Cupriavidus sp. strain Ni‑2 resistant to high concentration of nickel and its genes responsible for the tolerance by genome comparison

1. Cupriavidus sp. strain Ni-2 resistant to high concentration of nickel and its
genes responsible for the tolerance by genome comparison
STHITAPRAJNA SAHOO

3. INTRODUCTION:
• Human activities release heavy metals from
chemical and metallurgical industries, water
disposal, textile industries etc.
• Because of their toxicity, excessive heavy metals
may cause many problems to environment as well
as humans.
• Even though heavy metals act as micronutrients in
bacterial cells, their excess concentration can cause
toxicity.
• The bacteria have thus evolved themselves to resist
higher concentrations of these heavy metals.
• Nickel (Ni) is one of the toxic heavy metal which
cause environmental pollution and it can hinder the
plant growth and can cause cancer in humans.
• Many microorganism requires Ni as essential trace
for constituting their enzymatic activities.

4. INTRODUCTION:
• Microorganisms resistant to high conc. of heavy metals tends to harbor mechanisms
for avoiding heavy metals by preventing them to enter cell through cell membrane.
• In this study, Ni-resistant bacteria were isolated from agricultural land with a high conc.
of heavy metal near to smelting area.
• Characterized the isolated strain.
• Analyzed the genome sequence of one strain among the isolates to suggest potential
mechanisms on the resistance to the high conc. of aqueous Ni.

5. Isolation and
identification of Ni-
resistant bacteria.
Metal resistant
experiment.
Antibiotic susceptibility
test
Electron microscopy Genome Sequencing Nucleotide Sequence
Accession number
METHODOLOGY:
• Soil samples collected
from agricultural fields.
• Inoculated the sample in
Nickel containing media
(20mM 𝑁𝑖2+
) and shaking
incubator at 130rpm at
28 °C.
• Serial dilution of the
culture were spread on
TSB agar media
containing 20mM 𝑁𝑖2+ .
• Phylogenetic analysis of
the isolates using 16S
rRNA genes.
• Isolated bacteria incubated
as seed cultures in 10 mL of
TSB medium having 10 mM
Ni at 28 °C with 160 rpm for
16 h
• Microbial growth was
monitored by OD (at 600
nm ) using
spectrophotometer.
• To examine minimum
inhibitory conc. (MIC) of
𝑁𝑖2+
, 3 different conc. Of
NiCl at 10,15,20 mM were
tested for the growth of the
strain Ni-2.
• Disk diffusion method
performed for 16 h at
28 °C for
vancomycin (30 µg),
erythromycin (15 µg),
chloramphenicol (30
µg), ampicillin (10 µg),
gentamicin (10 µg),
tetracycline (30 µg),
streptomycin (10 µg)
and kanamycin (30
µg)
Fresh cultures grown in
TSB transferred to the
electron microscopy grids
and examined at 120 kV
using a HITACHI Bio TEM
(H-7650)
• As strain Ni-2 showed relatively
stable resistance on high conc.
of Ni, it was selected for Genome
sequencing.
• DNA extracted using G-spin
Genomic DNA Extraction Kit.
• Sequence library constructed
according to Pacific Biosciences
RSII method.
• Genome assembly computed
using PacBio SMRT system
2.3.0.
• Annotated using NCBI-PGAP
(Prokaryotic Genome Annotation
Pipeline).
• Strain Ni-2 gene
sequence of 16s rRNA
is deposited in the
NCBI GenBank with
accession number of
MG948149 .
• Genome sequence of
the strain Ni-2
deposited in GenBank
under the accession
numberPRJNA432481

6. 1. Identification of the Ni-resistant isolate
RESULTS:
• From the soil culture, Three Ni-resistant bacterial strains
were isolated with morphological distinctiveness.
• Out of these three strains, strain Ni-2 was chosen for
further study.
• Rod-shaped Ni-2 strains,
cell diameter approx. 0.4 – 0.6μm ,
Lengths approx. 1-1.5μm

7. • 16s rRNA gene of strain Ni-2 was
sequenced and aligned with GenBank
database and SILVA database.
• Alignment with GenBank database
showed highest similarity of 99% to
Cupriavidus metallidurans CH34.
• Sequence comparison with SILVA
database indicated the closest neighbor
with C. metallidurans by generating
neighbor-joining phylogenetic tree.
1. Identification of the Ni-resistant isolate
RESULTS:
• C. metallidurans has been focused due to its extensive heavy metal
tolerance and its plasmid have been studied for characterization and
application of heavy metal resistance.

8. 2. Nickel-resistance of the strain Ni-2
RESULTS:
• Isolated strain Ni-2 was examined for its growth in the
presence of 10mM 𝑁𝑖2+
.
• Conc. of Ni and Optical Density were monitored.
• The bacterial growth showed gradual increase to reach
stationary phase and after that the conc. of Ni
remained relatively consistent.
• When Ni-2 strain is incubated at minimal medium of
M1 with different conc. of NiCl, Ni-2 strain was not
inhibited even by high conc. of 20mM, 𝑁𝑖2+
.
• Which results into high Nickel resistant ability of Ni-2
strain.
Vancomycin (30µg) (+ve) Gentamicin (10µg) (+ve)
Erythromycin (15µg) (+ve) Streptomycin (10µg) (+ve)
Chlorampheicol (30µg) (+ve) Kanamycin (30µg) (+ve)
Ampicillin (10µg) (+ve) Tetracycline (30µg) (-ve)

9. 3. Genomic and comparative genomic analysis
RESULTS:
Genomic size: 6,953,108bp
GC content ratio: 63.55%
No. of contigs: 4 (1+3)
No. of rRNA genes: 12
No. of tRNA genes: 62
No. of ORFs: 6494
3.13%
8.44%
6.29%
0.63%
3.11%
5.14%
1.26%
6.75%
4.67%
6.94%
3.23%
6.94%
1.54%
2.20%
4.60%
2.78%
0.00%
32.37%
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10. 3. Genomic and comparative genomic analysis
RESULTS:
• 12 putative CDS genes annotated for Ni-
resistance and transport.
• Which encodes Ni-Co resistance OR Ni-Co-Cd
resistance proteins of CnrA, CnrB, CnrY, NccB,
NccC, NccN, NccX and Ni-transport system
permease protein.
• Cnr operon encodes a phenotype of inducible
resistance to Ni & Co.
• Ncc operon encodes a phenotype resistance to
Ni, Co & Cd.
• These operons form membrane-bound cation-
efflux protein complexes and perform
regulations for the resistances to the toxic heavy
metals.

11. 3. Genomic and comparative genomic analysis
RESULTS:
• Genome seq of strain Ni-2
compared with 4 strains of C.
metallidurans, CH34, H1130,
SHE and NE12.
• Orthologous Avg. nucleotide
identify values and PCoA
indicates that CH34 showed
highest value of 98.9 and
showed a grouping of Ni-2 &
CH34.
• UPGMA phylogenetic dendrogram
was constructed for all 5 strains
using OrthoANI, which indicates
closer relationship of Ni-2 & CH34

12. 3. Genomic and comparative genomic analysis
RESULTS:
• Chromosome and megaplasmid
of strain CH34 were compared
to the strain Ni-2 genome.
• This comparison showed high
values of OrthoANIu values of
98.9% & 98.6% respectively
which also support the close
relationship between the strains.
• All the 12 putative CDSs retrieved from Ni-
2 strain genome seq. are compared with all
the 5 strains i.e., Ni-2, CH34, H1130, SHE,
NE12 of C. metallidurans.
• H1130 was found to be the closest strain
to Ni-2 strain with high similarities (> 99%).

13. Conclusion:
• By assuming overall similarities on the diverse heavy metal resistance of the strain Ni-2 to the strain
H1130 and CH34; it seems that strain Ni-2 could be tolerant also to diverse toxic heavy metals at high
concentration.
• Considering the genetic composition, strain Ni-2 has a well equipped & complex genetic makeup to
adopt or survive metal-rich environments.
• This newly reported strain Ni-2 may provide a wider option for bioremediation application in heavy
metal polluted environments.
• Some metal resistance systems of C. metallidurans were adopted for endophytic plant growth
promoting bacteria which leads to enhancement of heavy metal tolerance in their host plants, and then
possibly better phytoremediation opportunities in the metal contaminated soil.