MICs of zinc sulphate
Given that metal ions exert does-dependent effects on bacterial cells, comparing the cellular responses of different strains at the same zinc stress level is important. In this study, the MIC of ZnSO4 was used in the treatment. P. putida KT2440 was highly tolerant to zinc and exhibited a zinc MIC value of up to 1.1 mmol L− 1. P. fluorescence was the least zinc-tolerant strain with an MIC value of approximately 0.4 mmol L− 1. P. aeruginosa PAO1 had intermediate levels of zinc tolerance. A cation-defined medium (CDM) plate that contained more than 0.7 mmol L− 1 zinc completely inhibited its colony formation. Therefore, in the following RNA-seq and UPLC–MS/MS analysis, 0.7, 1.1, and 0.4 mmol L− 1 zinc sulphate were used to compare the zinc-induced cellular responses in P. aeruginosa PAO1, P. putida KT2440, and P. fluorescence ATCC13525.
Global transcription features of the three Pseudomonas strains
The numbers of differentially expressed genes (DEGs) during the zinc treatment of P. aeruginosa PAO1, P. putida KT2440, and P. fluorescence ATCC13525T are provided in Table 1. The comparison of the zinc-treated P. aeruginosa PAO1 with their control samples identified 529 genes that were differently transcribed. Amongst which, 406 genes, 7.1% of the total coding DNA sequence (CDS) in the genome, were upregulated, whereas 123 (2.2%) genes were downregulated. The comparison of the zinc-treated P. putida KT2440 with their control bioreplicates showed that, the expression of 607 genes was significantly altered, with the majority (344 genes, 6.0%) displaying significant induction in transcription compared with those (263 genes, 4.6%) demonstrating decreased transcription. The lowest number of DEGs was observed in P. fluorescence ATCC13525, with only 48 induced genes and 19 repressed genes. In general, KEGG analysis showed that large quantities of DEGs identified in P. aeruginosa PAO1 and P. putida KT2440 can be clustered into the metabolism class (Fig. 1a), particularly the carbohydrate and energy metabolism subclasses. As an opportunistic animal pathogen, P. aeruginosa PAO1 also regulated many genes belonging to the human disease class. The cellular response of P. fluorescence ATCC13525 differed from those of P. aeruginosa and P. putida, in which approximately 50% of DEGs with clear KEGG classifications were categorised into the environmental information processing group. To better compared the cellular responses occurred in the three Pseudomonas strains, the DNA sequence of the DEGs was extracted from each genome, and then blast against the DEGs identified in the other two genomes with a match cutoff of 70% and an E-value exponent cutoff of 1-e5. As shown in Fig. 1b, only four orthologues were commonly regulated by the three Pseudomonas strains, and 1044 genes were strain-specifically regulated. The transcriptional patterns of P. aeruginosa and P. putida presented high similarities with 58 commonly regulated DEGs. In accordance with the KEGG analysis, P. fluorescence stands out in in all pair-wise blast analysis. Only 19 orthologues were commonly regulated by P. fluorescence and P. aeruginosa, and this value was reduced to 7 in P. fluorescence versus P. putida analysis.
The quantity of DEGs in P. aeruginosa PAO1, P. putida KT2440, and P. fluorescence ATCC13525T.
P. aeruginosa PAO1
P. putida KT2440
P. fluorescens ATCC13525
|a Percentage of the protein encoding genes with changes more than 2-fold.|
Functional analysis of DEGs identified in more than one strain
All the four DEGs (cadA, cadR, czcR, and czcS) commonly upregulated by the three strains were involved in metal efflux. cadA has been recognised as an efficient P-type ATPase that moves several divalent heavy metal ions out of the cellular membrane , whilst czcRS has been proven as an important two-component system that connects metal efflux and entrance tunnels. In P. aeruginosa, unphosphorylated CzcR decreased the expression of outer membrane porins, OprD. By contrast, the presence of phosphorylated CzcR induced the most effective metal efflux system, czcCBA . DEGs mutually regulated by P. aeruginosa PAO1 and P. putida KT2440 are listed in Additional file 1 (Table S1). As expected, czcCBA operons, which are known to be induced by several divalent metal ions, were found to be highly responsive. The analysis of other DEGs with known functions showed that most of these DEGs can be manually clustered into three categories. The genes involved in membrane structure and channels constituted the largest functional category. In this group, seven genes, including desA, ompB, dgkA, warB, warA, amgR and PA4819 (PP_0034), were strongly upregulated (> 5 fold), whereas oprD and kguT were mutually downregulated. In the other two groups, genes associated with central metabolism and those responsible for protein folding and degradation were generally upregulated. For P. fluorescens, the most prominent response mutually identified in P. fluorescens and P. aeruginosa was the upregulation of acyl-CoA dehydrogenase (RS07420, RS06160) and protein–tyrosine–phosphatase (RS10450). A ton-dependent receptor (RS23050) and a non-ribosomal peptide synthatase were markedly down-regulated (RS27315) (Additional file 2: Table S2). In addition to the four DEGs commonly regulated by the three strains, P. fluorescens and P. putida shared three more transcripts (Additional file 3: Table S3). Two of them were responsible for active transmembrane transport (RS21740, RS19005). The induction of asparaginase (RS29965) was also identified. The genome locations of these genes that are regulated by more than one strain are spread throughout the genomes, no obvious genome island was observed (Fig. 1c).
DEGs specifically identified in P. aeruginosa PAO1
The DEGs specifically identified in P. aeruginosa PAO1 are shown in Additional file 4 (Table S4). The fold-change data indicated that more energy were required for P. aeruginosa to alleviate zinc-induced toxicity. The proton-translocating NADH:ubiquinone oxidoreductase complex (nuoGHIJLMN) and the pyruvate dehydrogenase complex (PA3416-3417), which transform pyruvate into acetyl-CoA were upregulated by approximately 2.3-fold, whilst the synthesis of ribosomal protein was significantly downregulated (rpsB, rpsS, rpsT, rpsR, rpsF, rplS, rplW, rplJ, and rplU). A distinct group of operons involved in lipid A synthesis/modification was highly induced (arnBCADEF), suggesting that the P. aeruginosa cells were in a cell-envelope-stressed state during growth. In support of this, there was also significant upregulation of other genes previously linked to bacterial cell envelop homeostasis, such as glmM (phosphoglucosamine mutase) and pagL (lipid A 3-O-deacylase). The direct raplacement of iron from their binding sites has been recognised as an important mechanism for zinc to exhibit cytotoxicity. Therefore, it is reasonable that the biosynthesis of pyoverdine was downregulated (pvdQAPNOFEDJ) to reduce the unexpected intrusion of zinc ions.
Given that P. aeruginosa is an opportunistic pathogen, monitoring the transcriptional variation of pathogenicity related genes is important. The MIC of zinc strongly enhanced the antibiotic resistance of P. aeruginosa. Two important multidrug efflux systems, mexRAB-oprM  and mexXY , were strongly upregulated. Moreover, one operon involved in alginate synthesis (algU-mucABC) was significantly activated. The overproduction of exopolysaccharide alginate caused mucoid conversion in P. aeruginosa [23, 24], which increased bacterial metal tolerance via metal chelation . Amongst all the DEGs specifically identified in P. aeruginosa, the most upregulated gene was ptrA. The function of PtrA was studied by two groups, but contrasting conclusions were reported. One group showed that PtrA directly binds to ExsA, which in turn, suppresses the expression of the type III secretion system (T3SS) . By contrast, the other group demonstrated that PtrA is a periplasmic protein, the expression of which increases the Cu tolerance of P. aeruginosa without affecting basal ExsA . Our data supported that PtrA is not a T3SS repressor because the transcription of the T3SS gene clusters remained unchanged when ptrA was considerably induced by zinc.
DEGs specifically identified in P. putida KT2440
In our previous study, the transcriptional response of P. putida KT2440 to stress-inducing concentrations of zinc was analysed. The results showed that different zinc stress levels strongly influenced (> 4 fold change) the transcription of genes from four functional groups, including metal transporting genes, genes associated with membrane homeostasis, antioxidant-encoding genes and genes involved in basic cellular metabolism . In this study, a twofold change was used as a criterion, and a total of 545 genes were identified as DEGs specifically regulated in P. putida KT2440. After analysing the function of differentially expressed operons, the results further showed that most of the nutrient uptake transporters (about 64% of the total downregulated operons) were downregulated (Additional 5: Table S5), indicating that P. putida KT2440 tended to decrease the permeability of the cell envelope. Only the transporters responsible for methoine, sulphate and sulfonates uptake were upregulated, suggesting that, unlike P. aeruginosa PAO1, P. putida KT2440 did not suffer from a severe iron metabolism perturbation, but the sulphur-containing molecules were considerably disrupted. The synthesis of ribosomal protein was another remarkable difference between P. aeruginosa PAO1 and P. putida KT2440. In P. aeruginosa PAO1, the transcription of ribosomal protein encoding genes was generally downregulated. Meanwhile, their orthologues in P. putida KT2440 were highly stable, and even the transcription of rpmH was increased. In addition, the MIC of zinc significantly increased the transcription of the nickel (nikABCDE) and arsenic (arsBR) efflux systems in P. putida KT2440. Such phenomenon was not observed in the other two strains.
DEGs specifically identified in P. fluorescens ATCC13525
Compared with the transcriptional responses of P. aeruginosa PAO1 and P. putida KT2440, the downregulation of type VI secretion system (T6SS) was the most remarkable feature of P. fluorescens ATCC13525 under zinc stresses (Additional file 6: Table S6). T6SS is widely distributed across diverse bacterial species; around one-third of the sequenced Gram-negative bacteria possess T6SS-associated genes. Bacterial T6SS functions as a contractile nanomachine that delivers effectors upon direct contact with a target cell . Given that these effectors have different functions but frequently disturb the cellular structure, such as the cell wall, nucleic acid, or membrane compartment, T6SS is currently perceived as an antibacterial weapon . In recent study, the T6SS4 in Burkholderia thailandenss was found to secrete a proteinaceous zincophore, which interacts with the outer membrane heme transporter to import zinc under oxidative stress . Given that the transcriptional changes of other zinc importers, such as znu and zip, were not observed in the current study, we postulated that T6SS also plays an important role in zinc transport in P. fluorescens, the downregulation of which reduced the transport of zinc across the outer membrane.
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) validation
To confirm the RNA-seq data, six genes were selected from each strain, and their transcription was analyzed via RT-qPCR with three biological replicates. As shown in Fig. 2, the dynamic transcription patterns of all the genes were consistent with the RNA-seq data (Fig. 2a), and the observed fold changes for each gene were moderately correlated (R2 ranged from 80.1–89.6%) (Fig. 2b). Therefore, the RT-qPCR results validated the accuracy of the RNA-seq data. Moreover, previous studies on the transcriptomic profiles of P. putida KT2440 were useful in further confirming the results obtained in this study.
Metabolite changes in the three Pseudomonas strains
Considering that large quantities of DEGs identified in the transcriptome analysis were genes associated with metabolism, we detected the concentrations of metabolites in these bacterial cells. Metabolic profiles of whole-cell extracts were obtained from zinc-treated and control samples exponentially cultured in CDM. Six replicates were performed for each sample. A total of 83 annotated metabolites were significantly changed, and approximately 76% of which were down-regulated. Only two metabolites were commonly regulated by the three Pseudomonas strains (pyroglutamic, 2-phosphoglycerate). The metabolite profiles of P. aeruginosa and P. putida shared more similarity, 18 metabolites were commonly regulated (Fig. 3).However, after referring to the KEGG database, the metabolic pathways affected by the MICs of zinc imposed to the three Pseudomonas strains exhibited higher similarity (Additonal files 7–9: Tables S7–S9). Several metabolic intermediates associated with central carbon metabolisms (the Entner–Doudoroff pathway, tricarboxylic cycle, and pentose phosphate pathway) were significantly downregulated. The concentrations of citrate, phosphoenolpyruvate, and 2-phosphoglycerate in the zinc-treated P. aeruginosa PAO1 was only approximately 20% of that detected in the control samples. Similar phenomena were also observed in P. putida KT2440 and P. fluorescens ATCC13525. Compared with the P. putida cells without zinc treatment, the concentrations of citrate, succinate, and 2-phosphoglycerate were reduced by approximately 60.4%, 72.6%, and 78.2%, respectively. These observations are consistent with the RNA-seq data. The RPKM values of mdh (malate dehydrogenase) and idh (isocitrate dehydrogenase) in P. putida were only about 70% of that calculated in the cells without zinc treatment, and the transcription of eda (2-keto-3-deoxy-6-phosphogluconate aldolase) were downregulated about 1.7 and 1.5-fold in P. putida and P. fluorescens, respectively. Central carbon metabolism is the main pathway that satisfies the energy requirement of cells. Moreover, these metabolic processes provide important precursors for primary and secondary metabolites. Therefore, the metabolites of many other pathways were also reduced. For example, a wide range of amino acids (ornithine, Lys, Pro, Asp, Trp, and His) or intermediates involved in amino acid biosynthesis were decreased in P. putida. And lower concentrations of metabolites involved in Arg, Glu, Lys, Cys, and Met metabolisms were observed in P. aeruginosa.