pH-responsive Genes Research Articles

Alkalinity modulates a unique suite of genes to recalibrate growth and pH homeostasis.

Alkaline soils pose a conglomerate of constraints to plants, restricting the growth and fitness of non-adapted species in habitats with low active proton concentrations. To thrive under such conditions, plants have to compensate for a potential increase in cytosolic pH and restricted softening of the cell wall to invigorate cell elongation in a proton-depleted environment. To discern mechanisms that aid in the adaptation to external pH, we grew plants on media with pH values ranging from 5.5 to 8.5. Growth was severely restricted above pH 6.5 and associated with decreasing chlorophyll levels at alkaline pH. Bicarbonate treatment worsened plant performance, suggesting effects that differ from those exerted by pH as such. Transcriptional profiling of roots subjected to short-term transfer from optimal (pH 5.5) to alkaline (pH 7.5) media unveiled a large set of differentially expressed genes that were partially congruent with genes affected by low pH, bicarbonate, and nitrate, but showed only a very small overlap with genes responsive to the availability of iron. Further analysis of selected genes disclosed pronounced responsiveness of their expression over a wide range of external pH values. Alkalinity altered the expression of various proton/anion co-transporters, possibly to recalibrate cellular proton homeostasis. Co-expression analysis of pH-responsive genes identified a module of genes encoding proteins with putative functions in the regulation of root growth, which appears to be conserved in plants subjected to low pH or bicarbonate. Our analysis provides an inventory of pH-sensitive genes and allows comprehensive insights into processes that are orchestrated by external pH.

Human Tooth as a Fungal Niche: Candida albicans Traits in Dental Plaque Isolates.

Candida albicans, a fungus typically found in the mucosal niche, is frequently detected in biofilms formed on teeth (dental plaque) of toddlers with severe childhood caries, a global public health problem that causes rampant tooth decay. However, knowledge about fungal traits on the tooth surface remains limited. Here, we assess the phylogeny, phenotype, and interkingdom interactions of C. albicans isolated from plaque of diseased toddlers and compare their properties to reference strains, including 529L (mucosal isolate). C. albicans isolates exhibit broad phenotypic variations, but all display cariogenic traits, including high proteinase activity, acidogenicity, and acid tolerance. Unexpectedly, we find distinctive variations in filamentous growth, ranging from hyphal defective to hyperfilamentous. We then investigate the ability of tooth isolates to form interkingdom biofilms with Streptococcus mutans (cariogenic partner) and Streptococcus gordonii (mucosal partner). The hyphal-defective isolate lacks cobinding with S. gordonii, but all C. albicans isolates develop robust biofilms with S. mutans irrespective of their filamentation state. Moreover, either type of C. albicans (hyphae defective or hyperfilamentous) enhances sucrose metabolism and biofilm acidogenicity, creating highly acidic environmental pH (<5.5). Notably, C. albicans isolates show altered transcriptomes associated with pH, adhesion, and cell wall composition (versus reference strains), further supporting niche-associated traits. Our data reveal that C. albicans displays distinctive adaptive mechanisms on the tooth surface and develops interactions with pathogenic bacteria while creating an acidogenic state regardless of fungal morphology, contrasting with interkingdom partnerships in mucosal infections. Human tooth may provide new insights into fungal colonization/adaptation, interkingdom biofilms, and contributions to disease pathogenesis. IMPORTANCE Severe early childhood caries is a widespread global public health problem causing extensive tooth decay and systemic complications. Candida albicans, a fungus typically found in mucosal surfaces, is frequently detected in dental plaque formed on teeth of diseased toddlers. However, the clinical traits of C. albicans isolated from tooth remain underexplored. Here, we find that C. albicans tooth isolates exhibit unique biological and transcriptomic traits. Notably, interkingdom biofilms with S. mutans can be formed irrespective of their filamentation state. Furthermore, tooth isolates commonly share dental caries-promoting functions, including acidogenesis, proteolytic activity, and enhanced sugar metabolism, while displaying increased expression of pH-responsive and adhesion genes. Our findings reveal that C. albicans colonizing human teeth displays distinctive adaptive mechanisms to mediate interkingdom interactions associated with a disease-causing state on a mineralized surface, providing new insights into Candida pathobiology and its role in a costly pediatric disease.

Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated “striated muscle contraction” as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.

PH-dependent transcriptional profile changes in iron-deficient Arabidopsis roots

BackgroundIron is an essential element for plants and abundantly present in most mineral soils. The mobility of iron is, however, dependent on the redox potential and hydrogen activity (pH) of the soil, factors that may limit its availability to plants in particular at alkaline pHs. Iron deficiency triggers pronounced changes in the transcriptional profile of plants, inducing processes that aid in the acquisition, uptake, and translocation of iron. How ambient pH impact the transcriptional iron deficiency response has not yet been elucidated in detail.ResultsHere, we provide an RNA-seq data set that catalogs global gene expression changes of iron-deficient plants grown at either optimal (5.5) or high (7.0) pH. A suite of 857 genes changed significantly and more than twofold in expression; only 54 genes of this suite were also differentially expressed between iron-deficient and iron-sufficient plants grown at pH 5.5. Among the high pH-responsive genes, 186 were earlier shown to be responsive to short-term transfer to low pH, 91 genes of this subset were anti-directionally regulated by high and low pH. The latter subset contained genes involved in cell wall organization, auxin homeostasis, and potential hubs of yet undefined signaling circuits. Growing iron-deficient plants at high pH also modulated the transcriptional iron deficiency response observed at pH 5.5 by compromising the enzymatic reduction of ferric chelates and favoring the production of iron-mobilizing coumarins.ConclusionsIt is concluded that ambient pH is an important determinant of global gene expression which tunes iron acquisition to the prevailing edaphic conditions.

Establishment of an Extracellular Acidic pH Culture System.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported. Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The effects of extracellular pH and of the transcriptional regulator PACI on the transcriptome of Trichoderma reesei.

BackgroundExtracellular pH is one of the several environmental factors affecting protein production by filamentous fungi. Regulatory mechanisms ensure that extracellular enzymes are produced under pH-conditions in which the enzymes are active. In filamentous fungi, the transcriptional regulation in different ambient pH has been studied especially in Aspergilli, whereas the effects of pH in the industrial producer of hydrolytic enzymes, Trichoderma reesei, have mainly been studied at the protein level. In this study, the pH-dependent expression of T. reesei genes was investigated by genome-wide transcriptional profiling and by analysing the effects of deletion of the gene encoding the transcriptional regulator pac1, the orthologue of Aspergillus nidulans pacC gene.ResultsTranscriptional analysis revealed the pH-responsive genes of T. reesei, and functional classification of the genes identified the activities most affected by changing pH. A large number of genes encoding especially transporters, signalling-related proteins, extracellular enzymes and proteins involved in different metabolism-related functions were found to be pH-responsive. Several cellulase- and hemicellulase-encoding genes were found among the pH-responsive genes. Especially, genes encoding hemicellulases with the similar type of activity were shown to include both genes up-regulated at low pH and genes up-regulated at high pH. However, relatively few of the cellulase- and hemicellulase-encoding genes showed direct PACI-mediated regulation, indicating the importance of other regulatory mechanisms affecting expression in different pH conditions. New information was gained on the effects of pH on the genes involved in ambient pH-signalling and on the known and candidate regulatory genes involved in regulation of cellulase and hemicellulase encoding genes. In addition, co-regulated genomic clusters responding to change of ambient pH were identified.ConclusionsAmbient pH was shown to be an important determinant of T. reesei gene expression. The pH-responsive genes, including those affected by the regulator of ambient pH sensing, were identified, and novel information on the activity of genes encoding carbohydrate active enzymes at different pH was gained.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0247-z) contains supplementary material, which is available to authorized users.

Differential gene expression in nearly isogenic lines with QTL for partial resistance to Puccinia hordei in barley

BackgroundThe barley-Puccinia hordei (barley leaf rust) pathosystem is a model for investigating partial disease resistance in crop plants and genetic mapping of phenotypic resistance has identified several quantitative trait loci (QTL) for partial resistance. Reciprocal QTL-specific near-isogenic lines (QTL-NILs) have been developed that combine two QTL, Rphq2 and Rphq3, the largest effects detected in a recombinant-inbred-line (RIL) population derived from a cross between the super-susceptible line L94 and partially-resistant line Vada. The molecular mechanism underpinning partial resistance in these QTL-NILs is unknown.ResultsAn Agilent custom microarray consisting of 15,000 probes derived from barley consensus EST sequences was used to investigate genome-wide and QTL-specific differential expression of genes 18 hours post-inoculation (hpi) with Puccinia hordei. A total of 1,410 genes were identified as being significantly differentially expressed across the genome, of which 55 were accounted for by the genetic differences defined by QTL-NILs at Rphq2 and Rphq3. These genes were predominantly located at the QTL regions and are, therefore, positional candidates. One gene, encoding the transcriptional repressor Ethylene-Responsive Element Binding Factor 4 (HvERF4) was located outside the QTL at 71 cM on chromosome 1H, within a previously detected eQTL hotspot for defence response. The results indicate that Rphq2 or Rphq3 contains a trans-eQTL that modulates expression of HvERF4. We speculate that HvERF4 functions as an intermediate that conveys the response signal from a gene(s) contained within Rphq2 or Rphq3 to a host of down-stream defense responsive genes. Our results also reveal that barley lines with extreme or intermediate partial resistance phenotypes exhibit a profound similarity in their spectrum of Ph-responsive genes and that hormone-related signalling pathways are actively involved in response to Puccinia hordei.ConclusionsDifferential gene expression between QTL-NILs identifies genes predominantly located within the target region(s) providing both transcriptional and positional candidate genes for the QTL. Genetically mapping the differentially expressed genes relative to the QTL has the potential to discover trans-eQTL mediated regulatory relays initiated from genes within the QTL regions.

Changes in external pH rapidly alter plant gene expression and modulate auxin and elicitor responses.

pH is a highly variable environmental factor for the root, and plant cells can modify apoplastic pH for nutrient acquisition and in response to extracellular signals. Nevertheless, surprisingly few effects of external pH on plant gene expression have been reported. We have used microarrays to investigate whether external pH affects global gene expression. In Arabidopsis thaliana roots, 881 genes displayed at least twofold changes in transcript abundance 8 h after shifting medium pH from 6.0 to 4.5, identifying pH as a major affector of global gene expression. Several genes responded within 20 min, and gene responses were also observed in leaves of seedling cultures. The pH 4.5 treatment was not associated with abiotic stress, as evaluated from growth and transcriptional response. However, the observed patterns of global gene expression indicated redundancies and interactions between the responses to pH, auxin and pathogen elicitors. In addition, major shifts in gene expression were associated with cell wall modifications and Ca(2+) signalling. Correspondingly, a marked overrepresentation of Ca(2+)/calmodulin-associated motifs was observed in the promoters of pH-responsive genes. This strongly suggests that plant pH recognition involves intracellular Ca(2+). Overall, the results emphasize the previously underappreciated role of pH in plant responses to the environment.

Histidine Residue 94 Is Involved in pH Sensing by Histidine Kinase ArsS of Helicobacter pylori

BackgroundThe ArsRS two-component system is the master regulator of acid adaptation in the human gastric pathogen Helicobacter pylori. Low pH is supposed to trigger the autophosphorylation of the histidine kinase ArsS and the subsequent transfer of the phosphoryl group to its cognate response regulator ArsR which then acts as an activator or repressor of pH-responsive genes. Orthologs of the ArsRS two-component system are also present in H. pylori's close relatives H. hepaticus, Campylobacter jejuni and Wolinella succinogenes which are non-gastric colonizers.Methodology/Principal FindingsIn order to investigate the mechanism of acid perception by ArsS, derivatives of H. pylori 26695 expressing ArsS proteins with substitutions of the histidine residues present in its periplasmic input domain were constructed. Analysis of pH-responsive transcription of selected ArsRS target genes in these mutants revealed that H94 is relevant for pH sensing, however, our data indicate that protonatable amino acids other than histidine contribute substantially to acid perception by ArsS. By the construction and analysis of H. pylori mutants carrying arsS allels from the related ε-proteobacteria we demonstrate that WS1818 of W. succinogenes efficiently responds to acidic pH.Conclusions/SignificanceWe show that H94 in the input domain of ArsS is crucial for acid perception in H. pylori 26695. In addition our data suggest that ArsS is able to adopt different conformations depending on the degree of protonation of acidic amino acids in the input domain. This might result in different activation states of the histidine kinase allowing a gradual transcriptional response to low pH conditions. Although retaining considerable similarity to ArsS the orthologous proteins of H. hepaticus and C. jejuni may have evolved to sensors of a different environmental stimulus in accordance with the non gastric habitat of these bacteria.

An activating mutation of the Sclerotinia sclerotiorum pac1 gene increases oxalic acid production at low pH but decreases virulence

SUMMARY The production of oxalic acid by Sclerotinia sclerotiorum is regulated by the ambient pH environment. This regulation and that of a few investigated pH-responsive genes is mediated in part by the zinc finger transcription factor encoded by pac1, an orthologue of the Aspergillus nidulans pacC gene. We manipulated the pac1 sequence by site-directed mutagenesis to create a dominant activating pac1(c) mutation and introduced this allele into a pac1 loss-of-function (Deltapac1) strain. Consistent with a constitutive activation of Pac1 function, oxalic acid accumulation in recovered Deltapac1-pac1(c) strains was largely independent of ambient pH. Likewise, all three Deltapac1-pac1(c) strains accumulated detectable pac1 transcripts in a pH 3 environment; however, accumulation of pac1 transcripts remained alkaline-inducible, but much reduced relative to wild-type in two of the three Deltapac1-pac1(c) strains. Surprisingly, the accumulation of pg1 and acp1 transcripts, normally favoured by low pH conditions, were up-regulated across the range of ambient pH conditions examined (pH 3.4-7.2). Accumulation of neutral pH-expressed endopolygalacturonase-6 (pg6) transcripts, however, did not differ from wild-type. In pathogenicity assays using Arabidopsis and detached tomato leaflets, Deltapac1-pac1(c) strains were reduced in virulence despite the ability to accumulate oxalic acid independent of the prevailing ambient pH environment. These results support the hypothesis that appropriate gene regulation in response to ambient pH is important for full S. sclerotiorum virulence independent of oxalic acid accumulation.

Transcriptional profiling in Candida albicans reveals new adaptive responses to extracellular pH and functions for Rim101p

The human pathogen Candida albicans grows and colonizes sites that can vary markedly in pH. The pH response in C. albicans is governed in part by the Rim101p pathway. In Saccharomyces cerevisiae, Rim101p promotes alkaline responses by repressing expression of NRG1, itself a transcriptional repressor. Our studies reveal that in C. albicans, Rim101p-mediated alkaline adaptation is not through repression of CaNRG1. Furthermore, our studies suggest that Rim101p and Nrg1p act in parallel pathways to regulate hyphal morphogenesis, an important contributor to virulence. To determine the wild-type C. albicans transcriptional response to acidic and alkaline pH, we utilized microarrays and identified 514 pH-responsive genes. Of these, several genes involved in iron acquisition were upregulated at pH 8, suggesting that alkaline pH induces iron starvation. Microarray analysis of rim101-/- cells indicated that Rim101p does not govern transcriptional responses at acidic pH, but does regulate a subset of transcriptional responses at alkaline pH, including the iron acquisition genes. We found that rim101-/- cells are sensitive to iron starvation, which suggests that one important aspect of the Rim101p-dependent alkaline pH response is to adapt to iron starvation conditions.

Diverged binding specificity of Rim101p, the Candida albicans ortholog of PacC.

The biology of Candida albicans, including dimorphism and virulence, is significantly influenced by environmental pH. The response to ambient pH includes the pH-conditional expression of several genes, which is directly or indirectly regulated by Rim101p. Rim101p is homologous to PacC, a transcription factor that regulates pH-conditional gene expression in Aspergillus nidulans. PacC binds 5'-GCCARG-3' sequences upstream of pH-responsive genes and either activates or represses transcription. The absence of pacC consensus binding sites upstream of PHR1, a RIM101-dependent, alkaline pH-induced gene of C. albicans, suggested either that PHR1 is indirectly regulated by Rim101p or that the binding specificity of Rim101p is different. In vitro binding studies demonstrated that Rim101p strongly bound two regions upstream of PHR1 that were only weakly bound by PacC. Deletion analysis and site-specific mutagenesis demonstrated that both sites were functionally significant, mutation of either site reduced RIM101-dependent induction, and expression was abolished in the double mutant. Furthermore, oligonucleotides containing these sites conferred pH-conditional expression when inserted upstream of a reporter gene. The consensus sequence of these sites, 5'-CCAAGAAA-3', was identical to the binding recognition sequence identified by in vitro selection of Rim101p binding oligonucleotides from a random pool. The functional significance of this binding sequence was reinforced by its observed presence upstream of a number of newly identified pH-conditional genes. We conclude that Rim101p acts as a transcription factor and directly regulates pH-conditional gene expression but has a binding specificity different from that of PacC.

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as β-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Δ yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Δ yeast to maintain expression of the temperature-inducible β-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.

Characterization of Pneumocystis carinii PHR1, a pH-regulated gene important for cell wall Integrity.

Pneumocystis carinii remains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region coding GSC-1, a gene mediating beta-glucan cell wall synthesis in P. carinii. The predicted amino acid sequence of this new gene, termed P. carinii PHR1, exhibited 38% homology to Saccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology to Candida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking beta-1,3- and beta-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression of P. carinii PHR1 was examined. As in C. albicans, P. carinii PHR1 expression was repressed under acidic conditions but induced at neutral and more alkaline pH. PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties with P. carinii culture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression of P. carinii genes in related fungi to address functional correlates of P. carinii-encoded proteins. Therefore, the potential role of P. carinii PHR1 in cell wall integrity was examined by assessing its ability to rescue an S. cerevisiae gas1 mutant with absent endogenous Phr1p-like activity. Interestingly, P. carinii PHR1 DNA successfully restored proliferation of S. cerevisiae gas1 mutants under lethal conditions of cell wall stress. These results indicate that P. carinii PHR1 encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.

Alkaline Response Genes of Saccharomyces cerevisiaeand Their Relationship to the RIM101 Pathway

Environmental pH exerts broad control over growth and differentiation, but the molecular responses to external pH changes are poorly understood. Here we have used open reading frame macroarray hybridization to identify alkaline response genes in Saccharomyces cerevisiae. Northern or lacZ fusion assays confirmed the alkaline induction of two ion pump genes (ENA1 and VMA4), several ion limitation genes (CTR3, FRE1, PHO11/12, and PHO84), a siderophore-iron transporter gene (ARN4/ENB1), two transcription factor genes (NRG2 and TIS11), and two predicted membrane protein genes (YAR068W/YHR214W and YOL154W). Unlike ENA1 and SHC1, these new alkaline response genes are not induced by high salinity. The known pH-responsive genes in other fungi depend on the conserved PacC/Rim101p transcription factor, but induction of several of these new genes relied upon Rim101p-independent pH signaling mechanisms. Rim101p-dependent genes were also dependent on Rim13p, a protease required for Rim101p processing. The Rim101p-dependent gene VMA4 is required for growth in alkaline conditions, illustrating how Rim101p may control adaptation. Because Rim101p activates ion pump genes, we tested the role of RIM101 in ion homeostasis and found that RIM101 promotes resistance to elevated cation concentrations. Thus, gene expression surveys can reveal new functions for characterized transcription factors in addition to uncovering physiological responses to environmental conditions.