Recombinant Photobacterium profundum S-ribosylhomocysteine lyase (luxS)

What is the biochemical function of S-ribosylhomocysteine lyase (luxS) in bacterial communication?

S-ribosylhomocysteine lyase (LuxS) catalyzes the conversion of S-ribosylhomocysteine (SRH) into 4,5-dihydroxy-2,3-pentanedione (DPD), which is the precursor of the interspecies quorum sensing signaling molecule autoinducer-2 (AI-2) . This enzyme serves a dual function in bacteria:

• It facilitates quorum sensing, a cell-to-cell communication process that coordinates bacterial behavior based on population density

• It forms an integral part of the activated methyl cycle, which is critical for metabolic functions

The biochemical pathway proceeds as follows:

• S-adenosylmethionine (SAM) is converted to S-adenosylhomocysteine (SAH) during various methyltransferase reactions

• SAH is detoxified by the Pfs enzyme to yield adenine and SRH

• LuxS then catalyzes the conversion of SRH to homocysteine and DPD

• DPD spontaneously cyclizes to form AI-2, which can be detected by various bacterial species

This dual role in signaling and metabolism makes luxS particularly important for understanding bacterial adaptation to different environments.

How does luxS genetic organization differ among Photobacterium species?

Genomic analyses have revealed significant variations in luxS organization across Photobacterium species:

In P. leiognathi, the first operon (lux-rib 1) is flanked by putA and a gene encoding a hypothetical multidrug resistance efflux pump (orf1), while the second operon (lux-rib 2) is flanked by transposase genes, suggesting different evolutionary origins . This merodiploidy (presence of two copies) appears to be stable, with strains collected over 20 years showing little sequence divergence .

What are the optimal conditions for expressing recombinant Photobacterium profundum luxS in heterologous systems?

Successful expression of recombinant P. profundum luxS requires careful optimization of multiple parameters:

For optimal enzyme activity, it's critical to ensure proper metal ion availability during purification. Studies in related species like Lactobacillus plantarum have shown that LuxS activity is affected by metal ions, with some stimulating and others inhibiting activity .

What methods are effective for measuring luxS enzymatic activity and AI-2 production?

Several complementary methods can be employed to assess luxS activity:

• AI-2 Bioluminescence Reporter Assay

  • Uses Vibrio harveyi BB170 strain as a biological reporter

  • Quantifies AI-2 activity as percentage of induction relative to positive control (V. harveyi BB152)

  • Procedure:
    a. Culture the test strain and collect cell-free supernatant
    b. Add supernatant to the reporter strain culture
    c. Measure luminescence after incubation
    d. Calculate relative induction compared to controls

• Enzyme Kinetics Analysis

  • Ellman's assay measures production of homocysteine (a product of the LuxS reaction)

  • Determines Km, Vmax, and Kcat values for substrate conversion

  • Example kinetic parameters from comparative studies:

• Direct Chemical Analysis

  • HPLC or LC-MS/MS to directly quantify DPD/AI-2 production

  • Provides absolute quantities rather than relative activities

  • Can differentiate between different structural forms of AI-2

What genomic evidence supports horizontal gene transfer of luxS in Photobacterium species?

Several genomic features suggest potential horizontal gene transfer (HGT) involving luxS in Photobacterium:

• Transposase Associations

  • In P. leiognathi, transposase genes flank the lux-rib 2 operon

  • Specifically, regions upstream of luxC contain sequences homologous to a putative transposase gene from P. profundum SS9

  • Regions downstream of ribA contain homologs to bacterial transposases of the IS66 family

• Genomic Island Features

  • The lux-rib 2 operon in P. leiognathi is located in a distinct chromosomal region

  • The presence of this operon varies among strains (present in lnuch.13.1, lelon.2.1, and lnuch.21.1, but absent in ATCC 25521 T)

• Phylogenetic Incongruence

  • Comparison of luxS gene trees with species phylogeny shows discrepancies

  • The two lux-rib operons in P. leiognathi are more closely related to each other than to operons in other species

  • This suggests intraspecific transfer rather than interspecific transfer

How does having multiple copies of luxS affect evolutionary fitness in Photobacterium species?

The presence of multiple luxS copies appears to confer several evolutionary advantages:

• Functional Redundancy and Specialization

  • In species with multiple copies (like P. leiognathi with lux-rib 1 and lux-rib 2), both operons contain complete and translatable genes

  • Similar to findings in L. plantarum, where two LuxS proteins (LuxS1 and LuxS2) have different temperature optima (45°C and 37°C, respectively)

  • This suggests potential specialization for different environmental conditions

• Stable Inheritance and Maintenance

  • Despite opportunities for recombination between duplicate operons, both are stably inherited

  • Extended growth of P. leiognathi lnuch.13.1 in continuous culture (~400 generations) did not lead to loss or altered location of lux-rib 2

• Resilience to Mutations

  • In P. leiognathi lelon.2.1, the luxC gene in lux-rib 2 has a single nucleotide deletion causing a premature stop codon

  • The presence of a functional copy in lux-rib 1 ensures continued functionality despite mutations

The divergence between duplicate copies suggests they might serve different adaptive functions rather than simply providing redundancy.

How does hydrostatic pressure affect luxS expression and function in Photobacterium profundum?

As a piezophilic bacterium, P. profundum shows distinct pressure-responsive patterns that likely affect luxS:

• Differential Protein Expression Under Pressure

  • Shotgun proteomic analysis of P. profundum grown at atmospheric vs. high pressure (28 MPa) shows differential expression of many proteins

  • Proteins involved in glycolysis/gluconeogenesis pathway are up-regulated at high pressure

  • Proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure

  • These metabolic shifts likely affect the activated methyl cycle involving LuxS

• Pressure-Sensing Mechanisms

  • P. profundum utilizes the ToxRS two-component regulatory system to sense pressure through membrane conformation changes

  • This system transmits signals that regulate gene expression in a pressure-responsive manner

  • LuxS-mediated pathways may interface with this pressure-sensing system

• Growth Condition Dependencies

  • P. profundum exhibits a more pronounced piezophilic phenotype when grown in minimal medium compared to rich medium

  • This suggests that nutrient availability interacts with pressure response

  • Since LuxS is involved in metabolic pathways related to methionine and cysteine, these interactions are likely significant

How does luxS contribute to biofilm formation in Photobacterium species?

LuxS plays a complex role in biofilm formation across Photobacterium and related species:

• Quorum Sensing-Dependent Mechanisms

  • Studies in L. rhamnosus GG showed that luxS mutation results in defects in monospecies biofilm formation

  • AI-2 signaling coordinates community behaviors that facilitate attachment and matrix production

• Metabolic Contributions

  • Experiments with chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione, coculture with wild type, and nutritional complementation suggest that biofilm defects in luxS mutants have a primarily metabolic nature

  • This indicates that the activated methyl cycle function may be more important than signaling in some contexts

• Nutrient-Dependent Effects

  • Cysteine, pantothenate, folic acid, and biotin can partially complement growth defects in luxS mutants

  • This suggests that metabolic imbalances affect biofilm formation indirectly through growth deficiencies

• Pressure-Related Adaptations

  • In deep-sea environments, biofilm formation may be an adaptive strategy for withstanding high pressure

  • The role of LuxS in coordinating this response through either metabolic or signaling functions remains to be fully explored

What are the methodological challenges in distinguishing between quorum sensing and metabolic roles of luxS?

One of the most significant challenges in luxS research is differentiating between its dual functions:

• Complementation Approaches

  • Chemical complementation with synthetic AI-2 can help isolate signaling effects

  • Metabolic complementation with key nutrients (cysteine, methionine, etc.) can address metabolic deficiencies

  • Complementation that restores growth but not biofilm formation (or vice versa) helps separate these functions

• Experimental Design Strategies

  • Cross-feeding experiments where wild-type and mutant strains are grown in co-culture

  • Conditioned medium experiments where cell-free supernatant from wild-type cultures is added to mutant cultures

  • Use of luxS mutants complemented with heterologous enzymes that restore only the metabolic function

• Molecular Verification Techniques

  • Transcriptomic analysis to identify genes responding to AI-2 versus metabolic changes

  • Proteomic studies comparing different complementation conditions

  • Reporter constructs specifically responsive to AI-2 signaling

This methodological challenge is exemplified by findings that suppressor mutations are likely to occur in luxS mutants, potentially masking or confounding experimental results .

What are the current gaps in understanding strain-specific variations in luxS function among Photobacterium species?

Despite significant research, several knowledge gaps remain:

• Strain-Specific Functional Differences

  • While genomic variations in luxS organization have been documented (particularly in P. leiognathi), functional implications remain underexplored

  • Limited understanding of how these differences affect adaptation to specific ecological niches

• Pressure-Responsive Regulation

  • While P. profundum is known to exhibit pressure-responsive gene expression, specific mechanisms regulating luxS under different pressure conditions remain unclear

  • The interplay between pressure, temperature, and nutrient availability in regulating luxS expression needs further study

• Evolutionary Dynamics of Multiple luxS Copies

  • The evolutionary forces maintaining multiple luxS copies in some species remain incompletely understood

  • Whether functional divergence between copies represents specialization or redundancy is not fully established

• Host-Microbe Interactions

  • Despite being bioluminescent symbionts, the role of luxS in symbiotic relationships of Photobacterium with host animals remains unclear

  • Comparative studies found no evidence that bioluminescent symbioses with specific host animals have played a role in diversification of the two lineages of P. leiognathi and P. mandapamensis

What are the optimal culture conditions for studying luxS function in Photobacterium profundum?

The unique piezophilic nature of P. profundum requires specific culture conditions:

The contrast between growth at atmospheric vs. high pressure provides valuable insights into pressure-responsive functions of luxS and related systems.

What considerations are important when designing gene knockout studies for luxS in Photobacterium profundum?

When designing gene knockout studies for luxS in P. profundum, researchers should consider:

• Genetic Redundancy Assessment

  • Before knockout, verify whether multiple copies of luxS exist in the strain

  • In P. leiognathi, the presence of two copies (lux-rib 1 and lux-rib 2) would require different knockout strategies than for a single-copy gene

• Knockout Strategy Selection

  • Homologous recombination-based methods using suicide vectors

  • Counter-selection markers for identifying double crossover events

  • CRISPR-Cas9 systems adapted for marine bacteria

• Verification Methods

  • PCR verification with primers spanning the expected deletion/insertion site

  • Southern blotting to verify single integration and absence of wild-type gene

  • AI-2 production assay using V. harveyi BB170 reporter strain

  • RT-PCR to confirm absence of luxS transcript

• Complementation Controls

  • Genetic complementation with wild-type luxS

  • Chemical complementation with synthetic AI-2

  • Nutritional supplementation (cysteine, pantothenate, folic acid, and biotin)

• Suppressor Mutation Monitoring

  • Regular sequencing of maintained mutant strains

  • Analysis of any phenotypic reversion

  • Comparative genomics of original and evolved mutant strains

Research in L. rhamnosus has demonstrated that suppressor mutations are likely to occur in luxS mutants, potentially confounding experimental results if not carefully monitored .

Comparative Analysis of LuxS Function Across Bacterial Species

Comparative genomics of luxS across Photobacterium species reveals several evolutionary patterns:

• Gene Duplication and Divergence

  • In P. leiognathi, the presence of two lux-rib operons that are phylogenetically distinct suggests gene duplication followed by sequence divergence

  • Similarly, in L. plantarum, two luxS genes with 70% sequence identity show different enzymatic properties, suggesting functional divergence after duplication

• Horizontal vs. Vertical Transmission

  • Though flanked by transposases, P. leiognathi's lux-rib 2 appears to have originated from within the species rather than through interspecific transfer

  • Phylogenetic analysis shows the two operons are more closely related to each other than to operons in other species

• Selective Pressures

  • Both operons in P. leiognathi are maintained over evolutionary time with little evidence of mutation or recombination

  • This suggests positive selection for maintaining both copies

  • In contrast, other Photobacterium species like P. mandapamensis have strains with nonsense mutations in certain lux genes (luxF), indicating relaxed selective pressure on those genes

• Clade-Specific Patterns

  • P. leiognathi belongs to clade 1 of the Photobacterium genus, which includes luminous and symbiotic species

  • Non-luminous species are grouped in clade 2, suggesting different evolutionary trajectories for luxS and related genes in these clades