Recombinant Xylella fastidiosa Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is the molecular function of MtgA in Xylella fastidiosa?

MtgA (Monofunctional peptidoglycan glycosyltransferase) in X. fastidiosa, as in other bacteria, catalyzes the polymerization of glycan chains during peptidoglycan synthesis, a critical component of bacterial cell wall assembly. The enzyme facilitates the formation of β-1,4-glycosidic bonds between N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues using lipid II as a substrate. In X. fastidiosa subsp. pauca specifically, mtgA has been identified as one of 96 subspecies-specific genes, suggesting it may contribute to the unique biological characteristics and host specificity of this subspecies . While its precise role in X. fastidiosa pathogenicity remains under investigation, evidence from studies in other bacteria indicates that MtgA may collaborate with other cell division proteins to synthesize peptidoglycan at the bacterial septum during division .

How does MtgA expression differ among Xylella fastidiosa subspecies?

The expression and presence of the mtgA gene varies significantly across X. fastidiosa subspecies. Genomic analyses have revealed that mtgA is predominantly found in X. fastidiosa subsp. pauca, where it appears to be conserved across multiple strains. In contrast, this gene is generally absent in X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex . This differential gene presence suggests that MtgA may contribute to the distinctive pathogenicity and host specificity of X. fastidiosa subsp. pauca, which is primarily associated with citrus variegated chlorosis and olive quick decline syndrome. This subspecies-specific distribution indicates that mtgA might have been acquired through horizontal gene transfer or retained due to selective pressures related to the subspecies' ecological niche and host adaptation . The expression patterns likely correlate with growth phase and environmental conditions, potentially increasing during active cell division when peptidoglycan synthesis is most active.

What is the evolutionary significance of mtgA in Xylella fastidiosa subspecies?

The presence of mtgA specifically in X. fastidiosa subsp. pauca but not in other subspecies highlights its evolutionary significance in bacterial adaptation and specialization. This distribution pattern suggests that mtgA may be part of the genetic toolkit that enables X. fastidiosa subsp. pauca to infect and colonize its specific host plants. Genomic diversity studies indicate that recombination plays a crucial role in shaping X. fastidiosa genomes, with each subspecies experiencing different selective pressures . The acquisition or retention of mtgA in X. fastidiosa subsp. pauca likely provides some adaptive advantage in its specific ecological niche. The gene may have been acquired through horizontal gene transfer events, which are known to contribute significantly to bacterial evolution by introducing novel functions. Alternatively, it may represent an ancestral gene that was lost in other lineages due to different selective pressures. Understanding the evolutionary trajectory of mtgA could provide insights into the divergence of X. fastidiosa subspecies and their adaptation to different host plants and environmental conditions .

What structural domains characterize the MtgA protein in Xylella fastidiosa?

The MtgA protein in X. fastidiosa, like its homologs in other bacteria, contains specific structural domains characteristic of monofunctional glycosyltransferases. Based on comparative analyses with better-characterized bacterial MtgA proteins such as those from E. coli, the X. fastidiosa MtgA likely possesses:

• A glycosyltransferase domain that belongs to the GT51 family (according to CAZy classification), which catalyzes the formation of β-1,4-glycosidic bonds.

• A transmembrane segment that anchors the protein to the cytoplasmic membrane, positioning the catalytic domain appropriately for peptidoglycan synthesis.

• A catalytic site containing conserved glutamate residues essential for the glycosyltransferase reaction.

The protein likely adopts a fold similar to other peptidoglycan glycosyltransferases, with a globular catalytic domain extending into the periplasmic space. While detailed crystallographic studies of X. fastidiosa MtgA are not yet available in the provided search results, structural predictions based on homology modeling would likely reveal similarities to the E. coli MtgA structure, which has been characterized as having an elongated shape with active site residues positioned to interact with the lipid II substrate . The structural features of MtgA enable its specific localization and interactions with other cell division proteins involved in peptidoglycan synthesis.

How does recombinant Xylella fastidiosa MtgA interact with other components of the cell division machinery?

Recombinant X. fastidiosa MtgA likely participates in a complex network of protein-protein interactions within the bacterial divisome, similar to what has been observed in E. coli. In E. coli, MtgA has been shown to interact with three key divisome components: FtsW (a lipid II flippase), FtsN (an essential cell division protein), and PBP3 (a transpeptidase essential for septal peptidoglycan synthesis) . These interactions suggest that MtgA works collaboratively with other enzymes to coordinate peptidoglycan synthesis during cell division.

The interaction with these proteins is likely mediated through specific domains, with the transmembrane segment of PBP3 being particularly important for the interaction with MtgA . For X. fastidiosa MtgA, similar interactions may occur, though the specific protein partners might differ due to evolutionary divergence.

To investigate these interactions in X. fastidiosa, researchers could employ:

• Bacterial two-hybrid assays using recombinant X. fastidiosa proteins to identify potential interaction partners

• Co-immunoprecipitation studies with tagged recombinant MtgA

• Fluorescence microscopy with labeled proteins to visualize co-localization patterns

• Surface plasmon resonance or isothermal titration calorimetry to quantify binding affinities

These interactions are particularly significant as they may reveal subspecies-specific mechanisms of cell division and wall synthesis that contribute to X. fastidiosa's pathogenicity and host adaptation .

What are the catalytic properties of purified recombinant Xylella fastidiosa MtgA?

The catalytic properties of purified recombinant X. fastidiosa MtgA can be characterized through various biochemical assays that assess its glycosyltransferase activity. Based on studies of MtgA from other bacterial species, X. fastidiosa MtgA is expected to catalyze the polymerization of peptidoglycan from lipid II precursors. Experimentally, this activity can be measured using assays that track the incorporation of radiolabeled or fluorescently-labeled lipid II into polymeric products.

In vitro enzymatic assays would likely reveal:

• Reaction kinetics (K_m, V_max) for lipid II utilization

• Metal ion dependencies, particularly divalent cations like Mg²⁺ or Ca²⁺

• pH and temperature optima for catalytic activity

• Susceptibility to inhibitors

• Processivity of the enzyme (number of glycan units added before dissociation)

From studies with E. coli MtgA, we know that a GFP-MtgA fusion protein demonstrated glycosyltransferase activity in vitro, with a 2.4-fold increase in peptidoglycan polymerization compared to control conditions (26% versus 11% of lipid II used) . X. fastidiosa MtgA likely exhibits similar activity, though potentially with different kinetic parameters reflecting its adaptation to X. fastidiosa's unique physiological conditions.

The catalytic activity might also be influenced by interactions with other proteins, suggesting that in vivo activity could differ from purified enzyme assays. Furthermore, subspecies-specific variations in MtgA structure might confer distinct catalytic properties that contribute to the pathogenicity and host range of X. fastidiosa subsp. pauca .

How does recombination shape the genetic diversity of mtgA within Xylella fastidiosa populations?

Recombination plays a significant role in shaping the genetic diversity of X. fastidiosa, including genes like mtgA. Genomic studies have demonstrated that recombination events contribute substantially to the evolution and adaptation of X. fastidiosa subspecies, creating mosaic genomes with segments derived from different lineages .

For mtgA specifically, which is predominantly found in X. fastidiosa subsp. pauca, recombination could influence:

• Sequence variation within the gene, potentially affecting protein function or regulation

• Acquisition or loss of the gene between different subspecies or strains

• Integration of the gene into different genomic contexts, affecting its expression patterns

Analysis of 72 X. fastidiosa genomes has revealed that each subspecies is under different selective pressures, which would impact the evolutionary trajectory of mtgA . In European isolates of X. fastidiosa, recombination has been detected in numerous genes, with some recombination events potentially predating the introduction of the bacterium to Europe .

The impact of recombination on mtgA diversity can be assessed through:

• Comparative sequence analysis across strains to identify potential recombination breakpoints

• Phylogenetic incongruence tests that compare mtgA phylogeny with whole-genome phylogeny

• Analysis of selection signatures to determine if recombination has introduced adaptive variants

Understanding how recombination shapes mtgA diversity could provide insights into the evolutionary mechanisms that drive X. fastidiosa adaptation to new hosts and environments, potentially informing strategies to manage diseases caused by this pathogen .

What methodological challenges exist in expressing and purifying recombinant Xylella fastidiosa MtgA?

Expressing and purifying recombinant X. fastidiosa MtgA presents several methodological challenges that researchers must address to obtain functional protein for structural and biochemical studies. These challenges stem from the protein's membrane association, potential toxicity when overexpressed, and requirements for proper folding.

Expression system selection:
The choice of expression system is critical. While E. coli is commonly used for heterologous protein expression, membrane-associated proteins like MtgA may not fold properly in this host. Alternative expression systems to consider include:

• Specialized E. coli strains designed for membrane protein expression (e.g., C41(DE3), C43(DE3))

• Homologous expression in a non-pathogenic Xylella or Xanthomonas strain

• Insect cell or yeast expression systems for eukaryotic post-translational processing

Solubilization and purification strategies:

• Fusion tags (His, GST, MBP) may improve solubility but must be carefully selected to not interfere with activity

• Detergent screening is essential for solubilizing membrane-associated MtgA (e.g., n-dodecyl-β-D-maltoside, CHAPS)

• Lipid nanodiscs or amphipols could provide a membrane-like environment to maintain protein structure

Activity preservation:
Ensuring the recombinant protein retains catalytic activity requires:

• Preserving native-like membrane environment during purification

• Including appropriate cofactors (divalent cations)

• Avoiding oxidation of catalytic residues

Validation approaches:

• Circular dichroism to confirm secondary structure

• In vitro activity assays using lipid II substrates

• Binding studies with known interaction partners (e.g., FtsW, FtsN)

These methodological considerations are particularly important when working with X. fastidiosa MtgA, as there may be subspecies-specific structural features that affect protein stability and function. Successful expression and purification strategies will enable detailed structural and functional characterization of this enzyme, potentially revealing targets for controlling X. fastidiosa infections .

What experimental approaches can be used to study the localization of MtgA in Xylella fastidiosa cells?

Studying the subcellular localization of MtgA in X. fastidiosa requires specialized techniques that can visualize protein distribution within these small bacterial cells. Based on approaches used to study MtgA in E. coli, several complementary methods can be employed:

Fluorescent protein fusion microscopy:

• Construction of translational fusions between MtgA and fluorescent proteins (e.g., GFP, mCherry)

• Expression of these fusions in X. fastidiosa under native promoter control

• Live-cell imaging using high-resolution fluorescence microscopy to track dynamic localization

• Co-localization studies with other divisome components labeled with spectrally distinct fluorophores

In E. coli, GFP-MtgA fusions have demonstrated localization to the division site in cells deficient in PBP1b and expressing thermosensitive PBP1a . Similar approaches in X. fastidiosa would reveal whether MtgA localizes to the division septum or has a different distribution pattern.

Immunofluorescence microscopy:

• Generation of specific antibodies against X. fastidiosa MtgA

• Fixation and permeabilization of X. fastidiosa cells

• Immunolabeling followed by fluorescence microscopy

• This approach avoids potential artifacts from fusion proteins but requires cell fixation

Correlative light and electron microscopy:

• Initial identification of MtgA-GFP localization by fluorescence microscopy

• Processing the same sample for electron microscopy

• Correlation of fluorescence signal with ultrastructural features

Challenges and controls:

• X. fastidiosa's small cell size requires super-resolution microscopy techniques

• Proper controls to ensure fusion proteins remain functional (complementation of mtgA deletion)

• Accounting for growth phase-dependent localization patterns

• Verification that tagged proteins retain their interaction capabilities with other divisome components

By implementing these approaches, researchers can determine whether X. fastidiosa MtgA shares the septal localization pattern observed in E. coli or exhibits subspecies-specific localization patterns that might contribute to its unique biology and pathogenicity .

How can researchers design effective gene knockout experiments to study mtgA function in Xylella fastidiosa?

Designing effective gene knockout experiments for mtgA in X. fastidiosa requires careful planning due to the bacterium's slow growth, limited genetic tools, and natural competence variability between strains. The following methodological approach outlines key considerations for successful functional analysis:

Knockout strategy design:

• Targeting method selection:

  • Homologous recombination with antibiotic resistance cassette

  • CRISPR-Cas9 system adapted for X. fastidiosa

  • Suicide vector delivery with counter-selection markers

• Homology arm design:

  • Minimum 500-1000 bp flanking regions for efficient recombination

  • Verification of uniqueness using whole genome sequence data

  • Consideration of potential polar effects on downstream genes

• Selectable marker choice:

  • Antibiotic resistance genes compatible with X. fastidiosa sensitivity profile

  • Promoters that function effectively in X. fastidiosa

Transformation protocol optimization:

• Competent cell preparation:

  • Growth phase optimization (early-mid log phase typically optimal)

  • Buffer composition adjustments for X. fastidiosa

  • Use of chemical facilitators (e.g., calcium chloride)

• DNA delivery methods:

  • Electroporation parameters optimized for X. fastidiosa

  • Natural competence induction

  • Conjugation using helper strains

Knockout verification:

• PCR verification strategies:

  • Primers spanning junction regions and internal regions

  • qPCR for copy number analysis

  • RT-PCR to confirm absence of transcription

• Whole genome sequencing:

  • To confirm clean deletion without off-target effects

  • To detect any compensatory mutations

Complementation controls:

• Complementation vector construction:

  • Native promoter-driven mtgA expression

  • Inducible system for controlled expression

  • Site-specific integration options

• Phenotypic rescue assessment:

  • Growth rate recovery

  • Cell morphology normalization

  • Pathogenicity restoration

Phenotypic characterization:

Table 1: Key phenotypic analyses for mtgA knockout in X. fastidiosa

Since the mtgA gene appears specific to X. fastidiosa subsp. pauca , complementary comparative analyses between subspecies that naturally contain or lack this gene would provide valuable insights into its biological significance. Additionally, researchers should be prepared for potential essentiality of mtgA, which would necessitate conditional knockout approaches using inducible promoters or antisense RNA strategies .

What analytical techniques can be used to characterize the enzyme kinetics of recombinant Xylella fastidiosa MtgA?

Characterizing the enzyme kinetics of recombinant X. fastidiosa MtgA requires specialized analytical techniques that can accurately measure glycosyltransferase activity. Based on approaches used for studying similar enzymes, including E. coli MtgA, the following methodological framework is recommended:

Substrate preparation:

• Lipid II synthesis:

  • Chemical synthesis of lipid II or extraction from bacterial sources

  • Radiolabeling with ¹⁴C or ³H for detection (e.g., ¹⁴C-GlcNAc-labeled lipid II)

  • Fluorescent labeling alternatives (dansyl, NBD, or BODIPY)

• Substrate quality control:

  • HPLC or thin-layer chromatography verification

  • Mass spectrometry confirmation

  • Determination of specific activity for radiolabeled substrates

In vitro activity assays:

• Reaction mixture composition:

  • Purified recombinant MtgA protein

  • Labeled lipid II substrate

  • Buffer optimization (HEPES pH 7.0-7.5)

  • Divalent cations (Ca²⁺, Mg²⁺)

  • Membrane-mimetic environment (detergent micelles, liposomes)

• Reaction monitoring methods:

  • Paper chromatography separation of products and substrates

  • SDS-PAGE analysis of polymerized material

  • Size-exclusion chromatography

  • HPLC analysis with radiochemical or fluorescence detection

• Product verification:

  • Lysozyme digestion to confirm glycan chain formation

  • Mass spectrometry of reaction products

  • Electron microscopy visualization of polymer formation

Kinetic parameter determination:

• Initial velocity measurements:

  • Determination of linear range for time and enzyme concentration

  • Substrate concentration series (typically 0.1-10× K_m)

  • Data fitting to Michaelis-Menten or appropriate kinetic models

• Key parameters to determine:

• Inhibition studies:

  • IC₅₀ determination for known glycosyltransferase inhibitors (moenomycin)

  • Mechanism of inhibition (competitive, non-competitive)

Specialized analytical considerations:

• Real-time monitoring options:

  • Fluorescence polarization for substrate consumption

  • FRET-based assays for glycan chain extension

  • Surface plasmon resonance for binding kinetics

• Advanced product analysis:

  • Atomic force microscopy of polymerized products

  • Light scattering for polymer size distribution

  • Solid-state NMR for structural characterization

When applying these methods to X. fastidiosa MtgA, researchers should consider potential subspecies-specific variations in optimal reaction conditions and substrate preferences that might reflect adaptation to different host environments . Comparative kinetic analyses between MtgA from different X. fastidiosa subspecies could reveal biochemical differences contributing to host specificity and pathogenicity .

How can researchers assess the role of MtgA in Xylella fastidiosa pathogenicity?

Assessing the role of MtgA in X. fastidiosa pathogenicity requires a multifaceted approach that combines molecular genetic manipulation, controlled plant infection studies, and detailed phenotypic characterization. The following methodological framework outlines effective strategies:

Genetic manipulation approaches:

• Gene knockout or knockdown:

  • Creation of mtgA deletion mutants in X. fastidiosa subsp. pauca

  • Construction of conditional expression strains if mtgA is essential

  • CRISPR interference for partial repression

• Complementation and overexpression:

  • Wild-type mtgA complementation in trans

  • Site-directed mutagenesis of catalytic residues

  • Heterologous expression of mtgA in subspecies that naturally lack it

Plant infection assays:

• Model plant systems:

  • Nicotiana tabacum or Arabidopsis for rapid screening

  • Host-specific plants for definitive assessment (citrus for subsp. pauca)

• Inoculation methods:

  • Needle inoculation with standardized bacterial suspensions

  • Insect vector-mediated transmission for natural infection process

  • Quantification of inoculum using optical density and viable counts

• Disease progression monitoring:

  • Symptom severity scoring using standardized scales

  • Time-course analysis of symptom development

  • Quantitative PCR to measure bacterial colonization and movement

  • Microscopy to visualize bacteria in plant tissues

Bacterial fitness assessments:

• In planta growth dynamics:

  • Bacterial recovery and enumeration from infected tissues

  • Competitive index assays (co-inoculation of wild-type and mutant)

  • Spatial distribution analysis within plant vasculature

• Stress resistance profiling:

  • Survival under oxidative stress conditions mimicking plant defense responses

  • Tolerance to antimicrobial compounds produced by plants

  • Resistance to osmotic fluctuations in xylem environment

Phenotypic characterization:

• Biofilm formation analysis:

  • Crystal violet quantification

  • Confocal microscopy with fluorescent stains

  • Comparison of extracellular polymeric substance composition

• Cell wall integrity assessments:

  • Muropeptide analysis by HPLC

  • Susceptibility to cell wall-targeting antibiotics

  • Morphological analysis by electron microscopy

• Virulence factor expression:

  • Transcriptomic analysis of known virulence genes

  • Secretion of plant cell wall-degrading enzymes

  • Type IV pili production and twitching motility

Comparative subspecies analysis:

Since mtgA is specifically found in X. fastidiosa subsp. pauca but absent in other subspecies , researchers can implement a natural comparative approach:

• Cross-subspecies complementation:

  • Introduction of pauca mtgA into fastidiosa or multiplex strains

  • Assessment of resulting changes in host range or virulence

• Chimeric protein analysis:

  • Construction of chimeric MtgA proteins with domains from different subspecies

  • Identification of regions conferring subspecies-specific properties

This comprehensive methodological framework enables researchers to thoroughly assess MtgA's contribution to X. fastidiosa pathogenicity, potentially revealing subspecies-specific mechanisms of host adaptation and identifying targets for disease management strategies .

How can recombinant Xylella fastidiosa MtgA be leveraged for developing new antimicrobial strategies?

Recombinant X. fastidiosa MtgA represents a promising target for developing novel antimicrobial strategies against this important plant pathogen. As a monofunctional peptidoglycan glycosyltransferase involved in cell wall biosynthesis, MtgA offers several advantages as a target for intervention, particularly given its subspecies-specific distribution in X. fastidiosa subsp. pauca . The following approaches outline how recombinant MtgA can be leveraged for antimicrobial development:

Target-based inhibitor screening:

• High-throughput screening platforms:

  • Fluorescence-based assays monitoring glycosyltransferase activity

  • Fragment-based screening to identify initial chemical scaffolds

  • In silico screening using structural models of X. fastidiosa MtgA

• Rational inhibitor design:

  • Structure-based approaches targeting the catalytic site

  • Allosteric inhibitors disrupting essential protein-protein interactions

  • Peptide inhibitors mimicking natural binding partners

Biochemical validation methodologies:

• In vitro inhibition assessment:

  • IC₅₀ and K_i determination for candidate compounds

  • Mode of inhibition analysis (competitive, non-competitive)

  • Time-dependency and reversibility characterization

• Cell-based validation:

  • Minimal inhibitory concentration (MIC) determination

  • Killing kinetics against X. fastidiosa cultures

  • Selectivity profiling against beneficial microorganisms

Delivery strategies for field application:

• Systemic delivery through plant vasculature:

  • Soil drench applications of water-soluble compounds

  • Trunk injection methods for established trees

  • Seed treatments for preventative protection

• Vector-targeting approaches:

  • Incorporation of inhibitors into insect trap systems

  • Development of compounds that reduce bacterial acquisition by vectors

Resistance management considerations:

• Combination therapies:

  • Dual-targeting of MtgA and other cell wall synthesis enzymes

  • Integration with different modes of action (membrane disruptors, metabolic inhibitors)

• Evolutionary constraint analysis:

  • Assessment of genetic barriers to resistance development

  • Identification of conserved catalytic residues with limited mutation potential

The subspecies-specific nature of mtgA in X. fastidiosa subsp. pauca offers the advantage of potentially developing control strategies with high specificity, targeting this particularly damaging subspecies while minimizing impacts on beneficial microorganisms. Additionally, because MtgA interacts with multiple divisome proteins , disrupting these interactions represents an alternative targeting strategy that could complement direct enzymatic inhibition. By leveraging the recombinant protein for these various screening and validation approaches, researchers can develop sustainable management strategies for X. fastidiosa-caused diseases like citrus variegated chlorosis and olive quick decline syndrome .

What protocols should be followed when sharing recombinant Xylella fastidiosa MtgA materials between research laboratories?

When sharing recombinant X. fastidiosa MtgA materials between research laboratories, adherence to proper material transfer protocols is essential to ensure regulatory compliance, maintain scientific integrity, and promote collaborative research. The following comprehensive protocol outlines best practices for material sharing:

Material Transfer Documentation:

• Material Transfer Agreements (MTAs):

  • Required for transfers between institutions, even for non-commercial research

  • Should clearly specify permitted uses and limitations

  • Must address intellectual property rights and publication permissions

  • Should include biosafety classification and handling requirements

• Documentation requirements:

  • Complete description of material (plasmid maps, sequence information)

  • Source organism and specific subspecies identification

  • Any modifications or tags added to the recombinant protein

  • Expression system details and antibiotic resistance markers

Regulatory Compliance:

• Biosafety considerations:

  • X. fastidiosa is a regulated quarantine organism in many countries

  • Material containing X. fastidiosa DNA requires appropriate permits

  • Recombinant proteins generally face fewer restrictions than viable organisms

  • Recipient must verify their institutional approval to receive materials

• Permit requirements:

  • USDA/APHIS permits for materials of plant pathogen origin

  • Import permits for international transfers

  • Additional state-level permits may be required in some locations

Material Preparation and Quality Control:

• Plasmid DNA preparations:

  • High-quality preparations with concentration ≥100 ng/μl

  • Verification by restriction analysis and sequencing

  • Sterility testing to ensure no viable X. fastidiosa contamination

• Recombinant protein preparations:

  • Purity assessment by SDS-PAGE (minimum 90% purity)

  • Activity verification before shipping

  • Stability testing under proposed shipping conditions

Shipping Procedures:

• Packaging requirements:

  • Leak-proof primary container

  • Secondary containment with absorbent material

  • Rigid outer packaging with appropriate labeling

  • Cold chain maintenance if required (dry ice or gel packs)

• Documentation for shipping:

  • Material safety data sheets

  • Declaration of non-hazardous material (for purified proteins)

  • Customs declarations for international shipments

  • Tracking information shared with recipient

Recipient Responsibilities:

• Material receipt confirmation:

  • Inspection of package integrity upon arrival

  • Verification of material quality by appropriate tests

  • Storage according to recommended conditions

  • Reporting any issues to the provider

• Usage limitations:

  • Adherence to agreed terms in the MTA

  • No transfer to third parties without permission

  • Proper acknowledgment in publications

  • Data sharing as specified in the agreement

By following these protocols, researchers can ensure smooth and compliant sharing of recombinant X. fastidiosa MtgA materials, facilitating collaborative efforts to understand this important plant pathogen and develop strategies for disease management. The formal material transfer process also helps protect intellectual property rights and ensures appropriate attribution in subsequent research publications .