The effect of zinc limitation on the transcriptome of Pseudomonas protegens Pf-5 (original) (raw)
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Zur: Zinc-Sensing Transcriptional Regulator in a Diverse Set of Bacterial Species
Pathogens, 2021
Zinc (Zn) is the quintessential d block metal, needed for survival in all living organisms. While Zn is an essential element, its excess is deleterious, therefore, maintenance of its intracellular concentrations is needed for survival. The living organisms, during the course of evolution, developed proteins that can track the limitation or excess of necessary metal ions, thus providing survival benefits under variable environmental conditions. Zinc uptake regulator (Zur) is a regulatory transcriptional factor of the FUR superfamily of proteins, abundant among the bacterial species and known for its intracellular Zn sensing ability. In this study, we highlight the roles played by Zur in maintaining the Zn levels in various bacterial species as well as the fact that in recent years Zur has emerged not only as a Zn homeostatic regulator but also as a protein involved directly or indirectly in virulence of some pathogens. This functional aspect of Zur could be exploited in the ventures for the identification of newer antimicrobial targets. Despite extensive research on Zur, the insights into its overall regulon and its moonlighting functions in various pathogens yet remain to be explored. Here in this review, we aim to summarise the disparate functional aspects of Zur proteins present in various bacterial species
Microbiology, 2012
We describe a hybrid transcriptomic and modelling analysis of the dynamics of a bacterial response to stress, namely the addition of 200 mM Zn to Escherichia coli growing in severely Zndepleted medium and of cells growing at different Zn concentrations at steady state. Genes that changed significantly in response to the transition were those reported previously to be associated with zinc deficiency (zinT, znuA, ykgM) or excess (basR, cpxP, cusF). Cellular Zn levels were confirmed by ICP-AES to be 14-to 28-fold greater after Zn addition but there was also 6-to 8-fold more cellular Fe 30 min after Zn addition. Statistical modelling of the transcriptomic data generated from the Zn shift focused on the role of ten key regulators; ArsR, BaeR, CpxR, CusR, Fur, OxyR, SoxS, ZntR, ZraR and Zur. The data and modelling reveal a transient change in the activity of the iron regulator Fur and of the oxidative stress regulator SoxS, neither of which is evident from the steady-state transcriptomic analyses. We hypothesize a competitive binding mechanism that combines these observations and existing data on the physiology of Zn and Fe uptake. Formalizing the mechanism in a differential equation model shows that it can reproduce qualitatively the behaviour seen in the data. This gives new insights into the interplay of these two fundamental metal ions in gene regulation and bacterial physiology, as well as highlighting the importance of dynamic studies to reverse-engineer systems behaviour.
Bacterial zinc uptake regulator proteins and their regulons
Biochemical Society Transactions, 2018
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 resea...
Microbiology, 2014
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.
Frontiers in Microbiology, 2021
Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to counteract bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn ...
FEMS Microbiology Letters, 2000
Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.
Journal of Biological Chemistry, 1998
Zinc is an essential trace element required for structural integrity and functional activity of numerous proteins, yet mechanisms by which cells regulate zinc concentration are poorly understood. Here, we identified a gene from Proteus mirabilis that encodes a 135-amino acid residue protein, PMTR (P. mirabilis transcription regulator), a new member of the MerR family of transcription activators. Transformation of Escherichia coli with PMTR-carrying vectors specifically increases cell tolerance to zinc, suggesting the role of PMTR in zinc homeostasis. In response to zinc, PMTR-containing cells robustly accumulate a 12-kDa protein, the amount of which correlates with the cells' ability to grow at high zinc concentrations. The 12-kDa protein is not induced in the presence of Ni 2؉ , Co 2؉ , Cd 2؉ , Mn 2؉ , or Fe 2؉ , indicating that the PMTR-dependent expression of the 12-kDa protein is specifically regulated by zinc. The 12-kDa protein was identified as the C-terminal fragment of E. coli protein YJAI, and was shown to contain two zincbinding motifs. Metal-affinity chromatography and 65 Zn blotting assay confirmed the ability of the 12-kDa protein to bind zinc specifically (zinc > cobalt > > cadmium). We propose that YJAI is an important component of the zinc-balancing mechanism in E. coli, the up-regulation of which with PMTR results in an increased tolerance to zinc.
Elevated zinc induces siderophore biosynthesis genes and azntA-like gene inPseudomonas fluorescens
Fems Microbiology Letters, 2000
Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.
Journal of Biological Chemistry, 2009
Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn 2؉ from the environment, making it exceptionally difficult to achieve Zn 2؉ deficiency, and so a comprehensive understanding of the importance of Zn 2؉ has not been attained. Reduction of the Zn 2؉ content of Escherichia coli growth medium to 60 nM or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn 2؉-deficient medium had a reduced growth rate and contained up to five times less cellular Zn 2؉. To understand global responses to Zn 2؉ deficiency, microarray analysis was conducted of cells grown under Zn 2؉-replete and Zn 2؉-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn 2؉-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn 2؉ level under Zn 2؉ limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn 2؉ or Cd 2؉. A further up-regulated gene, ykgM, is believed to encode a non-Zn 2؉ fingercontaining paralogue of the Zn 2؉ finger ribosomal protein L31. The gene encoding the periplasmic Zn 2؉-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn 2؉ than was originally added to the culture, presumably because of leaching from the culture vessel. Zn 2؉ elimination is shown to be a more precise method of depleting Zn 2؉ than by using the chelator N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine. Almost all biological interactions depend upon contacts between precisely structured protein domains, and Zn 2ϩ may be used to facilitate correct folding and stabilize the domain (1, 2). Zn 2ϩ also plays an indispensable catalytic role in many proteins (1). Although normally classed as a trace element, Zn 2ϩ accumulates to the same levels as calcium and iron in the Esch-erichia coli cell (3); predicted Zn 2ϩ-binding proteins account for 5-6% of the total proteome (4). However, despite its indispensable role in biology, as with all metals, Zn 2ϩ can become toxic if accumulated to excess. With no subcellular compartments to deposit excess metal, Zn 2ϩ homeostasis in bacteria relies primarily on tightly regulated import and export mechanisms (5). The major inducible high affinity Zn 2ϩ uptake system is the ABC transporter ZnuABC. ZnuA is important for growth (6) and Zn 2ϩ uptake (7) and is thought to pass Zn 2ϩ to ZnuB for transport through the membrane. Zn 2ϩ-bound Zur represses transcription of znuABC, whereas the addition of the metal chelator TPEN 3 de-represses expression from a promoterless lacZ gene inserted into znuA, znuB, and znuC (8). Zur can sense subfemtomolar concentrations of cytosolic Zn 2ϩ , implying that cellular Zn 2ϩ starvation commences at exceptionally low Zn 2ϩ concentrations (3). Outten and O'Halloran (3) found that the minimal Zn 2ϩ content required for growth in E. coli is 2 ϫ 10 5 atoms/cell, which corresponds to a total cellular Zn 2ϩ concentration of 0.2 mM, ϳ2000 times the Zn 2ϩ concentration found in the medium. A similar cellular concentration of Zn 2ϩ was found in cells grown in LB medium. Thus, E. coli has an impressive ability to acquire and concentrate Zn 2ϩ (3), making the task of depleting this organism of Zn 2ϩ very difficult. Nevertheless, during the course of this work, a paper was published (9) in which the authors conclude that ZinT (formerly YodA) "is involved in periplasmic zinc binding and either the subsequent import or shuttling of zinc to periplasmic zinc-containing proteins under zinc-limiting conditions." Surprisingly, this conclusion was drawn from experiments in which Zn 2ϩ levels in the medium were lowered only by reducing the amount of Zn 2ϩ added, without metal extraction or chelation. Only a few attempts have been made to study the global consequences of metal deficiency using "omic" technologies. A study using TPEN (10) found 101 genes to be differentially regulated in E. coli. However, the authors note that TPEN has been reported to bind Cd 2ϩ , Co 2ϩ , Ni 2ϩ , and Cu 2ϩ more tightly than it binds Zn 2ϩ , and indeed, 34 of the 101 differentially regulated * This work was supported by the Biotechnology and Biological Sciences Research Council, UK.
Zinc-Dependent Transcriptional Regulation in Paracoccus denitrificans
Frontiers in Microbiology, 2017
Zinc homeostasis is critical for bacterial survival and is mediated largely at the transcriptional level by the regulation of zinc uptake and efflux genes. Here we use RNA-seq to assess transcriptional changes as a result of zinc limitation in the denitrifying bacterium Paracoccus denitrificans. The results identify the differential expression of 147 genes, most of which were upregulated in zinc-depleted medium. Included in this set of genes are a large number of transition metal transporters, several transcription factors, and hypothetical proteins. Intriguingly, genes encoding nitric oxide reductase (norCB) and nitrite reductase (nirS) were also upregulated. A Zur consensus binding motif was identified in the promoters of the most highly upregulated genes. The zinc uptake regulator (Zur) from this organism was also characterized and shown to bind to the Zur motif in a zinc-dependent manner. This work expands our current understanding of the transcriptional response of gram-negative bacteria to zinc limitation and identifies genes involved in denitrification as part of the Zur regulon.