Genomic traits of Klebsiella oxytoca DSM 29614, an uncommon metal-nanoparticle producer strain isolated from acid mine drainages

Culture conditions

Cells of K. oxytoca DSM 29614 (ex BAS-10) strain were stored in cryovials at − 80 °C in 25% glycerol solution until they were retrieved using Difco Nutrient Broth (BD Bioscience, Italy) growth medium at 30 °C. Aerobic and anaerobic cultivations were performed as described in Buttacavoli et al. (2018) [24].

When the strain was grown in the presence of heavy metals either the NaC or FeC media were used. The NaC medium contains per liter of distilled water: 2.5 g NaHCO₃, 1.5 g NH₄Cl, 1.5 g MgSO₄.7H₂O, 0.6 g NaH₂PO₄, 0.1 g KCl, and 14.7 g Na(I)-citrate (Carlo Erba, Italy). The NaC medium was buffered at pH 7.8 with a solution of NaOH. In the FeC medium 50 mM Fe(III)-citrate replaces 50 mM Na-citrate [27]. The growth of the strain was monitored determining total protein content by using Protein Assay kit (Bio-Rad, Italy), based on Bradford dye-binding method (1976) [28], according to manufacturing instruction for micro-assay.

DNA isolation and whole genome sequencing

K. oxytoca DSM 29614 strain was grown at 30 °C using Difco Nutrient Broth (BD Bioscience, Italy) growth medium. The genomic DNA was extracted using the CTAB method [29] and the authenticity of the genomic DNA was confirmed by 16S rRNA gene sequencing (Ylichron Srl, Italy). Whole genome shot-gun sequencing was performed by using the Illumina HiSeq system (Illumina Inc., San Diego, CA) with a paired-end approach loading genomic DNA onto one single flowcell (BGI-HongKong Co. limited, Hong Kong).

Phylogenetic analysis

16S rRNA gene sequences from strains belonging to K. oxytoca were selected from NCBI ftp site to run a phylogenetic analysis. Sequence alignment and phylogenetic tree were obtained as previously described in Presta et al. (2017) [30].

Genome assembly and annotation

Poor quality bases were removed using the dynamic trimming algorithm embedded in the SolexaQA suite [31] selecting a Phred score threshold value of 13. Assembly was performed by using ABySS 1.3.7 software [32]. The resulting contigs were launched in a multidraft-based scaffolder MeDuSa [33] which starting from 307 initial contigs generated 62 scaffolds. The last ones were then annotated by using NCBI Automated Genome Annotation Pipeline.

Comparative genomics

All the 85 K. oxytoca representative (Additional file 1: Table S1) genomes sequenced up to date (i.e. September 2016) were collected from the NCBI ftp site (ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Klebsiella_oxytoca/all_assembly_versions/) and, alongside K. oxytoca DSM 29614 genome, they were analyzed using Roary software [34] to identify shared orthologous and strain-specific genes.

Identification of genes putatively involved in metal tolerance, secondary metabolite biosynthesis and antibiotic resistance

AntiSMASH software [35] for genome-wide identification, annotation and analysis of secondary metabolite biosynthetic gene clusters was used to scan the genome sequence of K. oxytoca DSM 29614 in order to identify gene clusters involved in secondary metabolite production.

BacMet database [36] was used to identify the genetic elements responsible for heavy metal resistance, by using BLASTp algorithm with a threshold e-value ≤0.0001 and percentage identity ≥30.

Protein functional enrichment analysis

Protein functional enrichment analysis was performed using the SmartTables tool at BioCyc database collection [37] with the option “Fisher Exact Parent-Child Union” (FEPCU) on. Two analyses were performed on the sets of differentially abundant proteins (Additional file 2: Table S2) obtained from the proteomic analyses described in Gallo et al. (2012) [26]. In particular, up-regulated proteins i) in FeC versus NaC in anaerobic condition and ii) in anaerobic versus aerobic FeC cultivations were used. For these analyses, the K. oxytoca 10–5243 (NCBI accession JH603143.1) was chosen as reference strain since it was the most related strain among the selectable ones at BioCyc database collection according to the phylogenetic tree based on K. oxytoca 16S rRNA gene sequences (Fig. 1).

EPS extraction and determination

Aliquots of 10 ml of of DSM 29614 anaerobic culture in FeC medium were sampled at different times. The samples were firstly centrifuged to eliminate bacterial cells, then precipitation of polysaccharides was induced by treating the supernatant with 8 ml of cooled ethyl alcohol (95%). The purification phase was repeated twice. The concentration of total carbohydrate in EPS was quantified spectrophotometrically (485 nm) as glucose (Fluka, Germany) equivalents after reaction with 96% sulfuric acid (Fluka, Germany) and 5% phenol (Fluka, Germany) [38]. Iron removal from polysaccharide was performed by treating the rusty gel with 100 mmol l⁻¹EDTA (Fluka, Germany) until it was colorless [10].

Determination of total iron and Fe(II)

In order to quantify iron species in K. oxytoca DSM 29614 cultivations, 3-ml aliquots were collected at different times and processed for colorimetric assay as previously described in Baldi et al. (2009) [10].

Struvite formation and determination

A 100 ml of NaC medium was inoculated with 1 ml of K. oxytoca DSM 29614 (OD_600nm = 0 1 ABS) and after 3 h of incubation at 30 °C, each culture was amended with 5 mg of Ag(NO₃), Hg(NO₃)₂ or Pd(NO₃)₂ (Sigma-aldrich, Germany). The cultures were incubated at 30 °C in static mode until colloidal material and the crystals precipitated. Crystals were easily separated by filtration from colloidal fraction and the determination of crystal was performed with X-ray-Diffractometry (Bruker D8, Italy) with 40 kV and 40 mA geometry with Cu Kα (λ = 0.154 nm) radiation. The micro-crystals formed in the cultures added with Pd²⁺ were observed by optical microscopy (Axio-Imager Z1stand, Zeiss, Italy) equipped with Axiocam MRm 2.8 in transmission and in fluorescence modes. The specimens were stained with DAPI (Fluka, Germany) to observe cells involved in crystal precipitation.

TEM observation of struvite

A Pd-EPS aliquot of 1 mg was suspended in 1 ml of milli-Q water and sonicated for 10 min. Then, in order to obtain TEM images a 5 μl aliquot of each suspension was processed as previously described in Battistel et al. (2015) [23].

Determination of metals in struvite

Total metals in 10 mg of crystals from K.oxytoca DSM 29614 culture was determined in triplicates. The sample was mineralized with aqua-regia for 4 h at 70 °C. The total metals were determined by flameless Atomic Absorption Spectroscopy (Varian SpectrAA 250 Plus, Italy).

Determination of alkaline phosphatase activity

The K. oxytoca DSM 29614 cells were harvested from aerobic and anaerobic NaC and FeC media without metal additions and with additions of AgNO₃, Hg(NO₃)₂ or Pd(NO₃)₂ (Sigma-aldrich, Germany). The cells were harvested by centrifuging, washed and then recovered in 5 ml of carbonate buffer constituted by 0.095 M NaHCO₃, 0.005 M Na₂CO₃, 0.1 M NaCl, 0.05 M MgCl₂, at pH 8.5.

Aliquots of 3 ml of cell suspension were lysed on ice bath by a Vibra-Cell VC50 sonicator (Sonics & Materials, Newtown, USA), equipped with a 3-mm microtip. Two cycles of 45 s of pulsed sonication (30 s of pulse and 15 s of pause) were performed for each sample. The sonicator was set to 20 kHz, with a power output of 50 W. Cytosol with phosphatase activity was collected after centrifugation (20 min. at 14,100 g). The supernatant was recovered, filtered (0.02 mm pore size) and stored at − 20 °C until phosphatase assay was performed [39]. The supernatant (cytosol) and the pellet (cell membranes) were characterized for total protein concentration by Coomassie Blue dye (Biorad, Italy) assay protocol [28].

The enzymatic activity was determined in the presence of different concentrations of the fluorescence substrate MUF-PO₄ (Sigma-Aldrich, Germany) [40] in order to determine V_max and K_m. The florescence intensity was measured by using a spectrofluorometer (Victor mod. X2, Perkin-Elmer). The fluorescence excitation (E_ex) and emission (E_em) wavelengths were set up at 380 and 480 nm, respectively. The calculation of phosphatase activity (PA) was performed within a range of MUF-PO₄ between 10 to 1000 μM. The PA activity is expressed as “IU”, namely the enzyme activity unit, which transforms 1 μM per minute of substrate. Than the PA, reported as international unit (IU), is normalized to the cell biomass calculated as grams of proteins (g_PRT).

K. oxytoca DSM 29614 genome

Comparative genomics analyses were performed between all the 85 previously named K. oxytoca representatives (Additional file 1: Table S1) and K. oxytoca DSM 29614; the phylogeny of the genus has been also investigated and the diversity and composition of the global gene repertoire has been studied through the pangenome approach analysis, as described in Materials and Methods.

Despite the phylogenetic analysis revealed that the 16S rRNA coding sequences can be included into three main clusters (Fig 1), the phylogenetic history of these organisms appears to be very complex. In this regard, the data obtained by evaluating the number of conserved genes vs. the number of total genes (plotted in Fig. 2) revealed that this group of microorganisms has an open pangenome - i.e. the number of unique and accessory genes increases with the number of genomes embedded into the analysis. Moreover, Fig. 3 shows the size of unique, accessory and core genomes. The core genome, here presented as soft-core and proper core, consists of 1009 genes (about 2% of the total), whereas there is a high fraction of unique (43,808 genes, about 87%) and accessory genes (5559 genes, about 11%).

A deeper functional characterization of K. oxytoca DSM 29614 genome has been obtained by mapping its open-reading frames (ORFs) to the COG database [41]. Data obtained are shown in Fig. 4 reporting the abundance of genes assigned to each category. Such analysis revealed that proteins belonging to COG categories “Carbohydrate transport and metabolism” (G), “Amino acid transport and metabolism” (E), “Coenzyme transport and metabolism” (H), “Inorganic ion transport and metabolism” (P), and “membrane biogenesis-related proteins” (M) are particularly abundant in the predicted proteome.

Insights from genomics for Fe(III)-citrate fermentation capability

According to previous data [12], medium acidification occurs when DSM 29614 strain is incubated in presence of citrate under anaerobic conditions while medium pH is stable when the strain is incubated aerobically in presence of citrate (Fig. 5a). Indeed, the DSM 29614 strain genome contains the genes encoding citrate lyase (CitEF), Na⁺-dependent membrane oxaloacetate decarboxylase (OadA), pyruvate formate lyase (PFL), phosphotransacetylase (PTA) and acetate kinase (ACK), that are the enzymes required for anaerobic citrate utilization [42]. Interestingly, the differential representation of these enzymes was already described in two differentially abundant protein sets in Gallo et al. (2012) [26]. In the present work, these two protein sets (Additional file 2: Table S2) were used for a BioCyc-based functional enrichment analysis whose results are available at the internet address https://biocyc.org/smarttables as BioCyc Public Smart Tables “Enriched with FEPCU from anaerobic Vs aerobic growth on FeC up-regulated proteins” and “Enriched with FEPCU from FeC Vs NaC during anaerobic growth up-regulated proteins”. In particular, these results revealed an up-regulation of enzymes belonging to i) a super pathway including glycolysis, pyruvate dehydrogenase, TCA and glyoxylate bypass, ii) fermentation pathways including the one forming acetate, and iii) anaerobic respiration during anaerobic utilization of Fe(III)-citrate as carbon and energy source. Thus, this result further supports the capability of the DSM 29614 strain to optimize energy production.

Another intriguing aspect of DSM 29614 strain is the massive reduction of 50 mM of Fe³⁺ to 22 mM Fe²⁺ during anaerobic growth on FeC medium (Fig. 5b). A BLAST interrogation revealed that the genome of DSM 29614 strain does not contain genes homologous to those found in Shewanella – like cymA, mtrABC operon, mtrF and omcA - or in Geobacter – such as omcS, omcT, dhc2 and pccF - involved in dissimilatory Fe(III) reduction [43–46]. However, the DSM 29614 strain possesses genes that can have a role in iron transport and reduction. Among them there are: fecABCDE operon (BI322_RS14055- BI322_RS14075) that is devoted to Fe(III)-citrate uptake in bacterial cells [47]; genes encoding flavin metal reductases, like the flavodoxin FldA (BI322_RS16850), the NADPH-dependent ferric siderophore reductase (BI322_RS21400), the succinate dehydrogenase flavoprotein subunit (BI322_RS16715), and the flavocytochrome C (BI322_RS10335); genes encoding ion efflux pumps, such as the cation transporter FieF (BI322_RS11975) [45, 48]. In particular, the NADPH-dependent ferric siderophore reductase is one of the 33 gene products involved in iron tolerance predicted in DSM 29614 strain genome (Fig. 6; Additional file 3: Table S3), and both the succinate dehydrogenase flavoprotein subunit and the flavocytochrome C are predicted to play a role in the anaerobic respiration of fumarate according to KEGG database. Indeed, the succinate dehydrogenase flavoprotein subunit was upregulated in anaerobic FeC cultivation in the respect of both aerobic FeC cultivation and anaerobic NaC cultivation as previously revealed by differential proteomics [26].

In addition, the ability to produce bioactive compounds capable of acquiring Fe(III) was revealed by antiSMASH analysis showing the presence of a turnerbactin biosynthetic gene cluster (from BI322_RS17265 to BI322_RS17505). Indeed, turnerbactin is a triscatecholate siderophore synthetized through a Non Ribosomal Peptide Synthetase, which was firstly isolated from the shipworm endosymbiont Teredinibacter turnerae T7901 [49].

Correlation between metal tolerance and genomic traits

A crucial contribution to tolerance to high levels of iron can be ascribed to the peculiar EPS produced by DSM 29614 strain which promotes metal ion entrapment and reduction. In fact, when the strain grows anaerobically in the presence of FeC, in concomitance to the Fe³⁺ reduction to Fe²⁺, iron also precipitates in the bacterial EPS (Fig. 5b; Additional file 4: Figure S1) [22]. In these growth conditions the pH is low and the citrate is consumed, but the increased ratio between polysaccharides (CHO) and cell proteins (PRT) in harvested cells at stationary phase demonstrated an overproduction of EPS (Fig. 5b).

In addition to tolerance to high levels of iron, the DSM 29614 strain possesses a high level of tolerance to diverse metals such as Pd, Rh, Cd, Pb, Zn, As, Hg and Ag as already mentioned. Therefore, in order to detect genetic elements putatively responsible for this capability, BacMet database [36] was used to scan the DSM 29614 strain genome. This investigation allowed to detect 149 genes putatively responsible for specific resistance to a total of 23 different kinds of metals (Fig. 6; Additional file 3: Table S3 and Additional file 5: Supporting Information).

Phosphatase activity and metal cation precipitation

K. oxytoca DSM 29614 genome contains typical pho regulon genes [50] - like phoA, phoR, phoB, phoE and pstSABC – and acid phosphatases – including a phosphatase PAP2 family protein which is part of cps1 gene cluster. The overall phosphatase activity (oPA) of DSM 29614 strain was investigated using samples collected from four different experimental sets: cultivations using FeC or in NaC media under anaerobic or aerobic conditions (Fig. 7). The highest oPA was observed in the NaC medium under anaerobic condition while the lowest oPA was revealed in FeC medium under aerobic condition. The V_max and K_m of oPA in cells cultivated under anaerobic conditions in NaC medium are by far the highest in respect to the other growing conditions (Table 4). The addition of increasing amounts of inorganic phosphate to NaC medium causes a modest but appreciable oPA decrement only in anaerobic condition, with a maximum reduction of approximately 22%comparing 25 and 500 μM (Fig. 8a). On the contrary, the addition of heavy metals to NaC growth medium strongly stimulates oPA in aerobiosis only (Fig. 8b). Indeed, the formation of struvite (MgNH₄PO₄.6H₂O) was revealed in K. oxytoca DSM 29614 cultivations performed using NaC growth medium under aerobic conditions with the addition of different heavy metals. In particular, the addition of 50 mg.l^− 1 Hg²⁺ or Ag⁺ induced the formation of millimetric struvite crystals (Additional file 4: Figure S2 A and B) as well as the addition of Pd²⁺ induced the formation micrometric struvite crystals (Additional file 4: Figure S2 C and D). The presence of Ag⁺ on struvite crystal surface, inferred from crystal face darkening, was confirmed by the spectrophotometric analysis revealing a content of 0.03 ± 0.01% (w/w).

Growth medium	Condition	Vmax (IU∙gPRT−1)		Km (μM)
FeC	Aerobic	0.19	±0.055	452	±30
	Anaerobic	0.73	±0.102	204	±87
NaC	Aerobic	0.83	±0.059	356	±75
	Anaerobic	4.02	±0.271	223	±15