Recombinant Mycobacterium smegmatis Trehalase (MSMEG_4535), partial

What is the functional significance of trehalase (MSMEG_4535) in Mycobacterium smegmatis?

Trehalase (MSMEG_4535) in M. smegmatis is an enzyme that hydrolyzes trehalose (α,α-1,1-glucosyl-glucose), a nonreducing disaccharide essential for mycobacterial growth and survival. The enzyme plays a critical role in controlling intracellular trehalose levels, which is particularly important during transitions between dormant and active states. Research has demonstrated that trehalose accumulation is significant in dormant cells, while trehalase activation during resuscitation leads to trehalose breakdown, suggesting its importance in the dormancy-resuscitation cycle. The enzyme shows high specificity for α,α-trehalose and does not hydrolyze α,β-trehalose, β,β-trehalose, trehalose dimycolate, or other glucosides .

How does MSMEG_4535 trehalase differ from other mycobacterial trehalases?

Table 1: Comparison of Trehalase Properties Between M. smegmatis and M. tuberculosis

What are the recommended methods for cloning and expressing recombinant MSMEG_4535?

For successful cloning and expression of recombinant MSMEG_4535, researchers should amplify the gene from M. smegmatis mc² 155 chromosomal DNA using specific primers. Based on published research, the recommended primers are F4535 (5′-TATCTGCAGAAGAGGAGAGCTGCATGGTTCTGCAACAGACCGA-3′) and R4535 (5′-CACAAGCTTCGGCCAACAGCAGCCTCACC-3′). The forward primer includes a PstI restriction site (underlined), an artificial ribosome-binding site (italicized), and the first 20 nucleotides of the gene with the ATG start codon (bold). The reverse primer contains a HindIII site (underlined) .

The amplified PCR product should be TA-cloned into an appropriate vector (such as pGEM-T easy), digested with PstI and HindIII enzymes, and then subcloned into an expression vector (like pES) pre-digested with the same restriction enzymes. The final construct can be transformed into M. smegmatis mc² 155 strain by electroporation. Expression systems including E. coli, yeast, mammalian, and insect cells are also viable options, with appropriate fusion tags (His, FLAG, MBP, GST, etc.) to aid in purification and detection .

What purification strategies yield the highest purity and activity for recombinant MSMEG_4535 trehalase?

Optimal purification of recombinant MSMEG_4535 trehalase requires a multi-step approach to achieve high purity while maintaining enzymatic activity. An effective purification strategy includes:

• Initial clarification of cell lysate by centrifugation to remove cellular debris

• Affinity chromatography using the appropriate tag (His-tag being commonly used)

• Size exclusion chromatography, noting that active trehalase elutes in the void volume of a Sephacryl S-300 column, suggesting a multimeric structure with a molecular mass of approximately 1500 kDa

• Maintaining phosphate buffer throughout purification to preserve enzyme stability

• Including Mg²⁺ in all buffers to maintain the catalytic competence of the enzyme

When assessing purity, SDS-PAGE should reveal a single 71 kDa band, while native gel electrophoresis or size exclusion chromatography will indicate the multimeric state. Importantly, researchers should include phosphate in all buffers, as it significantly enhances the heat stability of trehalase and appears to stabilize protein conformation .

How do phosphate and magnesium ions affect MSMEG_4535 trehalase activity, and what are the optimal assay conditions?

MSMEG_4535 trehalase exhibits an absolute requirement for both inorganic phosphate and Mg²⁺ ions for catalytic activity. The phosphate requirement is unusual among glycosyl hydrolases. Despite this requirement, there is no evidence for phosphorolytic cleavage or phosphorylated intermediates in the reaction. Rather, phosphate appears to bind to the enzyme, greatly increasing its heat stability, suggesting a role in stabilizing protein conformation and/or initiating protein aggregation .

Optimal assay conditions for MSMEG_4535 trehalase activity include:

• Buffer: Sodium phosphate (optimal concentration between 50-100 mM)

• pH: 6.5-7.0

• Temperature: 37°C

• Mg²⁺ concentration: 5-10 mM

• Substrate (α,α-trehalose) concentration: 10-20 mM

It's important to note that sodium arsenate can partially substitute for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates are inhibitory to enzyme activity. When designing assays, researchers should be aware that ATP (even at concentrations as low as 2 mM) prevents self-activation of trehalase in vitro and that the activated enzyme remains sensitive to ATP inhibition .

What mechanistic insights explain the multimeric structure of MSMEG_4535 trehalase?

The trehalase from M. smegmatis presents an intriguing structural organization. While it appears as a single 71 kDa band on SDS-PAGE gels, active enzyme elutes in the void volume of a Sephacryl S-300 column, suggesting a molecular mass of approximately 1500 kDa. This indicates a multimeric structure composed of approximately 20 or more subunits .

The multimeric structure likely contributes to enzyme stability and regulation. Evidence suggests that phosphate plays a role in this multimerization process, potentially by inducing conformational changes that promote subunit association. This phosphate-dependent multimerization may serve as a regulatory mechanism, allowing the enzyme to respond to changes in cellular phosphate levels. The precise arrangement of subunits and the molecular interactions driving assembly remain active areas of research, with implications for understanding enzyme regulation in the context of mycobacterial physiology .

How does trehalose metabolism change during M. smegmatis dormancy, and what role does MSMEG_4535 play?

During transition to dormancy under gradual acidification of growth medium, M. smegmatis undergoes significant changes in trehalose metabolism. NMR spectroscopy reveals that trehalose accumulates to remarkably high levels in dormant cells, comprising up to 64% of total organic substances, compared to only 15% in active cells from early stationary phase. This accumulation is facilitated by the activation of genes involved in two trehalose biosynthetic pathways: OtsA-OtsB and TreY-TreZ, as confirmed by RT-PCR analysis .

MSMEG_4535 (trehalase) plays a crucial regulatory role in this process. During dormancy, trehalase activity is suppressed, allowing trehalose to accumulate. Studies manipulating MSMEG_4535 expression have established a direct relationship between trehalose levels and cell viability: dormant cells with higher trehalose content demonstrate significantly better survival rates than those with lower levels. This suggests that trehalose accumulation serves as a protective mechanism during dormancy, potentially acting as a compatible solute, energy reserve, and stabilizer of cellular structures .

Table 2: Trehalose Content During Different Growth Phases of M. smegmatis

What molecular events involving MSMEG_4535 occur during resuscitation from dormancy?

During resuscitation of dormant M. smegmatis, a precise sequence of molecular events occurs involving MSMEG_4535 trehalase. Within the first 2 hours of resuscitation, a significant decrease in free trehalose concentration is observed, accompanied by a corresponding increase in glucose levels. This change is directly attributable to trehalase activity, which shows a transient but marked increase between 1-3 hours into the resuscitation process .

Importantly, this activation of trehalase is not due to de novo protein synthesis but rather results from self-activation of preexisting enzyme molecules transitioning from an inactive state in dormant cells. The regulatory mechanism appears to involve ATP: in vitro studies demonstrate that ATP at concentrations as low as 2 mM prevents trehalase self-activation, and the activated enzyme remains sensitive to ATP inhibition. This suggests that fluctuations in intracellular ATP concentration during early resuscitation temporarily relieve inhibition, allowing transient trehalase activation .

Further evidence for the critical role of trehalase in resuscitation comes from experiments with validamycin A, a trehalase inhibitor. Treatment with this compound negatively impacts the resuscitation of dormant cells, confirming that trehalose breakdown via trehalase activity is essential for successful reactivation. This mechanism mirrors similar processes observed in the germination of yeast and fungal spores, suggesting evolutionary conservation of trehalose metabolism in microbial dormancy-resuscitation cycles .

What is known about the ATP-mediated regulation of MSMEG_4535 trehalase activity?

ATP plays a sophisticated role in regulating MSMEG_4535 trehalase activity through multiple mechanisms. Research has demonstrated that ATP, even at concentrations as low as 2 mM, prevents the self-activation of trehalase in vitro. Once the enzyme is activated, it remains sensitive to ATP inhibition, suggesting a dual regulatory mechanism where ATP influences both the activation process and the activity of the already-activated enzyme .

During dormancy, intracellular ATP levels are presumably sufficient to maintain trehalase in an inactive state, allowing trehalose to accumulate. Upon initiation of resuscitation, changes in cellular energy status likely lead to fluctuations in ATP concentration. The transient decrease in ATP during early resuscitation (1-3 hours) appears to temporarily relieve inhibition, allowing trehalase to self-activate and initiate trehalose hydrolysis. As cellular metabolism fully restarts and ATP levels rise again, trehalase activity is once more suppressed, explaining the transient nature of the activation observed during resuscitation .

This ATP-mediated regulation creates a sophisticated control system that links trehalose metabolism directly to the cell's energy status, ensuring that trehalose breakdown occurs precisely when needed to support resuscitation from dormancy.

How do researchers distinguish between the three trehalose biosynthetic pathways in M. smegmatis and their relative contributions?

M. smegmatis possesses three distinct biosynthetic pathways for trehalose production, and distinguishing between these pathways and assessing their relative contributions requires a multi-faceted experimental approach:

• Gene Expression Analysis: RT-PCR targeting key genes from each pathway (OtsA-OtsB, TreY-TreZ, and TreS) during different growth phases has revealed that the OtsA-OtsB and TreY-TreZ pathways are specifically activated during transition to dormancy, suggesting their primary role in trehalose accumulation under these conditions .

• Metabolic Labeling: Incorporation of isotopically labeled precursors (¹³C-glucose or ¹⁴C-glucose) followed by analysis of labeled trehalose can trace the flow through different pathways.

• Genetic Manipulation: Construction of single, double, and triple pathway mutants by targeting key genes in each pathway enables assessment of the relative contribution of each pathway under different conditions. Previous research has shown that mutants devoid of all three pathways require exogenous trehalose for growth, confirming the essential nature of this metabolite .

• Enzymatic Assays: In vitro assays of key enzymes from each pathway under conditions mimicking different physiological states can provide insights into potential regulatory mechanisms.

The relative contribution of each pathway appears to be context-dependent, with the OtsA-OtsB and TreY-TreZ pathways predominating during dormancy induction, while the role of the TreS pathway (which interconverts trehalose and maltose) may be more significant under other conditions .

What are the implications of arginine phosphorylation on MSMEG_4535 function and regulation?

Recent proteomic studies have identified arginine phosphorylation in M. smegmatis proteins, suggesting a novel regulatory mechanism that may impact MSMEG_4535 trehalase. While direct evidence for arginine phosphorylation of trehalase is still emerging, this post-translational modification could represent an additional layer of regulation beyond the established ATP-mediated control .

Arginine phosphorylation could potentially affect:

• Protein-protein interactions within the multimeric trehalase complex

• Substrate binding affinity or catalytic efficiency

• Interactions with regulatory molecules or protein partners

• Conformational changes affecting enzyme activation/inactivation

Research into this area requires sophisticated phosphoproteomic approaches. Experimental designs should include at least three biological replicates to ensure statistical validity, with appropriate controls for phosphatase activity during sample preparation. Mass spectrometry analysis on Q Exactive Orbitrap interfaced with Ultimate 3000 Nano-LC systems offers the resolution necessary to identify specific phosphorylation sites .

Further investigation of arginine phosphorylation in MSMEG_4535 may reveal connections between protein phosphorylation networks and trehalose metabolism, potentially uncovering new strategies for modulating mycobacterial dormancy and resuscitation.

How can researchers exploit MSMEG_4535 trehalase to develop new strategies for controlling mycobacterial dormancy and resuscitation?

Understanding MSMEG_4535 trehalase offers several strategic approaches for controlling mycobacterial dormancy and resuscitation:

• Targeted Inhibition: Developing specific inhibitors of trehalase, building on the known effects of validamycin A, could potentially prevent resuscitation of dormant mycobacteria. This approach might be valuable for addressing persistent infections caused by dormant mycobacteria that are otherwise resistant to conventional antibiotics .

• Manipulation of Trehalose Levels: Genetic or pharmacological interventions that alter trehalose accumulation in dormant cells could impact long-term survival. Since higher trehalose levels correlate with better viability of dormant cells, reducing trehalose synthesis might compromise the ability of pathogenic mycobacteria to persist in a dormant state .

• ATP-Responsive Systems: Exploiting the ATP-sensitive regulation of trehalase could provide a means to trigger artificial resuscitation at inappropriate times, potentially rendering dormant cells vulnerable to concurrent antibiotic treatment.

• Structural Studies: Advanced structural analysis of MSMEG_4535, particularly its multimeric organization and the binding sites for phosphate and Mg²⁺, could inform the design of specific modulators with therapeutic potential.

Researchers pursuing these strategies should employ a combinatorial approach, integrating genetic manipulation, biochemical characterization, and in vivo infection models. Comparative studies with pathogenic mycobacteria like M. tuberculosis will be essential for translating findings from the M. smegmatis model system to clinically relevant applications .