Recombinant Lactobacillus johnsonii ATP synthase gamma chain (atpG)

What is the primary function of ATP synthase gamma chain (atpG) in Lactobacillus johnsonii?

The ATP synthase gamma chain (atpG) in Lactobacillus johnsonii, like in other bacteria, produces ATP from ADP in the presence of a proton gradient across the membrane. The gamma chain specifically plays a critical regulatory role in ATPase activity and controls the flow of protons through the CF(0) complex . In L. johnsonii, this energy production is particularly important given its limited biosynthetic capabilities, as the organism depends heavily on transport mechanisms and energy generation to acquire essential nutrients from its environment .

What is the molecular structure of L. johnsonii atpG and how does it compare to atpG in other species?

The ATP synthase gamma chain belongs to the ATPase gamma chain family . While the search results don't provide the specific sequence for L. johnsonii atpG, we can extrapolate from related species. For instance, in Geobacter metallireducens, the gamma chain consists of 287 amino acids with a molecular weight of approximately 31.9 kDa .

Comparative structural analysis methodology:

• Perform sequence alignment between L. johnsonii atpG and other bacterial species

• Conduct homology modeling using crystallographic data from related species

• Analyze conserved domains using tools like PFAM and InterPro

• Identify L. johnsonii-specific structural features that may relate to its probiotic functions

How does the genomic context of atpG in L. johnsonii influence its expression?

L. johnsonii NCC 533 has a 1.99-Mb genome that has been fully sequenced and analyzed . The genomic context of atpG should be examined in relation to the organism's metabolic capabilities. L. johnsonii demonstrates limited biosynthetic pathways, being auxotrophic for amino acids, nucleotides, and many cofactors . This genomic context suggests that expression of energy-generating proteins like atpG is likely under tight regulatory control.

The regulatory mechanisms may involve:

• Negative regulators from the GntR, LacI, RpiR, and ArsR families, which are numerically predominant in L. johnsonii

• Phospho-sugar responsive repressors, which are relatively abundant in L. johnsonii compared to other bacteria

• Potential coordination with the organism's extensive transport systems that compensate for its biosynthetic limitations

What are the optimal expression systems for producing recombinant L. johnsonii atpG?

While the search results don't provide specific information about atpG expression systems, researchers can consider established methodologies for expressing recombinant proteins from Lactobacillus species:

Heterologous Expression Methodology:

• E. coli-based systems: Similar to the approach used for L. acidophilus deoxynucleoside kinases where genes were expressed in transformed E. coli

• Lactobacillus-specific vectors: These may provide more appropriate post-translational modifications

• Codon optimization: Essential given the different codon preferences between Lactobacillus and common expression hosts

Expression Optimization Table:

What purification strategies yield the highest purity and activity for recombinant L. johnsonii atpG?

Purification of membrane-associated proteins like ATP synthase components requires specialized approaches:

Recommended Purification Protocol:

• Cell lysis under conditions that preserve protein structure (mild detergents, appropriate pH buffer)

• Affinity chromatography using His-tag or other fusion tags

• Ion exchange chromatography to separate based on charge differences

• Size exclusion chromatography for final polishing

• Activity assays at each purification step to track functional protein yield

The purification strategy should account for:

• The membrane association of ATP synthase components

• The need to maintain the native conformation for functional studies

• Potential requirement for lipid reconstitution to assess full activity

How can researchers verify the proper folding and activity of recombinant L. johnsonii atpG?

Verification of proper folding and activity requires multiple complementary approaches:

Activity Assessment Protocol:

• ATPase activity assay: Measure ATP hydrolysis rates using colorimetric phosphate detection

• Proton pumping assay: Assess using pH-sensitive fluorescent probes in reconstituted vesicles

• Circular dichroism spectroscopy: Analyze secondary structure elements

• Thermal shift assays: Evaluate protein stability under various conditions

• Limited proteolysis: Compare digestion patterns between recombinant and native protein

Researchers should compare the activity of the recombinant protein to the anticipated activity based on known characteristics of ATP synthase gamma chains in related bacterial species.

How does atpG contribute to the probiotic properties of L. johnsonii?

L. johnsonii is known for probiotic activities including pathogen inhibition, epithelial cell attachment, and immunomodulation . While the search results don't directly link atpG to these functions, we can propose research methodologies to investigate this relationship:

Experimental Approach:

• Generate atpG knockout mutants and assess changes in probiotic properties

• Perform comparative proteomics between wild-type and atpG-modified strains

• Assess energy status and its correlation with adhesion capabilities and immunomodulatory effects

• Investigate whether ATP production influences the synthesis or secretion of bioactive compounds

The energy production mediated by ATP synthase may be particularly important for L. johnsonii given its limited biosynthetic capabilities and reliance on transport mechanisms .

What is the relationship between L. johnsonii atpG and gut barrier function enhancement?

L. johnsonii has been shown to enhance gut barrier integrity through the interaction between its glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and mouse tight junction protein JAM-2 . While atpG is not directly implicated in this interaction, researchers could investigate:

• Whether ATP production by atpG indirectly supports the GAPDH-JAM-2 interaction

• If energy status influences the surface expression of GAPDH

• How modifications to atpG affect the organism's ability to repair damaged tight junctions

Experimental protocol for assessing barrier function:

• Measure transepithelial electrical resistance (TEER) in Caco-2 cell monolayers

• Assess fluorescent dextran permeability before and after treatment with wild-type or atpG-modified strains

• Quantify tight junction protein expression using Western blot and immunofluorescence

• Perform RNA sequencing to identify changes in host cell gene expression related to barrier function

What are the potential structural adaptations of L. johnsonii atpG that might reflect its adaptation to the intestinal environment?

ATP synthase components may have evolved specific adaptations for function in the gastrointestinal tract environment:

Research Methodology:

• Comparative sequence analysis between intestinal and non-intestinal Lactobacillus species

• Structure-function analysis focusing on regions facing the intestinal lumen

• pH-dependent activity profiling to assess adaptation to gut pH gradients

• Analysis of protein stability under bile salt and digestive enzyme exposure

Researchers might investigate whether L. johnsonii atpG has adaptations reflecting its probiotic lifestyle, considering that the genome analysis of L. johnsonii NCC 533 revealed "an unexpected number of genes that are not widely distributed among prokaryotes and hence may be important for the ability of L. johnsonii NCC 533 to persist and compete in the complex ecosystem of the GIT [gastrointestinal tract]" .

How does the regulation of atpG expression interact with L. johnsonii's nutritional requirements and transport systems?

L. johnsonii is auxotrophic for multiple nutrients including amino acids, nucleotides, and cofactors, compensating with enhanced transport capabilities . Researchers investigating the relationship between energy production (via atpG) and nutrient acquisition could:

• Map regulatory networks connecting energy status and transporter expression

• Assess how atpG expression changes in response to different nutrient limitations

• Investigate whether ATP synthase activity coordinates with specific transport systems

• Determine if atpG regulation involves the abundant negative regulators identified in L. johnsonii (GntR, LacI, RpiR, and ArsR families)

Experimental Design Table:

How does L. johnsonii atpG compare structurally and functionally to atpG in other probiotic bacteria?

This comparative analysis would help researchers understand conserved and specialized features:

Research Approach:

• Sequence alignment of atpG from L. johnsonii, L. acidophilus, L. gasseri, and other probiotics

• Phylogenetic analysis to understand evolutionary relationships

• Functional comparison through complementation studies

• Structural modeling to identify probiotic-specific adaptations

What methodologies are most effective for studying the role of atpG in host-microbe interactions involving L. johnsonii?

Investigating how atpG influences L. johnsonii's interaction with the host requires interdisciplinary approaches:

Recommended Methodologies:

• In vitro co-culture systems: Epithelial cell lines with wild-type or atpG-modified strains

• Ex vivo organ cultures: Intestinal tissue explants to assess physiological responses

• Gnotobiotic animal models: Mono-colonization with wild-type or atpG-mutant strains

• Host transcriptomics: Analyze host gene expression changes in response to different strains

• Metaproteomics: Identify proteins at the host-microbe interface

The search results indicate that L. johnsonii promotes barrier function integrity via GAPDH-JAM-2 binding . Similar protein-protein interaction studies could be designed to investigate whether atpG or ATP synthase complex components directly or indirectly interact with host factors.

What are the emerging technologies that could advance our understanding of L. johnsonii atpG structure and function?

Researchers should consider these cutting-edge approaches:

Advanced Methodologies:

• Cryo-electron microscopy: For high-resolution structural analysis of the ATP synthase complex

• Single-molecule FRET: To study conformational changes during the catalytic cycle

• CRISPR-Cas9 genome editing: For precise manipulation of atpG in L. johnsonii

• Biomolecular NMR: For dynamic studies of protein-protein interactions

• Microfluidic systems: To study ATP synthase function under controlled microenvironmental conditions

How might recombinant L. johnsonii atpG be utilized in synthetic biology applications for probiotic enhancement?

This question explores forward-looking research applications:

Research Directions:

• Engineering atpG variants with enhanced efficiency for improved probiotic fitness

• Creating synthetic regulatory circuits linking atpG expression to sensing of intestinal conditions

• Developing L. johnsonii strains with optimized energy production for enhanced probiotic functions

• Designing chimeric ATP synthase complexes with novel regulatory properties

• Exploring whether modified atpG could enhance the production of beneficial compounds

The extensive dependence of L. johnsonii on environmental nutrients suggests that optimizing energy production through atpG engineering could significantly impact its probiotic capabilities.