Functional characterization of Fur from the strict anaerobe Clostridioides difficile provides insight into its redox-driven regulatory capacity.

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur-mediated mechanism independent of iron, which affects Fur DNA-binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur-regulated genes in vivo, is specific and does not require co-regulator metal under reducing conditions. Fur treatment with H2O2 produces dose-dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH-thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection.

Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation?

Metal and redox homeostasis in cyanobacteria is tightly controlled to preserve the photosynthetic machinery from mismetallation and minimize cell damage. This control is mainly taken by FUR (ferric uptake regulation) proteins. FurC works as the PerR (peroxide response) paralog in Anabaena sp. PCC7120. Despite its importance, this regulator remained poorly characterized. Although FurC lacks the typical CXXC motifs present in FUR proteins, it contains a tightly bound zinc per subunit. FurC: Zn stoichiometrically binds zinc and manganese in a second site, manganese being more efficient in the binding of FurC: Zn to its DNA target PprxA. Oligomerization analyses of FurC: Zn evidence the occurrence of different aggregates ranging from dimers to octamers. Notably, intermolecular disulfide bonds are not involved in FurC: Zn dimerization, dimer being the most reduced form of the protein. Oligomerization of dimers occurs upon oxidation of thiols by H2O2 or diamide and can be reversed by 1,4-Dithiothreitol (DTT). Irreversible inactivation of the regulator occurs by metal catalyzed oxidation promoted by ferrous iron. However, inactivation upon oxidation with H2O2 in the absence of iron was reverted by addition of DTT. Comparison of models for FurC: Zn dimers and tetramers obtained using AlphaFold Colab and SWISS-MODEL allowed to infer the residues forming both metal-binding sites and to propose the involvement of Cys86 in reversible tetramer formation. Our results decipher the existence of two levels of inactivation of FurC: Zn of Anabaena sp. PCC7120, a reversible one through disulfide-formed FurC: Zn tetramers and the irreversible metal catalyzed oxidation. This additional reversible regulation may be specific of cyanobacteria.

Fur-like proteins: Beyond the ferric uptake regulator (Fur) paralog.

Proteins belonging to the FUR (ferric uptake regulator) family are the cornerstone of metalloregulation in most prokaryotes. Although numerous reviews have been devoted to these proteins, these reports are mainly focused on the Fur paralog that gives name to the family. In the last years, the increasing knowledge on the other, less ubiquitous members of this family has evidenced their importance in bacterial metabolism. As the Fur paralog, the major regulator of iron homeostasis, Zur, Irr, BosR and PerR are tightly related to stress defenses and host-pathogen interaction being in many cases essential for virulence. Furthermore, the Nur and Mur paralogs largely contribute to control nickel and manganese homeostasis, which are cofactors of pivotal proteins for host colonization and bacterial redox homeostasis. The present review highlights the main features of FUR proteins that differ to the canonical Fur paralog either in the coregulatory metal, such as Zur, Nur and Mur, or in the action mechanism to control target genes, such as PerR, Irr and BosR.

Repeated introduction and spread of the MRSA clone t304/ST6 in northern Europe.

OBJECTIVES: During the last decades several methicillin-resistant Staphylococcus aureus (MRSA) clones with the capability of global spread have emerged in the community. Here, we have investigated a large collection of clinical isolates belonging to MRSA clone t304/ST6, which has emerged in many European countries over the last years, in order to retrace its phylogeny and its spread. METHODS: We characterized 466 ST6 isolates from Denmark (n = 354), France (n = 10), Norway (n = 24), Sweden (n = 27) and the UK (n = 51). All had spa-type t304 (n = 454) or t304-related spa-types (n = 12) and whole genome sequencing (WGS) was carried out on Illumina Miseq or Hiseq with 100-300 bp reads. cgMLST was performed using Ridom SeqSphere. RESULTS: A minimum spanning tree (MST) of all 466 isolates showed one large cluster including 182 isolates collected only from Denmark and related to a long-term neonatal outbreak in Copenhagen. This cluster contrasted with numerous small clusters, including the remaining Danish isolates and isolates from the other countries that interspersed throughout the tree. Most isolates were Panton-Valentine leukocidin (PVL) negative (95%) and harboured SCCmec IVa. One genome was closed using Oxford Nanopore technology and Illumina MiSeq. It contained a plasmid of 19.769 bp including the blaZ gene. A similar plasmid was found in 78% of all isolates. DISCUSSION: t304/ST6 is a successful emerging clone and the fact that isolates from five countries are interspersed throughout the MST indicates a common origin. This clone is commonly described in the Middle East and its emergence in Europe coincides with influx of refugees from the Syrian Civil War.

Root cone angle is enlarged in docs1 LRR-RLK mutants in rice.

BACKGROUND: The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1-1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain. FINDINGS: We have generated a new loss-of-function mutation in the DOCS1 gene (docs1-2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1-1 present similar phenotypes suggesting the original docs1-1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles. CONCLUSIONS: All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

Serum hepatitis B core-related antigen is more accurate than hepatitis B surface antigen to identify inactive carriers, regardless of hepatitis B virus genotype.

OBJECTIVES: To investigate whether hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) levels are useful to identify inactive carriers among HBeAg-negative patients infected by different hepatitis B virus (HBV) genotypes. METHODS: In all, 202 consecutive HBeAg-negative patients with chronic hepatitis B, 135 inactive carriers and 67 with HBV activity, were prospectively followed for 1 year. RESULTS: In HBeAg-negative patients, HBsAg levels differed across the different genotypes (p <0.001). The highest levels were observed in genotypes F or H (4.2 ± 0.6 logIU/mL), followed by genotype E (3.4 ± 1.1 logIU/mL), genotype A (3.4 ± 0.8 logIU/mL), and the lowest in genotype D (2.7 ± 1.1 logIU/mL). Variations in HBsAg levels were similar in inactive carriers and patients with HBV activity. HBsAg <3 logIU/mL showed good performance for identifying genotype D inactive carriers: 76% of genotype D inactive carriers met this cut-off versus ≤31% for genotypes A, E, F or H. However, in patients with genotype A, HBsAg levels ≤3.7 logIU/mL better classified inactive carriers. The combination of a single measurement of HBcrAg ≤3 logU/mL plus HBV DNA ≤2000 IU/mL yielded a positive predictive value and diagnostic accuracy >85% in all HBV genotypes, except genotype H or F, with values of 62.5% and 72.7%, respectively, for the two parameters. CONCLUSIONS: HBsAg levels varied across genotypes in HBeAg-negative patients. HBsAg levels <3 logIU/mL were only useful for identifying genotype D inactive carriers. A single HBcrAg measurement ≤3 logU/mL plus HBV DNA ≤2000 IU/mL was highly accurate for identifying inactive carriers, regardless of their HBV genotype.

In vitro activity of ceftobiprole on 440 Staphylococcus aureus strains isolated from bronchopulmonary infections.

OBJECTIVE: We assessed the in vitro activity of ceftobiprole on 440 Staphylococcus aureus clinical strains isolated from bronchopulmonary infections (2010-2014). METHODS: S. aureus isolates were characterized for methicillin resistance, PVL status, and clonal complex. All isolates were tested for minimal inhibitory concentrations (MIC) determination by broth microdilution method for ceftobiprole, ceftaroline fosamil, and comparator antibiotics (linezolid, tigecycline, vancomycin, and daptomycin). RESULTS: A total of 325 (74%) strains were methicillin-susceptible S. aureus (MSSA) and 115 (26%) were methicillin-resistant S. aureus (MRSA); 105 (24%) S. aureus strains were PVL-positive, including 35.2% (37/105) MRSA and 64.8% (68/105) MSSA. Ceftobiprole was highly active against S. aureus with MIC90 of 1 mg/L, MICs ranging between 0.12 and 4mg/L (only one resistant strain, MIC of 4 mg/L). MIC50 and MIC90 were twice lower in MSSA than MRSA. Moreover, PVL+ MRSA were slightly more susceptible to ceftobiprole (MIC50 of 0.5 mg/L and MIC90 of 1 mg/L) than PVL- MRSA (MIC50 and MIC90 of 1 mg/L). The ceftobiprole-resistant strain was also resistant to ceftaroline fosamil and presented the D239L mutation in PBP2A. The comparator antibiotics were equally active on the strains tested, with MIC90 of 0.5 mg/L for ceftaroline fosamil, tigecycline, and daptomycin; 1 mg/L for vancomycin; and 2 mg/L for linezolid. CONCLUSIONS: Our results suggest that ceftobiprole is highly active against S. aureus and is an effective alternative to vancomycin or linezolid in the management of staphylococcal pneumonia. However, close monitoring of isolates should be maintained to prevent resistant strain diffusion.

MRSA infections among patients in the emergency department: a European multicentre study.

BACKGROUND: MRSA is a therapeutic concern worldwide, and a major agent of community-acquired skin and soft tissue infections (CA-SSTIs). While the US epidemiology of MRSA in CA-SSTIs is well described and reports the high prevalence of the USA300 clone, data on the European situation are lacking. OBJECTIVES: To determine the prevalence and clonal characteristics of MRSA in CA-SSTIs in seven European emergency departments. PATIENTS AND METHODS: From April to June 2015, patients presenting to the tertiary hospital emergency department with a Staphylococcus aureus CA-SSTI were prospectively enrolled. S. aureus isolates were characterized by antimicrobial susceptibility testing, detection of Panton-Valentine leucocidin encoding genes and spa-typing, MLST and/or DNA microarray. RESULTS: Two-hundred and five cases of S. aureus-associated CA-SSTIs were included, comprising folliculitis, furuncles, abscesses, paronychia, impetigo, carbuncles and cellulitis. Of the 205 cases, we report an MRSA prevalence rate of 15.1%, with a north (0%) to south (29%) increasing gradient. Fifty-one isolates were Panton-Valentine leucocidin-positive (24.9%), whether MSSA or MRSA, with a heterogeneous distribution between countries. Clonal distribution of MSSA and MRSA showed high diversity, with no predominant circulating clone and no archetypical USA300 CA-MRSA clone. CONCLUSIONS: This original prospective multicentre study highlights stark differences in European MRSA epidemiology compared with the USA, and that the USA300 CA-MRSA clone is not predominant among community-infected patients in Europe.

Pivotal Role of Iron in the Regulation of Cyanobacterial Electron Transport.

Iron-containing metalloproteins are the main cornerstones for efficient electron transport in biological systems. The abundance and diversity of iron-dependent proteins in cyanobacteria makes those organisms highly dependent of this micronutrient. To cope with iron imbalance, cyanobacteria have developed a survey of adaptation strategies that are strongly related to the regulation of photosynthesis, nitrogen metabolism and other central electron transfer pathways. Furthermore, either in its ferrous form or as a component of the haem group, iron plays a crucial role as regulatory signalling molecule that directly or indirectly modulates the composition and efficiency of cyanobacterial redox reactions. We present here the major mechanism used by cyanobacteria to couple iron homeostasis to the regulation of electron transport, making special emphasis in processes specific in those organisms.

Expanding the Role of FurA as Essential Global Regulator in Cyanobacteria.

In the nitrogen-fixing heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, the ferric uptake regulator FurA plays a global regulatory role. Failures to eliminate wild-type copies of furA gene from the polyploid genome suggest essential functions. In the present study, we developed a selectively regulated furA expression system by the replacement of furA promoter in the Anabaena sp. chromosomes with the Co2+/Zn2+ inducible coaT promoter from Synechocystis sp. PCC 6803. By removing Co2+ and Zn2+ from the medium and shutting off furA expression, we showed that FurA was absolutely required for cyanobacterial growth. RNA-seq based comparative transcriptome analyses of the furA-turning off strain and its parental wild-type in conjunction with subsequent electrophoretic mobility shift assays and semi-quantitative RT-PCR were carried out in order to identify direct transcriptional targets and unravel new biological roles of FurA. The results of such approaches led us to identify 15 novel direct iron-dependent transcriptional targets belonging to different functional categories including detoxification and defences against oxidative stress, phycobilisome degradation, chlorophyll catabolism and programmed cell death, light sensing and response, heterocyst differentiation, exopolysaccharide biosynthesis, among others. Our analyses evidence novel interactions in the complex regulatory network orchestrated by FurA in cyanobacteria.

Evaluation of the R-Biopharm RIDA®GENE Panton-Valentine leukocidin (PVL) kit for the detection of Staphylococcus aureus PVL from pus samples.

Staphylococcus aureus Panton-Valentine leukocidin (PVL) is associated with primary skin and soft-tissue infections (SSTI). We aimed to divert the RIDA®GENE PVL kit (RBiopharm) from its intended use on cultures to the detection of PVL-encoding genes directly from pus samples. Performance was compared with that of the in-house PCR method developed by the French National Reference Centre for Staphylococci. From June 2013 to May 2014, pus samples from S. aureus SSTI were tested. Our in-house PCR was performed on parallel cultures as the gold standard, while the RIDA®GENE PVL assay was used directly on pus samples from the sterile container, or a swab or an Eswab previously dipped in the pus. The kit specificity was also evaluated with pus samples that grew Streptococcus pyogenes. S. aureus reference strains harboring PVL-encoding genes, including known polymorphisms, were also tested. A total of 56 S. aureus-containing pus samples (28 PVL + and 28 PVL-) were collected and analyzed. Sensitivity and specificity of the commercial kit were 96.4 % and 100 % respectively, with equal performance whether tested directly from the sterile container or the Eswab. Sensitivity was lower (67.9 %) when the test was performed from a regular SSTI swab. None of the Streptococcus pyogenes pus samples scored positive (n = 5). Specificity was assessed using reference strains (n = 14); in all strains the PVL gene was correctly detected. This study identified the RIDA®GENE PVL kit as an efficient, sensitive, and specific tool for the rapid detection of PVL-encoding genes in pus samples.

Community-acquired infections due to Staphylococcus argenteus lineage isolates harbouring the Panton-Valentine leucocidin, France, 2014.

We describe two cases of human infections caused by Staphylococcus aureus clonal complex (CC) 75, also called Staphylococcus argenteus, harbouring the Panton-Valentine leucocidin (PVL). These two sporadic cases were community-acquired, and identified in France in 2014. Both had an epidemiological link with Mayotte, an overseas department of France located in the Indian Ocean off the south-eastern African coast. This report illustrates that, contrary to previous descriptions, S. argenteus can acquire important virulence factors and be responsible for severe infections.

Delta-toxin production deficiency in Staphylococcus aureus: a diagnostic marker of bone and joint infection chronicity linked with osteoblast invasion and biofilm formation.

Biofilm formation, intra-osteoblastic persistence, small-colony variants (SCVs) and the dysregulation of agr, the major virulence regulon, are possibly involved in staphylococcal bone and joint infection (BJI) pathogenesis. We aimed to investigate the contributions of these mechanisms among a collection of 95 Staphylococcus aureus clinical isolates from 64 acute (67.4%) and 31 chronic (32.6%) first episodes of BJI. The included isolates were compared for internalization rate, cell damage and SCV intracellular emergence using an ex vivo model of human osteoblast infection. Biofilm formation was assessed in a microbead immobilization assay (BioFilm Ring test). Virulence gene profiles were assessed by DNA microarray. Seventeen different clonal complexes were identified among the screened collection. The staphylococcal internalization rate in osteoblasts was significantly higher for chronic than acute BJI isolates, regardless of the genetic background. Conversely, no differences regarding cytotoxicity, SCV emergence, biofilm formation and virulence gene distribution were observed. Additionally, agr dysfunction, detected by the lack of delta-toxin production using whole-cell matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis (n = 15; 15.8%), was significantly associated with BJI chronicity, osteoblast invasion and biofilm formation. These findings provide new insights into MSSA BJI pathogenesis, suggesting the correlation between chronicity and staphylococcal osteoblast invasion. This adaptive mechanism, along with biofilm formation, is associated with agr dysfunction, which can be routinely assessed by delta-toxin detection using MALDI-TOF spectrum analysis, possibly providing clinicians with a diagnostic marker of BJI chronicity at the time of diagnosis.

The levels of antibodies to Panton-Valentine leukocidin (PVL) vary with PVL prevalence along a north-to-south gradient.

Recent research on Staphylococcus aureus vaccine development has focused on active immunization against Panton-Valentine leukocidin (PVL), a potent leukotoxin associated with both superficial and severe deep-seated infections. PVL prevalence is highly variable worldwide, but it is unknown to what extent immunity to PVL varies between patients from geographic areas with different PVL-positive S. aureus prevalences. We conducted a retrospective multicentric study of anti-PVL and anti-alpha-toxin (Hla) antibody levels in uninfected adult patients from France (low PVL prevalence; n = 200), Algeria (moderate prevalence; n = 143), and Senegal (high prevalence; n = 228). The antibody levels were quantified by an enzyme-linked immunosorbent assay (ELISA) procedure. Because Hla is present in virtually all S. aureus strains, its corresponding antibody levels were considered to reflect population exposure to S. aureus. Compared with French participants, the average anti-PVL antibody levels were 2.5-fold and 8.2-fold higher in Algerian and Senegalese participants, respectively (p < 0.001). Conversely, anti-Hla antibody levels did not differ between participants from the three countries, suggesting that the observed differences in anti-PVL antibody levels were not biased by variations in population exposure to S. aureus. Hence, anti-PVL antibody levels in the general populations of France, Algeria, and Senegal vary widely and match variations in PVL-positive S. aureus strain prevalence, with an increasing north-to-south gradient. To conclude, immunity to PVL in a given population correlates with local PVL prevalence. This finding can help to inform PVL vaccine strategies.

Detection of methicillin-resistant Staphylococcus aureus carrying the mecC gene in human samples in Slovenia.

SUMMARY Following the recognition of a mecC MRSA isolate from a patient hospitalized in the northeastern region of Slovenia, a national collection of 395 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates from 2006 to 2013 was screened. An additional six mecC MRSA strains were found and characterized as spa types t843, t9397 and t10009, and multilocus sequence type ST130. The low oxacillin minimum inhibitory concentrations and absence of the mecA gene make recognition of these MRSA strains problematical for diagnostic laboratories. In such strains the presence of mecC should be determined.

Early predictors of antiviral treatment response in liver transplant recipients with recurrent hepatitis C genotype 1.

The success of current antiviral treatment for hepatitis C virus (HCV) recurrence in liver transplant (LT) recipients remains limited. We aimed at evaluating the value of IL28B genotype and early viral kinetics to predict response to standard treatment in the transplant setting. We retrospectively evaluated 104 LT recipients treated for HCV genotype 1 recurrence between 2001 and 2010. Baseline variables, including IL28B genotype, and early viral kinetics were compared among patients who did or did not achieve a sustained virological response (SVR). Logistic regression analyses of candidate variables were conducted to generate a reliable predictive model based on the minimum set of variables. Twenty-nine (28%) achieved an SVR. On multivariate analysis, the magnitude of HCV RNA decline at 4 weeks (OR: 3.74, 95% CI: 1.64-9.39; P = 0.003) and treatment compliance (OR: 35.27, 95% CI: 3.35-365.54; P = 0.003) were the only independent predictors of SVR. Favourable recipient IL28B genotype significantly correlates with virological response at week 4 (OR 3.23; 95% CI, 1.12-9.15; P = 0.03). By logistic regression analysis, a model including donor age, recipient rs12979860 genotype and viral load at 4 weeks showed the best predictive value for SVR with an area under the receiver operating curve of 0.861. Favourable recipient IL28B genotype strongly correlates with the viral response at week 4 which is the strongest predictor of response. The combination of recipient IL28B genotype and donor age with the week 4 response reliably estimates the probability of SVR early on-treatment and may facilitate therapeutic strategies incorporating new antiviral agents.

Methicillin-susceptible Staphylococcus aureus clonal complex 398: high prevalence and geographical heterogeneity in bone and joint infection and nasal carriage.

The prevalence of clonal complex (CC) 398 methicillin-susceptible Staphylococcus aureus (MSSA) was unexpectedly high among bone and joint infections (BJIs) and nasal-colonizing isolates in France, with surprising geographical heterogeneity. With none of the major, most-known staphylococcal virulence genes, MSSA CC398 BJI was associated with lower biological inflammatory syndrome and lower treatment failure rates.

Unraveling the redox properties of the global regulator FurA from Anabaena sp. PCC 7120: disulfide reductase activity based on its CXXC motifs.

UNLABELLED: Cyanobacterial FurA works as a global regulator linking iron homeostasis to photosynthetic metabolism and the responses to different environmental stresses. Additionally, FurA modulates several genes involved in redox homeostasis and fulfills the characteristics of a heme-sensor protein whose interaction with this cofactor negatively affects its DNA binding ability. FurA from Anabaena PCC 7120 contains five cysteine residues, four of them arranged in two redox CXXC motifs. AIMS: Our goals were to analyze in depth the putative contribution of these CXXC motifs in the redox properties of FurA and to identify potential interacting partners of this regulator. RESULTS: Insulin reduction assays unravel that FurA exhibits disulfide reductase activity. Simultaneous presence of both CXXC signatures greatly enhances the reduction rate, although the redox motif containing Cys(101) and Cys(104) seems a major contributor to this activity. Disulfide reductase activity was not detected in other ferric uptake regulator (Fur) proteins isolated from heterotrophic bacteria. In vivo, FurA presents different redox states involving intramolecular disulfide bonds when is partially oxidized. Redox potential values for CXXC motifs, -235 and -238 mV, are consistent with those reported for other proteins displaying disulfide reductase activity. Pull-down and two-hybrid assays unveil potential FurA interacting partners, namely phosphoribulokinase Alr4123, the hypothetical amidase-containing domain All1140 and the DNA-binding protein HU. INNOVATION: A novel biochemical activity of cyanobacterial FurA based on its cysteine arrangements and the identification of novel interacting partners are reported. CONCLUSION: The present study discloses a putative connection of FurA with the cyanobacterial redox-signaling pathway.

Phosphate deficiency (N/P 40:1) induces mcyD transcription and microcystin synthesis in Microcystis aeruginosa PCC7806.

A real-time RT-PCR analysis of the transcriptional response to phosphate availability of the mcyD gene and microcystin-LR synthesis in Microcystis aeruginosa PCC7806 revealed that no significant changes were observed in the relative quantification of mcyD under excess phosphate (N/P = 1:1), whereas in deficiency of this nutrient (N/P = 40:1), a steady increase of mcyD during the exponential growth phase was detected, showing a maximal level on the 7th day of growth with a 6.8-fold increase over the control cells. The microcystin content in phosphate deficient cells correlates with the trend of mcyD transcription observed. Also, in this work we demonstrate that under phosphate deficiency conditions with a ratio of 40:1 N/P, the growth of M. aeruginosa PCC7806 was not affected when compared to control and phosphate excess samples. When blooms occur, the nutrients become exhausted and therefore phosphate availability will be scarce. In such a complex scenario, microcystin synthesis could be a response to phosphate deficiency, among other stress parameters.