Kadriu, E. | bioRxiv Science

bioRxiv2025

The interplay between glucose and aromatic compound regulation by two IclR-type transcription factors, LigR1 and LigR2, in Pseudomonas putida KT2440

The rhizosphere is a hotspot of microbial activity where plants release a diverse array of aromatic compounds, including shikimate pathway intermediates and monolignols. Pseudomonas putida KT2440, renowned for its metabolic versatility in this niche, uses largely uncharacterized regulatory and enzymatic strategies to utilize these compounds. We investigated two IclR-type transcriptional regulators, LigR1 and LigR2, that control expression of the uncharacterized lig1 and lig2 operons. We demonstrate that ligR1 deletion caused growth defects on glucose and 4-hydroxybenzoate accompanied by cell elongation and aggregation. Structural and functional analyses reveal that LigR1 and LigR2 activate the lig1 operon but repress the lig2 operon. LigR1 binding of 4-hydroxybenzoate induced repression by triggering tetramerization and increasing DNA-binding activity. In contrast, LigR2 responded to quinate, protocatechuate and 4-hydroxybenzoate to potently induce lig2 operon expression by relieving repression. While both operons cooperate in metabolizing these compounds, we propose the lig1 operon mediates influx through its major facilitator superfamily (MFS) transporter (PP_2604), whereas the lig2 operon catalyzes breakdown via a protocatechuate intermediate and the meta-cleavage pathway, supplying oxaloacetate to the TCA cycle. Importantly, we show that P. putida repurposes shikimate pathway intermediates for energy production. These findings challenge the canonical biosynthetic view of the shikimate pathway and redefine the metabolic flexibility of soil pseudomonads. We reveal a novel mechanism enabling P. putida to thrive in the chemically complex rhizosphere and open new avenues for exploring alternate roles of the shikimate pathway, emphasizing transcriptional regulators as tools to deconvolute complex metabolic landscapes. HighlightsO_LILigR1 and LigR2 transcriptionally regulate the lig1 and lig2 operons C_LIO_LILig1 operon is required for import of glucose and shikimate-derived compounds C_LIO_LILig2 operon metabolizes shikimate pathway compounds C_LIO_LIDysregulated LigR1/LigR2 expression impacts bacterial physiology C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/670189v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@9b461aorg.highwire.dtl.DTLVardef@175c73org.highwire.dtl.DTLVardef@1b6bd90org.highwire.dtl.DTLVardef@574e55_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

The coordinated regulatory roles of two LysR-Type Transcriptional Regulators balance chorismate and protocatechuate partition in Listeria organisms

Listeria monocytogenes is an economically deleterious foodborne pathogens that continually challenges the global food supply chain. Listeria species in general, synthesize protocatechuate from saprophytically-derived quinate and shikimate utilizing a novel class of bacterial dehydroshikimate dehydratase. Paradoxically, Listeria species are unable to metabolically utilize protocatechuate, as such, it was proposed that this compound is used as a currency to Listeria interactions with other microorganisms to improve their environmental proliferation. Therefore, an understanding of the regulatory mechanism for the metabolic pathway for protocatechuate biosynthesis is of great importance. Two LysR Type Transcriptional Regulators (LTTR), annotated QuiR and in this study QuiR2, are found upstream of genomic operons, qui1 and qui2, which transcribe genes for protocatechuate synthesis. QuiR, has been shown to activate the expression of genes from both operons with shikimate as a coinducer. However, the role of QuiR2, Lmo2233, is not clear. In this study, we conducted structural, biochemical and bioinformatics analyses of QuiR2 and demonstrated that it functions as a negative regulator of protocatechuate biosynthesis in Listeria species. Moreover, we determined that protocatechuate functions in modulating QuiR2 repressive properties through our mobility shift assay and LacZ reporter activity studies. Furthermore, phylogenetic analyses reveal that QuiR2 clusters closely but independently from QuiR thus supporting their distinct regulatory roles. We propose that QuiR2 prevents metabolic commitment of dehydroshikimate to protocatechuate when elevated and in limiting shikimate condition. In this study we revisited the biological role of the shikimate pathway in microbes and demonstrated that in addition to it producing chorismite for aromatic compound metabolism it is also important in allowing organisms to shuttle shikimate and quinate to produce protocatechuate which can be used as an energy source and more importantly in Listeria it is used to facilitate microbial interactions.

molecular biology↗