Recombinant Lactobacillus plantarum Putative phosphotransferase lp_1974 (lp_1974)

What is the putative phosphotransferase lp_1974 in Lactobacillus plantarum and its functional role?

The putative phosphotransferase lp_1974 in L. plantarum likely functions as a component of the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS). Similar to other PTS enzymes, it may be involved in the simultaneous translocation and phosphorylation of sugar substrates across the cell membrane. PTS enzyme I transfers phosphoryl groups from phosphoenolpyruvate to the phosphoryl carrier protein HPr, which is critical for carbohydrate metabolism in many bacteria . While specific research on lp_1974 is limited in the provided context, its classification suggests involvement in L. plantarum's distinctive carbohydrate utilization capabilities, which contribute to its adaptability across diverse ecological niches including fermented foods and the human gastrointestinal tract .

How does the taxonomic reclassification of Lactobacillus affect research on lp_1974?

The recent taxonomic reclassification of Lactobacillus plantarum to Lactiplantibacillus plantarum (occurring in April 2020) requires researchers to be vigilant about nomenclature when reviewing literature and designing experiments . When conducting database searches and literature reviews on lp_1974, researchers should include both taxonomic designations to ensure comprehensive coverage of relevant studies. This reclassification reflects phylogenetic refinements within what was previously the Lactobacillus genus, which has now been divided into 25 different genera . Despite this taxonomic update, many product labels and research papers may still use the former designation, potentially creating confusion in research continuity and meta-analyses of functional studies on lp_1974.

What expression systems are most effective for recombinant production of lp_1974?

For recombinant expression of L. plantarum proteins such as lp_1974, the pSIP vector system has demonstrated significant efficacy in previous research. The inducible vector pSIP-409 has been successfully employed for heterologous protein expression in L. plantarum NC8, as evidenced in studies with other recombinant proteins . When expressing lp_1974, researchers should consider:

• Selection of appropriate promoters: Inducible promoters offer advantages for controlled expression

• Codon optimization: Adapting the lp_1974 sequence to the codon usage bias of the expression host

• Signal peptide selection: For proper localization if the native protein is membrane-associated

• Purification strategy: Incorporation of affinity tags that do not interfere with protein function

The expression system should be designed considering whether the research aims to study the protein in isolation or within the context of the living L. plantarum cells, as the latter may provide insights into natural regulatory mechanisms and interactions with other components of the phosphotransferase system .

What are the optimal conditions for genomic isolation and sequencing of L. plantarum for lp_1974 characterization?

Effective genomic DNA extraction from L. plantarum for subsequent lp_1974 analysis requires careful methodology. Published protocols indicate successful extraction using commercial kits such as the Qiagen DNeasy blood and tissue kit, followed by quality assessment using calibrated Nano-drop spectrophotometry and Qubit fluorometry . For whole genome sequencing to identify and characterize lp_1974, the Illumina MiSeq 300 × 2 Platform has proven effective, as demonstrated in studies of L. plantarum strains DHCU70 and DKP1 .

Researchers should pay particular attention to:

• Cell wall disruption: L. plantarum has a robust cell wall requiring effective lysis procedures

• RNA contamination removal: RNase treatment is crucial for pure genomic DNA preparation

• Sequencing depth: Aim for sufficient coverage (>30x) to ensure accurate assembly and annotation

• Bioinformatic analysis: Post-sequencing, gene identification should employ both BLASTP for sequence similarity searches and specialized databases (e.g., BAGEL4 for bacteriocin-encoding genes)

For targeted analysis of lp_1974, regions flanking the gene should be included in primer design to ensure complete capture of regulatory elements that may influence expression and function.

How can reporter assays be optimized to study lp_1974 function in L. plantarum?

Reporter assays represent powerful tools for functional characterization of lp_1974. High-throughput screening with targeted cell-based assays using reporters such as β-galactosidase or luciferase genes can provide reliable insights into protein activity and regulation . For lp_1974 functional studies, researchers should:

• Design constructs placing reporter genes under control of lp_1974 promoter regions

• Establish appropriate controls including inactive mutants of lp_1974

• Optimize induction conditions specific to the phosphotransferase system

• Develop clear readout parameters (e.g., colorimetric changes in X-Gal supplemented media)

In published studies with L. plantarum, reporter strains containing lacZ have been successfully used to evaluate gene expression, with blue coloration indicating β-galactosidase production . These systems can be adapted to study lp_1974 regulation under various conditions, providing insights into the environmental factors and metabolic states that influence phosphotransferase activity.

What are the critical considerations when designing knockout or complementation studies for lp_1974?

When designing knockout or complementation studies for lp_1974 in L. plantarum, researchers must address several methodological challenges:

• Genetic manipulation strategy:

  • CRISPR-Cas9 systems adapted for Lactobacillus

  • Homologous recombination approaches with selection markers

  • Antisense RNA strategies for conditional knockdowns

• Phenotypic assessment:

  • Growth kinetics in media with different carbon sources

  • Carbohydrate utilization profiles comparing wildtype and mutant strains

  • Metabolomic analysis to detect changes in phosphorylated intermediates

  • Stress response evaluation, particularly under conditions requiring rapid metabolic adaptation

• Complementation design:

  • Vector selection with appropriate copy number

  • Promoter choice (native vs. constitutive)

  • Expression timing that mimics natural regulation

The interpretation of phenotypic changes must account for potential compensatory mechanisms within the phosphotransferase system, as functional redundancy may exist among PTS components . Additionally, researchers should consider the impact of lp_1974 modification on L. plantarum stress tolerance and probiotic attributes, as PTS systems often influence these characteristics .

How does lp_1974 compare to phosphotransferase systems in other bacterial species?

Comparing lp_1974 to well-characterized phosphotransferase systems in other bacterial species reveals important evolutionary and functional insights. The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) in E. coli features Enzyme I, which transfers phosphoryl groups from phosphoenolpyruvate to the phosphoryl carrier protein HPr . Structural analysis of PTS Enzyme I from E. coli (PDB: 2hwg) shows two catalytic domains (CATH: 3.20.20.60 and 3.50.30.10) and requires magnesium as a cofactor .

To conduct meaningful comparative analyses, researchers should:

• Perform phylogenetic analysis of lp_1974 against homologs in diverse bacterial species

• Compare conserved domains and active site residues

• Evaluate substrate specificity differences

• Assess the genomic context of phosphotransferase genes

Functional differences may reflect adaptation to specific ecological niches, as L. plantarum's phosphotransferase system likely evolved to optimize nutrient acquisition in environments like fermented foods and the gastrointestinal tract . Computational models predicting the structural differences between lp_1974 and E. coli PTS Enzyme I could provide valuable insights into substrate specificity and regulatory mechanisms.

What role might lp_1974 play in L. plantarum's probiotic and metabolic activities?

The putative phosphotransferase lp_1974 may significantly contribute to L. plantarum's probiotic properties through its involvement in carbohydrate metabolism. L. plantarum is recognized for its probiotic attributes including the ability to break down food, absorb nutrients, and inhibit pathogenic organisms . The phosphotransferase system likely enables efficient utilization of diverse carbon sources, contributing to the organism's competitive fitness in complex environments like the human gut.

Recent research has identified various probiotic genes in L. plantarum strains through genome analysis and sequence similarity searches using BLASTP . While lp_1974 has not been specifically highlighted in these analyses, phosphotransferase components generally influence:

• Carbon source utilization flexibility

• Colonization capacity in competitive environments

• Biofilm formation capabilities

• Acid and bile tolerance

Experimental approaches to investigate these connections include:

These investigations would help establish whether lp_1974 contributes to L. plantarum's documented probiotic effects, including its applications in treating conditions like irritable bowel syndrome, inflammatory bowel disease, and respiratory infections .

How does environmental pH and bile salt concentration affect lp_1974 expression and function?

The expression and function of phosphotransferase systems in L. plantarum are likely influenced by environmental stressors such as acidic pH and bile salts, which are relevant to both food fermentation and gastrointestinal transit. Standard protocols for evaluating L. plantarum responses to these conditions involve exposure to pH 3 (adjusted with HCl) and 0.3% (w/v) bile salts, with viability assessed through serial dilution plating and spectrophotometric measurements .

For specific analysis of lp_1974 regulation under these conditions, researchers should:

• Design qRT-PCR assays targeting lp_1974 mRNA levels under stress conditions

• Develop reporter fusions linking lp_1974 promoter to measurable outputs

• Perform proteomic analysis to assess post-transcriptional regulation

• Evaluate enzymatic activity using phosphorylation assays with purified components

The connection between stress response and phosphotransferase activity is particularly relevant as L. plantarum strains exhibit variable acid and bile tolerance, which correlates with their probiotic efficacy . Understanding how lp_1974 contributes to these tolerance mechanisms could inform strain selection for specific applications and explain strain-specific differences in metabolic adaptability.

How can recombinant L. plantarum expressing modified lp_1974 be applied in metabolic engineering?

Recombinant L. plantarum strains with engineered lp_1974 present opportunities for metabolic engineering applications. The phosphotransferase system's central role in carbohydrate uptake and metabolism makes it an attractive target for modifying substrate utilization profiles. Potential applications include:

• Redirecting carbon flux toward valuable metabolites

• Enhancing utilization of non-preferred carbon sources

• Developing strains with improved fermentation characteristics

• Creating biosensors for specific carbohydrates

Previous research has demonstrated the feasibility of expressing recombinant proteins in L. plantarum using inducible expression systems like pSIP-409 . When engineering lp_1974 modifications, researchers should consider:

The successful engineering of recombinant L. plantarum for therapeutic applications, such as expressing angiotensin-converting enzyme inhibitory peptides for hypertension treatment , provides a methodological framework that could be adapted for lp_1974 modification.

What challenges arise in structural characterization of membrane-associated phosphotransferases like lp_1974?

Structural characterization of membrane-associated phosphotransferases presents significant technical challenges requiring specialized approaches. While the structure of phosphorylated Enzyme I of the PTS system has been determined for E. coli (PDB: 2hwg at 2.7 Å resolution) , similar studies with lp_1974 would face several obstacles:

• Expression challenges:

  • Achieving sufficient protein yields for structural studies

  • Maintaining protein stability during purification

  • Preserving native conformation and activity

• Purification considerations:

  • Selection of appropriate detergents for membrane protein extraction

  • Optimization of buffer conditions to maintain structural integrity

  • Development of purification strategies that preserve phosphorylation state

• Structural determination methods:

  • X-ray crystallography requirements for membrane protein crystallization

  • Cryo-EM sample preparation for membrane proteins

  • NMR spectroscopy limitations for larger membrane-associated proteins

• Functional validation:

  • Designing activity assays compatible with purified components

  • Confirming that structural insights correlate with functional properties

  • Analyzing dynamic changes associated with substrate binding and catalysis

Researchers might consider reconstitution of lp_1974 into nanodiscs or liposomes to maintain a membrane-like environment during structural studies. Alternatively, focusing on soluble domains first may provide initial insights while avoiding some technical difficulties associated with full-length membrane proteins.

How can systems biology approaches integrate lp_1974 function into broader metabolic networks in L. plantarum?

Systems biology approaches offer powerful frameworks for understanding how lp_1974 integrates into L. plantarum's metabolic networks. Comprehensive analysis requires multi-omics strategies and computational modeling:

• Multi-omics integration:

  • Genomics: Identifying regulatory elements and genetic context of lp_1974

  • Transcriptomics: Mapping co-expression networks under various conditions

  • Proteomics: Quantifying protein levels and post-translational modifications

  • Metabolomics: Tracking metabolic flux through phosphotransferase-dependent pathways

  • Interactomics: Identifying protein-protein interaction partners

• Computational modeling:

  • Genome-scale metabolic models incorporating lp_1974 function

  • Kinetic models of phosphotransferase activity

  • Regulatory network reconstructions

  • Flux balance analysis to predict metabolic shifts

• Experimental validation:

  • Targeted metabolite analysis before and after lp_1974 modification

  • 13C metabolic flux analysis to quantify changes in carbon distribution

  • Growth phenotype arrays under diverse nutrient conditions

This integrated approach would reveal how lp_1974 contributes to L. plantarum's remarkable metabolic flexibility, which underlies its success in diverse environments from fermented foods to the human gastrointestinal tract .

What potential exists for using recombinant L. plantarum with modified lp_1974 in vaccine development?

The application of recombinant L. plantarum in vaccine development represents an emerging research direction with significant potential. Previous studies have successfully used recombinant L. plantarum as an oral vaccine carrier against bacterial infections, such as Aeromonas hydrophila in fish models . This approach could potentially be extended to vaccine development involving modified lp_1974.

Key considerations for this application include:

• Antigen delivery system design:

  • Surface display vs. secretion vs. intracellular expression

  • Fusion protein design for optimal immunogenicity

  • Stability during gastrointestinal transit

• Immunological assessment:

  • Mucosal immune response evaluation

  • Systemic antibody production measurement

  • Cell-mediated immunity characterization

• Protection efficacy:

  • Challenge studies with target pathogens

  • Duration of immunity evaluation

  • Cross-protection against variant strains

In fish models, recombinant L. plantarum expressing A. hydrophila TPS maltoporin (Malt) significantly enhanced IgM levels and non-specific immune responses, providing 55% relative percent survival after pathogen challenge compared to 0% in control groups . Similar approaches could be developed using lp_1974 as part of fusion constructs designed to enhance immune responses against specific antigens.

How might CRISPR-Cas9 technologies advance precise modification of lp_1974 for functional studies?

CRISPR-Cas9 technologies offer unprecedented precision for genetic modification of lp_1974 in L. plantarum, enabling sophisticated functional studies. While not explicitly mentioned in the provided search results, these methodologies have revolutionized bacterial genetics and could be applied to lp_1974 research.

Advanced CRISPR-based approaches for lp_1974 studies might include:

• Precise genomic modifications:

  • Single nucleotide substitutions to alter specific amino acids

  • Domain swapping with homologous phosphotransferases

  • Promoter replacements to alter expression patterns

  • Insertion of regulatory elements to enable conditional expression

• High-throughput functional screening:

  • CRISPR interference (CRISPRi) for tunable repression

  • CRISPR activation (CRISPRa) for enhanced expression

  • Multiplex targeting of lp_1974 along with interacting partners

  • Creation of comprehensive mutation libraries for structure-function analysis

• In situ tagging for localization and interaction studies:

  • Fluorescent protein fusions at native loci

  • Affinity tags for in vivo pulldown experiments

  • Split-protein complementation systems for interaction mapping

These approaches would provide unprecedented insights into lp_1974 function while maintaining chromosomal context and natural regulation, overcoming limitations of traditional overexpression or knockout strategies.

What role might comparative genomics play in understanding lp_1974 evolution and specialization across L. plantarum strains?

Comparative genomics approaches offer valuable insights into the evolution and specialization of lp_1974 across diverse L. plantarum strains isolated from different ecological niches. Whole genome sequence analysis of L. plantarum strains from various sources, such as ethnic fermented foods, has revealed significant genetic diversity .

For investigating lp_1974 evolution, researchers should:

• Sequence acquisition and analysis:

  • Collect lp_1974 sequences from diverse L. plantarum isolates

  • Perform multiple sequence alignments to identify conserved and variable regions

  • Construct phylogenetic trees to infer evolutionary relationships

  • Calculate selection pressures (dN/dS ratios) across the gene length

• Genomic context examination:

  • Analyze synteny of regions flanking lp_1974

  • Identify potential horizontal gene transfer signatures

  • Evaluate presence of mobile genetic elements nearby

  • Compare regulatory elements across strains

• Functional correlation:

  • Link sequence variants to phenotypic differences in carbohydrate utilization

  • Correlate lp_1974 variants with strain ecological adaptations

  • Identify strain-specific post-translational modifications

This comparative approach could reveal how lp_1974 has evolved to optimize L. plantarum's adaptation to specific niches, from traditional fermented foods to the human gut microbiome, potentially explaining the remarkable ecological versatility of this bacterial species .