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Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu2+ and Zn2+ resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu2+ and Zn2+ resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu2+ and Zn2+ resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu2+ resistance systems do not show decreased fitness relative to the wild type when exposed to Cu2+. In addition, new genes are identified that have no known connection to Zn2+ (corB, corC, Psest_3226, Psest_3322, and Psest_0618) or Cu2+ resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB, corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu2+- and Zn2+-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu2+ and Zn2+ efflux and resistance and their effects on denitrifying metabolism.
IMPORTANCE In this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca2+/H+ antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg2+ transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB, corC, or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.
The responses of microorganisms to metal toxicity have been well studied (1). In brief, metal ions can be toxic by binding to essential proteins or other molecules, causing them to become nonfunctional or function incorrectly. This occurs, for example, where the toxic metal ion binds to a binding site that requires a different metal ion to function. Toxic metal ions can also catalyze reactions that are detrimental to the cell, such as hydroxyl radical generation catalyzed by free iron (Fe3+/2+) (2) or iron-sulfur cluster degradation by copper (3).
Understanding modes of toxicity and metal resistance systems is important, as the information gained from bacteria can be applied to human physiology and medicine. For example, Wilson's disease and Menkes disease are two human diseases caused by mutations in copper transporters (4). Likewise, acrodermatitis enteropathica, while less well-studied at a mechanistic level, is caused by a mutation of the SLC39A4 zinc transporter gene (5). Wilson's disease results in copper accumulation, which is known to cause tissue damage due to the generation of reactive oxygen species (4). Similarly, copper and zinc are known to be involved in neurodegenerative diseases, such as Alzheimer's and Parkinson's, again with a connection to oxidative stress, although their precise roles are still unclear (6). In addition, copper has long been used as an antimicrobial agent in agriculture, where the evolution of copper-resistant strains can cause problems for food production (7). Much of the current knowledge about the transport protein families and toxic effects relevant in each of these cases has been gained from studies in bacteria (8).
The resistance of microorganisms to potentially toxic metal ions generally involves decreasing the intracellular concentration of free metal ions through active transport to the periplasm or extracellular milieu (9) or sequestering metal ions in a tightly bound form (10). Several classes of transporters are involved, each with members that are specific for the removal of certain toxic metal ions (or groups of ions). In Gram-negative bacteria, metal resistance can be achieved through the expression of two separate systems. One transports metal ions across the plasma membrane, while the other transports them across the outer membrane (9, 11). Plasma membrane transporters are often P-type ATPase pumps (8) or members of the cation diffusion facilitator (CDF) protein family (e.g., CzcD, a Zn2+ transporter), driven by the proton motive force (12). The resistance-nodulation-division (RND) efflux pumps, named after the protein family of the A component (13), span the periplasm and are formed by proteins from the RND, membrane fusion protein (MFP), and outer membrane factor (OMF) families—the A, B, and C components, respectively (e.g., CzcCBA and CusCBA) (9, 14, 15). The RND protein is integrated in the cytoplasmic membrane, the MFP is in the periplasmic space, and the OMF is integrated in the outer membrane (13). Such transporters primarily serve to remove toxic metal ions from the periplasm. They can transport ions across both membranes, although this may be a signaling mechanism rather than a main transport pathway (9, 11, 13, 16). There are structural models for all three of these transporter classes (P-type ATPase, CDF, and RND type) available in the Protein Data Bank (13, 17, 18).
Generally, ion transporters or other metal-binding proteins are not absolutely specific in the metal ion or ions that they transport. For example, the CzcCBA (19) and CzcD (12) transporters are proposed to transport Co2+, Zn2+, and Cd2+, and corresponding regulatory systems respond to these cations, as well as other metal ions in some cases (20,–22). In addition, transporters that primarily import Mg2+ are a common source of various intracellular transition metals (23). Thus, transporters are not perfectly specific, due to similarities in the size, charge, and to some extent, coordination preferences of metal ions, particularly among the divalent late d-block metals (Fe-Zn). In some cases, lower-specificity transporters may even provide a competitive advantage (24, 25). However, the specificity (or generality) of a given transporter is currently very difficult to determine bioinformatically. For example, the InterPro protein family that contains czcA (IPR004763) contains RND proteins that are involved in the transport of any metal cation (26).
While bacteria frequently have several hundred or even a thousand transporter genes (14), in many cases, it is still unclear from sequence information alone which species each of them transports. Even transporters for organic molecules can be quite similar to metal ion transporters (15). Thus, gene annotations are often vague, and recognized protein families often include transporters of many different chemical species (IPR004763) or, sometimes, a very small collection of proteins for which the transported species is known (IPR005695) (26). In the case of copper and zinc, Cu2+ and Zn2+ are physically similar, but they have very different functions in cellular metabolism and are treated differently by transporters, since Cu2+ is readily reduced to Cu+ in the cytoplasm but Zn2+ is not redox active in the biological range (27). The transport of these ions has both agricultural and medical relevance (see above) and is the focus of this study.
Pseudomonas stutzeri is a model denitrifying gammaproteobacterium (28). Strain RCH2 was isolated from Hanford, a U.S. Department of Energy (DOE) Superfund site with metal contamination, particularly chromium, from a retired nuclear reactor complex (29). Strain RCH2 was originally characterized for its chromate-reducing activity (29), and a random barcode transposon site sequencing (RB-TnSeq) library is now available (30). In this study, we used this library to determine which RCH2 efflux systems were truly involved in Cu2+ and Zn2+ resistance during denitrifying growth (24). Since some Zn2+ transport systems can also transport Co2+ and Cd2+ (14), RB-TnSeq analysis was also conducted in the presence of toxic concentrations of each of these two metal ions for comparison. This work both extends results from well-characterized metal-resistant bacteria like Cupriavidus metallidurans (14) to test their generality and discovers some new determinants of metal resistance (14).
The RB-TnSeq technique provides a way to quantitatively measure, by multiplexed short-read DNA sequencing, the fitness of hundreds of thousands of single-insertion transposon mutant strains simultaneously (30). Fitness is measured by the abundance of an individual mutant relative to the overall mutant library population. The fitness values for mutants with insertions in the same gene are averaged to yield gene fitness values for nearly every gene in the genome. While single gene insertions may not always result in low fitness under conditions that make use of that gene, due to genetic redundancy (such as the presence of a paralog), the genome-wide fitness survey that is obtained can still reveal many key players in a given process. We grew the RCH2 RB-TnSeq library anaerobically with nitrate as the terminal electron acceptor and with and without the addition of toxic levels of Zn2+, Cu2+, Co2+, or Cd2+. Fitness values were assigned to 3,315 coding genes (out of 4,265 in the 4.6-Mb genome) (30) via parallel sequencing of strain barcodes. Certain genes that resulted in Zn2+-sensitive phenotypes were also selected for the construction of individual deletion strains to confirm the phenotypes observed via the RB-TnSeq library and to enable further study.
P. stutzeri RCH2 was cultured anaerobically in 100-well plates as previously described (24). Lactate (20 mM) was used as a carbon source, with nitrate (20 mM) as the terminal electron acceptor. The medium also contained 4.7 mM ammonium chloride, 1.3 mM potassium chloride, 2 mM magnesium sulfate, 0.1 mM calcium chloride, 0.3 mM sodium chloride, 29.7 mM sodium bicarbonate, and 5 mM sodium dihydrogen phosphate. Vitamins and minerals were added as described by Widdel and Bak (31). The medium was supplemented with various concentrations (1 to 1,500 μM) of ZnCl2, CuCl2, CoCl2, or CdCl2 to determine the concentration which yielded about 50% maximum optical density (OD) in 8 to 12 h compared to the OD of the untreated control.
The P. stutzeri RCH2 RB-TnSeq mutant library, containing 166,448 single transposon insertions with known, sequence-identified genome locations (30), was grown as previously described with nitrate (20 mM) as the electron acceptor (24). Zn2+, Cu2+, Co2+, or Cd2+ toxicity was induced by supplementing the growth medium with 500 μM ZnCl2, 100 μM CuCl2, 100 μM CoCl2, or 6 μM CdCl2. Yeast extract (0.5 g/liter) was added in all cases to avoid eliminating insertion mutants from the population due to nutrient auxotrophy. The addition of yeast extract also enhances growth, which expands the possible dynamic range of fitness values.
Sample processing, DNA sequencing, and analysis of sequence data were conducted as described previously; however, DNA samples were processed only by the BarSeq98 method (30). The PCR products were sequenced using the Illumina HiSeq system. Strain fitness values were calculated for each individual transposon insertion strain. Fitness is defined as the binary logarithm of the ratio of postgrowth and pregrowth relative abundances for that strain. Gene fitness values (w) were calculated, as previously described (30), by averaging the fitness values for strains with insertions in a given gene. The full set of gene fitness values, including values for each replicate, is reported in Table S1 in the supplemental material. Quality control and normalization of data were performed as previously reported (30), with only data from the sequencing runs mentioned here. Quality metrics for the fitness data are reported per growth condition in Table S2.
The construction of the deletion mutation strains used in this study (see Table S3 in the supplemental material) was accomplished by conjugating an unstable marker exchange plasmid (see Table S3) into P. stutzeri RCH2. Each plasmid was constructed by assembling either two or four PCR products together in α-select cells (Bioline) by the sequence- and ligation-independent cloning (SLIC) technique (32). These PCR products were amplified with Herculase II DNA polymerase (Stratagene) with primers (see Table S4) obtained from Integrated DNA Technologies. Each SLIC assembly reaction mixture contained a PCR product that included the plasmid backbone (spectinomycin resistance gene, pUC origin of replication, and origin of transfer), a PCR product containing a kanamycin resistance gene, and two PCR products flanking the gene to be deleted. For three of the marker exchange plasmids (plasmids for the deletion of the Psest_3322, Psest_0529, and Psest_3136 genes), the kanamycin resistance gene and the two homologous regions flanking the gene of interest were joined by sequence overlap extension (33) prior to the SLIC reaction. The PCR products of chromosomal regions flanking the gene to be deleted were the locations for double homologous recombination to exchange the gene of interest with the kanamycin resistance gene, npt. These PCR products were sequenced at the DNA core facility at the University of Missouri, Columbia, and compared with the published sequence for RCH2. The constructed mutagenic plasmid was then isolated and transformed into an Escherichia coli strain (WM3064) expressing the transfer function of RP4 (34), containing a dapA deletion, and capable of conjugation with RCH2. Following conjugation (35), RCH2 exconjugants were selected with 50 μg kanamycin/ml. Putative exconjugants were initially screened to check their sensitivity to spectinomycin (100 μg/ml) so that no kanamycin-resistant isolates containing the targeted gene (resulting from a single recombination event and therefore containing the spectinomycin resistance gene) were carried forward. The spectinomycin-sensitive isolates were further confirmed by Southern blotting to determine the presence or absence of the gene. After growth to early stationary phase, all strains were frozen in a 10% (vol/vol) glycerol solution. All E. coli cultures were grown in SOC or LC medium (36). When appropriate, diamino-pimelic acid (0.1 mM) was added to permit the growth of E. coli WM3064-derived strains.
Individual mutant strains and wild-type RCH2 were grown under the same conditions as the mutant library and with and without 1 mM ZnCl2 to verify Zn2+-sensitive phenotypes. Growth was monitored with a Bioscreen-C automated growth curve analysis system enclosed in a Plas-Labs La Petite anaerobic glove box under an argon atmosphere with <5% hydrogen for catalytic removal of oxygen.
Known target genes czcC, cueA, and copA for the respective metal-responsive regulatory systems CzcRS (37), CueR (38), and CopRS (39) were used as starting points for the identification of transcription factor (TF) binding motifs. We used the RegPredict Web server (40) for motif reconstruction. Sets of sequences upstream from czcC, cueA, and copA2 orthologs (from −400 to +50 with respect to the translation start site) from Pseudomonas species genomes were selected, and a common motif with the highest information content was identified in each set with the “Discover profiles” tool of the RegPredict Web server. These motifs were used as position-weight matrices for the reconstruction of regulons in the GenomeExplorer software package (41). Briefly, a position-weight matrix was used for a whole-genome search in the upstream regions of coding genes (from −400 to +50 with respect to the translation start site), with a threshold equal to a minimal score among all sites in a training set of the matrix.
The RCH2 mutant library was grown anaerobically, with lactate (20 mM) as the carbon source and electron donor and with nitrate (20 mM) as the electron acceptor. Toxic concentrations of Cu2+ (100 μM), Zn2+ (500 μM), Co2+ (100 μM), or Cd2+ (6 μM), which give approximately 50% growth inhibition, were added to this control medium, and cells from all conditions were analyzed with the RB-TnSeq technique. The focus of this study was to identify genes crucial for Cu2+ and Zn2+ resistance under denitrifying conditions. Co2+ and Cd2+ data were included where relevant to Zn2+ transport.
As previously described (24), the data are presented as gene fitness values (w), which reflect the growth of mutants with disruptions of individual genes relative to the overall mutant library population such that an increase in the relative abundance of mutants in which a gene is mutated shows positive fitness for that gene and a decrease shows negative fitness (30). Gene fitness values are interpreted as the effect of the absence of the gene product; however, in some cases, transposon insertion can affect the expression of downstream genes as well (so-called “polar effects”) (42). The original report of the P. stutzeri strain RCH2 RB-TnSeq library included analyses of the influence of polar effects on the data and concluded that they did not have a major influence (30). A major contributing factor to this is that only data from insertion strains in which the insertion occurs within the central 80% of the gene are used. In addition, Table S5 in the supplemental material gives a per-gene analysis of whether polar effects may influence the data and conclusions discussed here. Gene fitness values for growth in the control medium are designated wctrl, and wctrl values are subtracted from gene fitness values for growth in the presence of a toxic metal to obtain corrected fitness values that only reflect changes in fitness due to the metal stress. Hence, “wZn” is used to describe gene fitness values for growth in medium with Zn2+ that have been corrected by the wctrl value. Likewise, “wCu,” “wCo,” and “wCd” refer to values for gene fitness in medium supplemented with Cu2+, Co2+, or Cd2+, respectively, that have had wctrl subtracted to isolate the effect due to the added metal ion. The Zn2+ and Cu2+ RB-TnSeq studies produced phenotypes over similar ranges (−3.8 < wZn < 1.9 and −3.2 < wCu < 2.5) and with similar standard deviations (0.36 and 0.33, respectively). There were 72 genes with |wZn| values of >1 (see Table S6) and 56 genes with |wCu| values of >1 (see Table S7).
The most severe wZn values for genes previously known to be involved in Zn2+ transport were observed for the genes encoding an RND-type transporter, czcCBA (Psest_0614 to Psest_0616), and a gene that has been observed in gene clusters with czcCBA (43), czcI (Psest_0613) (wZn values of −3.6 ± 0.1 [mean ± standard deviation]) (Fig. 1). There are three czcCBA homologs present in the RCH2 genome, bearing the same UniProt annotation (Psest_1614 to Psest_1616, Psest_2265 to Psest_2263, and Psest_0598 to Psest_0596) (44). In order to determine how well annotations are likely to correspond to the specific ion that is transported, we generated a phylogenetic tree of the 4,253 sequences in the InterPro protein family (IPR004763), which contains CzcA and other RND family metal ion transporters (26). The set of proteins that bear annotations containing “copper,” “Cu,” or “cusA” was compared with those annotated by “zinc,” “Zn,” or “czcA,” but not “family” (see Fig. S1 in the supplemental material). Annotations including “family” tended to be scattered across the phylogenetic tree. For additional categorization, the ScanProsite tool was used to search these sequences for motifs established by Nies for CzcA/CnrA versus CusA subfamilies (see Fig. S1) (9, 45). This analysis showed that the Nies motifs cluster proteins according to clades on the tree, whereas annotations correspond only weakly to phylogenetic clustering, lending confidence to Nies' assignments. Nies' motifs also correctly assigned CzcCBA from strain RCH2 as a divalent cation transporter. We are unaware of any similar test of Nies' predictions against phylogeny.
Although czcICBA are named for the ability to transport Co2+, Zn2+, and Cd2+, the wZn values for these genes in strain RCH2 were 2.8 lower than the wCo values (−0.8). These four genes are among the 8 genes with the most negative wZn values but are only among the 89 genes with the most negative wCo values. All czc genes had wCd values between 0 and 0.2, so the CzcCBA system is not required for Cd2+ detoxification in RCH2 under denitrifying conditions. This could be due in part to the Zn2+ specificity of CzcS (46). However, the wZn values for czcRS were higher than those of the transporter genes (see Table S6 in the supplemental material), so it more likely reflects an altered specificity of the transporter from RCH2 relative to the specificities of other characterized CzcCBA transporters. It should be noted that the Cd2+ concentration used (for 50% growth inhibition) was only 6 μM, compared with 500 μM for Zn2+. There was no analogous transport system encoded by genes with wCd values of less than −1. The effects of czc genes on Zn2+, Co2+, and Cd2+ resistance in C. metallidurans have been studied extensively (43).
The czcI gene is not widely distributed in nature, occurring in only 127 sequences, predominantly betaproteobacterial (IPR020487) (26). The true family is likely to be larger, as a BLAST search based on CzcI from strain RCH2 (E < 10−10) identifies similar-length homologs with no InterPro match, and a few of them were included in IPR021333 (DUF2946). The CzcI protein is uncharacterized, and CzcCBA transporters are known to function in its absence (47). However, the negative wZn value for czcI observed in this study (−3.7) indicates that CzcI is an important component of the CzcCBA transporter in strain RCH2 (Fig. 1). It is predicted to localize to the periplasm and, thus, may play a role analogous to that of CusF, a periplasmic component of some CusCBA transport systems that is proposed to bind Cu+ and promote its efflux through CusCBA (13). The somewhat sporadic taxonomic distribution of czcI among organisms often isolated from contaminated environments, combined with its relatively rare occurrence (26), suggests that it is a marker for high zinc resistance and is spread via horizontal gene transfer.
Homologs to the czcSR genes (Psest_0610 and Psest_0611), which are associated with CzcCBA regulation in Pseudomonas aeruginosa (37), also had negative wZn values (−2.2 ± 0.1). The CzcR DNA binding motif had not been described previously in Pseudomonas species, so we identified it in upstream regions of divergent czcRS and czcCBA operons from P. aeruginosa, P. mendocina, P. syringae, and P. fluorescens. A conserved 38-bp imperfect inverted repeat was found in all the sequences and is a candidate CzcR binding motif (see Fig. S2 in the supplemental material). We used this motif for a whole-genome search in P. stutzeri RCH2 and found five occurrences. Putative CzcR binding sites were predicted in an upstream region common to the czcICBA operon and the divergent Psest_0612 gene, encoding an outer membrane porin, indicating CzcRS-dependent expression of czcICBA in strain RCH2. Another binding site was identified upstream from the czcRS operon, suggesting autoregulation. The final two putative CzcR binding sites were found in an upstream region common to two divergent genes, Psest_0618 and Psest_0619. Psest_0618 encodes a hypothetical protein, whereas Psest_0619 encodes a CDF family transporter. Although the fitness data indicate that Psest_0619 is not required for Zn2+ resistance (wZn value of −0.1), regulon reconstruction indicates that it may be a redundant Zn2+ (or other metal ion) efflux pump. The number of sequencing reads for the two strains with Psest_0618 mutations was too low to assign a confident wZn value; however, the results do indicate strong Zn2+ sensitivity (wZn value of approximately −3), implicating this hypothetical protein in Zn2+ resistance (Fig. 1) and as a likely contributor to czcRS fitness, along with czcICBA. Close homologs to Psest_0618 are only found in Pseudomonas species, except for one close homolog (BLAST value, E < 10−27) in Streptococcus pneumoniae (UniProt accession number A0A0T8LNT2) (44).
Members of the CDF family of cation/proton antiporters export metal ions into the periplasm. The CDF InterPro family (IPR002524) contains nearly 35,000 sequences with wide sequence variety, such that it is particularly difficult to subcategorize them bioinformatically in terms of metal ion specificity (26). RCH2 contains a total of six such CDF proteins, but only one of these (Psest_2759), which we refer to as CzcD after the established convention (12), was encoded by a gene with a negative wZn value (−2.0). While a remote location of czcD relative to the position of czcICBA is not very surprising due to its functional independence, other homologs located quite close to the czcICBA operon, Psest_0607 (wZn of 0.0) and Psest_0619 (see above), would appear to be Zn2+ related a priori. Values of −0.2 for wCo and 0.3 for wCd were observed for Psest_2759, suggesting that the gene product is predominantly involved in Zn2+ tolerance. The positive wCd values for czcICBA and czcD may result from higher intracellular Zn2+ levels in the mutants, since Zn2+ can compete for binding sites that are targets for Cd2+ toxicity.
In addition to genes predicted and annotated as part of transport systems, there were genes predicted to encode membrane-bound hypothetical proteins that resulted in strongly negative metal-specific phenotypes. These included Psest_3226, which had the second lowest wZn value (−3.7) (Fig. 1). Homologs of this gene can be found in all three domains of life. Defects in the human homolog have recently been shown to cause a newly discovered congenital glycosylation disorder (48). Subsequent patch clamp studies in human cells and phenotypic studies in yeast (49, 50) indicate that the protein functions as a H+/Ca2+ antiporter, although no bacterial homologs have been characterized. According to the domain-based classification previously proposed, Psest_3226 from strain RCH2 is clearly a fusion of two UPF0016-type domains, as is found in eukaryotes, including two of the consensus EΨGDKT motifs (Ψ is most often I, L, or M) (50,–52). The results presented here indicate that some proteins in this family can transport Zn2+ and, likely, other types of metal ions as well. The fitness values indicate that Psest_3226 is the primary Zn2+ efflux pump in the plasma membrane in strain RCH2, which should encourage further study of this protein in the future.
Another hypothetical membrane protein encoded by a gene with a high negative wZn value (−3.2) is Psest_3322 (Fig. 1). This large (138-kDa) protein belongs to the IPR011836 family of CHP02099 conserved hypothetical proteins, with homologs in the gamma- and betaproteobacteria, and contains a DUF3971 domain (IPR025263). It generally occurs in a six-gene operon, as in RCH2, encoding a predicted zinc-dependent protease (Psest_3320), an aminohydrolase (Psest_3321), an Rne/Rng family RNase (Psest_3323), and a septum formation inhibitor (Psest_3324). The other operon members did not result in significant phenotypes under the conditions tested, except for Psest_3323 (wZn of −1.0). Although the specific function of the protein encoded by Psest_3322 is not quite clear, it is apparently required for Zn2+ resistance. This phenotype could serve as a starting point for elucidating the function of this uncharacterized protein family.
Psest_3341 also had a high negative wZn value (−1.3). The protein encoded by this gene is a member of the large family of outer membrane TonB-dependent receptors and is likely involved in high-affinity siderophore-bound Fe3+ uptake, suggesting that Zn2+ toxicity is due in part to interference with iron uptake and metabolism. Mutation of this transporter likely eliminates a means of highly selective Fe3+ uptake, thus forcing the cell to rely on other means (that may leak more Zn2+) or, possibly, to reduce the intracellular iron concentration such that Zn2+ can more effectively compete for Fe2+ binding sites.
Three Salmonella genes were previously designated corB, corC, and corD because mutation of these genes led to enhanced Co2+ resistance (53), as did disruption of corA, which encodes a well-studied Mg2+ channel (54). The corB and corC homologs in strain RCH2 are remote from each other in the genome (Psest_3136 and Psest_0529) but exhibit strong cofitness (0.78), a measure of fitness correlation (55, 56), over many growth conditions (30), and they had very negative wZn values (−3.5 and −3.6, respectively) (Fig. 1). In Salmonella, CorBCD were proposed to enhance Mg2+ efflux through CorA (53). Strain RCH2 does contain homologs of corA (Psest_2314) and corD (Psest_3669); however, neither resulted in a significant phenotype under Cu2+ or Zn2+ toxicity conditions in strain RCH2. Hence, we propose that in strain RCH2, CorB and CorC work together, based on their high cofitness, but independently of CorA or CorD. Furthermore, CorB and CorC appear to function in Zn2+ efflux in strain RCH2 (Fig. 1). No results relating to Zn2+ efflux have been reported previously for these proteins, as they were thought to have a role only in efflux of Mg2+ (53). Assigning a role for CorB and CorC in CorA-independent efflux, however, leaves open the question of why corB and corC mutants have increased cobalt resistance (53). We believe this to be connected to CorA. If CorBC function in Mg2+ efflux under the conditions of wild-type Mg2+ efflux, the corB or corC deletion mutants would likely have elevated intracellular Mg2+ due to entry through the CorA Mg2+ channel. Elevated intracellular Mg2+ could cause enhanced Co2+ resistance, because Mg2+ enzymes are known intracellular targets of Co2+ toxicity (53, 57). The higher Km of Co2+ import observed in the mutants (53) could likewise result from inhibition due to high intracellular Mg2+ favoring an inward-facing Mg2+-bound conformation, thus reducing the effective enzyme concentration like a competitive inhibitor. It has long been believed that CorA itself mediates Mg2+ efflux, as well as uptake (54, 58). According to our reasoning, CorA is unlikely to be involved in efflux. Lack of Mg2+ efflux in the Salmonella enterica corA mutant (58) could occur simply because without CorA, intracellular Mg2+ does not reach concentrations high enough to induce efflux through CorBC. In addition, the presence of the CorA protein has regulatory effects in S. enterica that are not well understood, which would cause unanticipated effects in the corA mutant (59).
Independent confirmation of the phenotypes observed in the RB-TnSeq experiments was obtained via the construction of individual deletion mutants for several poorly characterized or conserved hypothetical genes that were shown in the present work to be involved in Zn2+ resistance; namely, czcI, Psest_3226, Psest_3322, corB, and corC. Individual deletion mutants were constructed for each of these genes and, as shown by the results in Fig. 2, each strain was much more sensitive than the wild-type strain to growth inhibition by Zn2+. For czcI in particular, these confirmatory results reduce the likelihood that the negative wZn value that was observed was due to polar effects of transposon insertions (see Table S5 in the supplemental material), although it is still possible that the genetic changes in the czcI mutant could influence the expression of the czcCBA genes downstream. Further study of these mutants and of novel factors in Zn2+ resistance is promising for the overall study of metal resistance and homeostasis but outside the scope of this study.
The microbial response to Cu2+ toxicity has been well studied and was recently reviewed (1). However, our RB-TnSeq results reveal that few of the genes in the characterized copper export systems are essential to resistance in strain RCH2, while one uncharacterized gene, not previously associated with copper resistance, is essential. Some in the former category may still be used for copper resistance under other growth conditions or may not result in significant phenotypes due to genetic redundancy (such as the presence of a paralog); however, there were still surprisingly few genes predicted to be involved in copper resistance that had significantly negative fitness values.
Of the three RND-type transporter subunits encoded in the RCH2 genome, two contain CusA-type motifs (HME4), as defined by Nies (9), and are found in the associated region of the phylogenetic tree in Fig. S1 in the supplemental material. Thus, we will refer to the cus genes in the two associated operons as cusC1B1A1F (Psest_0598 to Psest_0595) and cusC2B2A2 (Psest_2265 to Psest_2263) (Fig. 3). Psest_2262 is the last gene in the cusC2 operon and is annotated in UniProt as “Co/Zn/Cd efflux system component.” It is a member of the IPR002524 family of (mostly Zn2+) efflux proteins that does not have a known association with the cus system. One might expect the cusF-containing cusC1 operon to have a more-negative fitness value, since cusF has been shown in E. coli to improve copper resistance (15); however, of the two cus operons, only cusC2B2A2 had wCu values of less than −1 (−1.9, −1.4, and −1.5, respectively), indicating that the cusC2 operon is primarily used under these conditions (Fig. 3). In E. coli, CusF is a periplasmic Cu+ binding protein that delivers Cu+ to CusB for export (11, 13). In contrast with E. coli, however, the cusB genes in P. stutzeri RCH2 contain sequence homology that also places them in the CusF protein family (IPR021647) (60), indicating that this gene fusion may functionally replace a separate cusF gene to some extent in the cusC2 operon. The existence of such gene fusions has been recognized previously (60). A total of 31% of sequenced CusF proteins (1,001 proteins) exist as fusions with RND-type efflux domains such as CusB (IPR032317) (see Fig. S3). Roughly a third of organisms that have such fusions lack separate cusF genes, further indicating that the fusion protein is able to replace the two acting independently, even though the fusion CusF domain will be less diffusible than a separate CusF. P. stutzeri RCH2 does not contain homologs of the regulatory genes cusR and cusS found in the cus locus in E. coli, and sequence analysis of upstream regions of both cus operons did not reveal putative regulatory sequences similar to the E. coli CusR box (61); thus, it is unclear how or whether the cus operons are regulated.
Strain RCH2 also contains homologs of the cue genes, cueZ and cueAR (Psest_0739 to Psest_0737), that were characterized in E. coli (62). Other cueZ and cueA homologs are also present, but not as adjacent genes (Fig. 3). No cueO homolog (63) is located adjacent to other cue genes. CueA is a Cu+ P-type ATPase efflux pump, generally regulated by CueR (64), and CueZ is a Cu+ binding protein (65). There was insufficient data to assign a confident fitness value for cueZ, and cueAR resulted in weak phenotypes (wCu values of −0.9 and −0.6) (Fig. 3) relative to the phenotype resulting from cusA2. This is in contrast to what is reported for E. coli, where the Cue system is dominant under both aerobic and anaerobic conditions (62). However, the CueA ATPase still appears to cooperate with the periplasmic Cus efflux system in strain RCH2, just as in anaerobically grown E. coli. The reduced severity of the phenotype resulting from cueA (called copA in reference 62) may indicate the presence of some other system that concomitantly exports copper across the plasma membrane in strain RCH2.
For CueR regulon reconstruction, the binding motif reported in P. aeruginosa (38) was refined by comparative analysis of regions upstream from cueA in Pseudomonas species P. aeruginosa, P. fluorescens, P. entomophila, P. stutzeri, P. syringae, and P. putida. In all cases except P. aeruginosa, cueA and cueR genes are cotranscribed, and thus, CueR-dependent regulation of the cueAR operon was expected. Indeed, a conserved 23-bp inverted repeat was identified (see Table S8 in the supplemental material), corresponding to the motif from P. aeruginosa. A search for the refined binding motif in P. stutzeri RCH2 (see Fig. S2) identified two putative CueR target genes in addition to the cueAR operon binding sites (see Table S8). The first of these genes is cueZ. The second is Psest_1595, which is likely cotranscribed with downstream genes Psest_1594 and Psest_1593. The Psest_1595 gene product belongs to COG2608, which also includes CueZ and other copper chaperones, but the gene did not exhibit a wCu value of less than −1. Thus, the observed phenotype resulting from cueR was likely caused by the lack of expression of cueA.
RCH2 contains two sets of homologs to copper resistance cop genes that have been characterized primarily in Pseudomonas syringae (10). These are located at Psest_0581 (copR1), Psest_0583, Psest_0585, and Psest_0586 (copA1B1M1), Psest_1598 and Psest_1599 (copR25), Psest_1590, Psest_1588, and Psest_1587 (copA2B2M2), and Psest_3400 (copD). Both copA operons contain noncanonical genes (Psest_0582, Psest_0584, and Psest_1589). The only cop genes with wCu values of less than −0.6 were copR1 (−1.2) and Psest_0584 (−0.9). copM1 actually had positive fitness (wCu of 0.4) (Fig. 3). While this value is not very large, positive fitness values are rather unusual in these types of analysis, and copM has been previously reported to contribute to copper resistance in Xanthomonas species (7). The role of these cytochrome c proteins has yet to be elucidated, but it appears that they can either enhance or decrease copper resistance. In addition, the outer membrane protein CopB has been shown to enhance copper resistance in P. syringae and Xanthomonas species (7, 66) but has a minimal effect in strain RCH2. Psest_0584 is a hypothetical protein of 100 amino acids with homologs (E < 10−5) found only in members of the Pseudomonas genus, and thus, it may represent a genus-specific copper resistance factor. Given the homology to copper resistance operons in P. syringae and Xanthomonas, it is surprising that the more widespread copABM genes had fitness values rather close to zero and that the most negative fitness value in these operons is from a gene encoding a hypothetical protein. Clearly, this system is not crucial to copper resistance in strain RCH2 under denitrifying conditions. This may be related to the fact that CopA, as a multicopper oxidase, is likely only active under aerobic conditions.
Interestingly, of the two CopR response regulators in strain RCH2, only copR1, which lacks the accompanying copS gene, had a wCu value of less than −1. Reconstruction of the CopRS regulon in P. stutzeri RCH2 demonstrated that both copA operons have predicted CopR binding sites in the upstream regions (see Table S8 and Fig. S2 in the supplemental material). CopR binding sites were also identified upstream from copR1 and the copR2S operon, suggesting autoregulation, and upstream from Psest_0600, encoding a P-type ATPase transporter. A final CopR binding site was found upstream from the Psest_1595-to-Psest_1593 operon; thus, this operon is coregulated by two copper-responsive regulatory systems, CueR and CopRS. There is no striking difference between the CopR binding sites found upstream of copR1 and copR2 that could explain the different fitness values of the regulatory mutants. However, copR1 is colocalized with the Psest_0582-to-Psest_0586 operon, which contains the only CopR target gene, Psest_0584, with a notably negative wCu value (−0.9) (Fig. 3). We hypothesize that the locally synthesized CopR1 is more important for activation of Psest_0582 to Psest_0586 than the more distantly synthesized CopR2. Finally, some predicted members of the CopR (or CueR) regulon may indeed be copper regulated but perform functions unrelated to copper, such as response to other toxic compounds that often co-occur with elevated copper concentrations, as has been reported previously in P. aeruginosa (67).
While additional studies of the regulation and the expression levels of the genes related to copper resistance would be needed to complete the picture of how strain RCH2 responds to and resists copper toxicity, the fitness values and regulatory analysis presented here provide several new insights. For example, while a high degree of functional redundancy might be expected from the high degree of genetic redundancy among copper resistance systems in strain RCH2, certain proteins, such as CusA2B2C2 and, to a lesser extent, CopR1, Psest_0584, and CueA, may be regarded as the “front line” of defense in that they are individually essential to effective copper resistance (Fig. 3). Since CusA1B1C1F is unable to replace CusA2B2C2, the former complex seems to have some function other than copper export or is downregulated by a regulatory mechanism that could not be recognized by the methods used here.
In addition to many genes that would be expected to be among the top transport-related genes required for growth in the presence of copper, there were several that bear no clear connection to copper that had very negative fitness values. In particular, Psest_2850, whose product is annotated as an Mrp Na+/H+ antiporter subunit, has among the most negative transport-related wCu values (−1.8) (Fig. 3b). This gene is in a predicted six-gene operon (Psest_2846 to Psest_2851) encoding six putative subunits of such an antiporter; however, only Psest_2850 results in a significant phenotype (for all of the other genes, the wCu value was more than −0.2). Hence, it seems unlikely that the complex as a whole performs some vital function under these conditions. Such complexes are known to facilitate the export of alkali metal monovalent cations, such as Li+, Na+, and K+, but have also been proposed to serve other functions due to their large size and multisubunit composition (68), given that there are much simpler antiporters that perform the known functions of Mrp antiporters. Our data suggest that the c subunit of this particular antiporter may facilitate the export of Cu+ either alone or in combination with the products of other genes (redundancy could eliminate single mutant phenotypes). Indeed, based on the stronger phenotype, the antiporter would be the primary means of Cu+ efflux from the cytoplasm, and CueA-mediated efflux would be less critical (though the cueA paralog Psest_0600 could also reduce the effects of the cueA mutation). Such a proposal is in agreement with prior suggestions both that Mrp antiporters may have other unidentified substrates (68) and also that some alkali metal ion transporters may allow some passage of Cu+ (62, 69). The latter has been discussed in terms of entry of Cu+ into the cell under conditions of copper toxicity, and both proposals are without direct evidence for any specific examples. Indeed, Cu+ export by an Mrp complex may take only subtle adjustment, as Li+ and Cu+ differ by only 1 pm in ionic radius (76 and 77 pm, respectively, for an effective ionic radius of a 6-coordinate ion). However, their ligand preferences and chemistry are very different, as Cu+ is a redox-active soft acid and extremely thiophilic, whereas Li+ is redox inactive, a hard acid, and oxophilic. Psest_2850 does contain five methionine residues, which could be used to select for Cu+ transport, as in the Cus transporter (13).
As in all full denitrifiers, denitrifying growth of strain RCH2 begins with nitrate reduction, catalyzed by the molybdenum cofactor (Moco) in nitrate reductase (NarGHI). Nitrite is produced from this reaction and is subsequently reduced by nitrite reductase (NirS), nitric oxide reductase (NorBC), and nitrous oxide reductase (NosZ) to produce nitric oxide, nitrous oxide, and dinitrogen gas, respectively, although nitrate reduction can accept enough electrons to sustain growth in the absence of the other steps (24). Nitric oxide reductase, however, is required for the detoxification of nitric oxide even if nitric oxide is not needed for its electron-accepting potential (24). In addition to NirS, which is a heme-using cytochrome, there is a phylogenetically unrelated nitrite reductase, NirK, which uses copper for catalysis. RCH2 contains genes for both, but we have only observed negative fitness for NirS (24).
Surprisingly, the transporter genes with the most negative wCu values have nothing at all to do with copper transport but, rather, encode the well-known transporter for molybdate (MoO42−), ModABC (wCu of −2.8 ± 0.3 for modAB) (Fig. 4). These data point to a major target for copper toxicity related to molybdenum. In agreement with this, there were negative wCu values for several genes involved in Moco biosynthesis (see Table S7 in the supplemental material) and nitrate reduction (wCu of −2.0 ± 0.3 for NarGHI) (Fig. 4). While enhanced demand for molybdenum may, to some extent, simply reflect greater energy demands on the cell due to the need to continuously remove Cu+, we also measured wCu values of 0.4 ± 0.1 for nirBCDEFJS and 0.8 ± 0.3 for norBCDQ (Fig. 4). The decreased nar fitness combined with improved nir and nor fitness suggest that nitrite reductase may be inhibited by copper. In normal medium, nar mutants grown in the mutant pool can survive and grow to some extent by using nitrite released by other mutants in the pool. However, if copper inhibits Nir, this would no longer be an option, which would decrease the fitness values for genes involved in nitrate reduction, Moco biosynthesis, and MoO42− uptake, as was observed. Mutations in nir and nor would no longer be detrimental because Nir is ineffective even when present, due to copper inhibition, and in the absence of properly functioning Nir, nitric oxide toxicity is reduced, so the disadvantage of nor mutations is also reduced. If this is true, it would mean that the use of the copper-dependent nitrite reductase would be favored over iron-dependent nitrite reductase under high copper conditions, as well as under iron-limited conditions. Finally, copper is highly thiophilic and has been reported to bind to the dithiolene moiety of pyranopterin (70), which could compete for molybdenum insertion, particularly under conditions of low intracellular MoO42−, as in modABC mutants. This factor likely contributed to the negative wCu values observed for modABC but does not appear to have been a dominant effect, because the pattern of fitness values for nir and nor genes does not follow that of molybdenum limitation (24), a metabolic state similar to that of weak Moco biosynthesis.
Zn2+ also caused negative fitness values for the nitrate reductase narGHI genes (wZn of −2.5 ± 0.2) and those involved in Moco biosynthesis (−3.2 ± 0.5, excluding moaA1 and -2), and yet, the wZn values were less negative than the wCu values for the Mo transport modABC genes (−1.3 ± 0.0 for modAB) (Fig. 5). Zinc is less thiophilic than copper and, consequently, would be expected to cause less interference with molybdenum insertion into pyranopterin than was reported for copper (70), perhaps lowering the required intracellular MoO42− concentration to that which can be satisfied by sulfate and, possibly, other transporters (24). In addition, although the metal ion concentrations were chosen in an effort to achieve uniform growth defects for both toxic metals, the Zn2+-supplemented growth was slower. Slower growth kinetics may enable the rate of MoO42- uptake through the sulfate transporter to more closely match the Mo demands of the cell; however, increased energy demands and likely some inhibition of Nir combine to reduce Moco biosynthesis and nar mutant fitness and leave nir mutant fitness near zero in this case.
While Cu2+ and Zn2+ toxicity have been well studied in certain organisms, RB-TnSeq analysis in Pseudomonas stutzeri RCH2, a species with little previous characterization of metal resistance mechanisms, has shown that there are systems involved and impacts of elevated metal ion concentrations that are still unexplored. The well-known Zn2+ exporters CzcD and CzcCBA were identified from among other similar CDF- and RND-type transporter homologs, and phylogenetic analysis helped to more clearly delineate between czcA and cusA RND subfamilies. The CzcI component of this transporter has not been characterized like the rest of the CzcCBA transporter, but the wZn value of czcI was the most negative in the czcICBA operon, indicating that it is required for the proper function of this transporter in strain RCH2. A putative CzcR binding motif was identified and the corresponding regulon described, including Psest_0618. Psest_0618 mutant strains showed zinc sensitivity, implicating this hypothetical protein in zinc resistance. In addition, several other genes that had no previous connection with zinc toxicity exhibited wZn values of less than −1. Based on fitness values, cofitness, and previous characterization of mutants (53), we propose that CorB and CorC cooperate in metal ion efflux, including efflux of Mg2+ and Zn2+, independently of the CorA Mg2+ transporter. Two annotated hypothetical proteins, Psest_3226 and Psest_3322, also had very high negative wZn fitness values. Psest_3226 is found across all three domains of life, including in humans, where a homolog was recently connected with a congenital glycosylation disorder (48). Our data extend the function of transporters in this family to Zn2+ efflux in the first report on a bacterial homolog. Psest_3322 and Psest_0618, on the other hand, are more restricted taxonomically, with no characterized homologs. The Zn2+ sensitivity of mutants lacking these genes gives a starting point for characterization of members of these uncharacterized protein families. Finally we constructed individual mutant strains with deletions of czcI, corB, corC, Psest_3226, and Psest_3322 and confirmed the resulting phenotypes shown by the RB-TnSeq data.
There are five predicted copper efflux systems in strain RCH2, but only two of them—one of the two cusCBA operons and the cueA gene—had wCu values of less than −0.9 for known transporter-encoding genes under anaerobic growth conditions. Curiously, the cus operon that lacks cusF, which has been implicated in Cu2+ efflux through CusCBA in E. coli, was the one with the more negative wCu value. The cusB genes, however, both contain cusF signatures. Thus, these CusB-CusF fusion proteins may be able to carry out both functions to some extent. No evidence of cus regulation could be identified using the methods reported here. The two cop operons, which are homologous to copper resistance operons in both P. syringae and Xanthomonas species, did not contain any known transporter-encoding genes with a wCu value more negative than −0.6; however, copR1 and Psest_0584, a gene with unknown function, had wCu values of −1.2 and −0.9, respectively. Psest_0584 is not found outside the Pseudomonas genus but is likely to increase Cu2+ resistance among pseudomonads. Regulon reconstruction and analysis for CueR and CopR yielded a refined CueR binding motif and identified several regulon members outside the recognized copper transport operons, including the coregulated Psest_1595-to-Psest_1593 operon, but none with negative wCu values. Lastly, a subunit of an Mrp-type Na+/H+ antiporter had an unexpectedly negative wCu value of −1.8. We suggest that this subunit may allow this antiporter to transport Cu+. This is in accord with the suggestion that Mrp antiporters perform more functions than have been characterized so far, which would justify their size and complexity relative to those of other classes of Na+/H+ antiporters (68).
Cu2+ and Zn2+ induced decreases in fitness values for several genes involved in denitrification, Moco biosynthesis, and molybdenum uptake (Fig. 4 and and5).5). Some of these effects may be due to the increased energetic demands on the cell to continuously pump out metal ions; however, the pattern of fitness values was not simply an amplification of what is seen under typical denitrifying conditions. Instead, particularly for Cu2+, the fitness values for nar genes, as well as those involved in Moco biosynthesis and molybdenum uptake, decreased, while the values for nir and nor genes increased. Nitric oxide reduction, which is facilitated by the Nor system, is needed for detoxification of nitric oxide during denitrifying growth, so these changes indicate a reduced rate of nitric oxide production, which is consistent with copper inhibition of NirS, the active nitrite reductase. The fitness values for modABC, encoding the MoO42− ABC transporter, were very negative, which could be a sign of copper interference with molybdenum insertion into pyranopterin. The pattern of fitness values was similar under conditions of Zn2+ toxicity, except that the fitness values for modABC were closer to those for the control and the fitness values for Moco biosynthesis genes were much more negative. We hypothesize that zinc toxicity caused a similar inhibition of NirS, but the combination of a lower growth rate and the absence of significant competition for the pyranopterin dithiolene relaxed the requirement for functioning ModABC and emphasized the effect of a lack in any of the Moco biosynthesis genes. In summary, we both extend the number of genes known to be involved in zinc and copper efflux and resistance and expand the understanding of important intracellular targets for metal binding and toxicity.
We thank Morgan N. Price for assistance in data processing and Farris L. Poole, Jr., for helpful discussion concerning bioinformatic analysis.
This material by ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies; http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research, under contract number DE-AC02-05CH11231.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01845-16.