Recombinant Lactobacillus plantarum Probable rRNA maturation factor (lp_1969)

What is the genomic context of the lp_1969 gene in L. plantarum WCFS1?

The lp_1969 gene is part of the 3,308,274-bp chromosome of Lactobacillus plantarum strain WCFS1, which contains 3,052 predicted protein-encoding genes. L. plantarum has five rRNA operons that are distributed evenly around the chromosome, and the rRNA maturation factor would likely be functionally associated with these operons . The gene is part of the complete genome sequence that has been determined for this strain, which was originally isolated from human saliva .

How does the expression of lp_1969 compare to other genes in L. plantarum?

While specific expression data for lp_1969 is not directly provided in the search results, research on L. plantarum has identified various expression patterns for different genes. Studies have employed techniques such as resolvase-based in vivo expression technology (R-IVET) to identify genes whose expression is induced under specific conditions, such as passage through the gastrointestinal tract . Comparative analysis using proteomic approaches, such as those employed in the CMCC-P0002 strain study, could help determine the relative abundance of lp_1969 among the 434 proteins identified through tandem mass spectrometry .

What predictive functional analysis can be performed for lp_1969?

As a probable rRNA maturation factor, lp_1969 likely plays a role in the processing and maturation of ribosomal RNA. Functional prediction can be performed by comparing the lp_1969 sequence with known rRNA maturation factors in related organisms. Of the 3,052 protein-encoding genes in L. plantarum WCFS1, biological functions have been assigned to approximately 70% (2,120) of the predicted proteins . Researchers should employ bioinformatic tools to analyze conserved domains, predict secondary structure, and identify potential functional motifs to understand the likely role of lp_1969 in ribosome biogenesis.

What are the optimal promoter systems for recombinant expression of lp_1969 in L. plantarum?

Several constitutive promoters have been tested in L. plantarum CD033, including P11, Ptuf33, and Ptuf34, with varying expression strengths. The P11 promoter, originally derived from L. plantarum WCSF1, has been confirmed feasible for strong constitutive protein expression . Transcription elongation factor promoters isolated from L. plantarum CD033 and L. buchneri CD034 have also shown effectiveness. For optimal expression of lp_1969, researchers should consider testing these promoters, as promoter strength is a key factor controlling gene expression .

How do plasmid copy numbers affect the expression levels of recombinant proteins in L. plantarum?

Studies have demonstrated that high copy number origins of replication increase protein expression approximately twofold compared to low copy number origins in L. plantarum. Specifically, constructs containing a high copy number origin of replication derived from the L. buchneri CD034 plasmid pCD034-1 showed significantly higher product yields . When expressing lp_1969, researchers should consider using the pCDLbu-1ΔEc-based constructs with a high copy number origin for maximum protein yield, while low copy constructs may be preferable if the overexpression causes cellular stress .

What is the optimal spacing between the Shine-Dalgarno sequence and start codon for efficient translation of lp_1969?

Research on L. plantarum CD033 has determined that the optimal spacer between the Shine-Dalgarno sequence and the start codon consists of 8 nucleotides. Both elongation and shortening of this sequence gradually down-regulates gene expression . For fine-tuning translational efficiency of lp_1969, researchers should consider optimizing this spacing, as variations in the range between 5 and 12 nucleotides have been analyzed to determine their impact on protein expression levels .

What proteomic approaches are suitable for detecting and quantifying lp_1969 expression?

Two-dimensional electrophoresis methods have been successfully used to separate whole-cell proteins and secretory proteins of L. plantarum, with subsequent identification by tandem mass spectrometry . For studying lp_1969, researchers should consider employing similar proteomic techniques, particularly if the protein is among the more abundant cellular proteins. In the proteomic database of L. plantarum CMCC-P0002, information on the first 20 highest abundance proteins has been documented, which can serve as a reference for expression-level comparison .

How can protein-protein interactions of lp_1969 be characterized?

The Blue-Native/SDS-PAGE technique has been successfully applied to establish interaction maps in L. plantarum, identifying both heterodimeric and homodimeric complexes . As an rRNA maturation factor, lp_1969 likely interacts with ribosomal components and possibly other maturation factors. Researchers should consider employing this technique, coupled with mass spectrometry, to identify potential interaction partners of lp_1969, which would provide insights into its functional role in ribosome biogenesis.

What methods can be used to study the in vivo function of lp_1969 in L. plantarum?

To study the in vivo function of lp_1969, researchers can use homologous recombination approaches to create gene knockouts or modifications. A methodology involving a loxP-ery-loxP cassette has been described for L. plantarum WCFS1, allowing for homologous recombination via a double-crossover event . This approach involves constructing an integration vector, selecting primary single-crossover integrants using antibiotic markers, and confirming the desired chromosomal organization by PCR and Southern blotting . Application of this technique to lp_1969 would help determine its essentiality and functional importance in L. plantarum.

How can RT-PCR be optimized to study lp_1969 expression in complex environments?

Real-time RT-PCR methodologies have been developed to study L. plantarum gene expression in complex environments, such as the mouse digestive tract. PCR amplification protocols typically involve initiation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 55°C for 60 s . When studying lp_1969 expression in complex samples, researchers should include appropriate controls to detect background contamination (no-template control) and remaining chromosomal DNA (RT reactions without reverse transcriptase). Spiking experiments with L. plantarum RNA added to sample-derived RNA can confirm that amplification is not inhibited by sample components .

What approaches can determine if lp_1969 expression is induced during gastrointestinal passage?

Resolvase-based in vivo expression technology (R-IVET) has been successfully applied to identify L. plantarum genes induced during passage through the gastrointestinal tract compared to laboratory media . This approach identified 72 genes with induced expression in the GI tract environment. To determine if lp_1969 is similarly regulated, researchers could apply this R-IVET approach or develop targeted expression assays using RT-PCR with primers specific to lp_1969. Understanding environmental induction patterns could provide insights into the physiological relevance of this rRNA maturation factor under different growth conditions .

How can comparative genomics be used to understand the evolutionary significance of lp_1969?

The complete genome sequence of L. plantarum WCFS1 provides a foundation for comparative genomic analysis of lp_1969 across different strains and related species . Researchers should examine genome databases to identify orthologs of lp_1969 in other Lactobacillus species and related lactic acid bacteria. Analysis of sequence conservation, synteny (gene order), and potential horizontal gene transfer events could provide insights into the evolutionary history and importance of this gene. Special attention should be paid to examining whether lp_1969 resides within the 600-kb region near the origin of replication that contains many genes involved in environmental interactions, as this region has been identified as a lifestyle adaptation region in the L. plantarum chromosome .

How can codon optimization improve the expression of lp_1969?

Codon adaptation is an important factor in determining gene expression levels in L. plantarum. The genome-wide analysis of L. plantarum WCFS1 has identified codon usage patterns for potentially highly expressed genes, such as those encoding glycolysis and phosphoketolase pathway enzymes . For optimal expression of lp_1969, researchers should analyze its codon adaptation index and consider optimizing the coding sequence accordingly. Codon optimization should aim to match the codon usage of highly expressed genes in L. plantarum while maintaining appropriate mRNA secondary structure to ensure efficient translation.

What are common issues in purification of recombinant proteins from L. plantarum and how can they be addressed?

When purifying recombinant proteins like lp_1969 from L. plantarum, researchers may encounter challenges such as low yield, protein insolubility, or contamination with host proteins. The proteomic database of L. plantarum CMCC-P0002 has identified 434 proteins, including information on high-abundance proteins that might co-purify with the target . Researchers should consider implementing affinity tags that allow specific purification, optimizing lysis conditions based on predicted protein properties, and employing multi-step purification strategies that take advantage of unique physicochemical properties of lp_1969. Protein solubility can be improved by expressing lp_1969 as a fusion with solubility-enhancing partners or by optimizing growth and induction conditions to minimize inclusion body formation.

What are the best practices for confirming the functionality of recombinant lp_1969?

As an rRNA maturation factor, the functionality of recombinant lp_1969 should be assessed through its ability to participate in ribosome biogenesis. Researchers should consider complementation assays in lp_1969-deficient strains to determine if the recombinant protein can restore normal growth and ribosome profiles. In vitro assays using purified components of the ribosome assembly machinery could also be developed to assess specific biochemical activities, such as RNA binding, processing, or chaperone functions. Additionally, structural characterization through techniques like circular dichroism or limited proteolysis can confirm proper folding of the recombinant protein, which is a prerequisite for functionality.

How should researchers analyze differential expression data for lp_1969 across experimental conditions?

When analyzing differential expression of lp_1969 across conditions, researchers should employ robust statistical methods appropriate for the specific experimental platform used (e.g., RNA-seq, microarray, or qRT-PCR). For qRT-PCR data, appropriate reference genes should be selected based on their stable expression across the conditions being compared. Studies in L. plantarum have established methodologies for RNA extraction and analysis from complex samples, including host tissues . Researchers should consider both statistical significance and biological significance (fold-change thresholds) when interpreting expression differences, and validate findings using independent biological replicates and complementary techniques.

What bioinformatic pipelines can predict structural features of lp_1969 relevant to its function?

To predict structural features of lp_1969 relevant to its function as an rRNA maturation factor, researchers should employ a multi-faceted bioinformatic approach. This should include sequence alignment with characterized rRNA maturation factors, prediction of secondary structure elements, identification of conserved domains or motifs, and if possible, homology modeling based on crystal structures of related proteins. Particular attention should be paid to identifying potential RNA-binding motifs, as these would be essential for the predicted function in rRNA processing. Integration of structural predictions with available experimental data from related proteins can guide hypothesis generation for functional studies.