The Zinc-Responsive Regulator Zur Controls a Zinc Uptake System and Some Ribosomal Proteins in Streptomyces coelicolor A3(2)

ABSTRACT In various bacteria, Zur, a zinc-specific regulator of the Fur family, regulates genes for zinc transport systems to maintain zinc homeostasis. It has also been suggested that Zur controls zinc mobilization by regulating some ribosomal proteins. The antibiotic-producing soil bacterium Streptomyces coelicolor contains four genes for Fur family regulators, and one (named zur) is located downstream of the znuACB operon encoding a putative zinc uptake transporter. We found that zinc specifically repressed the level of znuA transcripts and that this level was derepressed in a Δzur mutant. Purified Zur existing as homodimers bound to the znuA promoter region in the presence of zinc, confirming the role of Zur as a zinc-responsive repressor. We analyzed transcripts for paralogous forms of ribosomal proteins L31 (RpmE1 and RpmE2) and L33 (RpmG2 and RpmG3) for their dependence on Zur and found that RpmE2 and RpmG2 with no zinc-binding motif of conserved cysteines (C's) were negatively regulated by Zur. C-negative RpmG3 and C-positive RpmE1 were not regulated by Zur. Instead, they were regulated by the sigma factor σR as predicted from their promoter sequences. The rpmE1 and rpmG3 genes were partially induced by EDTA in a manner dependent on σR, suggesting that zinc depletion may stimulate the σR regulatory system. This finding reflects a link between thiol-oxidizing stress and zinc depletion. We determined the Zur-binding sites within znuA and rpmG2 promoter regions by footprinting analyses and identified a consensus inverted repeat sequence (TGaaAatgatTttCA, where uppercase letters represent the nucleotides common to all sites analyzed). This sequence closely matches that for mycobacterial Zur and allows the prediction of more genes in the Zur regulon.

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Coordinated Zinc Homeostasis Is Essential for the Wild-Type Virulence of Brucella abortus

Journal of Bacteriology ◽

10.1128/jb.02543-14 ◽

2015 ◽

Vol 197 (9) ◽

pp. 1582-1591 ◽

Cited By ~ 15

Author(s):

Lauren M. Sheehan ◽

James A. Budnick ◽

R. Martin Roop ◽

Clayton C. Caswell

Keyword(s):

Mouse Model ◽

Brucella Abortus ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Zinc Toxicity ◽

Uptake System ◽

Bacterial Cells ◽

Gene Encoding ◽

Content Type ◽

Genes Encoding

ABSTRACTMetal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.

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Control of zinc homeostasis in Agrobacterium tumefaciens via zur and the zinc uptake genes znuABC and zinT

Microbiology ◽

10.1099/mic.0.082446-0 ◽

2014 ◽

Vol 160 (11) ◽

pp. 2452-2463 ◽

Cited By ~ 16

Author(s):

Sakkarin Bhubhanil ◽

Panida Sittipo ◽

Paweena Chaoprasid ◽

Sumontha Nookabkaew ◽

Rojana Sukchawalit ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Dominant Role ◽

Zinc Content ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Dependent Manner ◽

Transporter Family ◽

Zinc Depletion ◽

Zinc Uptake Regulator ◽

Dose Dependent

The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein. The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens.

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Zinc depletion induces ribosome hibernation in mycobacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804555115 ◽

2018 ◽

Vol 115 (32) ◽

pp. 8191-8196 ◽

Cited By ~ 19

Author(s):

Yunlong Li ◽

Manjuli R. Sharma ◽

Ravi K. Koripella ◽

Yong Yang ◽

Prem S. Kaushal ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Ribosomal Proteins ◽

Specific Protein ◽

Zinc Homeostasis ◽

Aminoglycoside Resistance ◽

Zinc Depletion ◽

Binding Pockets ◽

Mouse Model Of Infection ◽

Decoding Region ◽

Mycobacterial Protein

Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C−) paralogues. Consequences of this process beyond zinc homeostasis are unknown. Here, we show that the C− ribosome in Mycobacterium smegmatis is the exclusive target of a bacterial protein Y homolog, referred to as mycobacterial-specific protein Y (MPY), which binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. MPY binding is dependent on another mycobacterial protein, MPY recruitment factor (MRF), which is induced on zinc depletion, and interacts with C− ribosomes. MPY binding confers structural stability to C− ribosomes, promoting survival of growth-arrested cells under zinc-limiting conditions. Binding of MPY also has direct influence on the dynamics of aminoglycoside-binding pockets of the C− ribosome to inhibit binding of these antibiotics. Together, our data suggest that zinc limitation leads to ribosome hibernation and aminoglycoside resistance in mycobacteria. Furthermore, our observation of the expression of the proteins of C− ribosomes in Mycobacterium tuberculosis in a mouse model of infection suggests that ribosome hibernation could be relevant in our understanding of persistence and drug tolerance of the pathogen encountered during chemotherapy of TB.

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The dasABC Gene Cluster, Adjacent to dasR, Encodes a Novel ABC Transporter for the Uptake of N,N′-Diacetylchitobiose in Streptomyces coelicolor A3(2)

Applied and Environmental Microbiology ◽

10.1128/aem.02612-06 ◽

2007 ◽

Vol 73 (9) ◽

pp. 3000-3008 ◽

Cited By ~ 38

Author(s):

Akihiro Saito ◽

Tomonori Shinya ◽

Katsushiro Miyamoto ◽

Tomofumi Yokoyama ◽

Hanae Kaku ◽

...

Keyword(s):

Streptomyces Coelicolor ◽

Integral Membrane Protein ◽

Repeat Sequence ◽

Open Reading Frames ◽

Uptake System ◽

Surface Plasmon Resonance Analysis ◽

Rate Of Decline ◽

Equilibrium Dissociation

ABSTRACT N,N′-Diacetylchitobiose [(GlcNAc)2] induces the transcription of chitinase (chi) genes in Streptomyces coelicolor A3(2). Physiological studies showed that (GlcNAc)2 addition triggered chi expression and increased the rate of (GlcNAc)2 concentration decline in culture supernatants of mycelia already cultivated with (GlcNAc)2, suggesting that (GlcNAc)2 induced the synthesis of its own uptake system. Four open reading frames (SCO0531, SCO0914, SCO2946, and SCO5232) encoding putative sugar-binding proteins of ABC transporters were found in the genome by probing the 12-bp repeat sequence required for regulation of chi transcription. SCO5232, named dasA, showed transcriptional induction by (GlcNAc)2 and N,N′,N‴-triacetylchitotriose [(GlcNAc)3]. Surface plasmon resonance analysis showed that recombinant DasA protein exhibited the highest affinity for (GlcNAc)2 (equilibrium dissociation constant [KD ] = 3.22 × 10−8). In the dasA-null mutant, the rate of decline of the (GlcNAc)2 concentration in the culture supernatant was about 25% of that in strain M145. The in vitro and in vivo data clearly demonstrated that dasA is involved in (GlcNAc)2 uptake. Upstream and downstream of dasA, the transcriptional regulator gene (dasR) and two putative integral membrane protein genes (dasBC) are located in the opposite and same orientations, respectively. The expression of dasR and dasB, which seemed independent of dasA transcription, was also induced by (GlcNAc)2 and (GlcNAc)3.

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The Zinc Uptake Regulator Zur Is Essential for the Full Virulence of Xanthomonas campestris pv. campestris

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0652 ◽

2005 ◽

Vol 18 (7) ◽

pp. 652-658 ◽

Cited By ~ 40

Author(s):

Dong-Jie Tang ◽

Xiang-Jiang Li ◽

Yong-Qiang He ◽

Jia-Xun Feng ◽

Baoshan Chen ◽

...

Keyword(s):

Type Strain ◽

Xanthomonas Campestris ◽

Wild Type Strain ◽

Extracellular Polysaccharide ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Uptake System ◽

Rich Medium ◽

Wild Type ◽

Xanthomonas Campestris Pv Campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 μM and in nonrich medium supplemented with Zn2+ at a concentration of 110 μM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 μM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.

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Functional traits of particle-associated microbial assemblages in the low-latitude Pacific Ocean

10.21203/rs.2.21628/v1 ◽

2020 ◽

Author(s):

Hideto Takami ◽

Yuya Tada ◽

Takashi Okubo ◽

Wataru Arai ◽

Yoshitoshi Ogura ◽

...

Keyword(s):

Organic Matter ◽

Functional Traits ◽

Ribosomal Proteins ◽

Inorganic Nitrogen ◽

Regulatory System ◽

Bacterial Transport ◽

Global Ocean ◽

Transport Systems ◽

Nutrient Regeneration ◽

Dicarboxylate Transport

Abstract Background Organic particles are hotspots for microbial activity and serve as sites of organic matter mineralisation in the water column of marine systems. In nutrient-limited surface water, degradation of organic matter and nutrient regeneration by marine microbes is crucial. Although free-living (FL) bacteria vastly outnumber those on particles, particle-associated (PA) bacteria can reach locally higher concentrations. Accordingly, to achieve a better understanding of marine microbial ecosystems, it is important to elucidate the differences in not only microbial community structures, but also functional traits, between PA and FL environmental sample fractions. In a previous study, we demonstrated that the Genomaple (formerly MAPLE) system could successfully differentiate the functional potentials and diversity of contributors to each function in four metagenomic datasets generated by the Global Ocean Sampling expedition. Hence, we also used this system to highlight functional traits in PA microbial assemblages. Results The PA and FL fractions could be distinguished from one another by their taxonomic compositions, inferred from ribosomal proteins and relative abundance of module functions. Module functions that were more abundant among PA assemblages than FL assemblages were shared between both subtropical gyres, and their taxonomic compositions were similar. Bacterial transport systems associated with adhesive molecules used for forming microbial assemblages through particulate organic matter were more abundant in the PA fractions. Bacterial regulatory system elements for C 4 -dicarboxylate transport and B-vitamin biosynthesis were also abundant among PA assemblages, suggesting mutual relationships between bacteria and algae involved in exchange of nutrient sources. On the other hand, module functions related to amino acid biosynthesis and bacterial transport systems for inorganic nitrogen, phosphorus, and urea were significantly more abundant in the PA assemblages of more oligotrophic North and South Pacific subtropical gyres than eastern equatorial Pacific regions. Conclusions Comprehensive functional metagenomic analyses based on functional abundance revealed some notable functional traits in PA assemblages related to cell adhesion and nutrient acquisition, enabling the microbes to survive in subtropical regions that are more oligotrophic than the equatorial regions.

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Zinc uptake system ZnuACB is essential for maintaining pathogenic phenotype of F4ac+ enterotoxigenic E. coli (ETEC) under a zinc restricted environment

Veterinary Research ◽

10.1186/s13567-020-00854-1 ◽

2020 ◽

Vol 51 (1) ◽

Author(s):

Guomei Quan ◽

Pengpeng Xia ◽

Siqi Lian ◽

Yunping Wu ◽

Guoqiang Zhu

Keyword(s):

Sharp Decrease ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Data Sets ◽

Uptake System ◽

E Coli ◽

High Affinity ◽

Restricted Environment ◽

Living Organisms

Abstract Zinc is the second trace element of living organisms after iron. Given its crucial importance, mammalian hosts restrict the bioavailability of Zinc ions (Zn2+) to bacterial pathogens. As a countermeasure, pathogens utilize high affinity Zn2+ transporters, such as ZnuACB to compete with the host for zinc. It is essential for bacteria to maintain zinc homeostasis and thus maintain their physiology and pathogenesis. In an attempt to uncover the zinc transporter in F4+ enterotoxigenic E. coli (ETEC) C83902, we analyzed two RNA-seq data sets of bacteria samples when different zinc treatments (restriction or abundance) were applied. Considering data revealing that the high affinity zinc uptake system ZnuACB acts as the main transporter in ETEC C83902 to resist zinc deficiency, we deleted znuACB genes to study the role of them in ETEC C83902. The deletion of znuACB genes results in growth perturbation and a sharp decrease in the ability of biofilm formation and adhesion of bacteria in vitro. Taking the data together, this study demonstrates that the ZnuACB system is required for ETEC C83902 to acquire zinc, which highly contributes to ETEC pathogenicity as well.

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Bacterial zinc uptake regulator proteins and their regulons

Biochemical Society Transactions ◽

10.1042/bst20170228 ◽

2018 ◽

Vol 46 (4) ◽

pp. 983-1001 ◽

Cited By ~ 23

Author(s):

Alevtina Mikhaylina ◽

Amira Z. Ksibe ◽

David J. Scanlan ◽

Claudia A. Blindauer

Keyword(s):

Intracellular Trafficking ◽

Ribosomal Proteins ◽

Pathogenic Bacteria ◽

Transcriptional Regulators ◽

Research Area ◽

Essential Elements ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Dna Binding Affinity ◽

Zinc Uptake Regulator

All organisms must regulate the cellular uptake, efflux, and intracellular trafficking of essential elements, including d-block metal ions. In bacteria, such regulation is achieved by the action of metal-responsive transcriptional regulators. Among several families of zinc-responsive transcription factors, the ‘zinc uptake regulator’ Zur is the most widespread. Zur normally represses transcription in its zinc-bound form, in which DNA-binding affinity is enhanced allosterically. Experimental and bioinformatic searches for Zur-regulated genes have revealed that in many cases, Zur proteins govern zinc homeostasis in a much more profound way than merely through the expression of uptake systems. Zur regulons also comprise biosynthetic clusters for metallophore synthesis, ribosomal proteins, enzymes, and virulence factors. In recognition of the importance of zinc homeostasis at the host–pathogen interface, studying Zur regulons of pathogenic bacteria is a particularly active current research area.

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Identification and Characterization of Mycobacterium smegmatis and Mycobacterium avium subsp. paratuberculosis Zinc Transporters

Journal of Bacteriology ◽

10.1128/jb.00049-21 ◽

2021 ◽

Author(s):

Elke Goethe ◽

Ayla Gieseke ◽

Kristin Laarmann ◽

Janita Lührs ◽

Ralph Goethe

Keyword(s):

Mycobacterium Smegmatis ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Zinc Transporters ◽

Transport Systems ◽

Marker Genes ◽

Growth Media ◽

Fast Growing ◽

Growth Defects ◽

Genes Encoding

Zinc uptake in bacteria is essential to maintain cellular homeostasis and survival. ZnuABC is an important zinc importer of numerous bacterial genera, which is expressed to restore zinc homeostasis when the cytosolic concentration decreases beyond a critical threshold. Upon zinc limitation the fast-growing nonpathogenic organism Mycobacterium smegmatis (MSMEG) as well as the ruminant pathogen M. avium subsp. paratuberculosis (MAP) increases expression of genes encoding ZnuABC homologues, but also of genes encoding other transporters. This suggests an involvement of these transporters in zinc homeostasis. Here we characterized the putative zinc transporters of MSMEG (ZnuABC and ZnuABC2) and MAP (ZnuABC, MptABC, and MAP3774-76). Deletion of either ZnuABC or ZnuABC2 in MSMEG did not lead to growth defects, but to an increased expression of zinc marker genes in MSMEGΔznuABC, indicating cytosolic zinc limitation. However, chromatin immunoprecipitation proved direct binding of the global zinc regulator Zur to promoter regions of both znuABC and znuABC2. Simultaneous deletion of both transporters caused severe growth defects, which could be restored either by homologous complementation with single ZnuABC transporters or supplementation of growth media with zinc but not iron, manganese, cobalt, or magnesium. Heterologous complementation of the double mutant with MAP transporters also resulted in reconstitution of growth. Nonradioactive FluoZinTM-3AM zinc uptake assays directly revealed the competence of all transporters to import zinc. Finally, structural and phylogenetic analyses provided evidence of a novel class of ZnuABC transporters represented by the ZnuABC2 of MSMEG, which is present only in actinobacteria, mainly in the genera Nocardia, Streptomyces and fast growing Mycobacteria. IMPORTANCE Zinc is necessary for bacterial growth but simultaneously toxic when in excess. Hence, bacterial cells have developed systems to alter intracellular concentration. Regulation of these systems is primarily executed at transcriptional level by regulator proteins which sense femtomolar changes in the zinc level. In environmental and pathogenic mycobacteria zinc starvation induces expression of common zinc import systems such as the ZnuABC transporter, but also of other additional not yet characterized transport systems. In this study, we characterized the role of such systems in zinc transport. We showed that transport systems of both species whose transcription is induced upon zinc starvation can exchangeably restore cellular zinc homeostasis in transporter deficient mutants by transporting zinc into the cell.

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Zinc-Responsive Regulation of Alternative Ribosomal Protein Genes in Streptomyces coelicolor Involves Zur and σR

Journal of Bacteriology ◽

10.1128/jb.01901-06 ◽

2007 ◽

Vol 189 (11) ◽

pp. 4078-4086 ◽

Cited By ~ 46

Author(s):

Gillian A. Owen ◽

Ben Pascoe ◽

Dimitris Kallifidas ◽

Mark S. B. Paget

Keyword(s):

Sigma Factor ◽

Ribosomal Proteins ◽

Elevated Level ◽

Streptomyces Coelicolor ◽

Zinc Homeostasis ◽

Zinc Transport ◽

Ribosomal Protein Genes ◽

High Affinity ◽

Transcriptional Units ◽

Responsive Regulation

ABSTRACT Streptomyces coelicolor contains paralogous versions of seven ribosomal proteins (S14, S18, L28, L31, L32, L33, and L36), which differ in their potential to bind structural zinc. The paralogues are termed C+ or C− on the basis of the presence or absence of putative cysteine ligands. Here, mutational studies suggest that the C− version of L31 can functionally replace its C+ paralogue only when expressed at an artificially elevated level. We show that the level of expression of four transcriptional units encoding C− proteins is elevated under conditions of zinc deprivation. Zur controls the expression of three transcriptional units (including rpmG2, rpmE2, rpmB2, rpsN2, rpmF2, and possibly rpsR2). Zur also controls the expression of the znuACB operon, which is predicted to encode a high-affinity zinc transport system. Surprisingly, the zinc-responsive control of the rpmG3-rpmJ2 operon is dictated by σR, a sigma factor that was previously shown to control the response to disulfide stress in S. coelicolor. The induction of σR activity during zinc limitation establishes an important link between thiol-disulfide metabolism and zinc homeostasis.

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