Multidrug Transporters and Alterations in Sterol Biosynthesis Contribute to Azole Antifungal Resistance in Candida parapsilosis

ABSTRACTWhile much is known concerning azole resistance inCandida albicans, considerably less is understood aboutCandida parapsilosis, an emerging species ofCandidawith clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistantC. parapsilosisclinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genesCDR1andMDR1and that ofERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators,TAC1,MRR1, andUPC2, respectively. We also sequenced the sterol biosynthesis genesERG3andERG11in these isolates to find mutations that might contribute to this phenotype in thisCandidaspecies. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution inERG11is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance inC. parapsilosis.

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Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00651-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 14

Author(s):

Jeffrey M. Rybak ◽

C. Michael Dickens ◽

Josie E. Parker ◽

Kelly E. Caudle ◽

Kayihura Manigaba ◽

...

Keyword(s):

Clinical Isolate ◽

Desaturase Activity ◽

Candida Parapsilosis ◽

Prosthetic Valve Endocarditis ◽

Bloodstream Infections ◽

Resistant Isolate ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Content Type ◽

High Level

ABSTRACT Among emerging non-albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2. Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

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A Zinc Cluster Transcription Factor Contributes to the Intrinsic Fluconazole Resistance of Candida auris

mSphere ◽

10.1128/msphere.00279-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 5

Author(s):

Eva-Maria Mayr ◽

Bernardo Ramírez-Zavala ◽

Ines Krüger ◽

Joachim Morschhäuser

Keyword(s):

Transcription Factor ◽

Efflux Pumps ◽

Azole Resistance ◽

Drug Resistant ◽

Candida Auris ◽

Pathogenic Yeast ◽

Fluconazole Resistance ◽

Content Type ◽

Zinc Cluster ◽

Genes Encoding

ABSTRACT The recently emerged pathogenic yeast Candida auris is a major concern for human health, because it is easily transmissible, difficult to eradicate from hospitals, and highly drug resistant. Most C. auris isolates are resistant to the widely used antifungal drug fluconazole due to mutations in the target enzyme Erg11 and high activity of efflux pumps, such as Cdr1. In the well-studied, distantly related yeast Candida albicans, overexpression of drug efflux pumps also is a major mechanism of acquired fluconazole resistance and caused by gain-of-function mutations in the zinc cluster transcription factors Mrr1 and Tac1. In this study, we investigated a possible involvement of related transcription factors in efflux pump expression and fluconazole resistance of C. auris. The C. auris genome contains three genes encoding Mrr1 homologs and two genes encoding Tac1 homologs, and we generated deletion mutants lacking these genes in two fluconazole-resistant strains from clade III and clade IV. Deletion of TAC1b decreased the resistance to fluconazole and voriconazole in both strain backgrounds, demonstrating that the encoded transcription factor contributes to azole resistance in C. auris strains from different clades. CDR1 expression was not or only minimally affected in the mutants, indicating that Tac1b can confer increased azole resistance by a CDR1-independent mechanism. IMPORTANCE Candida auris is a recently emerged pathogenic yeast that within a few years after its initial description has spread all over the globe. C. auris is a major concern for human health, because it can cause life-threatening systemic infections, is easily transmissible, and is difficult to eradicate from hospital environments. Furthermore, C. auris is highly drug resistant, especially against the widely used antifungal drug fluconazole. Mutations in the drug target and high activity of efflux pumps are associated with azole resistance, but it is not known how drug resistance genes are regulated in C. auris. We have investigated the potential role of several candidate transcriptional regulators in the intrinsic fluconazole resistance of C. auris and identified a transcription factor that contributes to the high resistance to fluconazole and voriconazole of two C. auris strains from different genetic clades, thereby providing insight into the molecular basis of drug resistance of this medically important yeast.

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GH51 Arabinofuranosidase and Its Role in the Methylglucuronoarabinoxylan Utilization System in Paenibacillus sp. Strain JDR-2

Applied and Environmental Microbiology ◽

10.1128/aem.01684-14 ◽

2014 ◽

Vol 80 (19) ◽

pp. 6114-6125 ◽

Cited By ~ 5

Author(s):

Neha Sawhney ◽

James F. Preston

Keyword(s):

Abc Transporters ◽

Transcriptional Regulators ◽

Fermentable Sugars ◽

Efficient Utilization ◽

Content Type ◽

Thermochemical Pretreatment ◽

Reverse Transcription Pcr ◽

Paenibacillus Sp ◽

Intracellular Enzymes ◽

Genes Encoding

ABSTRACTMethylglucuronoarabinoxylan (MeGAXn) from agricultural residues and energy crops is a significant yet underutilized biomass resource for production of biofuels and chemicals. Mild thermochemical pretreatment of bagasse yields MeGAXnrequiring saccharifying enzymes for conversion to fermentable sugars. A xylanolytic bacterium,Paenibacillussp. strain JDR-2, produces an extracellular cell-associated GH10 endoxylanse (XynA1) which efficiently depolymerizes methylglucuronoxylan (MeGXn) from hardwoods coupled with assimilation of oligosaccharides for further processing by intracellular GH67 α-glucuronidase, GH10 endoxylanase, and GH43 β-xylosidase. This process has been ascribed to genes that comprise a xylan utilization regulon that encodes XynA1and includes a gene cluster encoding transcriptional regulators, ABC transporters, and intracellular enzymes that convert assimilated oligosaccharides to fermentable sugars. Here we show thatPaenibacillussp. JDR-2 utilized MeGAXnwithout accumulation of oligosaccharides in the medium. ThePaenibacillussp. JDR-2 growth rate on MeGAXnwas 3.1-fold greater than that on oligosaccharides generated from MeGAXnby XynA1. Candidate genes encoding GH51 arabinofuranosidases with potential roles were identified. Following growth on MeGAXn, quantitative reverse transcription-PCR identified a cluster of genes encoding a GH51 arabinofuranosidase (AbfB) and transcriptional regulators which were coordinately expressed along with the genes comprising the xylan utilization regulon. The action of XynA1on MeGAXngenerated arabinoxylobiose, arabinoxylotriose, xylobiose, xylotriose, and methylglucuronoxylotriose. Recombinant AbfB processed arabinoxylooligosaccharides to xylooligosaccharides and arabinose. MeGAXnprocessing byPaenibacillussp. JDR-2 may be achieved by extracellular depolymerization by XynA1coupled to assimilation of oligosaccharides and further processing by intracellular enzymes, including AbfB.Paenibacillussp. JDR-2 provides a GH10/GH67 system complemented with genes encoding intracellular GH51 arabinofuranosidases for efficient utilization of MeGAXn.

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Fluconazole and Voriconazole Resistance in Candida parapsilosis Is Conferred by Gain-of-Function Mutations inMRR1Transcription Factor Gene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00842-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6629-6633 ◽

Cited By ~ 24

Author(s):

Joana Branco ◽

Ana P. Silva ◽

Raquel M. Silva ◽

Ana Silva-Dias ◽

Cidália Pina-Vaz ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Candida Parapsilosis ◽

Bloodstream Infections ◽

Azole Resistance ◽

Transcription Factor Gene ◽

Gain Of Function ◽

Content Type ◽

Factor Gene ◽

Resistant Strains

ABSTRACTCandida parapsilosisis the second most prevalent fungal agent causing bloodstream infections. Nevertheless, there is little information about the molecular mechanisms underlying azole resistance in this species. Mutations (G1747A, A2619C, and A3191C) in theMRR1transcription factor gene were identified in fluconazole- and voriconazole-resistant strains. Independent expression ofMRR1genes harboring these mutations showed that G1747A (G583R) and A2619C (K873N) are gain-of-function mutations responsible for azole resistance, the first described inC. parapsilosis.

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A 1,3-1,4-β-Glucan Utilization Regulon in Paenibacillus sp. Strain JDR-2

Applied and Environmental Microbiology ◽

10.1128/aem.03526-15 ◽

2016 ◽

Vol 82 (6) ◽

pp. 1789-1798 ◽

Cited By ~ 6

Author(s):

Virginia Chow ◽

Young Sik Kim ◽

Mun Su Rhee ◽

Neha Sawhney ◽

Franz J. St. John ◽

...

Keyword(s):

Catalytic Domain ◽

Transcriptional Regulators ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Expression Of Genes ◽

Paenibacillus Sp ◽

Genes Encoding ◽

Marked Preference ◽

Intracellular Metabolism ◽

Polymeric Substrates

ABSTRACTPaenibacillussp. strain JDR-2 (PaenibacillusJDR-2) secretes a multimodular cell-associated glycoside hydrolase family 10 (GH10) endoxylanase (XynA10A1) that catalyzes the depolymerization of methylglucuronoxylan (MeGXn) and rapidly assimilates the products of depolymerization. Efficient utilization of MeGXnhas been postulated to result from the coupling of the processes of exocellular depolymerization and assimilation of oligosaccharide products, followed by intracellular metabolism. Growth and substrate utilization patterns with barley glucan and laminarin similar to those observed with MeGXnas a substrate suggest similar processes for 1,3-1,4-β-glucan and 1,3-β-glucan depolymerization and product assimilation. ThePaenibacillusJDR-2 genome includes a cluster of genes encoding a secreted multimodular GH16 β-glucanase (Bgl16A1) containing surface layer homology (SLH) domains, a secreted GH16 β-glucanase with only a catalytic domain (Bgl16A2), transporter proteins, and transcriptional regulators. Recombinant Bgl16A1and Bgl16A2catalyze the formation of trisaccharides, tetrasaccharides, and larger oligosaccharides from barley glucan and of mono-, di-, tri-, and tetrasaccharides and larger oligosaccharides from laminarin. The lack of accumulation of depolymerization products during growth and a marked preference for polymeric glucan over depolymerization products support a process coupling extracellular depolymerization, assimilation, and intracellular metabolism for β-glucans similar to that ascribed to the GH10/GH67 xylan utilization system inPaenibacillusJDR-2. Coordinate expression of genes encoding GH16 β-glucanases, transporters, and transcriptional regulators supports their role as a regulon for the utilization of soluble β-glucans. As in the case of the xylan utilization regulons, this soluble β-glucan regulon provides advantages in the growth rate and yields on polymeric substrates and may be exploited for the efficient conversion of plant-derived polysaccharides to targeted products.

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Relative Contribution of the ABC Transporters Cdr1, Pdh1, and Snq2 to Azole Resistance inCandida glabrata

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01070-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 10

Author(s):

Sarah G. Whaley ◽

Qing Zhang ◽

Kelly E. Caudle ◽

P. David Rogers

Keyword(s):

Multidrug Resistance ◽

Minor Role ◽

Azole Resistance ◽

Content Type ◽

Azole Antifungals ◽

Zinc Cluster ◽

Relative Contribution ◽

Genes Encoding ◽

High Level ◽

A Minor

ABSTRACTThe utility of the azole antifungals for the treatment of invasive candidiasis is severely hampered by azole resistance inCandida glabrata. This resistance is mediated almost exclusively by activating mutations in the zinc cluster transcription factor Pdr1, which controls the genes encoding the multidrug resistance transporters Cdr1, Pdh1, and Snq2. However, the specific relative contributions of these transporters to resistance are not known. To address this question, theSAT1flipper method was used to deleteCDR1,PDH1, andSNQ2in a strain ofC. glabrataengineered to carry a clinically relevant activating mutation inPDR1. Susceptibility testing was performed according to the CLSI guidelines, with minor modifications, and confirmed with Etest strips. Of the single-transporter-deletion strains, only theCDR1deletion resulted in a decreased azole MIC. The deletion ofPDH1in combination withCDR1resulted in a moderate decrease in MIC compared to that observed with the deletion ofCDR1alone.SNQ2deletion only decreased the MIC in the triple-deletion strain in the absence of bothCDR1andPDH1. The deletion of all three transporters in combination decreased the MIC to the level observed in thePDR1deletion strains for some, but not all, azoles tested, which indicates that additional Pdr1 targets likely play a minor role in this process. These results indicate that while Cdr1 is the most important Pdr1-mediated multidrug resistance transporter for azole resistance in this clinical isolate, all three of these transporters contribute to its high-level resistance to the azole antifungals.

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A Transcriptomics Approach To Unveiling the Mechanisms ofIn VitroEvolution towards Fluconazole Resistance of aCandida glabrataClinical Isolate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00995-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 8

Author(s):

Mafalda Cavalheiro ◽

Catarina Costa ◽

Ana Silva-Dias ◽

Isabel M. Miranda ◽

Can Wang ◽

...

Keyword(s):

Fungal Pathogen ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Antifungal Drugs ◽

Azole Resistance ◽

Fluconazole Resistance ◽

Content Type ◽

Antifungal Drug Resistance ◽

Genes Encoding ◽

Multidrug Resistance Transporters

ABSTRACTCandida glabratais an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of aC. glabrataclinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in theC. glabrataPDR1gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of aC. glabratapopulation toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.

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Triazole Evolution of Candida parapsilosis Results in Cross-Resistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal Drug-Dependent Manner

mSphere ◽

10.1128/msphere.00821-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Csaba Papp ◽

Flóra Bohner ◽

Katica Kocsis ◽

Mónika Varga ◽

András Szekeres ◽

...

Keyword(s):

Amino Acid ◽

Candida Parapsilosis ◽

Treatment Strategies ◽

Antifungal Drugs ◽

Antifungal Treatment ◽

Cross Resistance ◽

Azole Resistance ◽

Resistance Development ◽

Content Type ◽

Invasive Infections

ABSTRACT The number of invasive infections caused by Candida species is increasing worldwide. The incidence of candidiasis cases caused by non-albicans Candida species, such as Candida parapsilosis, is also increasing, and non-albicans Candida species are currently responsible for more invasive infections than C. albicans. Additionally, while the development of azole resistance during invasive disease with C. albicans remains uncommon, azole-resistant C. parapsilosis strains are frequently isolated in the hospital setting. In this study, we applied direct selection to generate azole-adapted and azole-evolved C. parapsilosis strains in order to examine the effect of azole resistance development on fungal viability and pathogenesis progression. Depending on the drug applied, the different evolved strains developed distinct cross-resistance patterns: the fluconazole-evolved (FLUEVO) and voriconazole-evolved (VOREVO) strains gained resistance to fluconazole and voriconazole only, while posaconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POSEVO) strains showed higher echinocandin MIC values than the FLUEVO and VOREVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLUEVO and VOREVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POSEVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLUEVO and the VOREVO strains, along with the altered sterol composition of the POSEVO strain, we now highlight the impact of the above-mentioned amino acid changes in C. parapsilosis azole resistance development. We further revealed that the virulence of this species was only slightly or partially affected by fluconazole and voriconazole adaptation, while it significantly decreased after posaconazole adaptation. Our results suggest that triazole adaptation can result in azole cross-resistance and that this process may also result in virulence alterations in C. parapsilosis, depending on the applied drug. IMPORTANCE Candida parapsilosis causes life-threatening fungal infections. In the last 2 decades, the increasing number of azole-resistant C. parapsilosis clinical isolates has been attributable to the overuse and misuse of fluconazole, the first-line antifungal agent most commonly used in several countries. To date, the range of applicable antifungal drugs is limited. As a consequence, it is essential to understand the possible mechanisms of antifungal resistance development and their effect on virulence in order to optimize antifungal treatment strategies in the clinical setting. Our results revealed that the prolonged exposure to azoles resulted not only in azole resistance but also in cross-resistance development. Our data further indicate that resistance development may occur through different mechanisms that can also alter the virulence of C. parapsilosis. These results highlight the consequences of prolonged drug usage and suggest the need for developing alternative antifungal treatment strategies in clinical practice.

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Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements

Mammalian Genome ◽

10.1007/s00335-020-09854-0 ◽

2020 ◽

Author(s):

Maik Hintze ◽

Sebastian Griesing ◽

Marion Michels ◽

Birgit Blanck ◽

Lena Wischhof ◽

...

Keyword(s):

Hair Growth ◽

Transcriptional Regulators ◽

Mutant Mice ◽

Wild Type ◽

Protein Levels ◽

Expression Of Genes ◽

Genes Encoding ◽

Keratinocyte Cell ◽

Apoptosis Inducing Factor ◽

Hair Cortex

AbstractWe investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

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D-galactose catabolism in archaea: operation of the DeLey–Doudoroff pathway in Haloferax volcanii

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa029 ◽

2020 ◽

Vol 367 (1) ◽

Author(s):

Julia-Beate Tästensen ◽

Ulrike Johnsen ◽

Andreas Reinhardt ◽

Marius Ortjohann ◽

Peter Schönheit

Keyword(s):

Transcriptional Regulator ◽

High Specificity ◽

Comprehensive Analysis ◽

Haloferax Volcanii ◽

Knock Out ◽

Genes Encoding ◽

Functional Involvement ◽

Genome Analyses ◽

Galactose Dehydrogenase ◽

Substrate Binding Protein

ABSTRACT The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey–Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey–Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey–Doudoroff pathway in the domain of archaea.

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