Mutational analyses reveal PLP-independent functions at PipY, the cyanobacterial paradigm for pyridoxal-phosphate binding proteins

Antonio Llop; Lorena Tremiño; Asunción Contreras

doi:10.1038/s41598-026-43837-6

. 2026 Mar 12;16:13255. doi: 10.1038/s41598-026-43837-6

Mutational analyses reveal PLP-independent functions at PipY, the cyanobacterial paradigm for pyridoxal-phosphate binding proteins

Antonio Llop ¹, Lorena Tremiño ¹, Asunción Contreras ^1,^✉

PMCID: PMC13102913 PMID: 41820517

Abstract

Pyridoxal-phosphate binding proteins (PLPBP) are involved in the homeostasis of B₆ vitamers and amino/keto acids, share a high degree of sequence conservation and are represented in all three domains of life. Despite the obligate presence of the catalyst cofactor PLP, attempts to show enzymatic activity have been unsuccessful. Instead, evidence of RNA binding activity has been provided for several members of the family. Here we use PipY, one of the few PLPBP members studied so far, as a model system to address the phenotypic impact in the cyanobacterium Synechococcus elongatus of mutations K26A, P63L and R210Q, which respectively prevent PLP binding or are equivalent to those conferring B₆-dependent epilepsy in humans with a recessive inheritance pattern. We found that while mutation K26A at the PLP-binding residue abrogated all phenotypes associated to PipY overexpression and toxicity, P63L and R210Q behaved as dominant gain-of-function mutations that inhibited bacterial growth. We provide in vivo evidence of PipY performing PLP-independent functions, for which mutant variant PipY^K26A but not PipY^P63L or PipY^R210Q would be defective. A model integrating our observations with previous data from other organisms and PLPBP variants is discussed.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-43837-6.

Keywords: Synechococcus, PipX, B₆ homeostasis, RNA-binding, PLPHP

Subject terms: Biochemistry, Genetics, Molecular biology

Introduction

The Pyridoxal phosphate (PLP)-binding proteins (COG0325/PLPBP) are involved in vitamin B₆ and amino acid homeostasis^1–6. PLPBP/PLPHP (for PLP homeostasis proteins) family members are found in all kingdoms of life^6,7 exemplified by the proteins YBL036C (yeast), YggS (Gram-negative bacteria), YlmE (Gram-positive bacteria), PipY (cyanobacteria) and PLPHP (previously called PROSC in humans or plants). They are all single domain proteins exhibiting the type-III fold of PLP-holoenzymes^8–12 whose deficiency alters the B₆ pool in the different systems studied^2,6,13–16 or confer PLP-related phenotypes such increased sensitivity to pyridoxine or to the antibiotics D-cycloserine and β-chloro-D-alanine¹. In humans, the severity of PLPBP mutations causing vitamin B₆-dependent epilepsy appears to correlate with decreased ability of the PLPHP variants to bind the PLP cofactor¹⁴ although only some of the pathogenic mutations found in patients complement the pyridoxine-sensitive phenotype of the E. coli yggS null mutant¹³. In zebrafish the complete absence of PLPHP triggers epilepsy seizures and, importantly, early death, indicating the essentiality of the protein in development¹⁴ and further suggesting an important regulatory complexity.

Despite the obligate presence of the catalyst cofactor PLP, no enzymatic activity has been associated to the PLPBP family. Instead, a recent work shows that PLPBP members are RNA binding proteins and that the apo-forms of YggS and human PLPHP bind RNA with much greater affinity than the holo-forms¹⁷, and the current view is that they would have purely regulatory functions as components of signal transduction pathways^4,17. In bacteria PLPBP genes cluster with cell division and cell wall biosynthesis (dcw, in Gram-positive bacteria and mycobacteria¹⁸⁾, PLP salvage, surface motility, secretion, amino acid metabolism, and translation genes^4,6. In cyanobacterial genomes the sepF gene (involved in cell division and restricted to gram-positive bacteria and cyanobacteria) is typically located downstream pipY. In the cyanobacterium Synechococcus elongatus PCC7942 (hereafter S. elongatus) and in most cyanobacteria pipY is part of a bicistronic pipXpipY operon with relatively short or non-existent intergenic distances suggestive of tight co-regulation and functional interactions between their products in this bacterial phylum^1,19. Both PipX (PII interacting protein X) and PipY regulate the levels of a common set of transcripts and affect the susceptibility to PLP-targeting antibiotics D-cycloserine and β-chloro-D-alanine, further supporting their involvement in the same genetic pathway(s)¹.

PipX, a hallmark of cyanobacteria^20,21, is a small protein that, in response to intracellular signals of carbon/nitrogen or the energy status, forms complexes with the widely distributed signal transduction protein PII^22–25 and the cyanobacterial transcriptional regulator NtcA^23,25,26. PipX stabilizes NtcA conformation and helps local recruitment of RNA polymerase²⁷. PipX can also interact, in response to additional signals, with the poorly characterized cyanobacterial transcriptional regulator PlmA²⁸ or the essential ribosome-assembly GTPase EngA^29–31. The binding of PII to PipX modulates PipX levels and PipX interactions with its different partners^23,32–37. PipX and PII are the hubs of a yet expanding cyanobacterial interaction network involved in metabolic regulation^21,38.

While deciphering the mechanisms of action and regulatory connections of PLPBP members remains very challenging, the inferred functional association between PipY with PipX provides a unique opportunity to investigate PLPBP functions in the context of a signalling pathway that is highly conserved in an important biological group of organisms. Neither PipX or PipY are required for growth of S. elongatus under standard laboratory conditions^1,19,36, although their overexpression or certain gain-of-function mutations at PipX prevent growth^1,30,39,40. Overexpression of either PipX or PipY also induce a specific stress program termed chlorosis or bleaching^30,40, a complex adaptative response by which cyanobacteria degrade their light-harvesting antenna, the phycobilisome, and reduce their chlorophyll content^41–43. PipY overexpression also confers specific phenotypic traits in S. elongatus including increase of cell length, decrease of cell viability and photosynthesis activity, accompanied by a dramatic and unprecedented accumulation of giant polyphosphate (hereafter polyP) granules^1,40.

PipY is one of the best-characterized PLPBP members and can be regarded as a paradigm for these proteins^1,4,10,40. Figure 1 illustrates the structural parallelism between cyanobacterial PipY and human PLPHP, highlighting the PipY residues Lys26, Pro63 and Arg210, whose mutations are analysed in this work. The PLP cofactor, which is found in the same position and orientation in all deposited structures of PLPBP family members, is covalently bound to Lys26 in PipY (Lys47 in human PLPHP). Recombinantly produced PipY has been used to mimic the effect on protein function of pathogenic mutations that differed in their ability to complement the pyridoxine-sensitive phenotype of the E. coli yggS null mutant¹⁰. These were human mutation P87L and R241Q, of which P87L but not R241Q complemented the pyridoxine-sensitive phenotype of the E. coli yggS null mutant¹³. While the P63L mutation did not impair any of the parameters assayed at PipY, including protein yield, thermal stability, proper fold or PLP-content, mutation R210Q conferred a very low yield of the recombinant protein that, although well folded, contained hardly any PLP and showed decreased stability at high temperature¹⁰. Those results support the importance of cofactor binding in the context of B₆-dependent homeostasis. However, the instability of the PipY^R210Q protein, added to the fact that no PLPBP mutations affecting the key PLP-binding lysine have been characterised in vivo in any model system, or described in human patients, call for caution in the interpretation. The aim of this work was to gain insights into the functional complexities of the PLPBP family, investigating the role of the PLP cofactor and of residues of interest. To this end, and taking advantage of the already existing structural and functional information on PipY, we address the phenotypic effects conferred in S. elongatus or E. coli strains by PipY variants expressing point mutations K26A, P63L and R210Q that together prevent PLP binding or mimic pathogenic changes found in the orthologous human protein.

Fig. 1 — Conservation between cyanobacterial and human PLPBP orthologs and location of relevant residues mutated in this work. A) Structure of PipY from *S. elongatus* (PDB ID: 5NM8), colored according to the evolutionary conservation score of residues between PipY and its human ortholog (PLPHP). Color coding was defined based on the conservation score of an automatic Clustalω alignment of both protein sequences, as indicated in the legend. Relevant residues are mapped as spheres. PLP is shown in stick representation and labeled, with C, O, N, and P atoms colored yellow, red, blue, and orange, respectively. B, C) Superimposition of the I-TASSER structural model of PLPHP over PipY colored purple and orange, respectively, with the residues of interest and secondary-structure elements indicated.

Results

PipY mutations P63L or R210Q, but not K26A, prevent growth of S. elongatus

In contrast to PipY deficiency, PipY overexpression results in rather dramatic phenotypes that include growth arrest, chlorosis and alteration of several cell parameters^1,40. Strain 1^SPtrc-PipY, used in the previous studies, carries an extra copy of pipY at the neutral site I (NSI) of the S. elongatus chromosome under the control of the IPTG-inducible promoter Ptrc. The LacI repressor, also encoded within the NSI (see Fig. 2A) allows inducible overexpression of PipY from the ectopic pipY gene. In the absence of IPTG transcription leakage increase 3–4 fold the levels of PipY in S. elongatus¹.

Fig. 2 — Constructs to overexpress PipY derivatives in *S. elongatus* and segregation analysis. **(A)** Schematic representation of the *NSI* at the *S. elongatus* genomic region and of the constructs to express the PipY derivatives under the control of the *Ptrc* promoter in *pipY* or *pipXpipY* null backgrounds. Primer positions for PCR analysis are depicted by blue arrows, with the size of products on the right. **(B)** Representative agarose gels showing PCR products obtained in the indicated strains and backgrounds after up to ten selective transfers, with the expected amplicon depicted by arrows on the right. M, 100 bp size marker. The number of colonies obtained in two independent transformations was > 10³ for PipY or PipY^K26A, and < 15 for PipY^P63L or PipY^R210Q, with no significative differences between both backgrounds. * Refers to the WT, K26A, P63L, and R210Q versions of PipY.

To determine the effect of relevant point mutations on PipY activity in S. elongatus we first attempted construction of variants of strain 1^SPtrc-PipY, so the phenotypic effects of wild type and mutant PipY derivatives can be studied in parallel both at just above physiological levels and at very high levels of expression. With this in mind we first generated mutations encoding K26A, P63L or R210Q variants in plasmid pUAGC294, giving plasmids pUAGC1133, pUAGC1130 or pUAGC1129, respectively (Table 1). To avoid possible interference and/or gene conversion between the wild type and mutant alleles of pipY, we performed all experiments in pipY null strains. To explore a possible contribution of PipX to the phenotypes studied here while strengthening the robustness of the data we also obtained and studied pipXpipY derivatives in parallel by precisely replacing their ORF by the cat (chloramphenicol-acetyltransferase) gene as described in¹.

Table 1.

Plasmids.

Plasmid	Description, relevant characteristics	Source or reference
pUAGC127	pipY replaced with cat, Ap^R Cm^R	¹
pUAGC128	pipXpipY replaced with cat, Ap^R Cm^R	¹
pUAGC280	(cs3 lacI Ptrc) into NSI, Ap^R Sm^R	⁵²
pUAGC294	(cs3 lacI Ptrc: pipY) into NSI, Ap^R Sm^R	¹
pUAGC1129	(cs3 lacI Ptrc: pipY^{628cga> caa}) into NSI, Ap^R Sm^R	This work
pUAGC1130	(cs3 lacI Ptrc: pipY^{199ccc> ctg}) into NSI, Ap^R Sm^R	This work
pUAGC1133	(cs3 lacI Ptrc: pipY^{76aag> gcg}) into NSI, Ap^R Sm^R	This work

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The pipY and pipXpipY mutants were next used as recipients in independent transformations with plasmids containing wild type, the three mutant alleles of pipY or, to provide a negative control for PipY activity, carrying no pipY gene (plasmid pUAGC280) (Table 2). Surprisingly, while constructs encoding PipY or PipY^K26A yield thousands of streptomycin-resistant transformants, the plasmids encoding PipY^P63L or PipY^R210Q produced just a few. In each case transformants clones were PCR-analysed for the presence of the insert at NSI. For comparison the unmodified NSI was amplified from the WT strain.

Table 2.

Strains.

Strain	Genotype, relevant characteristics	Source or reference
E. coli XL1-Blue	recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’proAB lacI ^q Z∆M15 Tn10 (Tet ^R )]	⁵³
E. coli MG1655	F⁻ λ- ilvG- rfb-50 rph-1	⁵⁴
WT	Synechococcus elongatus PCC7942	Pasteur culture collection
pipY	ΔpipY::cat, Cm^R	¹
pipXY	ΔpipXpipY::cat, Cm^R	¹
pipY 1^SPtrc	ΔpipY::cat NSI::(cs3 lacI P_trc), Sm^R	This work
pipY 1^SPtrc-PipY	ΔpipY::cat NSI::(cs3 lacI P_trc:pipY), Cm^R Sm^R	¹
pipY 1^SPtrc-PipY^K26A	ΔpipY::cat NSI::( (cs3 lacI P_trc:pipY^{76aag> gcg}), Cm^R Sm^R	This work
pipY 1^SPtrc-PipY^P63L	ΔpipY::cat NSI::(cs3 lacI P_trc:pipY^{199ccc> ctg}), Cm^R Sm^R	This work
pipY 1^SPtrc-PipY^R210Q	ΔpipY::cat NSI::(cs3 lacI P_trc:pipY^{628cga> caa}), Cm^R Sm^R	This work
pipXpipY 1^SPtrc	ΔpipXpipY::cat NSI::(cs3 lacI P_trc), Cm^R Sm^R	This work
pipXpipY 1^SPtrc-PipY	ΔpipXpipY::cat NSI::(cs3 lacI P_trc:pipY), Cm^R Sm^R	¹
pipXpipY 1^SPtrc-PipY^K26A	ΔpipXpipY::cat NSI::(cs3 lacI P_trc:pipY^{76aag> gcg}), Cm^R Sm^R	This work
pipXpipY 1^SPtrc-PipY^P63L	ΔpipXpipY::cat NSI::(cs3 lacI P_trc:pipY^{199ccc> ctg}), Cm^R Sm^R	This work
pipXpipY 1^SPtrc-PipY^R210Q	ΔpipXpipY::cat NSI::(cs3 lacI P_trc:pipY^{628cga> caa}), Cm^R Sm^R	This work

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As shown in Fig. 2B (left panels), PCR products of the expected size were found when the transformations involved 1^SPtrc-PipY, 1^SPtrc-PipY^K26A or 1^SPtrc constructs in both genetic backgrounds. In contrast, the PCR products of some of the few transformants obtained for 1^SPtrc-PipY^P63L and 1^SPtrc-PipY^R210Q constructs were indistinguishable from those of the WT control strain in the two genetic backgrounds (Fig. 2B, right panels). Since the unmodified NSI allele was the only one detected in those transformants, the results indicate that, even at just 3–4 fold above the normal levels of PipY, expression of PipY^P63L or PipY^R210Q, but not of PipY or PipY^K26A, prevents the appearance of S. elongatus colonies, independently of the pipX gene. Therefore, the results indicate that PipY^P63L or PipY^R210Q, but not PipY^K26A, behave as gain-of-function proteins that prevent growth of S. elongatus in our experimental conditions.

Heterologous expression of PipY variants in E. coli parallels their behaviour in S. elongatus

The failure to obtain viable transformants expressing the PipY^P63L or PipY^R210Q variants (Figs. 2 and S1) raised questions on the mechanism(s) by which each of these proteins prevent S. elongatus growth. Since PipY is not required for S. elongatus viability, it follows that mutations P63L or R210Q are behaving as gain-of-function mutations and thus both PipY^P63L and PipY^R210Q must be interfering with cellular processes required for culture growth. To distinguish if this interference affects signalling pathways restricted to cyanobacteria or more universal processes conserved in phylogenetically distant bacterial groups, we next investigated the effect of overexpressing each of the PipY variants in E. coli. We reasoned that the expression of the PipY ortholog YggS at physiological levels in the E. coli strains would not be enough to mask the phenotypic effects of expressing PipY from high copy number plasmids.

It is worth noting that all plasmids constructed in this work were obtained in E. coli XL1-Blue and that this strain carries a lacI^q gene to prevent expression of the cloned genes. Therefore, to unveil a possible phenotypic impact of the PipY variants in E. coli the LacI repressor must be inactivated. To this goal, plasmids pUAGC294, pUAGC1133, pUAGC1130 or pUAGC1129 were independently introduced into E. coli XL1-Blue and their corresponding transformants were selected in parallel in the absence or presence of the IPTG inducer.

As shown in Fig. 3A, multiple ampicillin-resistant transformants were obtained in all cases in the absence of IPTG. However, colonies derived from the plasmid bearing the P63L variant were noticeably smaller than those obtained with the other constructs (Fig. 3B), suggesting greater toxicity for PipY^P63L than for PipY^R210Q. Since the latter protein is recovered at lower yield than the former from E. coli extracts¹⁰, differences in expression level may, at least in part, explain their different toxicities. Upon IPTG addition (0.5 mM), the number of transformants remained unchanged only for the construct expressing PipY^K26A but decreased by 98% for PipY and by 100% for both PipY^P63L and PipY^R210Q (Fig. 3C). Thus, the results show that the PipY variants confer different effects when expressed in E. coli, confirming the suitability of the heterologous assays to discriminate between PipY variants.

Independent corroboration of these results was next provided by transforming the same four plasmids into E. coli MG1655, a strain lacking the lacI^q and therefore unable to provide enough repression of the promoter driving expression of the PipY variants (Fig. 3D). The substantially higher number of transformant obtained for PipY^K26A relative to PipY is also consistent with a negative impact of PipY overexpression on E. coli viability that can be attenuated by the K26A mutation. No colonies were obtained when the proteins expressed were PipY^P63L or PipY^R210Q, confirming the higher toxicity of both variants in E. coli.

Taken together, our results show that the expression of PipY^R210Q and PipY^P63L variants inhibit growth in both S. elongatus and E. coli. Therefore, while the behaviour of PipY^K26A is consistent with loss-of-function for toxicity (see also below), that of the “pathogenic” variants reveals gain-of-function in both bacterial model systems.

Lys26 is required for PipY overexpression phenotypes on growth, chlorosis, cell size and overaccumulation of polyP

To gain insights into the factors affecting PipY overexpression phenotypes in S. elongatus, we addressed the impact of mutation K26A on previously studied traits, which include growth, chlorosis, cell size and overaccumulation of polyP⁴⁰. The S. elongatus strains compared were pipY 1^SPtrc, pipY 1^SPtrc-PipY, pipY 1^SPtrc-PipY^K26A, pipXpipY 1^SPtrc, pipXpipY 1^SPtrc-PipY, and pipXpipY 1^SPtrc-PipY^K26A.

The impact on the different strains of IPTG induction on growth and pigment composition was followed by performing drop-plate assays or according to the visual appearance and absorbance spectra of liquid cultures (Fig. 4). As expected, no differences in growth were found amongst otherwise isogenic 1^SPtrc-PipY and 1^SPtrc strains in the absence of IPTG (left panel in Fig. 4A). However, addition of 50 µM IPTG prevented growth of 1^SPtrc-PipY but not of 1^SPtrc-PipY^K26A or 1^SPtrc strains (Fig. 4A and B), indicating that mutation K26A abrogates the inhibitory effects of PipY overexpression on growth.

Fig. 4 — Effects of K26A mutation or *pipX* inactivation on PipY overexpression phenotypes in *S. elongatus*. Strains Ptrc (-) and derivatives expressing PipY (Y) or PipY^K26A (K26A) in the indicated backgrounds, grown in BG11 and, when indicated, supplemented with 50 µM IPTG. **(A)** Drop-plate assay (5 µL; initial OD₇₅₀= 0.5) with the dilutions indicated at the top. **(B)** Growth curves (mean ± standard deviation). **(C)** Whole absorbance spectra, indicating the peaks of phycocyanin (PC) and chlorophyll a (Chl a), and insets showing cultures after 48 h of IPTG induction. **(D)** Representative confocal micrographs of autofluorescence signal (+ 40% brightness, − 40% contrast; *left*) and scatterplot of cells length (n = 180 cells; *right*). Means are indicated by black lines and numbers inside the graphs. **(E)** Representative confocal micrographs of DAPI-polyP channel. **(F)** Ratio of the polyP signal (48 / 0 h) referred to the Ptrc strain (mean ± standard deviation). Pairwise comparisons using the Mann–Whitney U test with Bonferroni correction were performed. Significance levels were denoted as p ≤ 0.01 (**) or ≤ 0.001 (***). Effect size r was interpreted as large according to Cohen’s thresholds (r > 0.5). Data from two independent experiments are presented.

Next, we compared the dynamic of pigment loss induced by PipY overexpression in the different strains. As previously shown, addition of IPTG to pipY 1^SPtrc-PipY cultures triggered the yellow color and the progressive decrease in phycocyanin and chlorophyll a peaks characteristic of chlorosis (Fig. 4C, left panels). In contrast, pipY 1^SPtrc-PipY^K26A cultures maintained the normal green appearance and pigment composition throughout the 48 h experiment, exactly as the negative control strain pipY 1^SPtrc.

The impact of IPTG induction on cell size was subsequently analysed by confocal microscopy at 0 and 48 h after IPTG induction (Figs. 4D, S2, and S3). Representative micrographs showed that cell appearance and cell size were indistinguishable amongst the strains before IPTG addition (0), while only 1^SPtrc-PipY cells were significantly longer, in the two backgrounds, after induction (48 h; Fig. 4D). The lower autofluorescence of 1^SPtrc-PipY cells paralleled the loss of photosynthetic pigments shown in Fig. 4C. Scatter-plot representation of cell lengths for all six strains confirmed the significant increase in 1^SPtrc-PipY cells after IPTG induction (Fig. 4D, right).

PolyP accumulation, another relevant phenotype associated with PipY overexpression⁴⁰, was subsequently analysed by confocal microscopy in the same six strains at 0 and 48 h after IPTG induction. Representative micrographs of DAPI-stained cells are shown in Fig. 4E. To quantify the polyP signal avoiding possible differences in cell number per field, the total signal in the DAPI–polyP channel (ex405, em470–617) was normalized against the corresponding DAPI channel (ex405, em410–470). No differences between IPTG treated or untreated cells were observed in strain 1^SPtrc-PipY^K26A (Fig. 4F), indicating that the K26A mutation abolished the over accumulation of polyP resulting from PipY overexpression.

In summary, all four PipY overexpression-related phenotypic features analyzed here are abolished by the K26A mutation, indicating that they require the PLP cofactor and/or the integrity of Lys26 at PipY.

Inactivation of pipX affects overaccumulation of polyP but has no effect on other PipY overexpression phenotypes

Comparison between pipY and pipXpipY strains expressing PipY or PipY^K26A showed no phenotypic differences attributable to the presence or absence of pipX regarding growth, chlorosis or cell size (Fig. 4a–d). However, although pipXpipY 1^SPtrc-PipY cells showed significant accumulation of polyP after 48 h of induction, it was not as large as in pipY 1^SPtrc-PipY cells (Fig. 4E). PolyP signals were at least 1.7-fold lower from pipXpipY 1^SPtrc-PipY than from pipY 1^SPtrc-PipY cells (Fig. 4F), thus indicating that the pipX gene is required for the very high levels of polyP associated with PipY overexpression.

In summary, our results indicate that PipX plays a positive regulatory role in polyP accumulation, suggesting that both PipX and PipY may be required to increase polyP synthesis under certain environmental conditions. On the other hand, we cannot yet exclude regulation by PipX of the cellular targets involved in the other phenotypic traits analysed here in S. elongatus cells or cultures. Since we studied the effect of the presence or absence of pipX gene in conditions in which the PipX/PipY ratio is abnormally low giving that we provoked overexpression of just the PipY protein, a regulatory role of PipX in the studied traits could have been missed, particularly if it was a negative role.

Mutation K26A increase the levels of PipY in S. elongatus in a PipX independent manner

Given that the phenotypes discussed above depend on PipY overaccumulation in S. elongatus^1,40 (Fig. 4), it was important to exclude the possibility that mutation K26A impaired the stability of PipY therefore preventing accumulation of very high levels of PipY^K26A in the presence of IPTG. To investigate that possibility, samples of pipY 1^SPtrc-PipY, pipXpipY 1^SPtrc-PipY, pipY 1^SPtrc-PipY^K26A, and pipXpipY 1^SPtrc-PipY^K26A strains were taken at 0, 6, and 24 h timepoints after IPTG induction, and subsequently analysed by Western blot with anti-PipY (Figs. 5 and S1).

Fig. 5 — Effects of K26A mutation or *pipX* inactivation on PipY levels. **(A)** Representative immunodetections (α-PipY) and loading control (LC) of Ptrc strains expressing PipY (Y) or PipY^K26A (K26A) in the indicated null mutant grown in the presence of 1 mM of IPTG. **(B)** Ratio of PipY^K26A/PipY protein levels, normalized to loading control. **(C)** *pipXpipY/pipX* ratio at 24 h. Data are presented as means and error bars (standard deviation) from two biological replicates.

Although no differences between strains could be observed at timepoint 0, upon IPTG addition the signal from the PipY band was higher at both 6 and 24 h timepoints for PipY^K26A than for PipY (Fig. 5A). Quantification of band signals confirmed a ≈ 2-fold increase in PipY^K26A levels with respect to PipY (Fig. 5B) regardless of the presence or absence of the pipX gene (Fig. 5C).

Therefore, the failure of PipY^K26A to trigger any one of the PipY overexpression phenotypes in S. elongatus analysed here is not the result of lower accumulation of PipY^K26A in the presence of IPTG, indicating that either the PLP cofactor or Lys26 itself is required for PipY-triggered growth arrest, chlorosis, increased cell length and overaccumulation of polyP in S. elongatus.

Discussion

In this work we have gained insights into the PLPBP family by focussing on the phenotypic effects conferred by mutations at PipY that prevent the covalent binding of PLP (K26A) or that mimic pathogenic changes found in the orthologous human protein (P63L and R210Q). While the invariant residue Arg210/241 forms a stabilizing hydrogen bond with PLP in the PipY structure, Pro63/87 is located at the tip of the turn between α3-β3, facing outwards and well away from the PLP cofactor (Fig. 1).

The apparent correlation between the severity of PLPBP mutations causing vitamin B₆-dependent epilepsy and the decreased ability of some of the corresponding PLPHP variants to bind PLP¹⁴ speaks of the importance of the PLP cofactor for PLPBP function in the homeostasis of B₆ vitamers. For instance, both PipY^P63L and PLPHP^P87L were respectively less impaired than PipY^R210Q and PLPHP^R241Q in vitro^10,11, while PLPHP^P87L but not PLPHP^R241Q complemented the pyridoxine-sensitivity of the yggS null mutant¹³. Furthermore, the P63L mutation did not impair any of the parameters assayed at PipY, including protein yield, thermal stability, proper fold or PLP-content, whereas the R210Q change resulted in a lower yield of the recombinant protein that contained hardly any PLP and showed decreased stability at high temperature¹⁰.

Consistent with the idea that proteins with obvious defects in PLP binding were less functional in vivo, phenotypic analyses in S. elongatus indicated that mutation K26A caused complete loss of function. Despite the high levels of PipY^K26A achieved in S. elongatus (Fig. 5A and B) this protein did not confer toxicity, or indeed any of the phenotypes associated with PipY overexpression in S. elongatus (Figs. 2 and 4). Furthermore, while heterologous expression of PipY decreased viability in E. coli, expression of PipY^K26A did not (Fig. 3), indicating that the differential behaviour of wild type and mutant proteins is maintained across bacterial species. Since the equivalent mutation (K36A) at the E. coli homolog YggS also increases the stability of the protein⁴⁴, our results emphasise the importance and common roles of this invariant lysine within bacterial PLPBPs. Not surprisingly, PipX, a protein unique to cyanobacteria, appears to play no role in the different stability of PipY and PipY^K26A proteins (Fig. 5C).

Given the stabilising contacts of Arg210 with PLP and the low PLP content found for the recombinant PipY^R210Q protein¹⁰, it would be expected that PipY^R210Q would confer a similar and presumably less drastic phenotype than the PipY^K26A variant. However, in sharp contrast to PipY^K26A, the PipY^R210Q variant did not decrease PipY toxicity. On the contrary, its overexpression conferred toxicity in both S. elongatus and E. coli (Figs. 2 and 3) and thus the opposite phenotypes caused by two mutations impairing PLP binding argues in favour of the integrity of Lys26 and against the presence of the PLP cofactor as a requirement for PipY toxicity.

Consistent with the idea that PipY toxicity does not require PLP binding, two mutations that have very different effects on the PLP content of PipY (P63L and R210Q) conferred gain-of-function phenotypes for PipY toxicity in both S. elongatus and E. coli cells (Figs. 2 and 3). Remarkably, PipY^P63L conferred higher toxicity (that is, more severe gain-of-function phenotype) than PipY^R210Q in E. coli (Fig. 3B) while the P63L/P83L mutation conferred less severe loss-of-function phenotypes than R210Q/R241Q^10,13.

To explain the results obtained with the PipY variants, we propose that toxicity is an alternative function of apo-PipY that requires the presence of the Lys26 residue. It is worth noting that RNA binding is so far the only PLP-independent function characterised at the molecular level in the PLPBP family and that the residues predicted to bind RNA are among the most conserved and they are situated at the edges of the PLP binding site¹⁷. Results from the geometry-aware protein binding site predictor GPSite (https://bio-web1.nscc-gz.cn/app/GPSite;⁴⁵) at the Biochemical HPC + AI platform confirm the close relationship between RNA and PLP binding sites at PipY, supporting an important regulatory interplay between the two ligands that would be a hallmark of the PLPBP family. The location of predicted RNA binding residues and of PLP binding residues at PipY is illustrated in Fig. 6.

Fig. 6 — Structural overlap between PLP- and predicted RNA-binding residues of PipY. **(A)** Cartoon representation of PipY (PDB 5NM8) in yellow, with PLP-binding residues in pink, predicted RNA-binding residues in violet, and residues overlapping between the PLP-binding and predicted RNA-binding in red. Nitrogen, oxygen, and sulfur atoms are colored blue, red, and yellow, respectively. **(B)** Surface representation of panel A.

Interestingly, the apo-forms of YggS and human PLPHP bind RNA with much greater affinity than the holo-forms or the YggS^K36A variant, which adopts a holo-like conformation despite the absence of PLP¹⁷. The use of PipY mutant variants as seed in the GPSite predicts that mutation K26A, but not R210Q or P63L, impair RNA binding (Table S1).

PLP binding appears to induce rather subtle structural changes at PipY¹⁰, involving helices α1, α2, α6, and α9, as well as the β5–α6, β6–α7, and β7–α8 loops (PLP-bound PipY, PDB: 5NM8, versus apo PipY, PDB: 5NLC). In addition, use of KVFinder web tool (https://kvfinder-web.cnpem.br/;^46,47) revealed differences in the solvent-accessible PLP-binding cavity, with its volume increasing from 144.94 Å³ to 161.57 Å³ upon PLP release, suggesting the existence of relatively closed and open surface conformations associated with the holo and apo forms of PipY, respectively (Fig. 7A–C).

Fig. 7 — PLP driven changes and model for PipY functions based on alternative conformations. **(A)** Superposition of PipY-apo (blue) onto PipY-PLP (yellow). **(B)** PipY–PLP. **(C)** PipY-apo. The dashed ellipses mark solvent-accessible regions. **(D)** Schematic representation of the holo and apo forms bound to their respective ligands, with indication of functions. **(E)** Predicted effect of the indicated mutations on protein conformation. See text for additional details.

A working model in which PLP-dependent and independent functions of PipY can be respectively associated to the holo or apo forms of PipY is schematically illustrated in Fig. 7D and E. We propose that insufficient binding of the PLP cofactor would displace the equilibrium towards the apo-form of PipY^R210Q and that PipY^K26A would adopt a holo-like conformation with low affinity for RNA, as it was shown for YggS^{[K36A 17}. PipY^P63L is not impaired in PLP binding, and thus the proposed displacement of the equilibrium towards the apo-form of PipY by mutation P63L requires a different explanation. We speculate that Pro63 may play a role in maintaining the holo-form by imposing rigidity to the α3-β3 loop. Based on the different levels of PipY^R210Q and PipY^P63L detected upon heterologous overexpression in E. coli¹⁰, the prediction is that there would be enough amount of the minoritarian holo-form to function on vitamer B₆ homeostasis just in the case of the more stable protein PipY^P63L.

Finally, it has been proposed that PLPBP may be part of a regulatory mechanism of translation linked to amino acid levels¹⁷. Since PipX, a multifunctional protein co-expressed with PipY, is also involved in carbon/nitrogen homeostasis, transcription, and translation regulation^{1,19,20,25,28,30,35}, understanding the regulatory connections between PipX and PipY in cyanobacteria should provide additional insights into the yet enigmatic functions of PLPBP and of its pathogenic variants.

Methods

Plasmid construction

The plasmids and primers used in this study are listed in Tables 1 and S2, respectively. Cloning procedures were carried out in E. coli XL1-Blue. All constructs were verified by automated Sanger sequencing.

Plasmids pUAGC1133, pUAGC1130, and pUAGC1129 carrying mutant alleles pipY^K26A, pipY^P63L, and pipY^R210Q, respectively, were generated by QuickChange site-directed mutagenesis using pUAGC294 as template with primer pairs PipY-K26A-F/PipY-K26A-R, PipY-P63L-F/PipY-P63L-R, and PipY-R210Q-F/PipY-R210Q-R, respectively.

E. coli assays

Transformations of E. coli XL1-Blue and MG1655 (Table 2) were performed by heat shock, essentially as described in⁴⁸. 65 µL of competent cells were incubated with 50 ng of the plasmids. After the heat shock, 1 mL LB was added and cells were incubated at 37 °C for 1 h with agitation. A volume of 100 or 900 µL of XL1-Blue and MG1655 cells, respectively, were spread on LB agar (1.5% w/v) plates supplemented with 75 µg/mL ofampicillin. When indicated, the appropriate amount of propyl β-D-1-thiogalactopyranoside (IPTG) was added. Pictures were taken after 24- or 48-hours incubation at 37 °C.

Cyanobacteria culture conditions and strain generation

S. elongatus cultures were routinely grown in blue–green algae BG11 medium (BG11₀ supplemented with 17.5 mM sodium nitrate (NaNO₃) and 10 mM HEPES/NaOH (pH 7.8;⁴⁹) at 30 °C under constant cool white fluorescent light, either in liquid cultures (150 rpm, 70 µmol photons m^–2 s^–1; two mixed clones) or on plates (50 µmol photons m^–2 s^–1; individual clones). Solid media contained 1.5% (w/v) agar and 0.5 mM sodium thiosulfate (Na₂S₂O₃). When appropriate, chloramphenicol (Cm, 3.5 µg/mL) or streptomycin (Sm, 15 µg/mL) were added.

For liquid growth, cultures of 30 mL were grown in BG11 using baffled flasks. Growth was monitored by measuring the optical density at 750 nm (OD₇₅₀) in 1 mL samples using an Ultrospec 2100 Pro UV-Vis Spectrophotometer (Amersham Biosciences, Amersham, UK). All experiments were performed on mid-exponential phase (OD₇₅₀ = 0.4–0.8). To overexpress proteins the indicated concentration of IPTG was added.

S. elongatus strains used in this study are listed in Table 2. Transformations were performed essentially as described in⁵⁰, and correct allele replacement verified by PCR. The primer pairs used were inter2060-1 F/2059-Cm-1R and inter2060-1 F/PipX-5R-129 for pipY, PipX-126-F/2060-Cm-1R for pipXpipY, and 7942NSIA-F/ NSI-1R for NSI.

Determination of growth and pigment content

For growth measurements in liquid experiments, cultures were adjusted to an initial OD₇₅₀ of 0.1 and then recorded at 0, 24 and 72 h after addition of 50 µM IPTG. For drop-plate assays, liquid cultures were adjusted to an OD₇₅₀ of 0.5, and 5 µL samples and serial dilutions of 1/2, 1/10 and 1/100 were spotted on BG11 plates without or with 50 µM IPTG. Pictures were taken after 5 days growing under standard conditions.

Cell staining and confocal microscopy parameters

To detect polyP granules in S. elongatus, 4′,6-diamidino-2-phenylindole (DAPI) staining was used adapting the protocol described by⁴⁰. Samples (1 mL) were fixed with 1% formaldehyde for 20 min at room temperature, washed once with deionized water, and frozen at -20 °C for 24 h. Samples were thawed at room temperature and incubated with 0.25 mg/mL of DAPI for 15 min at room temperature in the dark, followed by three washes with deionized water.

Micrographs were taken using a Zeiss LSM800 confocal laser scanning microscope. 5 µL of samples were placed on 2% low-melting-point agarose pads. The microscopy settings were as follows: Ex 640 nm/em 650 + nm for auto-fluorescence, ex 405 nm/Em 470–617 nm for polyP, and ex 405 nm/em 470–617 nm for DAPI-control signals. Micrographs were coloured as red (auto-fluorescence) and light blue (polyP) to improve visualization contrast.

Protein extraction, immunodetection, and band quantification

For protein extraction, 10 mL samples of S. elongatus liquid cultures were collected at 0, 6–24 h after addition of 1 mM IPTG. Cells were pelleted at 7300× g, flash frozen in liquid nitrogen, and stored at − 20 °C until needed. Pellets were resuspended in 60 µL of lysis buffer (10 mM Tris/HCl pH 7.5, 0.5 mM EDTA, 1mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF)), and cells were disrupted with 1 spoonful of 0.1 mm glass beads (≈ 30 µL) in a high-speed homogenizer Minibeadbeater (three pulses of 1 min each, separated by 1-minute intervals on ice; speed of 5 m/s). Samples were centrifuged at 5500× g, and the supernatant fractions (crude protein extracts) were transferred to a new tube. Protein concentrations were estimated via the Bradford method (Bradford, 1976) using the PierceTM detergent-compatible Bradford assay kit (ThermoScientific, Waltham, MA, USA) in a VICTOR3^™ 1420 Multilabel Plate Reader (PerkinElmer). Protein extracts were stored at − 20 °C until needed.

For immunodetection, 50 µg of total protein extracts were loaded into a sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE; 15% polyacrylamide). Electrophoresis was followed by immunoblotting onto 0.2 μm polyvinylidene fluoride membranes (from GE Healthcare Technologies, Inc., Chicago, IL, USA), and the membranes were subsequently blocked with 5% non-fat dry milk in phosphate-buffered saline with 0.1% Tween 20 (PBS-Tween) for 1 h at room temperature. Membranes were then incubated overnight in PBS-Tween with 5% non-fat dried milk with a 1:300 dilution of PipY primary antibody and for 1 h at room temperature with a 1:150,000 dilution of ECL rabbit IgG and an HRP-linked F(ab’)2 fragment (from a donkey, GE Healthcare). Visualization of bands was performed using SuperSignal WestFemto reagent (Thermo Fisher Scientific, Waltham, MA, USA) in a Biorad ChemiDoc Imager using the automatic exposure mode and avoiding pixel saturation.

Computational methods

To measure cell length and protein band intensities images were analyzed using ImageJ v1.54 g. The length was determined with the “Straight” tool and the “Measure” button. The specific bands from immunoblots were selected using the “Rectangle” function, and their corresponding profiles were measured with the “Wand” tool.

To quantify the polyP signal, whole fluorescence intensities from the polyP and DAPI-control channels in the same confocal images were measured using ImageJ v1.54 g with the “Measure” button. PolyP intensities were normalized to the DAPI-control signal to account for differences in cell numbers and DAPI uptake.

To quantify E. coli colony sizes, Feret’s diameter was measured using ImageJ v1.54 g. Images typed as “8-bit” were thresholded by Otsu’s method after background subtraction and subjected to a “watershed” process to separate colonies. The resultant images were adjusted to an ellipse and the particles filtered with a minimum size of 0.3 mm before measuring Feret’s diameter.

To perform statistical analyses, RStudio v2025.09.2 Build 418 was used⁵¹.

To generate graphical representations of protein structures PyMOL v1.7.1.7 was used (The PyMOL Molecular Graphics System, Schrödinger, LLC).

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(3.6MB, tif)}

Supplementary Material 2^{(4.4MB, tif)}

Supplementary Material 3^{(5MB, tif)}

Supplementary Material 4^{(21.1KB, docx)}

Supplementary Material 5^{(21.4KB, docx)}

Acknowledgements

The authors thank S. Bibak for Western blots, R. Cantos, T. Mata-Balaguer, P. Salinas, and L. Fuertes-García for technical contributions or advice.

Author contributions

A.C. designed research; A.L. and L. T. performed research; A. C. wrote the manuscript. All authors analyzed data, reviewed the manuscript and approved the submitted version.

Funding

This work was supported by grant PID2023-149456NB-I00, funded by MCIN/AEI/10.13039/501100011033 from the Spanish Government, and grants VIGROB23-126 and GRE20-04-C from the University of Alicante to A.C., and grant UAUSTI24-27 from the University of Alicante to L.T.

Data availability

The original contributions presented in the study are included in the article/Supplementary material. Further inquiries can be directed to the corresponding author.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

7/3/2026

The original online version of this Article was revised: The Funding section in the original version of this Article was incorrect. The Funding section now reads: "This work was supported by grant PID2023-149456NB-I00, funded by MCIN/AEI/10.13039/501100011033 from the Spanish Government, and grants VIGROB23-126 and GRE20-04-C from the University of Alicante to A.C., and grant UAUSTI24-27 from the University of Alicante to L.T.".

The original Article has been corrected.

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Associated Data

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Supplementary Materials

Supplementary Material 1^{(3.6MB, tif)}

Supplementary Material 2^{(4.4MB, tif)}

Supplementary Material 3^{(5MB, tif)}

Supplementary Material 4^{(21.1KB, docx)}

Supplementary Material 5^{(21.4KB, docx)}

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary material. Further inquiries can be directed to the corresponding author.

[CR1] 1.Labella, J. I. et al. PipY, a member of the conserved COG0325 family of PLP-binding proteins, expands the cyanobacterial nitrogen regulatory network. Front. Microbiol.8, 1244 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Mutational analyses reveal PLP-independent functions at PipY, the cyanobacterial paradigm for pyridoxal-phosphate binding proteins

Antonio Llop

Lorena Tremiño

Asunción Contreras

Abstract

Supplementary Information

Introduction

Fig. 1.

Results

PipY mutations P63L or R210Q, but not K26A, prevent growth of S. elongatus

Fig. 2.

Table 1.

Table 2.

Heterologous expression of PipY variants in E. coli parallels their behaviour in S. elongatus

Fig. 3.

Lys26 is required for PipY overexpression phenotypes on growth, chlorosis, cell size and overaccumulation of polyP

Fig. 4.

Inactivation of pipX affects overaccumulation of polyP but has no effect on other PipY overexpression phenotypes

Mutation K26A increase the levels of PipY in S. elongatus in a PipX independent manner

Fig. 5.

Discussion

Fig. 6.

Fig. 7.

Methods

Plasmid construction

E. coli assays

Cyanobacteria culture conditions and strain generation

Determination of growth and pigment content

Cell staining and confocal microscopy parameters

Protein extraction, immunodetection, and band quantification

Computational methods

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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