyltransferase from Amycolatopsis mediterranei (32.3% identity in 124 aa overlap) . The protein is predicted to be involved in several metabolic pathways, including histidine metabolism, tyrosine metabolism, selenocompound metabolism, polycyclic aromatic hydrocarbon degradation, and microbial metabolism in diverse environments . Additionally, Rv1498c is predicted to be co-regulated in modules bicluster_0108 with residual 0.29 and bicluster_0149 with residual 0.40 .

Is Rv1498c essential for Mycobacterium tuberculosis viability?

Multiple experimental approaches have demonstrated that Rv1498c is a non-essential gene for in vitro growth of M. tuberculosis H37Rv. This determination is based on:

• Himar1 transposon mutagenesis in H37Rv strain in rich medium (MtbYM)

• Analysis of saturated Himar1 transposon libraries

• Independent validation by several research groups (Sassetti et al., 2003; Griffin et al., 2011; DeJesus et al. 2017; Minato et al. 2019)

What are the best methods to express and purify recombinant Rv1498c protein?

The recombinant Rv1498c protein can be expressed in several expression systems, including:

• E. coli

• Yeast

• Baculovirus

• Mammalian cells

For laboratory purification, the following protocol is recommended:

• Cloning and expression vector selection:

  • Clone the Rv1498c gene (sequence: VLDVGCGSGRMALPLTGYLNSEGRYAGFDISQKAIAWCQEHITSAHPNFQFEVSDIYNSLYNPKGKYQSLDFRFPYPDASFDVVFLTSVFTHMFPPDVEHYLDEISRVLKPGGRCLCTYFLLNDESLAHIAEGKSAHNFQHEGPGYRTIHKKRPEEAIGLPETFVRDVYGKFGLAVHEPLHYGSWSGREPRLSFQDIVIATKTAS) into an expression vector with an appropriate tag (His-tag is commonly used)

  • Consider codon optimization for the chosen expression system

• Protein expression:

  • For E. coli: Transform the plasmid into a suitable strain (BL21(DE3) or its derivatives)

  • Induce with IPTG (typically 0.5-1mM) when culture reaches OD600 of 0.6-0.8

  • Express at lower temperatures (16-25°C) overnight to enhance solubility

• Purification:

  • Lyse cells in a buffer containing Tris/PBS (pH 8.0)

  • Purify using affinity chromatography (Ni-NTA for His-tagged protein)

  • Consider additional purification steps such as ion-exchange or size-exclusion chromatography

  • Aim for purity greater than 85% as determined by SDS-PAGE

• Storage:

  • Store in Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • For long-term storage, reconstitute to 0.1-1.0 mg/mL and add glycerol to a final concentration of 5-50%

  • Store aliquots at -20°C/-80°C to avoid repeated freeze-thaw cycles

What analytical methods are optimal for studying the methyltransferase activity of Rv1498c?

To characterize the methyltransferase activity of Rv1498c, researchers should consider the following methodological approaches:

• Radioactive methylation assay:

  • Incubate purified Rv1498c with potential substrates and [³H]-S-adenosylmethionine or [¹⁴C]-SAM

  • Quantify methyl group transfer using scintillation counting

  • This method is highly sensitive but requires radioisotope handling facilities

• SAM-dependent methyltransferase activity assay:

  • Use coupled enzyme assays measuring S-adenosylhomocysteine (SAH) production

  • Commercial kits are available that convert SAH to a fluorescent or colorimetric readout

• Mass spectrometry-based approaches:

  • Identify methylated products using LC-MS/MS

  • Characterize substrate specificity by analyzing mass shifts (+14 Da per methyl group)

  • This approach was successfully used to identify Rv1498c in whole cell lysates

• Differential sequence variant analysis:

  • Apply techniques similar to those described by Zeck et al. (2012) to detect and quantify sequence variants

  • Use a two-stage analytical approach with multi-clone comparison followed by in-depth analysis

• In silico substrate prediction:

  • Given that Rv1498c contains an ATP/GTP-binding motif , analyze potential nucleotide-related substrates

  • Consider metabolic pathway analysis to identify potential substrates in the histidine and tyrosine metabolism pathways

How does Rv1498c contribute to Mycobacterium tuberculosis pathogenesis?

While the direct role of Rv1498c in M. tuberculosis pathogenesis remains partially characterized, several lines of evidence suggest its potential contributions:

• Regulation by Mce3R:

  • Rv1498c is among the genes controlled by Mce3R, a regulatory protein

  • Mutations in Mce3R were identified in resistant mutants in studies of 7-phenyl benzoxaborole compounds, suggesting a role in drug response mechanisms

• Potential involvement in persistent infections:

  • A study of patients with delayed culture conversion identified a non-synonymous mutation in Rv1498c (patient P12/L4), with variant frequencies changing from undetectable in the initial sample to 1.1% in the last sample and 21% after rifampicin exposure

  • This suggests Rv1498c mutations may contribute to persistence during antibiotic treatment

• Evolutionary acquisition through horizontal gene transfer:

  • Phylogenetic analysis has identified methyltransferases, including potentially Rv1498c, as being acquired through horizontal gene transfer during the evolution of M. tuberculosis as a pathogen

  • This acquisition may have contributed to the adaptive capabilities of the bacterium

• Potential involvement in metabolic adaptations:

  • As Rv1498c is involved in multiple metabolic pathways, it may contribute to the bacterium's ability to adapt to different host environments and stress conditions

  • Its presence in histidine and tyrosine metabolism pathways suggests a role in amino acid metabolism, which is crucial during host infection

Is Rv1498c associated with drug resistance in clinical isolates of Mycobacterium tuberculosis?

The relationship between Rv1498c and drug resistance appears complex based on available data:

• Clinical isolate variation:

  • In a study of patients with delayed culture conversion after two months of well-conducted anti-TB treatment, a non-synonymous SNP in Rv1498c was identified in one patient (P12/L4)

  • The variant frequency increased from undetectable in the initial sample to 1.1% in the last sample and 21% after rifampicin exposure

• Association with rifampicin tolerance:

  • While not directly implicated in canonical rifampicin resistance mechanisms, mutations in Rv1498c were detected after rifampicin exposure in clinical isolates

  • This suggests potential involvement in drug tolerance rather than direct resistance

• Regulatory network considerations:

  • Rv1498c is regulated by Mce3R, a regulatory protein also associated with resistance to 7-phenyl benzoxaborole compounds

  • This suggests Rv1498c may be part of a broader regulatory network involved in drug response

• Absence of direct drug targeting:

  • According to the combatTB Explorer database, "Rv1498c doesn't seem to be targeted by any drug" and "doesn't seem to be involved in any pathway" directly associated with drug resistance

These findings indicate that while Rv1498c mutations may contribute to persistence during antibiotic treatment, they are not established as primary drivers of drug resistance in the classical sense.

What is the predicted protein structure of Rv1498c and how does it relate to function?

Based on sequence analysis and comparison with other methyltransferases, the following structural features of Rv1498c can be inferred:

• Functional domains:

  • Contains a methyltransferase domain typical of S-adenosylmethionine-dependent methyltransferases

  • Features the ATP/GTP-binding site motif A (PS00017) , which is likely involved in substrate binding

• Sequence-based features:

  • The protein sequence (205 amino acids) shows similarity to methyltransferases from other organisms, particularly a 32.3% identity in a 124 amino acid overlap with a methyltransferase from Amycolatopsis mediterranei

  • The full sequence is: VLDVGCGSGRMALPLTGYLNSEGRYAGFDISQKAIAWCQEHITSAHPNFQFEVSDIYNSLYNPKGKYQSLDFRFPYPDASFDVVFLTSVFTHMFPPDVEHYLDEISRVLKPGGRCLCTYFLLNDESLAHIAEGKSAHNFQHEGPGYRTIHKKRPEEAIGLPETFVRDVYGKFGLAVHEPLHYGSWSGREPRLSFQDIVIATKTAS

• Structure-function relationship:

  • The GCGSG motif present in the sequence is typical of SAM-binding sites in methyltransferases

  • The C-terminal region likely contains the substrate binding domain

  • Structure prediction would be expected to show the characteristic methyltransferase fold with alternating α-helices and β-strands

• Evolutionary considerations:

  • Phylogenetic analysis suggests Rv1498c may have been acquired through horizontal gene transfer, which could explain unique structural features that differentiate it from other mycobacterial proteins

For detailed structural analysis, researchers should consider:

• Homology modeling based on related methyltransferases

• X-ray crystallography or cryo-EM studies of the purified protein

• Molecular dynamics simulations to predict substrate binding and catalytic mechanisms

How do sequence variants of Rv1498c affect its function in different Mycobacterium tuberculosis lineages?

Analysis of Rv1498c across M. tuberculosis lineages reveals important insights into sequence variation and potential functional implications:

• Lineage-specific variations:

  • A study comparing M. tuberculosis complex lineage 5 genomes with H37Rv (lineage 4) identified differences in gene content, which might include variations in Rv1498c

  • These differences could impact interpretation of transmission studies when using H37Rv as the reference genome

• Clinical relevance of variants:

  • In patients with delayed culture conversion, a non-synonymous SNP in Rv1498c was detected with varying frequencies:

    • Undetectable in initial sample

    • 1.1% in last sample

    • 21% after rifampicin exposure

  • This suggests selection for specific variants under antibiotic pressure

• Functional impact of sequence variations:

  • Non-synonymous mutations may affect:

    • Substrate specificity

    • Catalytic efficiency

    • Protein stability

    • Interaction with regulatory proteins like Mce3R

• Methodological approaches to study variants:

  • Low-level sequence variant analysis, as described by Zeck et al. (2012), can detect variants down to sub-percentage levels

  • This approach allows for detection of:

    • Single amino acid substitutions in unmodified peptides

    • Substitutions in posttranslationally modified peptides

    • Double amino acid substitutions

    • Truncated or elongated sequence variants

Understanding these variants is particularly important when considering that Rv1498c may be involved in metabolic adaptations that contribute to persistence during infection and treatment.

What recent advances have been made in understanding the role of Rv1498c in Mycobacterium tuberculosis biology?

Recent research has provided several new insights into the role of Rv1498c in M. tuberculosis biology:

• Genomic diversity studies:

  • Comparative genomics of M. tuberculosis lineages has revealed differences in gene content that may affect Rv1498c

  • Understanding these differences is important for accurate genomic analysis, including transmission studies

• Persistence and drug tolerance:

  • Research on rifampicin-exposed M. tuberculosis from patients with delayed culture conversion identified mutations in Rv1498c that may contribute to persistence

  • The variant frequency of Rv1498c mutation increased significantly after rifampicin exposure, suggesting a role in drug tolerance

• Regulatory networks:

  • Rv1498c has been identified as part of the Mce3R regulon, along with other genes such as:

    • A putative epoxide hydrolase (Rv1938)

    • A monooxygenase (Rv1936)

    • A succinate-semialdehyde dehydrogenase

  • This regulatory network may play a role in the response to antibiotics like 7-phenyl benzoxaborole compounds

• Evolutionary acquisition:

  • Phylogenetic analyses have suggested that methyltransferases, potentially including Rv1498c, were acquired through horizontal gene transfer during the evolution of pathogenic mycobacteria

  • This acquisition may have contributed to the pathogenic potential of M. tuberculosis

What are the potential applications of recombinant Rv1498c in tuberculosis research and diagnostics?

Recombinant Rv1498c offers several promising applications for tuberculosis research and diagnostics:

• Drug target validation and screening:

  • As a methyltransferase potentially involved in drug tolerance, Rv1498c could serve as a target for developing new anti-tuberculosis compounds

  • Recombinant protein can be used in high-throughput screening assays to identify potential inhibitors

• Biomarker development:

  • Given its detection in whole cell lysates by proteomics , Rv1498c could potentially serve as a biomarker

  • The presence of specific Rv1498c variants might indicate persistent infections or drug-tolerant populations

• Vaccine development:

  • Recombinant Rv1498c is being offered as a research tool for vaccine development

  • Understanding its immunogenicity and role in pathogenesis could inform subunit vaccine design

• Diagnostic applications:

  • Detection of Rv1498c variants could help identify persistent infections

  • Antibodies against Rv1498c could be used in serological assays

• Understanding treatment failure:

  • Research suggests Rv1498c mutations may be associated with persistent infections despite treatment

  • Recombinant protein could be used to study mechanisms of persistence and develop countermeasures

• Studying the evolution of pathogenicity:

  • As a protein potentially acquired through horizontal gene transfer , recombinant Rv1498c could be used to study how M. tuberculosis evolved as a pathogen

  • Functional studies could reveal how this acquisition contributed to virulence

How does the expression of Rv1498c differ under various environmental conditions relevant to TB infection?

Understanding the differential expression of Rv1498c under various conditions provides insights into its role during infection:

Research methodologies to study these expression patterns include:

• Transcriptomic analysis:

  • RNA-seq under different growth conditions

  • Single-cell transcriptomics from infected tissues

• Proteomic approaches:

  • Quantitative proteomics using labeled amino acids in cell culture (SILAC)

  • Targeted proteomics using selected reaction monitoring (SRM)

• Genetic approaches:

  • Construction of reporter strains with promoter fusions

  • Conditional knockdown systems to assess impact on fitness

• In vivo expression studies:

  • Analysis of expression in animal models at different stages of infection

  • Examination of Rv1498c in clinical isolates from different disease presentations