Recombinant Photorhabdus luminescens subsp. laumondii DNA-binding protein fis (fis)

What is the biological significance of the Fis protein in Photorhabdus luminescens?

Fis (Factor for Inversion Stimulation) is a nucleoid-associated protein (NAP) that plays a crucial role in gene expression regulation in P. luminescens, particularly in fast-growing cells. As shown in research, Fis functions as a global regulator involved in multiple cellular processes, including DNA organization, replication, and transcriptional regulation .

In P. luminescens, Fis is particularly important for:

• Regulation of genes associated with virulence and symbiosis

• Adaptation during host switching between nematodes and insects

• Control of phenotypic heterogeneity between primary (1°) and secondary (2°) cell types

• Modulation of bioluminescence and pigmentation pathways

The binding of Fis to specific DNA sequences creates architectural changes in the DNA topology, which subsequently affects the accessibility of promoter regions to RNA polymerase and other transcription factors, ultimately influencing gene expression patterns critical for the complex lifecycle of P. luminescens .

How can researchers distinguish between P. luminescens strain TT01 and strain DJC when working with recombinant Fis protein?

When working with recombinant Fis protein from P. luminescens, it's crucial to accurately identify the source strain, as genomic and phenotypic differences between strains TT01 and DJC (formerly known as TT01-Rifᴿ) significantly impact experimental outcomes.

Distinguishing characteristics:

• Genomic differences: Strain DJC contains approximately 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions compared to TT01

• Phenotypic differences: Strains vary in bioluminescence, pigmentation, biofilm formation, haemolysis, and growth rates

• Rifampicin resistance: DJC strain contains a mutation in the rpoB gene (H526Y) within the rifampicin-resistance hotspot, while TT01 lacks this mutation

For accurate strain verification when working with recombinant Fis, researchers should:

• Sequence the fis gene and adjacent regions to confirm strain identity

• Verify the expected phenotypic traits of the source strain

• Always clearly document the strain designation in publications to avoid ambiguity

• Consider potential differences in Fis-binding properties between strains when interpreting experimental results

What expression systems are recommended for producing functional recombinant P. luminescens Fis protein?

For optimal expression of recombinant P. luminescens Fis protein, E. coli-based expression systems have proven most effective due to compatibility with the target protein and high yield potential.

Recommended expression protocol:

• Vector selection:

  • pBAD18-Cm: Provides arabinose-inducible and glucose-repressible promoter control

  • pGEMT: Suitable for initial cloning before transfer to expression vectors

• Expression conditions:

  • Grow cultures for 18h at 37°C in LB medium with appropriate antibiotics

  • Use 0.2% glucose for repression of the araC promoter

  • Dilute 1:100 in fresh medium without glucose and grow to OD600 of 0.2

  • Induce with 0.2% L-arabinose and continue growth for 8h

• Purification approach:

  • Apply affinity chromatography using appropriate tags

  • Typical purity should exceed 85% as verified by SDS-PAGE

• Storage recommendations:

  • For liquid preparations: Store at -20°C/-80°C (6-month shelf life)

  • For lyophilized preparations: Store at -20°C/-80°C (12-month shelf life)

  • Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

What methods are available for characterizing the DNA-binding specificity of recombinant P. luminescens Fis?

Several complementary approaches can be employed to characterize the DNA-binding specificity of recombinant P. luminescens Fis:

How does the interaction between Fis and IHF contribute to complex gene regulation in P. luminescens?

The interplay between Fis and Integration Host Factor (IHF) in P. luminescens creates sophisticated regulatory networks that control diverse cellular processes through complex promoter architectures. This interaction has been studied using synthetic combinatorial promoters that reveal emergent regulatory properties .

Key findings on Fis-IHF interactions:

• Promoter architecture dictates regulatory logic:

  • Moving a perfect Fis binding site by just 20 bp (from position -121 to -101) can convert a Fis-specific repressed promoter into one repressed by both Fis and IHF

  • This modification transforms a NOT logic gate into a NOR logic gate

• Position-dependent effects:

  • Two IHF-binding sites at positions -121 and -101 create IHF-specific repression

  • Changing the second binding site to position -61 results in repression by both IHF and Fis

  • This demonstrates how spatial arrangement, not just binding site presence, determines regulatory outcome

• Binding site combinations produce emergent properties:

  • A promoter with 2 IHF-BS (positions -121 and -61) that is repressed by both Fis and IHF

  • Adding a third Fis binding site at position -81 creates a promoter strongly activated by both proteins

  • This single change converts a NOR logic gate into an OR logic gate responsive to the same transcription factors

• Binding specificity is context-dependent:

  • Some promoter architectures allow promiscuous interactions between transcription factors and binding sites

  • Other configurations completely abolish non-specific interactions

This complex interplay demonstrates that bacterial promoters can display emergent properties where final behavior cannot be predicted from individual component characterization .

What role does Fis play in mediating phenotypic heterogeneity in P. luminescens?

P. luminescens exhibits phenotypic heterogeneity with two distinct cell types: primary (1°) and secondary (2°) forms. Research indicates that Fis and associated transcriptional regulators play crucial roles in controlling this phenotypic switching process.

Key aspects of Fis involvement in phenotypic heterogeneity:

• Regulation of heterogeneity-associated genes:

  • Comparative transcriptome analysis between P. luminescens DJC 1° and 2° cells reveals differential expression of genes associated with bioluminescence, antibiotic production, and pigmentation

  • Many of these differentially expressed genes are regulated by Fis and other nucleoid-associated proteins

• Regulatory network with XreR1 and XreR2:

  • Evidence suggests that two novel transcriptional regulators, XreR1 and XreR2, constitute an epigenetic switch

  • XreR2 appears to induce and maintain the 2° phenotype

  • This regulatory network likely involves Fis-mediated control of gene expression

• Connection to toxin-antitoxin systems:

  • XreR2 binds to the promoter region of an operon encoding a putative toxin-antitoxin system (TAS)

  • This CcdAB-like system is upregulated in 2° cells

  • The putative toxin is C-terminally truncated, suggesting an alternative function possibly related to persister cell formation

• Environmental adaptation:

  • 2° cells show metabolic changes, increased motility, and enhanced chemotactic activity toward molecules derived from the rhizosphere

  • These adaptations suggest 2° cells are specialized for life outside the host

  • Fis likely participates in regulating these adaptive responses through its role as a global regulator

Understanding Fis involvement in phenotypic heterogeneity could provide insights into P. luminescens' complex lifecycle and potential applications in biocontrol.

How can Fis binding sites be engineered to create synthetic promoters with predictable behavior in P. luminescens?

Engineering synthetic promoters with predictable behavior in P. luminescens requires strategic design of Fis binding sites based on their position, arrangement, and interaction with other regulatory elements.

Design principles for Fis-responsive synthetic promoters:

• Position-dependent effects:

  • Fis binding sites at positions 3 and 4 (-101 and -81 relative to transcription start) often result in repression

  • A single Fis-BS at position 1 (-121) with IHF binding sites can produce activation

  • The specific regulatory outcome is highly dependent on the precise architecture

• Binding site combinations:

  Position combinations	Typical regulatory outcome
  Fis-BS at position 4	Repression by Fis
  Fis-BS at positions 1 & 4	Strong repression by Fis
  Fis-BS at position 2 with IHF-BS at positions 1 & 4	Activation by both Fis and IHF
  Multiple Fis-BS at positions 2 & 3 with IHF-BS at positions 1 & 4	Enhanced activation
  Fis-BS at position 3 with IHF-BS at positions 1 & 4	No activation effect

• Sequence considerations:

  • Avoid the -4A/+4T nucleotide combination in Fis binding sites as it reduces binding by >2000-fold

  • Consider using deoxyuridine instead of thymine at position +4 if -4A is required

  • Symmetrical combinations -4C/+4G, -4G/+4C, and -4T/+4A are well-tolerated

• Methodology for synthetic promoter construction:

  • Use consensus binding sites for Fis placed at different promoter positions

  • Generate complex promoters by combining Fis sites with other regulatory elements like IHF binding sites

  • Test promoter function using reporter systems such as GFP or mCherry

  • Measure relative expression levels after 4h of cell growth in different genetic backgrounds (wild-type, Δfis, ΔihfA)

• Boolean logic perspective:

  • Different architectures create distinct logic gates (NOT, NOR, OR)

  • A single modification in cis-element architecture can completely change the regulatory logic

  • The final behavior of complex promoters exhibits emergent properties that cannot be predicted from individual components

What is the role of Fis in regulating virulence and symbiosis genes in P. luminescens?

Fis plays a critical role in orchestrating the expression of virulence and symbiosis genes in P. luminescens, facilitating its complex lifestyle as both an insect pathogen and nematode symbiont.

Fis-mediated regulation of virulence factors:

• Toxin production:

  • P. luminescens produces numerous toxins including PirAB, which shows similarity to δ-endotoxins from B. thuringiensis

  • These toxins are crucial for insect pathogenicity, with as little as 40 ng sufficient to kill tobacco hornworm larvae

  • Fis likely regulates toxin expression in response to environmental cues during host infection

• Symbiosis factors:

  • Fis controls genes involved in maintaining the mutualistic association with Heterorhabditis nematodes

  • This includes regulation of the Photorhabdus clumping factor (PcfA), which is specifically expressed in primary (1°) cells

  • The expression of the pcfABCDEF operon is activated by the LuxR solo PluR, potentially through Fis-mediated mechanisms

• Host switching response:

  • When P. luminescens transitions between its nematode host and insect prey, significant transcriptional reprogramming occurs

  • Differential Induction in vivo (DIV) experiments identified genes specifically induced upon insect infection

  • Among the promoters activated during insect infection were those of plu1950, plu3608, plu3688, and the agaZSVCD operon

  • Fis likely contributes to this host-specific gene regulation

• Secretion systems:

  • P. luminescens utilizes sophisticated secretion systems for delivering virulence factors

  • Fis regulates genes encoding these systems, with different binding patterns observed in primary versus secondary cells

• Antimicrobial compounds:

  • The production of antibiotics, antifungals, and other antimicrobial compounds is controlled by complex regulatory networks

  • These compounds help eliminate competing microorganisms during insect infection

  • Fis participates in regulating their biosynthesis genes in coordination with other transcription factors

Understanding the Fis regulon in P. luminescens provides insights for potential applications in biocontrol and development of novel antimicrobial compounds.

How does the function of P. luminescens Fis compare to homologous proteins in other bacterial species?

Comparing P. luminescens Fis with homologs from other bacterial species reveals important evolutionary adaptations that reflect the unique lifestyle of this bacterium.

Comparative analysis of Fis proteins:

Understanding these differences provides valuable insights for biotechnological applications and potential development of targeted biocontrol strategies.

What are the latest methodological advances in studying Fis-DNA interactions in P. luminescens?

Recent methodological advances have significantly enhanced our ability to study Fis-DNA interactions in P. luminescens, providing deeper insights into its regulatory mechanisms.

Cutting-edge approaches:

• Complete genome sequencing using PacBio long-read technology:

  • Provides accurate reference genomes for P. luminescens strains

  • Enables precise mapping of Fis binding sites across the genome

  • Reveals strain-specific differences in regulatory regions

  • Example: The complete genome sequence for P. luminescens DJC was generated using the PacBio RSII sequencer, resulting in a single contig with an average 194-fold coverage

• Synthetic promoter analysis:

  • Construction of complex synthetic promoters with various combinations of Fis binding sites

  • Reporter gene systems using fluorescent proteins like mCherry

  • Quantitative assessment of promoter activity in different genetic backgrounds

  • Example methodology: Cloning the mCherry reporter gene into plasmid pBR322 under control of various promoter constructs containing Fis binding sites

• Advanced binding site characterization:

  • Systematic mutational analysis of binding sites

  • Nucleotide modification studies (e.g., deoxyuridine substitution)

  • High-throughput binding affinity measurements

  • These approaches have revealed that the -4A/+4T nucleotide combination severely hinders Fis binding, with the C5 methyl group on +4T being responsible for this effect

• Comparative transcriptomics:

  • RNA-seq analysis comparing gene expression between:

    • Wild-type and Fis-deficient strains

    • Primary (1°) and secondary (2°) cell types

    • Different growth conditions mimicking host environments

  • This approach has identified Fis-regulated genes involved in phenotypic heterogeneity

• In vivo infection models:

  • Differential induction in vivo (DIV) methodology to identify genes induced during insect infection

  • Injection of P. luminescens strains carrying promoter-trap libraries into the hemocoel of live Galleria mellonella larvae

  • Analysis of hemolymph for fluorescent cells expressing the reporter gene

  • This technique has successfully identified Fis-regulated promoters specifically activated during insect infection

• Boolean logic modeling of regulatory networks:

  • Abstraction of gene regulation patterns into formal logic gate definitions

  • Classification of promoters as NOT, NOR, or OR gates based on response to Fis and other regulators

  • This approach has revealed how slight changes in promoter architecture can drastically alter regulatory logic