Recombinant Lactobacillus acidophilus Glycogen synthase (glgA)

What is glycogen synthase (glgA) in L. acidophilus and what is its significance?

Glycogen synthase (glgA) in Lactobacillus acidophilus is a key enzyme responsible for glycogen biosynthesis. The enzyme catalyzes the formation of α-1,4-glycosidic bonds essential for glycogen polymer construction. The significance of glgA lies in its central role in the glycogen metabolic pathway, which contributes to the survival and probiotic functionality of L. acidophilus in the gastrointestinal (GI) tract. The presence of intact glycogen metabolic gene clusters in L. acidophilus NCFM and other Lactobacillus species commonly found in mammalian host environments suggests that glycogen metabolism is an important adaptation for these bacteria in natural habitats . Research indicates that the capability to synthesize intracellular glycogen provides a competitive advantage for L. acidophilus retention in the mouse GI tract, highlighting the physiological importance of this enzyme in host colonization .

How is the glg operon organized in L. acidophilus and what factors affect its expression?

The glycogen metabolism genes in L. acidophilus are organized in the glg operon, which contains genes encoding proteins necessary for glycogen synthesis and degradation. Expression of the glg operon and subsequent glycogen accumulation in L. acidophilus is influenced by several factors, most notably the type and availability of carbon sources and growth phase. Studies have demonstrated that carbohydrate conditions significantly affect the expression patterns of glycogen metabolism genes . The regulation appears to be coordinated with the bacterium's metabolic state, where glycogen acts as an energy reserve polysaccharide. Detailed expression analysis indicates that the transcription of glg genes can be modulated by environmental conditions, suggesting a sophisticated regulatory mechanism that allows L. acidophilus to optimize its energy storage based on nutritional availability .

What CRISPR systems are available for engineering glgA in Lactobacillus species?

A portable CRISPR-Cas9N (nickase) system has been developed specifically for genome engineering in Lactobacillus species, including L. acidophilus, L. gasseri, and L. paracasei. This system, designated pLbCas9N (pTRK1203), is composed of a Gram-positive-E. coli shuttle vector harboring cas9N under the regulation of the P6 Lactobacillus promoter, with sgRNA driven by the endogenous promoter of elongation factor-Tu gene (tuf) . The system includes an editing template consisting of 1-kb homologous arms flanking the editing site and a chloramphenicol selection marker. This CRISPR-Cas9N system has been successfully used to create gene deletions in various Lactobacillus species, including a 1,299-bp in-frame deletion of glgA in L. paracasei Lpc-37 . The efficiency of this system makes it a valuable tool for researchers working with Lactobacillus species that have traditionally been difficult to genetically manipulate.

How can I create and confirm a glgA knockout in Lactobacillus species?

Creating a glgA knockout in Lactobacillus species using the pLbCas9N system involves several key steps:

• Design a target-specific sgRNA for the glgA gene

• Construct an editing template with 1-kb homologous regions flanking the targeted site

• Clone these components into the pLbCas9N vector

• Transform the resulting plasmid into your Lactobacillus strain

• Select transformants using chloramphenicol resistance

• Screen for deletion mutants using PCR with primers flanking the deletion target
  For example, in L. paracasei, a 1,299-bp in-frame deletion within glgA was achieved using the pTRK1257 plasmid constructed with this system . Confirmation of successful deletion can be performed using PCR screening with glgA-specific primers (such as lpc_glgA-F and lpc_glgA-R for L. paracasei) to verify the size difference between wild-type and mutant strains . Phenotypic confirmation is also essential - an iodine staining assay can be used to detect intracellular glycogen, with ΔglgA mutants showing deficiency in glycogen accumulation compared to wild-type strains .

What transformation efficiencies can be expected when targeting glgA in Lactobacillus species?

Transformation efficiencies when using the pLbCas9N system vary depending on the Lactobacillus species and the specific gene being targeted. For the pLbCas9N control vector without targeting components, transformation efficiency in L. acidophilus NCFM was reported to be ≥2 × 103 CFU/μg of vector DNA . When targeting specific genes, the transformation efficiencies were typically lower. For example, transformation efficiencies for editing plasmids targeting other genes in L. acidophilus were 1.1 to 1.9 log lower than that for the pLbCas9N control vector . While specific data for glgA transformation efficiency in L. acidophilus is not provided in the search results, researchers should expect some reduction in efficiency when using editing plasmids compared to control vectors. The larger size of editing plasmids containing both sgRNA and editing template contributes to the lower transformation efficiencies .

How can glycogen accumulation be detected in wild-type versus glgA mutants?

Glycogen accumulation in wild-type versus glgA mutants can be effectively detected using an iodine staining assay. This colorimetric method is based on the formation of a colored complex between iodine and the helical structure of glycogen. The procedure involves:

• Growing wild-type and ΔglgA mutant cultures under conditions that promote glycogen synthesis

• Harvesting cells at appropriate time points

• Exposing the cells to iodine solution

• Observing color development - wild-type cells capable of synthesizing glycogen will develop a brown-purple color, while ΔglgA mutants will show little to no color development
  As demonstrated in L. paracasei, iodine staining clearly showed a deficiency of glycogen accumulation in the ΔglgA mutant compared to the parent strain . This simple yet effective assay provides a rapid visual confirmation that glgA inactivation disrupts the glycogen biosynthetic pathway, resulting in the inability to synthesize intracellular glycogen. The assay serves as both a phenotypic confirmation of successful gene deletion and a functional assessment of the glycogen synthesis capability.

How does glycogen metabolism contribute to the competitive fitness of L. acidophilus in vivo?

Glycogen metabolism significantly contributes to the competitive fitness of L. acidophilus in the gastrointestinal tract. In vivo studies using mouse models have demonstrated that the capability of synthesizing intracellular glycogen enhances the competitive retention of L. acidophilus in the GI tract . This competitive advantage likely stems from several mechanisms:

• Energy storage capability: Glycogen serves as an endogenous energy reserve that can be mobilized during nutrient limitation, providing a survival advantage in the competitive and fluctuating gut environment.

• Stress resistance: Glycogen reserves may contribute to better survival during transit through harsh GI conditions and adaptation to new microenvironments.

• Ecological fitness: The ability to store and utilize glycogen may influence niche occupation and interactions with other microbiota members and host cells.
  Research suggests that the glg operon in L. acidophilus has evolved to optimize energy storage and utilization in mammalian host environments, reflecting its adaptation as a commensal organism . The contribution of glycogen metabolism to competitive fitness underscores its importance not only for basic bacterial physiology but also for probiotic functionality in therapeutic applications.

What is the relationship between carbon source availability and glgA expression in L. acidophilus?

The relationship between carbon source availability and glgA expression in L. acidophilus reveals sophisticated regulatory mechanisms that link environmental sensing to energy storage. Studies indicate that both the type and availability of carbon sources significantly affect glg operon expression and glycogen biosynthesis in L. acidophilus . Key aspects of this relationship include:

• Differential expression: The glg operon expression varies with different carbohydrate sources, suggesting a prioritization system for carbon utilization and storage.

• Growth phase dependence: Expression patterns of glycogen metabolism genes change throughout the growth phases, with potential upregulation during transition to stationary phase when nutrient limitation becomes imminent.

• Regulatory integration: The glycogen metabolism pathway appears to be integrated with other metabolic networks, allowing coordinated responses to nutritional status.
  This dynamic relationship enables L. acidophilus to optimize its energy storage strategy based on environmental conditions, contributing to its ecological success in variable environments like the GI tract . Understanding these regulatory patterns provides insights into the metabolic adaptability of L. acidophilus and potential targets for modulating its probiotic functions.

What are common challenges in genetic manipulation of glgA in Lactobacillus species?

Genetic manipulation of glgA in Lactobacillus species presents several challenges that researchers should anticipate and address:

• Vector stability issues: For example, during development of the Cas9N system, delivery of Cas9N based on the pGK12 plasmid was unsuccessful due to instability of the cas9N expression cassette in various E. coli cloning hosts and in L. acidophilus . Similar stability issues may arise when manipulating glgA.

• Transformation efficiency constraints: Transformation efficiencies with editing plasmids are typically lower than with control vectors, potentially due to their larger size . When targeting glgA, researchers may need to optimize transformation protocols or screen more colonies to identify successful transformants.

• Mixed-genotype populations: As observed with some gene deletions in L. acidophilus, targeting glgA may result in mixed-genotype populations where only some cells contain the desired mutation . Purification of mutants may require additional steps such as streaking and isolating pure deletion mutants on selective media.

• Species-specific variations: Different Lactobacillus species may require adjustments to protocols. While the pLbCas9N system has been successful across multiple species, researchers should be prepared to optimize conditions for their specific Lactobacillus strain when targeting glgA .

How can I verify the complete removal of CRISPR-Cas9 plasmids after successful glgA mutation?

After successful glgA mutation, complete removal of CRISPR-Cas9 plasmids is essential for subsequent experiments and strain characterization. A systematic verification approach includes:

• Antibiotic sensitivity testing: Subculture mutants in media without antibiotics, then plate to obtain isolated colonies. Perform replica plating on both antibiotic-free and antibiotic-containing media. Successfully cured strains will be sensitive to the antibiotic (e.g., chloramphenicol for pLbCas9N) .

• PCR verification: Perform PCR using primers specific for the plasmid backbone. For pLbCas9N, primers such as P6-F and NC-R can be used to verify plasmid curing . The absence of PCR amplicons indicates successful plasmid elimination.

• Genomic confirmation: Perform whole-genome sequencing or targeted sequencing of the modified region to confirm both the intended mutation and absence of vector sequences.

• Functional testing: Verify the phenotypic consequences of glgA deletion using functional assays such as iodine staining for glycogen accumulation .
  Experience with the pLbCas9N system in Lactobacillus species has shown that 70-100% of mutant colonies become antibiotic-sensitive after just one passage in media without antibiotics, indicating efficient plasmid curing . This high rate of plasmid loss facilitates successive rounds of editing for creating multiple mutations if desired.

What are the most reliable phenotypic assays to confirm glgA function in recombinant Lactobacillus strains?

Several reliable phenotypic assays can confirm glgA function in recombinant Lactobacillus strains:

• Iodine staining assay: This is the most direct and widely used method to detect glycogen accumulation. Wild-type strains with functional glgA develop a brown-purple color when exposed to iodine solution, while ΔglgA mutants show minimal staining. This assay was successfully used to confirm glgA deletion in L. paracasei .

• Growth curve analysis under various carbon sources: Comparing growth profiles of wild-type and recombinant strains under different carbohydrate conditions can reveal physiological consequences of glgA modification, as glycogen metabolism affects carbon utilization strategies.

• Stress resistance testing: Since glycogen serves as an energy reserve during stressful conditions, assays measuring survival under acid stress, bile stress, or starvation conditions can indirectly assess glgA functionality.

• In vivo colonization studies: Mouse colonization models can assess the contribution of glgA to competitive fitness in the GI tract, as glycogen metabolism has been shown to enhance the competitive retention of L. acidophilus in the mouse GI tract .

• Biochemical quantification of glycogen: Direct measurement of intracellular glycogen content using enzymatic assays or analytical techniques provides quantitative data on glycogen synthesis capacity in wild-type versus recombinant strains. These complementary approaches provide robust confirmation of glgA function and insights into the physiological consequences of genetic modifications to the glycogen synthesis pathway in Lactobacillus species.