Recombinant Prochlorococcus marinus subsp. pastoris Maf-like protein PMM1159 (PMM1159)

What is PMM1159 and what organism does it come from?

PMM1159 is a Maf-like protein encoded by the genome of Prochlorococcus marinus subsp. pastoris (strain CCMP1986/NIES-2087/MED4). Prochlorococcus is a unicellular marine cyanobacterium and the most abundant photosynthetic organism in oligotrophic oceanic gyres . The protein is classified as a Maf-like protein based on sequence homology to the Maf (multicopy associated filamentation) protein family, originally identified in other bacterial species. PMM1159 is encoded by the PMM1159 gene in the Prochlorococcus genome and has the UniProt accession number Q7V0U3 .

What is the relationship between Prochlorococcus marinus MED4 and Prochlorococcus marinus subsp. pastoris?

Prochlorococcus marinus subsp. pastoris strain CCMP1986 is the current taxonomic designation for what was previously known as Prochlorococcus marinus MED4 . This reclassification reflects improved understanding of the taxonomy of marine cyanobacteria. When reviewing literature about this organism, researchers should be aware that both names refer to the same strain, and older publications will use the MED4 designation while newer publications may use the updated taxonomic name.

What protein family does PMM1159 belong to and what are the general functions of this family?

PMM1159 belongs to the Maf (multicopy associated filamentation) protein family. Maf proteins are found across diverse bacterial species and have been implicated in various cellular processes. In other organisms, Maf proteins function as:

• Transcription factors, as seen with the mammalian Maf proteins (like c-Maf) that regulate gene expression by binding to Maf recognition elements (MAREs) in DNA

• Regulators of cell division and chromosome segregation

• Potential nucleotide-binding proteins involved in DNA repair mechanisms

What expression systems are suitable for producing recombinant PMM1159?

The most documented successful expression system for PMM1159 is Escherichia coli. According to the product datasheet, commercial recombinant PMM1159 is expressed in E. coli . For researchers producing their own recombinant protein, several E. coli expression strains can be considered:

• BL21(DE3): Standard strain for protein expression with T7 promoter systems

• Rosetta: Enhances expression of proteins containing rare codons

• Arctic Express: Enables low-temperature expression to improve protein folding

Yeast expression systems have also been used for producing recombinant Prochlorococcus proteins when E. coli systems yield poor results or when post-translational modifications are required .

What are the optimal conditions for expressing PMM1159 in E. coli?

Based on protocols used for similar Prochlorococcus proteins, the following conditions are recommended for expressing PMM1159 in E. coli:

• Growth temperature: Initially culture at 37°C until reaching target OD600, then reduce to 18-20°C for induction

• Induction: 0.5-1.0 mM IPTG

• Expression time: 16-60 hours at reduced temperature (18°C) for optimal folding

• Media: LB medium supplemented with appropriate antibiotics

• Cell lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, with protease inhibitors

This approach resembles the protocol described for other Prochlorococcus proteins, which were "cultured overnight at 37°C in 100 ml of LB medium containing 75 μg ml−1 of ampicillin. The expression culture was diluted in 1 liter of LB medium and incubated for 5 h at 18°C. After the addition of 1 mM IPTG, cells were incubated at 18°C for 60 h before harvest" .

What purification strategies can be used for PMM1159?

Based on established protocols for similar proteins, the following purification strategy is recommended:

• Affinity chromatography: Use a GST-fusion tag or His-tag approach. For GST-tagged PMM1159:

  • Harvest cells and resuspend in cold extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl)

  • Lyse cells by sonication or French press

  • Clarify lysate by centrifugation

  • Pass through Glutathione Sepharose column

  • Elute with reduced glutathione buffer or cleave the tag with PreScission protease

• Size exclusion chromatography:

  • Further purify the protein using size exclusion chromatography

  • Recommended buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT

• Ion exchange chromatography:

  • If additional purification is needed, consider ion exchange chromatography

The purification process should be optimized based on the specific properties of PMM1159 and the requirements of downstream applications.

How can I verify the purity and integrity of purified PMM1159?

Multiple complementary approaches should be used to verify the purity and integrity of purified PMM1159:

• SDS-PAGE: >85% purity should be confirmed by SDS-PAGE

• Western blotting: Using anti-PMM1159 antibodies or tag-specific antibodies

• Mass spectrometry:

  • Confirm the identity through peptide mass fingerprinting

  • Assess intact mass to verify full-length protein

• Circular dichroism (CD) spectroscopy:

  • Evaluate secondary structure content

  • Compare with theoretical predictions based on Maf protein family

• Dynamic light scattering:

  • Assess protein homogeneity and detect aggregation

  • Measure hydrodynamic radius

When working with PMM1159 for functional studies, verification of correct folding through activity assays (e.g., DNA binding if applicable) is also recommended.

What are the known or predicted functions of PMM1159 in Prochlorococcus?

• Transcriptional regulation: Similar to mammalian Maf proteins, PMM1159 may function as a transcription factor regulating gene expression in response to environmental signals .

• DNA binding and genome maintenance: Maf-like proteins can interact with DNA and may be involved in DNA repair or regulation of DNA replication.

• Response to environmental stressors: Maf proteins in other organisms are involved in stress responses, suggesting PMM1159 might play a role in adapting Prochlorococcus to changing environmental conditions .

• Metabolic regulation: Given that Prochlorococcus strains have evolved specialized adaptations to low-nutrient environments, PMM1159 might be involved in regulating metabolic pathways specific to oceanic conditions .

To determine the specific function of PMM1159, a combination of genetic, biochemical, and structural approaches would be necessary.

How can I experimentally determine if PMM1159 binds to DNA?

To experimentally investigate the DNA-binding properties of PMM1159, several complementary approaches can be employed:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified PMM1159 with labeled DNA probes

  • Include competitive EMSA with unlabeled probes to confirm specificity

  • Consider using Maf recognition element (MARE) consensus sequences as positive controls

  • The protocol described for c-Maf protein can be adapted: "EMSA, competitive EMSA, and supershifts were performed. The following oligonucleotides were used as probes: MARE consensus, 5′-GGAATTGCTGACTCAGCATTACT-3′"

• Chromatin Immunoprecipitation (ChIP):

  • Generate antibodies against PMM1159 or use tagged versions

  • Identify genomic binding sites in vivo

  • Follow with sequencing (ChIP-seq) to map binding sites genome-wide

• DNA footprinting:

  • Identify specific DNA sequences protected by PMM1159 binding

  • Use DNase I protection assays or hydroxyl radical footprinting

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Quantify binding kinetics and affinity constants

  • Test different DNA sequences to determine binding specificity

• Fluorescence Anisotropy:

  • Measure binding of PMM1159 to fluorescently labeled DNA probes

  • Determine equilibrium binding constants

By combining these approaches, you can generate comprehensive data on PMM1159's DNA-binding capabilities and specificity.

What methods can be used to identify protein interaction partners of PMM1159?

To identify proteins that interact with PMM1159, several complementary approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Express tagged PMM1159 in Prochlorococcus or heterologous systems

  • Precipitate using antibodies against the tag

  • Identify co-precipitating proteins by mass spectrometry

• Yeast two-hybrid (Y2H) screening:

  • Use PMM1159 as bait to screen against a Prochlorococcus cDNA library

  • Verify positive interactions with alternative methods

• Pull-down assays:

  • Immobilize purified PMM1159 on a column or beads

  • Pass cell lysate through and identify retained proteins

  • Similar to the approach described for examining Bach1-MafK interactions: "FLAG-tagged MafK was incubated with or without GST-fused Bach1 and separated through non-denaturing polyacrylamide gels"

• Cross-linking mass spectrometry (XL-MS):

  • Use chemical cross-linkers to stabilize transient interactions

  • Digest and analyze by mass spectrometry

  • Identify cross-linked peptides to map interaction interfaces

• Protein microarrays:

  • Screen PMM1159 against arrays containing Prochlorococcus proteins

  • Identify specific binding partners for further validation

After identifying potential interaction partners, validate these interactions using orthogonal methods and assess their functional significance through genetic or biochemical approaches.

How can I genetically manipulate Prochlorococcus to study PMM1159 function?

Genetic manipulation of Prochlorococcus to study PMM1159 function can be accomplished using methods recently developed for this organism:

• Conjugal transfer system:

  • Use interspecific conjugation with Escherichia coli to transfer plasmid DNA into Prochlorococcus

  • "Conjugation, E. coli were removed from the Prochlorococcus cultures by infection with E. coli phage T7"

  • "We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus MIT9313"

• Expression of PMM1159 variants:

  • Create plasmids containing modified versions of PMM1159 (point mutations, deletions, etc.)

  • "When this plasmid was modified to contain green fluorescent protein (GFP) we detected its expression in Prochlorococcus by Western blot and cellular fluorescence"

• Transposon mutagenesis:

  • Use Tn5 transposition for random mutagenesis

  • "We applied these conjugation methods to show that Tn5 will transpose in vivo in Prochlorococcus"

• Reporter gene assays:

  • Fuse PMM1159 promoter to reporter genes to study regulation

  • Use GFP as a reporter, which has been successfully expressed in Prochlorococcus

These approaches provide "a means to experimentally alter the expression of genes in the Prochlorococcus cell" and can be applied to study PMM1159 function.

What growth conditions should be used when studying PMM1159 function in vivo?

When investigating PMM1159 function in Prochlorococcus, use the following culturing conditions:

• Standard growth media:

  • Natural seawater-based medium (PRO99)

  • Artificial seawater-based medium (AMP-J)

  • "We routinely grow Prochlorococcus in natural seawater-based (undefined) and artificial seawater-based (defined) media as well as an Instant Ocean-based medium"

• Light conditions:

  • Low to moderate light intensity (15-30 μEin/m²/sec)

  • Constant light or light:dark cycles depending on experimental goals

  • "Incubate cultures at 20-22°C under low continuous light (~16 μEin/m²/sec)"

• Temperature:

  • 20-24°C depending on strain

  • "Incubate cultures at 20-22°C"

• Experimental manipulations:

  • Include stress conditions (nutrient limitation, light stress, temperature shifts) to investigate PMM1159 response

  • Compare wild-type to PMM1159 mutant strains under various conditions

• Testing culture purity:

  • "Both 1/10X ProAC (rich) and PLAG (minimal) media should be used to routinely test the purity of the axenic Prochlorococcus cultures"

When studying gene expression changes, global mRNA expression profiles can be analyzed as described: "We thus studied the global mRNA expression profiles of these two Prochlorococcus strains on different N sources" .

How can I perform comparative analyses of PMM1159 across different Prochlorococcus strains?

To perform comparative analyses of PMM1159 across Prochlorococcus strains:

• Sequence alignment and phylogenetic analysis:

  • Extract PMM1159 sequences from available Prochlorococcus genomes

  • Perform multiple sequence alignment to identify conserved and variable regions

  • Construct phylogenetic trees to understand evolutionary relationships

  • Look for correlation between PMM1159 sequence variations and strain ecotype or habitat

• Expression pattern comparison:

  • Use quantitative PCR or RNA-seq to compare PMM1159 expression levels

  • Analyze expression under different conditions (light, nutrients, temperature)

  • "We thus studied the global mRNA expression profiles of these two Prochlorococcus strains on different N sources"

• Functional complementation:

  • Express PMM1159 variants from different strains in a single strain background

  • Assess functional differences through phenotypic analyses

  • Use the conjugation methods described for genetic transformation

• Protein structure prediction:

  • Generate structural models for PMM1159 variants

  • Compare predicted structure-function relationships

  • Identify strain-specific structural features

This comparative approach can reveal how PMM1159 may contribute to niche adaptation in different Prochlorococcus ecotypes, similar to how "Prochlorococcus strains are hypothesized to niche-partition the water column by utilizing different N sources" .

How do I analyze contradictory results when studying PMM1159 function?

When encountering contradictory results in PMM1159 functional studies, follow this systematic approach:

• Validate experimental controls:

  • Ensure positive and negative controls performed as expected

  • Verify protein expression and activity using multiple methods

  • Check for contamination in protein preparations or cultures

• Cross-validate with complementary techniques:

  • If DNA binding results are inconsistent, compare EMSA, ChIP, and footprinting data

  • For protein interaction studies, verify with multiple methods (Co-IP, Y2H, pull-downs)

  • "The task (DECODE) and a new conversational dataset containing both human-human and human-bot contradictory dialogues" provides an example of using multiple validation approaches

• Consider context-dependence:

  • Test functions under different conditions (pH, salt, temperature)

  • Evaluate effects of binding partners or cofactors

  • "The transactivation potential of c-Maf and MafB for the rat gammaD-crystallin Maf-responsive element (gammaD MARE) is dependent upon the cellular context"

• Examine strain-specific differences:

  • Verify you're working with the correct strain (MED4/CCMP1986)

  • Compare results across different Prochlorococcus strains

  • Consider evolutionary adaptations that might affect function

• Evaluate statistical validity:

  • Ensure adequate replication and appropriate statistical tests

  • Consider sources of biological and technical variability

  • "Three independent experiments, each carried out in duplicate, were performed, and the results were averaged and diagrammed with the standard errors"

Document all contradictions thoroughly to identify patterns that might suggest new hypotheses about PMM1159 function.

What are common challenges in expressing and purifying functional PMM1159?

Common challenges when working with recombinant PMM1159 and strategies to address them include:

• Poor expression yield:

  • Optimize codon usage for expression host

  • Test different expression strains (BL21, Rosetta, Arctic Express)

  • Adjust induction conditions (temperature, IPTG concentration, time)

  • Consider autoinduction media

• Protein solubility issues:

  • Lower induction temperature (16-18°C) to promote proper folding

  • Add solubility-enhancing tags (SUMO, MBP, TRX)

  • Include compatible solutes or mild detergents in lysis buffer

  • Consider on-column refolding if inclusion bodies form

• Protein instability:

  • Include protease inhibitors during purification

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Add stabilizing agents (glycerol, arginine, trehalose)

  • Store at appropriate temperature with flash-freezing in small aliquots

• Loss of functional activity:

  • Verify proper folding through CD spectroscopy

  • Test for cofactor requirements (metal ions, nucleotides)

  • Ensure reducing conditions are maintained if cysteine residues are present

  • Perform activity assays immediately after purification

• Aggregation during storage:

  • Optimize storage buffer composition

  • Use DLS to monitor aggregation state

  • Consider storage as ammonium sulfate precipitate

  • "The shelf life is related to many factors, storage state, buffer ingredients, storage temperature and the stability of the protein itself"

Maintaining functionality is critical: "Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C" .

How might PMM1159 contribute to Prochlorococcus adaptation to different environmental conditions?

PMM1159, as a Maf-like protein potentially involved in transcriptional regulation, may play significant roles in Prochlorococcus environmental adaptation:

• Nitrogen metabolism regulation:

  • May function similarly to other transcription factors in regulating nitrogen assimilation pathways

  • "Prochlorococcus strains are hypothesized to niche-partition the water column by utilizing different N sources"

  • Could interact with nitrogen-responsive regulators like PII protein, which "appears to function differently than other cyanobacteria in that it is not phosphorlyated in response to nitrogen deprivation"

• Light adaptation:

  • May regulate genes involved in photosynthesis and photoprotection

  • Could function in a manner similar to how "heme mediates derepression of Maf recognition element through heme-Bach1 interaction" by responding to cellular redox state

• Stress response coordination:

  • Potential role in regulating genes that respond to oxidative stress, similar to how "small MAF function, by virtue of their heterodimerization with the Cap 'n' Collar (CNC) family of transcription factors, to the stress response and detoxification pathways"

  • May be involved in temperature adaptation mechanisms

• Genomic plasticity:

  • Could participate in regulation of horizontal gene transfer events

  • "Transfer of photosynthesis genes to and from Prochlorococcus viruses" suggests mechanisms for rapid adaptation that might involve transcriptional regulators

Experimental approaches to test these hypotheses would include transcriptome analysis of PMM1159 mutants under different environmental conditions and ChIP-seq to identify PMM1159 binding sites in the genome under various stress conditions.

What techniques can be used to study the relationship between PMM1159 and extracellular vesicle production in Prochlorococcus?

Investigating the relationship between PMM1159 and extracellular vesicle (EV) production in Prochlorococcus requires specialized approaches:

• Comparative proteomics of EVs:

  • Isolate EVs from wild-type and PMM1159 mutant strains

  • Analyze protein composition using mass spectrometry

  • "Vesicles were produced by the marine cyanobacterium Prochlorococcus strains using a label-free, quantitative shotgun proteomics approach"

  • "During extraction and sample preparation, we utilized surfactants compatible with mass spectrometry to improve the recovery of more membrane-bound proteins"

• EV quantification and characterization:

  • Use nanoparticle tracking analysis to quantify EV production

  • Employ transmission electron microscopy to examine morphology

  • Compare EV size distributions and concentrations between strains

• Functional analysis of EVs:

  • Assess effects of EVs on recipient cells

  • Investigate gene transfer capabilities

  • Determine if PMM1159 affects EV cargo selection

• RNA content analysis:

  • Extract and sequence RNA from EVs

  • Compare RNA profiles between wild-type and mutant strains

  • Identify potential regulatory RNAs associated with PMM1159 function

• Environmental response studies:

  • Examine how environmental stressors affect EV production in relation to PMM1159 expression

  • Test if PMM1159 regulates EV production in response to specific signals

This investigation could reveal whether PMM1159 plays a role in the "functions, both for cells themselves and the emergent ecosystem" of extracellular vesicles, which currently "remain a mystery" .

How can systems biology approaches be applied to understand PMM1159's role in Prochlorococcus gene regulatory networks?

Systems biology approaches provide powerful tools for understanding PMM1159's role in Prochlorococcus regulatory networks:

• Integrated multi-omics analysis:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and PMM1159 mutant strains

  • Generate a comprehensive view of cellular changes associated with PMM1159 function

  • "Global gene expression of Prochlorococcus ecotypes under nitrogen starvation and on different nitrogen sources" provides a model for such studies

• Network inference:

  • Use computational algorithms to infer gene regulatory networks

  • Identify PMM1159 target genes and regulatory connections

  • Validate key predictions experimentally through ChIP-seq or reporter assays

• Dynamics and modeling:

  • Develop mathematical models of PMM1159-dependent regulatory circuits

  • Predict system behavior under various environmental conditions

  • Test predictions experimentally to refine models

• Comparative genomics integration:

  • Analyze PMM1159 conservation and variation across Prochlorococcus strains

  • Correlate with genomic adaptations to different oceanic niches

  • "Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation"

• Environmental context integration:

  • Incorporate oceanographic data to understand ecological relevance

  • Study how natural gradients of light, nutrients, and temperature affect PMM1159 function

  • Examine potential role in "niche-partition the water column by utilizing different N sources"

This systems approach can reveal how PMM1159 functions within the broader context of Prochlorococcus adaptation to oligotrophic oceanic environments, where it has evolved as "the most abundant phytoplankton in the oligotrophic, oceanic gyres" .