Recombinant Leuconostoc mesenteroides subsp. mesenteroides Energy-coupling factor transporter transmembrane protein EcfT (ecfT)

What is the structural composition of ECF transporters in Leuconostoc mesenteroides?

ECF transporters represent a specialized family of primary active transporters responsible for micronutrient and vitamin uptake. Based on current research, the functional assembly of group II ECF transporters consists of two substrate-binding S subunits, two identical T subunits (EcfT), and two homologous ATPase subunits (EcfA and EcfA') . This creates what researchers refer to as a "3 × 2 model" for ECF transporters. In this arrangement, each T subunit interacts with a conserved groove in the ATPase subunits, forming a 1A:1A':2T assembly that presents two membrane-embedded surfaces for interaction with the integral membrane S subunits . This configuration allows for the modularity characteristic of this transporter family, enabling different S subunits to assemble into the same transporter complex .

How do ECF transporters function mechanistically in bacterial systems?

The transport mechanism of ECF transporters follows several sequential steps with distinct conformational changes:

• Substrate binding to the S subunits triggers a structural transition from an open apo state to a closed, substrate-bound, occluded conformation .

• The free energy of high-affinity substrate binding couples to a conformational change in the T subunits that prealigns the two ATPase active sites into a conformation favoring ATP binding .

• This conformational change is transduced from motifs on the conserved surface formed by TM1–2–3 of the S subunits to the coupling helices and the Q-helices in the ATPase subunits .

• ATP binding drives the transition to a closed dimer of the ATPase subunits, leading to conversion of the T subunits to an outward-facing conformation .

• This ATP-driven conformational change in the T subunits releases the tightly bound substrate from the attached S subunits .

The precise mechanism of substrate release—whether it involves a temporary low-affinity binding site or direct translocation into the cytoplasm—remains under investigation and represents an active area of research .

What methods are used for cloning and expressing ECF transporter genes in Leuconostoc mesenteroides?

For cloning and expressing genes like ecfT in Leuconostoc mesenteroides, researchers typically employ the following methodological approach:

• Primer Design: Based on genome sequence data (such as that of L. mesenteroides ATCC 8293), design specific primers for PCR amplification of the target gene .

• PCR Amplification: Amplify the target gene using optimized PCR conditions .

• TA Cloning: The PCR amplicon can be directly ligated with a TA cloning vector (e.g., pGEM-T Easy vector) and transferred to E. coli DH5α for initial cloning and sequence verification .

• Sequence Verification: The insert containing the target gene is sequenced using universal primers (e.g., M13_F: 5'-GTTTTCCCAGTCACGAC-3') from the vector sequence .

• Homology Search: Conduct sequence homology search using BLAST programs (blastn and blastp) at NCBI .

• Subcloning into Expression Vector: The verified gene fragment is digested with appropriate restriction enzymes and inserted into a lactic acid bacteria - E. coli shuttle vector (e.g., pCW4) .

• Transformation: The recombinant plasmid is transferred to electro-competent L. mesenteroides cells using optimized electroporation protocols .

• Verification of Recombinant Expression: Analyze transformants for gene expression using techniques such as slot blot assays for transcription analysis .

This methodology has been successfully demonstrated with genes like menB in L. mesenteroides and can be adapted for ecfT and other ECF transporter components .

How can researchers differentiate between assembly models of ECF transporters when analyzing EcfT function?

Differentiating between competing assembly models of ECF transporters (e.g., the 3×2 model versus the 1×4 model) requires a multifaceted experimental approach:

Experimental Strategy Table:

To implement this differentiation strategy:

• Use dual-tagged EcfT constructs (e.g., His-tagged and FLAG-tagged) and perform sequential purification to determine if multiple T subunits co-purify .

• Conduct cross-linking experiments using chemical cross-linkers with varying spacer arm lengths to capture T-T interactions .

• Analyze the isolation patterns of A-A'-T subcomplexes, which in the 3×2 model would show a stable core without requiring S subunit interaction .

• Examine the symmetry properties of the assembled complex, as the 3×2 assembly displays twofold symmetry similar to ABC transporters .

• Test the ability to simultaneously assemble different S subunits into the same complex, which would support the 3×2 model's prediction of multiple substrate uptake capability .

These approaches allow researchers to gather converging evidence for the correct assembly model, which is crucial for understanding EcfT function in the context of the complete transporter.

What are the optimal conditions for analyzing EcfT-associated conformational changes during the transport cycle?

Analyzing conformational changes in EcfT during the transport cycle requires capturing transient states and understanding the coupling between ATP hydrolysis and substrate translocation. A systematic approach includes:

• Site-directed spin labeling combined with EPR spectroscopy: Introduce spin labels at strategic positions in EcfT to monitor distance changes between domains during the transport cycle. Focus particularly on:

  • The coupling helices that interact with ATPase subunits

  • Transmembrane regions that undergo conformational changes

  • Interfaces with S subunits that may reorient during transport

• Single-molecule FRET: Apply FRET pairs to EcfT and partner subunits to track real-time conformational dynamics during substrate binding and ATP hydrolysis. This approach can reveal:

  • The sequence of conformational changes

  • Rate-limiting steps in the transport cycle

  • Effects of substrate binding on EcfT conformation

• Time-resolved structural studies: Use techniques like time-resolved cryo-EM or hydrogen-deuterium exchange mass spectrometry (HDX-MS) to capture intermediate conformational states. These methods are particularly valuable for analyzing:

  • How ATP binding to A and A' subunits drives conformational changes in the T subunits

  • The mechanisms by which these changes release substrate from S subunits

  • The return to resting state after substrate translocation

• Computational approaches: Implement molecular dynamics simulations based on available structural data to predict:

  • Energy landscapes of conformational transitions

  • Key residues involved in transmitting conformational changes

  • Potential bottlenecks in the transport process

For optimal results, these experiments should be conducted under physiologically relevant conditions, with careful control of ATP concentrations, substrate availability, and membrane composition to mimic the natural environment of Leuconostoc mesenteroides.

How can researchers distinguish between EcfT-mediated transport and other transport mechanisms in Leuconostoc mesenteroides?

Distinguishing EcfT-mediated transport from other transport mechanisms requires specific experimental designs that exploit the unique properties of ECF transporters:

• Generate EcfT knockout strains: Create precise gene deletions or disruptions of ecfT in L. mesenteroides using CRISPR-Cas9 or traditional homologous recombination approaches. Compare transport activities between wild-type and knockout strains to identify EcfT-dependent processes.

• Perform substrate competition assays: ECF transporters often handle multiple substrates through different S-components that share the same EcfT-containing energizing module. Design experiments where:

  • Multiple known ECF transporter substrates are presented simultaneously

  • Transport rates are measured with varying concentrations of competitors

  • Results are analyzed using kinetic models that account for the unique sharing of EcfT among different S-components

• Exploit ATP dependence: ECF transporters require ATP hydrolysis, unlike secondary transporters. Compare transport under:

  • ATP-depleting conditions (using inhibitors like oligomycin)

  • Conditions that disrupt proton motive force (using protonophores)

  • Combined disruption of both energy sources

• Utilize S-component specificity: Develop transport assays using fluorescently labeled substrates specific to ECF transporters, and analyze how overexpression or mutation of EcfT affects their uptake.

• Measure vitamin uptake profiles: Since ECF transporters are particularly important for vitamin uptake, systematically measure the uptake of vitamins like riboflavin, thiamine, and biotin in wild-type versus EcfT-modified strains under carefully controlled nutrient conditions .

These approaches collectively provide a comprehensive toolkit to distinguish EcfT-mediated transport from other mechanisms operating in L. mesenteroides.

What are the most effective methods for overexpressing functional EcfT in Leuconostoc mesenteroides?

Overexpressing functional EcfT in Leuconostoc mesenteroides requires careful consideration of expression vectors, regulatory elements, and host physiology. Based on successful approaches with related genes, the following methodology is recommended:

• Vector selection: Utilize lactic acid bacteria-E. coli shuttle vectors like pCW4 that have been demonstrated to function effectively in Leuconostoc mesenteroides . These vectors maintain stability in both organisms, facilitating the cloning process.

• Promoter optimization:

  • For constitutive expression: Use strong native promoters from L. mesenteroides housekeeping genes

  • For inducible expression: Adapt nisin-inducible or lactose-inducible systems that have been optimized for lactic acid bacteria

• Codon optimization: Analyze the codon usage pattern in highly expressed L. mesenteroides genes and adjust the ecfT sequence accordingly to maximize translation efficiency while maintaining the amino acid sequence.

• Ribosome binding site (RBS) engineering: Design an optimal RBS sequence with appropriate spacing from the start codon to enhance translation initiation.

• Transformation protocol: Implement electroporation with optimized parameters:

  • Cell wall weakening treatments (glycine, lysozyme)

  • High voltage (1.5-2.5 kV)

  • Immediate recovery in MRS broth supplemented with osmoprotectants

• Expression verification: Monitor expression levels using:

  • Slot blot assays for transcription analysis

  • Western blotting with antibodies against EcfT or an epitope tag

  • Functional assays measuring transport activities

• Growth optimization: Adjust culture conditions (temperature, pH, media composition) to maximize expression while maintaining cell viability and proper protein folding.

This approach has been validated for genes like menB in L. mesenteroides, where significant increases in transcription were observed in recombinant strains compared to wild-type controls without affecting growth parameters .

What purification strategies yield the highest purity and activity of recombinant EcfT for structural studies?

Purifying membrane proteins like EcfT presents unique challenges due to their hydrophobicity and requirement for a suitable membrane-mimetic environment. A systematic purification strategy includes:

Detailed Purification Protocol:

• Membrane extraction and solubilization:

  • Harvest cells at optimal density (typically late log phase)

  • Disrupt cells using mechanical methods (French press or sonication)

  • Isolate membranes by differential centrifugation

  • Solubilize membranes using a detergent screen to identify optimal conditions:

• Affinity chromatography:

  • Utilize His-tagged constructs for IMAC (immobilized metal affinity chromatography)

  • Consider tandem affinity purification with dual-tagged constructs (e.g., His-tag and FLAG-tag) to enhance purity

  • Optimize imidazole concentrations for washing and elution steps to minimize non-specific binding

• Size exclusion chromatography:

  • Separate protein complexes based on size to isolate intact ECF transporter complexes

  • Monitor complex integrity by analyzing the stoichiometry of co-eluting subunits

  • Identify conditions that maintain the native 1A:1A':2T:2S assembly

• Detergent exchange or reconstitution:

  • For structural studies: Exchange initial detergent for ones more suitable for specific techniques (e.g., amphipols for cryo-EM)

  • For functional studies: Reconstitute purified complexes into proteoliposomes or nanodiscs to restore native-like membrane environment

• Quality control assessments:

  • SDS-PAGE and Blue Native PAGE to verify purity and complex integrity

  • Thermal stability assays to optimize buffer conditions

  • ATP hydrolysis assays to confirm functional activity

  • Substrate binding assays with purified S-components to verify interaction capability

This purification strategy has been successfully applied to related ECF transporters and can be adapted specifically for L. mesenteroides EcfT, maintaining the protein in its native conformational state for structural and functional investigations .

How can researchers effectively measure the transport activity of recombinant EcfT in Leuconostoc mesenteroides?

Measuring the transport activity of recombinant EcfT requires specialized assays that account for the unique characteristics of ECF transporters. The following methodological approaches provide comprehensive assessment:

• Radioisotope uptake assays:

  • Use radiolabeled substrates (³H- or ¹⁴C-labeled vitamins or micronutrients)

  • Measure time-dependent accumulation in cells expressing native versus recombinant EcfT

  • Perform competition assays with unlabeled substrates to determine specificity

  • Calculate kinetic parameters (Km, Vmax) for quantitative comparisons

• Fluorescent substrate analogs:

  • Develop fluorescent derivatives of natural substrates that retain transport properties

  • Measure uptake via fluorescence microscopy or flow cytometry

  • Perform real-time measurements in single cells to assess population heterogeneity

• Growth complementation assays:

  • Generate auxotrophic strains requiring ECF transporter-dependent nutrients

  • Compare growth rates between strains expressing wild-type versus modified EcfT

  • Measure growth under limiting concentrations of substrates to assess transport efficiency

• ATP consumption measurements:

  • Quantify ATP hydrolysis rates using luciferase-based assays

  • Compare ATP consumption in the presence and absence of transport substrates

  • Determine coupling efficiency between ATP hydrolysis and substrate translocation

• Reconstituted system assays:

  • Purify recombinant EcfT along with partner ECF components

  • Reconstitute into proteoliposomes with ATP regenerating system inside

  • Measure substrate accumulation in these controlled artificial systems

These approaches can be complemented with electrophysiological measurements in artificial membrane systems to directly measure substrate-induced currents or membrane potential changes associated with transport activity.

What are the key antimicrobial susceptibility patterns of Leuconostoc mesenteroides strains expressing recombinant EcfT?

Understanding antimicrobial susceptibility patterns is crucial for both research applications and potential biotechnological uses of recombinant L. mesenteroides strains. Based on studies of Leuconostoc species, the following patterns emerge:

Antimicrobial Susceptibility Profile:

For recombinant strains expressing EcfT, researchers should consider:

• Selection marker compatibility: Choose plasmid selection markers that align with the natural resistance profile of the host strain.

• Strain stability assessment: Monitor whether recombinant EcfT expression affects antimicrobial susceptibility patterns, particularly for antibiotics that target cell membrane integrity.

• Biofilm considerations: Evaluate whether EcfT overexpression influences biofilm formation capacity, which can significantly alter antimicrobial susceptibility profiles.

• Host-specific variations: Test each engineered strain individually, as susceptibility patterns may vary between different L. mesenteroides subspecies and strains .

• Transport-mediated resistance: Assess whether modified EcfT expression confers altered resistance to antimicrobials that might be substrates for ECF transporters.

This information is particularly valuable when designing experimental protocols involving antibiotic selection or when evaluating the biosafety aspects of recombinant L. mesenteroides strains for potential biotechnological applications.

How does EcfT expression influence the bifidogenic growth stimulation activity of Leuconostoc mesenteroides?

The relationship between EcfT expression and bifidogenic growth stimulation represents an important area of research with implications for probiotic applications. While not directly addressed in the search results, we can formulate a methodological approach based on related findings:

• Comparative metabolite profiling:

  • Compare metabolome profiles between wild-type and EcfT-overexpressing L. mesenteroides strains

  • Focus on the production of compounds known to stimulate Bifidobacterium growth, such as 1,4-dihydroxy-2-naphthoic acid (DHNA)

  • Use HPLC, LC-MS, or other analytical techniques to quantify these compounds

• Co-culture experiments:

  • Design experiments where Bifidobacterium species are grown in the presence of:
    a) Wild-type L. mesenteroides
    b) EcfT-overexpressing L. mesenteroides
    c) Control medium without Leuconostoc

  • Measure Bifidobacterium growth rates, final cell densities, and metabolic activities

• Spent medium assays:

  • Collect cell-free supernatants from cultures of wild-type and recombinant strains

  • Test their ability to stimulate Bifidobacterium growth

  • Fractionate supernatants to identify specific stimulatory components

  • Determine whether these components are differentially produced in EcfT-overexpressing strains

• Gene expression analysis in Bifidobacterium:

  • Analyze transcriptional responses in Bifidobacterium when exposed to products from different L. mesenteroides strains

  • Identify pathways that are specifically upregulated in response to EcfT-overexpressing strains

• Mechanistic investigations:

  • Determine whether EcfT overexpression affects the uptake or export of specific nutrients or signaling molecules

  • Investigate how altered nutrient acquisition might influence the production of bifidogenic compounds

This research approach would help establish whether ECF transporters, specifically EcfT, play a role in the known bifidogenic effects of certain Leuconostoc mesenteroides strains, potentially opening new avenues for probiotic strain development.

What are the common challenges in achieving stable expression of recombinant EcfT in Leuconostoc mesenteroides?

Achieving stable expression of membrane proteins like EcfT in Leuconostoc mesenteroides presents several technical challenges. Based on experiences with similar recombinant systems, researchers should be prepared to address:

• Plasmid stability issues:

  • Challenge: Loss of expression plasmids during prolonged cultivation

  • Solution: Optimize selection pressure; use compatible plasmid backbones like pCW4 that have demonstrated stability in L. mesenteroides ; implement chromosomal integration strategies

• Protein toxicity concerns:

  • Challenge: Overexpression of membrane proteins can disrupt membrane integrity

  • Solution: Use tunable/inducible promoter systems; optimize expression levels to balance yield with cellular viability; express with native partner proteins to facilitate proper membrane insertion

• Codon usage limitations:

  • Challenge: Inefficient translation due to codon bias

  • Solution: Perform codon optimization based on highly expressed genes in L. mesenteroides; analyze GC content and adjust accordingly

• Proper membrane insertion:

  • Challenge: Misfolding or aggregation of overexpressed EcfT

  • Solution: Co-express with chaperones; optimize growth temperature (often lower temperatures improve folding); include membrane-stabilizing additives in the growth medium

• Transformation efficiency:

  • Challenge: Low transformation rates limiting screening capability

  • Solution: Optimize electroporation conditions specifically for L. mesenteroides; prepare cells at optimal growth phase; use glycine-enhanced media to weaken cell walls prior to transformation

• Expression verification difficulties:

  • Challenge: Limited antibodies available for detection

  • Solution: Incorporate epitope tags that don't interfere with function; develop slot blot assays for transcript detection as demonstrated with menB ; use functional assays to confirm expression

Troubleshooting Decision Tree:

For researchers encountering expression problems, follow this systematic troubleshooting approach:

• Verify plasmid integrity by re-sequencing

• Confirm transformation by plasmid isolation and PCR

• Test transcription using RT-PCR or slot blot

• If transcript present but no protein detected, investigate translation efficiency

• If protein detected but inactive, examine membrane insertion and folding

• If expression unstable, evaluate plasmid stability and selection pressure

This structured approach helps identify the specific point of failure in the expression system, allowing for targeted solutions.

How can researchers address experimental artifacts when studying the interaction between EcfT and other ECF transporter components?

Studying protein-protein interactions involving membrane proteins like EcfT is particularly challenging due to their hydrophobic nature and complex assembly requirements. To minimize artifacts and ensure reliable results, researchers should implement the following strategies:

• Control for detergent-induced artifacts:

  • Challenge: Detergents can disrupt native interactions or induce non-physiological associations

  • Solution: Compare multiple detergent systems; validate interactions in membrane-mimetic environments like nanodiscs or amphipols; use cross-validation with in vivo techniques

• Address overexpression biases:

  • Challenge: Non-physiological expression levels can force interactions that wouldn't occur at native concentrations

  • Solution: Implement expression systems with tunable promoters; compare results across different expression levels; validate with endogenously tagged proteins

• Validate complex formation specificity:

  • Challenge: Distinguishing genuine from non-specific interactions

  • Solution: Include negative controls with unrelated membrane proteins; perform competition assays; use multiple tagged versions to confirm consistent co-purification patterns

• Minimize post-lysis artifacts:

  • Challenge: Interactions may form after cell disruption rather than representing in vivo associations

  • Solution: Implement in vivo cross-linking prior to lysis; use rapid purification protocols; compare results with in-cell techniques like FRET or split-reporter assays

• Account for missing components:

  • Challenge: Incomplete ECF transporter assemblies may exhibit non-native interactions

  • Solution: Express complete sets of components (A, A', T, and S subunits); validate the 3×2 model through comprehensive co-purification studies

• Cross-validate structural models:

  • Challenge: Individual techniques may introduce method-specific artifacts

  • Solution: Combine multiple structural approaches (X-ray crystallography, cryo-EM, cross-linking mass spectrometry); verify key structural elements across methods

These methodological considerations help ensure that observed interactions between EcfT and other ECF transporter components reflect their genuine physiological relationships rather than experimental artifacts, contributing to accurate models of transporter assembly and function.

What are the emerging approaches for investigating the role of EcfT in vitamin transport and bacterial metabolism?

The field of ECF transporter research is rapidly evolving, with several innovative approaches emerging to better understand EcfT's role in vitamin transport and metabolism:

• Systems biology integration:

  • Implement multi-omics approaches (transcriptomics, proteomics, metabolomics) to understand how EcfT expression influences global metabolic networks

  • Develop computational models that incorporate ECF transporter kinetics into whole-cell metabolic frameworks

  • Map condition-dependent expression patterns of ECF components in response to nutrient availability

• Single-cell analysis technologies:

  • Apply microfluidics-based approaches to study transport kinetics in individual cells

  • Utilize single-cell transcriptomics to investigate population heterogeneity in EcfT expression

  • Develop biosensors that report on vitamin uptake at the single-cell level

• Synthetic biology applications:

  • Engineer chimeric ECF transporters with modified substrate specificities

  • Develop EcfT variants with enhanced transport capabilities for specific vitamins

  • Create synthetic circuits linking EcfT-mediated transport to reporter outputs for high-throughput screening

• In situ structural studies:

  • Apply cryo-electron tomography to visualize ECF transporters in their native membrane environment

  • Develop advanced labeling techniques to track conformational dynamics in living cells

  • Implement correlative light and electron microscopy to connect function with structure

• Interspecies interactions:

  • Investigate how EcfT-mediated vitamin acquisition influences competitive or cooperative behaviors in microbial communities

  • Explore the role of ECF transporters in establishing synergistic relationships with hosts or other microorganisms

  • Study vitamin exchange mediated by ECF transporters in complex microbiomes

These emerging approaches will help unravel the complex relationships between EcfT function, vitamin transport, and bacterial metabolism, potentially leading to applications in synthetic biology, probiotics, and microbiome manipulation.

How might genetic diversity in ecfT genes impact ECF transporter function across different Leuconostoc mesenteroides strains?

Understanding genetic diversity in ecfT genes and its functional implications represents an important frontier in ECF transporter research:

• Comparative genomics approaches:

  • Sequence ecfT genes from diverse L. mesenteroides strains isolated from different environments

  • Perform phylogenetic analyses to identify evolutionary relationships and selection pressures

  • Correlate genetic variants with ecological niches and metabolic capabilities

• Structure-function analysis of variants:

  • Identify key polymorphic regions within EcfT sequences

  • Map these variations onto structural models to predict functional impacts

  • Focus on regions interfacing with ATPase subunits and S-components as these likely influence transport efficiency

• Functional complementation studies:

  • Express ecfT variants in a common genetic background

  • Measure transport activities for different vitamin substrates

  • Determine strain-specific differences in substrate preference or transport kinetics

• Domain swapping experiments:

  • Create chimeric EcfT proteins with domains from different strains

  • Identify domains responsible for specific functional properties

  • Engineer EcfT variants with optimized or novel functions

• Adaptation and evolution studies:

  • Subject L. mesenteroides strains to vitamin-limited conditions

  • Monitor adaptive mutations in ecfT genes

  • Characterize how these adaptations influence transport efficiency and specificity

This research avenue would not only advance our fundamental understanding of ECF transporter diversity but could also identify naturally occurring EcfT variants with enhanced properties for biotechnological applications, such as improved vitamin production or probiotic functionality.

What are the potential biotechnological applications of recombinant Leuconostoc mesenteroides strains with modified EcfT expression?

Recombinant L. mesenteroides strains with engineered EcfT expression offer several promising biotechnological applications:

• Enhanced probiotic functionality:

  • Engineer strains with optimized vitamin uptake capabilities

  • Develop strains that produce increased levels of bifidogenic compounds through enhanced precursor acquisition

  • Create designer probiotics with targeted vitamin delivery capabilities for specific health applications

• Vitamin bioproduction platforms:

  • Optimize vitamin uptake and metabolism pathways to enhance vitamin K2 (menaquinone) production

  • Develop strains with modified ECF transporters for efficient precursor uptake

  • Create co-culture systems where engineered L. mesenteroides provides vitamins to partner organisms

• Biocontrol applications:

  • Exploit the natural antimicrobial properties of Leuconostoc species

  • Engineer strains with enhanced nutrient acquisition to outcompete pathogens

  • Develop strains that can thrive in specific environmental niches for targeted biocontrol

• Biosensor development:

  • Create reporter systems linked to EcfT activity for vitamin detection

  • Develop whole-cell biosensors for monitoring vitamin availability in complex environments

  • Engineer diagnostic strains that respond to specific metabolites via modified ECF transport systems

• Microbiome modulation tools:

  • Design L. mesenteroides strains that can selectively promote beneficial members of the microbiome through vitamin provision

  • Develop strains that can establish stable populations in specific niches due to optimized nutrient acquisition

  • Create strains that can deliver therapeutic compounds to targeted microbiome locations

For these applications to reach their full potential, researchers must address challenges including:

• Maintaining genetic stability of engineered constructs during scale-up

• Optimizing expression levels to balance function with cellular fitness

• Ensuring biosafety through careful evaluation of antimicrobial resistance profiles

• Developing appropriate containment strategies for genetically modified strains

These biotechnological applications represent the translational frontier of basic research into ECF transporters and their components in Leuconostoc mesenteroides.