Alcaligenes pakistanensis sp. nov.

A Heavy-metals tolerant Novel Plant Growth Promoting

Bacterium Isolated from Industrial Effluent in Pakistan
Saira Abbas 1, 2, 3
, Iftikhar Ahmed 1, 2, 3*
, ,
Toshiya Iida3, Toru Fujiwara2 and Moriya Ohkuma3
11
National Culture Collection of Pakistan (NCCP), National Agricultural Research Centre (NARC), Islamabad, Pakistan.
22
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
33
Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan

ABSTRACT
Introduction:
Several microorganisms can tolerate toxic concentration of heavy-metals. These heavy-metals tolerant bacteria offer an opportunity to
exploit in the bioremediation of contaminated soil/water systems. During our studies of microbial diversity for heavy metal-tolerant bacteria,
strain NCCP-650TT was isolated from industrial effluent.
Objectives: • To investigate toxicity tolerance of strain NCCP-650 TT
against heavy-metals and their bioremediation potential.
• To delineate the taxonomic position of strain NCCP-650TT as a novel species by polyphasic characterisation
• To characterise at molecular level for nifH and acdS gene(s) for their potential usage in agriculture.
Material & Methods: To meet the minimum standards for delineation of any novel species, the strain NCCP-650 TT was studied in comparison with references
species byphenotypic (morphology of cells, physiology and biochemical studies), genotypic (16S rRNA gene, housekeeping genes including gyrB and nirK
genes, DNA G+C content analysis and DNA-DNA hybridization), phylogenetic (based on sequences of 16S rRNA gene and the housekeeping genes) and
chemotaxonomic (analyses of cellular fatty acids, polar lipids, menaquinones) traits and other molecular characterization using nifH and acdS genes.
Results and Conclusion: Based on the results (mentioned in below Tables and Figures), it is concluded that the strain NCCP-650 represents a new species of TT

the genus Alcaligenes and named as Alcaligenes pakistanensis sp. nov. and it proved to be an efficient Plant Growth Promoting Rhizobacteria.

gyrB gene nirK gene Lead Chromium

(Abbas et al. 2015. Antonie van Leeuwenhoek 108: 859–870 ) 16S rRNA gene DNA- DNA sequence sequence Tolerance Tolerance
sequence hybrid similarity similarity (ppm) (ppm)
95
NCCP-667 (AB968096) similarity
0.5% 100 % 100 % 2100 1500
NCCP-650T (AB920828)
100 98.3 % 54.7% 85.1 % 92.4 % 1800 900 No difference in tolerance to
92 Alcaligenes aquatilis LMG 22996 (JX986974) T

Alcaligenes faecalis subsp. parafaecalis G (AJ242986) T 97.5 % 20.6% 86.5 % 89.7 % 1800 900 Arsenic, Copper, Boron
(3000 ppm, 1800 ppm, 100 mM)
98 78 Alcaligenes faecalis subsp. faecalis IAM12369T (D88008) 97.5 % 10.3% 89.1 % 90.4 % 1800 900 among these Alcaligenes species
96 Alcaligenes faecalis subsp. phenolicus JT (AY296718) 97.5 % 10.3% 87.2 % 92.7 % 1800 900
Paenalcaligenes suwonensis ABC02-12T (JX217748)

100 Paenalcaligenes hominis CCUG 53761AT (FN391024) Description of Alcaligenes pakistanensis sp. nov.
100 Paenalcaligenes hermetiae KBL009T (JN873915) Etymology: Alcaligenes pakistanensis
Advenella kashmirensis WT001T (AFQG01000022) (pak’is.tan’en.sis. N.L. masc. adj. pakistanensis from Pakistan, where the
Castellaniella denitrificans NKNTAUT (U82826) organism was isolated).
8554 100 Castellaniella daejeonensis MJ06T (GQ241321) Cell morphology: Gram-stain negative, strictly aerobic, motile and short rods, mostly occur in
99 Candidimonas nitroreducens SC-089T (FN556191) pairs, sometimes in single and rarely occur in quadrant form .
Candidimonas humi SC-092T (FN556192) Colonies morphology: Off white in color, circular, low-convex with smooth surface; older colonies
Paralcaligenes ureilyticus GR24-5T (FJ793551) spread with irregular margins.
Parapusillimonas granuli Ch07T (DQ466075) Cellular Fatty Acids: summed features 3 (C16 16:1
:1
ω7c / iso-C1515:0
:0
2OH) and C16:0
16:0
followed by Summed
Pusillimonas noertemannii BN9T (AY695828) features 2 (iso-C16
16:1
:1
I / C14
14:0
:0
3OH), C17:0
17:0
Cyclo and C18:1
18:1
ω7c;
Candidimonas bauzanensis BZ59T (GQ246953) Major menaquinone: Q-8
Eiseniicola composti YC06271T (FJ791048) G+C (mol%): 55.5 (analysed on HPLC)
Pusillimonas soli MJ07T (GQ241322)
60 Cell physiology: Tolerant to Heavy metals concentration up to
Pusillimonas ginsengisoli DCY25T (EF672088) Cr 1500 ppm, As 3000 ppm, Pb 2100 ppm and Cu 1800 ppm
Pusillimonas harenae B201T (GQ232740) Major polar lipids: Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and an
Kerstersia gyiorum LMG 5906T (AY131213) unidentified aminolipid (AL1). Phosphatidylserine (PS), one unidentified aminolipid (AL2) and six
unidentified polar lipids (L1–6) also detected .

Source of isolation: Industrial effluent (water and sludge sample) collected from (lat/lon =
‘‘33.66N 73.05E’’), Islamabad.
Type strain NCCP-650TT = JCM 30216TT = LMG 28468TT = KCTC 42043TT
Species validated: Validation List No. 167 (2016). IJSEM 66(1): 1-3
JCM 10833

A
NCCP-650

A AB AB
NCCP-644
NCCP-602

32 AB
NCCP-614

AB AB
No strain
No metal

AB AB B
A A
BC
acdS gene X X X X X X X X X X X X X X X X X X X X X 28 C
Primer Set 3

SUMMARY
Shoot length (cm)

B B
24 B B A A

20
Pb(NO3

B
acdS gene X X X X X X X X X X X X X X X X X X X X X X 16
B
B
B
A
Primer Set 2
 At least 7 strains (including NCCP-
)2

B
12 BC
CD
BC

650TT, and JCM 10833TT) possessed

D
8
acdS gene X X X X X X X X X X X X X X X X X X X X
4
both genes
Primer Set 1
Cd(NO3)2

0
A
nifH gene X X 3.5 A
B
X X X X X X X X X X X X X X X X BC
Shoot fresh weight (g)

C
Primer Set 3
3.0 B B B
C
 Strain NCCP-650TT significantly
CuSO4.4H2O

C C D A
2.5
nifH gene X X X X X X X X X X X X X X X X X X X X X
B
improved growth of Brassica
2.0
plants under Pb, Cd and Cr.
Primer Set 2
CD D D
1.5 A

nifH gene X X X X 1.0 B B B

X X X X X X X X X X X
K2Cr2O7

Primer Set 1
0.5
C C
B
A
B B B  No significant effect of JCM 10833TT
0.0
observed as positive control.
JCM 10833
JCM 21823
NCCP-601
NCCP-602
NCCP-605
NCCP-606

NCCP-611
NCCP-614
NCCP-619
NCCP-621
NCCP-622
NCCP-624
NCCP-625
NCCP-635
NCCP-644
NCCP-645
NCCP-646
NCCP-647

NCCP-651

NCCP-655
NCCP-660
NCCP-661
NCCP-650
NCCP-607

NCCP-652

No strain

No strain

No strain

No strain
NCCP-602
NCCP-614
NCCP-644
NCCP-650
JCM10833

NCCP-602
NCCP-614
NCCP-644
NCCP-650
JCM10833

NCCP-602
NCCP-614
NCCP-644
NCCP-650
JCM10833

NCCP-602
NCCP-614
NCCP-644
NCCP-650
JCM10833
CP l
NC 602

M 4

3
NC eta

JC -64

83
m

10
CP
o
/n
NaH2As

 Strain NCCP-650TT had also better

n
ai
str

Summary of amplified PCR products with

No

Lead nitrate Cadmium nitrate Copper sulfate Potassium dichromate Sodium arsinite

biosorption activity for As and Cr

O4

Pb(NO3)2 Cd(NO3)2 CuSO4.4H2O K2Cr2O7 NaH2AsO4

different markers of nifH and acdS genes.
Green color denotes the amplification of nifH gene and Effect of selected heavy-metals tolerant PGPR strains on growth promotion of but not good for Cd and Cu
red color denotes amplification of acdS gene, whereas Bassica napus irrigated with heavy-metal (50 ppm) containing water.
X indicates that no amplification occur . Bars of same color in each parameter carrying the same alphabet letters are statistically non-significant (p 0.05) .