Neighbor - joining phylogenetic tree showing inter - relationship of strain NCCP - 644 T with the closely related type species of genera Acinetobacter inferred from sequences of the 16S rRNA gene. Nodes indicated by empty circles were recovered by at least two algorithms, whereas nodes with solid circles were recovered by three algorithms. Acinetobacter pakistanensis sp. nov. A Heavy - metals tolerant and Psychrotolerant Bacterium Isolated from a Textile Dyeing Waste Water Treatment Pond Saira Abbas 1, 2, 3 , Iftikhar Ahmed 1, 2, 3* , Ghulam Muhammad Ali 1 , Takuji Kudo 3 , Toshiya Iida 3 , Toru Fujiwara 2 and Moriya Ohkuma 3 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-644 T was isolated from a textile dyeing wastewater treatment pond, Kohnoor Mills, Islamabad. Objective : To investigate the taxonomic position of the strain NCCP-644 T as a novel species by polyphasic characterisation. Materials & Methods : To meet the minimum standards for delineation of any novel species, the strain NCCP-644 T was studied in comparison with references species by phenotypic (morphology of cells, physiology and biochemical studies), genotypic (16S rRNA gene, housekeeping genes including gyrB, rpoB and atpD 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) characterisation. Results and Conclusion : Based on the results (mentioned in below Tables and Figures), it is concluded that the strain NCCP-644 T represents a new species of the genus Acinetobacter and named as Acinetobacter pakistanensis sp . nov . ABSTRACT Analysis of Housekeeping gene(s) in Acinetobacter pakistanensis strain NCCP - 644 T in comparison to the reference species and their accession numbers published in DNA database Etymology: Acinetobacter pakistanensis (pak’is.tan’en.sis. N.L. masc. adj. pakistanensis from Pakistan, where the organism was isolated). Cell morphology: Gram-stain negative, cocci to short rod (coccobacillus), occur primarily in pairs and sometimes triplet form, which are non-motile and strictly aerobic. Colonies morphology: Moist, raised, off-white in colour with entire margins and are 1–2 mm in size after 1–2 days when grown on TSA (Difco) medium at 25°C. Cellular Fatty Acids: Summed feature 3 (iso-C 15 :0 2OH / C 16 :1 ω7c), followed by C 18:1 ω9c, C 16:0 , C 12:0 3-OH, C 12:0 and C 10:0 . Major menaquinone: Q-9 G+C (mol%): 40.6 (analysed on HPLC) Cell physiology: Tolerant to Heavy metals (Cr, As, Pb, Cu) Major polar lipids: Phosphatidyl monomethyl ethanol (PME), phosphatidyl inositol (PI), diphosphatidyl glycerol (DPG), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS), phosphatidyl glycerol (PG) and one unknown phospholipid. Source of isolation: Textile dyeing wastewater treatment pond, Kohnoor Mills, Islamabad, Pakistan Type strain NCCP-644 T = JCM18977 T = LMG 28467 T = KCTC 42081 T Species validated: Validation List No. 161 ( 2015 ). IJSEM 65 ( 1 ): 1 - 4 1 National Institute for Genomics and Advanced Biotechnology, National Agricultural Research Centre, Islamabad-45500, PAKISTAN. 2 Laboratory of Plant Nutrition and Fertilizers, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan 3 Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan 20.6% 54.7% DNA- DNA hybrid 16 S rRNA gene sequence similarity Description of Acinetobacter pakistanensis sp. nov. Major differentiating characteristics of strain NCCP-644 T from the closely related species Characteristics NCCP - 644 T " A. kyonggiensis " JCM 17071 T A. harbinensis KCTC32411 T A. bouvetii JCM 18991 T A. beijerinckii JCM 18990 T A . johnsonii JCM 20194 T A. calcoaceticus JCM 6842 T Temperature (ºC) range, (optimum) 3-37 (20-25) 5-30 (20-25) 2-35 (8-20) † 5-37 (20-30) 5-40 (25-33) 10-33 (20-30) 5-37 (20-30) pH range (optimum) 6-10 (7-8) 6-9 (7-8) 6-8.5 (7.2) † 6-9 (6-8) 6-10 (7-8) nd nd NaCl range (%), (optimum) 0-3 (0-1) 0-3 (0) 0-4 (0) † 0-3 (0-2) 0-4 (0-2) 0-4 (1-2) 0-2 (0-1) Tolerance to heavy metals: Chromium (ppm) 2700 1200 nd 600 1800 300 300 Arsenic (ppm) 3000 2400 3000 3000 3000 900 1200 Lead (ppm) 2100 900 900 1500 1500 300 900 Copper (ppm) 2700 1200 900 1200 2100 300 300 Utilisation of citrate – – – – + – – Nitrate reduction to N 2 + + – + + + + Voges-Proskauer reaction + – + + + + + Acid from N-acetyl glucosamine – + – – – – – Assimilation of: Capric acid + – + + + + – Adipic acid – – – – – – + Malate + + – + + + + Trisodium citrate – – – w+ + + – Phenyl acetic acid – – – – – – w+ Enzyme activity (API-Zym and Rapid 32 ID) Alkaline phosphatase +++ ++ – ++ w+ w+ +++ Lipase (C 14) + + w+ ++ +++ ++ w+ Cysteine arylamidase + + w+ + + + w+ Acid phosphatase + w+ + ++ ++ + +++ Napthol-As-BI-phosphohydrolase +++ – w+ + ++ + ++ Proline arylamidase + + + – + + + Glutamyl glutamic acid arylamidase + – – – + – + HIG HER EDUC ATIO N C O M MISSIO N Acinetobacter soli B1 T (EU290155) Acinetobacter baylyi DSM 14961 T (APPT01000006) Acinetobacter boissieri SAP 284.1 T (JQ771141) Acinetobacter towneri DSM 14962 T (APPY01000064) Acinetobacter indicus A648 T (HM047743) Acinetobacter puyangensis BQ4-1 T (JN664255) Acinetobacter baumannii ATCC 19606 T (ACQB01000091) Acinetobacter junii CIP 64.5 T (APPX01000010) Acinetobacter gerneri DSM 14967 T (APPN01000041) Acinetobacter marinus SW-3 T (AY633607) Acinetobacter rudis G30 T (EF204258) Acinetobacter guillouiae CIP 63.46 T (APOS01000028) Acinetobacter bereziniae LMG 1003 T (AIEI01000248) Acinetobacter schindleri CIP 107287 T (APPQ01000011) Acinetobacter haemolyticus CIP 64.3 T (APQQ01000002) Acinetobacter brisouii 5YN5-8 T (DQ832256) Acinetobacter oleivorans DR1 T (CP002080) Acinetobacter pittii CIP 70.29 T (APQP01000001) Acinetobacter calcoaceticus DSM 30006 T (AIEC01000170) Acinetobacter nosocomialis NIPH 2119 T (APOP01000014) Acinetobacter pakistanensis NCCP-644 T (AB916465) “Acinetobacter kyonggiensis” KSL5401-037 T (FJ527818) Acinetobacter johnsonii CIP 64.6 T (APON01000005) Acinetobacter oryzae B23 T (GU954428) Acinetobacter bouvetii DSM 14964 T (APQD01000004) Acinetobacter gyllenbergii 1271 T (AJ293694) Acinetobacter tjernbergiae DSM 14971 T (ARFU01000016) Acinetobacter parvus DSM 16617 T (AIEB01000124) Acinetobacter kookii 11-0202 T (JX137279) Acinetobacter beijerinckii 58a T (AJ626712) Acinetobacter venetianus RAG-1 T (AKIQ01000085) Acinetobacter radioresistens DSM 6976 T (BAGY01000082) Acinetobacter harbinensis HITLi 7 T (KC843488) Prolinoborus fasciculus CIP 103579 T (JN175353) Acinetobacter lwoffii NCTC 5866 T (AIEL01000120) Acinetobacter ursingii DSM 16037 T (AIEA01000080) Alkanindiges illinoisensis MVAB Hex1 T (AF513979) 82 55 70 72 87 84 98 78 94 89 81 100 82 0.5% 97.7 % 10.3% 97.5 % 97.5 % 97.5 % 98.3 % Symbols: +++, Very strongly positive; ++, strongly positive; +, Positive; w+, weakly positive; –, negative; Marker (100 bp) a - NCCP-644 a - JCM17071 a – JCM18990 a - JCM118991 b - NCCP-644 b - JCM17071 b – JCM18990 b - JCM118991 c - NCCP-644 c - JCM17071 c– JCM18990 c - JCM118991 d - NCCP-644 d - JCM17071 d – JCM18990 d - JCM118991 Marker (1-kb) Marker E - NCCP-644 E - JCM17071 F- NCCP-644 F - JCM17071 G - NCCP-644 G - JCM17071 Negative control Targeted Housekeeping Gene(s) gyrB rpoB atpD infB rpoB rpoB rpoB Primer pair for amplification gyrB 01-F (forward) gyrB 02-R (reverse) rpoB CM7- F (forward) rpoB CM31b-R (reverse) atpD 01-F (forward) atpD 02-R (reverse) infB 01-F (forward) infB 02-R (reverse) Ac696F (forward) Ac1093R (reverse) Ac1055F (forward) Ac1598R (reverse) Ac696F (forward) Ac1598R (reverse) Expected amplicon size 742 bp 558 bp 657 bp 615 bp 350 bp 450 bp 700 bp Reference Coenye et al., 1999 La Scola et al., 2006 Strain Name Name of housekeeping Gene Number of nucleotides housekeeping Gene Accession number of housekeeping Gene Closely related validly published taxa as per respective gene sequence similarity Similarity %age of housekeeping gene sequence with closely related species Similarity %age range of housekeeping gene sequence with all other species of the genus Acinetobacter pakistanensis NCCP-644 T gyrB 876 AB924048 Acinetobacter kyonggiensis (AB924049) 92.3 82.7 - 71.1 Acinetobacter pakistanensis NCCP-644 T rpoB 873 AB938199 Acinetobacter kyonggiensis (AB938200 ) 95.3 85.3 - 76.7 Acinetobacter pakistanensis NCCP-644 T atpD 756 AB924051 Acinetobacter kyonggiensis (AB924052) 96.6 --- 12.3% 19.6%
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Neighbor-joining phylogenetic tree showing inter-relationship of strain NCCP-644T
with the closely related type species of genera Acinetobacter inferred from sequences of the 16S rRNA
gene. Nodes indicated by empty circles were recovered by at least two algorithms, whereas nodes
with solid circles were recovered by three algorithms.
Acinetobacter pakistanensis sp. nov.A Heavy-metals tolerant and Psychrotolerant Bacterium
Isolated from a Textile Dyeing Waste Water Treatment Pond
Saira Abbas1, 2, 3, Iftikhar Ahmed1, 2, 3*, Ghulam Muhammad Ali1, Takuji Kudo3, Toshiya Iida3, Toru Fujiwara2 and Moriya Ohkuma3
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-644T
was isolated from a textile dyeing wastewater treatment pond, Kohnoor Mills, Islamabad.
Objective: To investigate the taxonomic position of the strain NCCP-644T as a novel species by polyphasic characterisation.
Materials & Methods: To meet the minimum standards for delineation of any novel species, the strain NCCP-644T was studied in comparison with
references species by phenotypic (morphology of cells, physiology and biochemical studies), genotypic (16S rRNA gene, housekeeping genes
including gyrB, rpoB and atpD 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) characterisation.
Results and Conclusion: Based on the results (mentioned in below Tables and Figures), it is concluded that the strain NCCP-644T represents a new
species of the genus Acinetobacter and named as Acinetobacter pakistanensis sp. nov.
ABSTRACT
Analysis of Housekeeping gene(s) in Acinetobacter pakistanensis strain NCCP-644T
in comparison to the reference species and their accession numbers published in DNA database
Etymology: Acinetobacter pakistanensis
(pak’is.tan’en.sis. N.L. masc. adj. pakistanensis from
Pakistan, where the organism was isolated).
Cell morphology: Gram-stain negative, cocci to short rod (coccobacillus),
occur primarily in pairs and sometimes triplet form, which
are non-motile and strictly aerobic.
Colonies morphology: Moist, raised, off-white in colour with entire margins and are
1–2 mm in size after 1–2 days when grown on TSA (Difco)
medium at 25°C.
Cellular Fatty Acids: Summed feature 3 (iso-C15 :0 2OH / C16 :1 ω7c), followed by
C18:1 ω9c, C16:0, C12:0 3-OH, C12:0 and C10:0.
Major menaquinone: Q-9
G+C (mol%): 40.6 (analysed on HPLC)
Cell physiology: Tolerant to Heavy metals (Cr, As, Pb, Cu)
Major polar lipids: Phosphatidyl monomethyl ethanol (PME), phosphatidyl
inositol (PI), diphosphatidyl glycerol (DPG), phosphatidyl
ethanolamine (PE), phosphatidyl serine (PS), phosphatidyl
glycerol (PG) and one unknown phospholipid.
Source of isolation: Textile dyeing wastewater treatment pond, Kohnoor
Mills, Islamabad, Pakistan
Type strain NCCP-644T = JCM18977T = LMG 28467T = KCTC 42081T
Species validated: Validation List No. 161 (2015). IJSEM 65(1): 1-4
1 National Institute for Genomics and Advanced Biotechnology, National Agricultural Research Centre, Islamabad-45500, PAKISTAN. 2 Laboratory of Plant Nutrition and Fertilizers, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan3 Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
20.6%
54.7%
DNA- DNA
hybrid
16S rRNA gene
sequence
similarity
Description of Acinetobacter pakistanensis sp. nov.Major differentiating characteristics of strain NCCP-644T from the closely related species
Characteristics NCCP-
644T
"A.
kyonggiensis"
JCM 17071T
A.
harbinensis
KCTC32411T
A.
bouvetii
JCM 18991T
A.
beijerinckii
JCM 18990 T
A.
johnsonii
JCM 20194 T
A.
calcoaceticus
JCM 6842T
Temperature (ºC) range,
(optimum)
3-37
(20-25)
5-30
(20-25)
2-35
(8-20) †5-37
(20-30)
5-40
(25-33)
10-33
(20-30)
5-37
(20-30)
pH range (optimum) 6-10 (7-8) 6-9 (7-8) 6-8.5 (7.2) † 6-9 (6-8) 6-10 (7-8) nd nd
NaCl range (%), (optimum) 0-3 (0-1) 0-3 (0) 0-4 (0)† 0-3 (0-2) 0-4 (0-2) 0-4 (1-2) 0-2 (0-1)
Tolerance to heavy metals:
Chromium (ppm) 2700 1200 nd 600 1800 300 300
Arsenic (ppm) 3000 2400 3000 3000 3000 900 1200
Lead (ppm) 2100 900 900 1500 1500 300 900
Copper (ppm) 2700 1200 900 1200 2100 300 300
Utilisation of citrate – – – – + – –Nitrate reduction to N2 + + – + + + +
Voges-Proskauer reaction + – + + + + +Acid from N-acetyl glucosamine – + – – – – –
Assimilation of:
Capric acid + – + + + + –Adipic acid – – – – – – +Malate + + – + + + +Trisodium citrate – – – w+ + + –Phenyl acetic acid – – – – – – w+
Enzyme activity (API-Zym and Rapid 32 ID)
Alkaline phosphatase +++ ++ – ++ w+ w+ +++
Lipase (C 14) + + w+ ++ +++ ++ w+
Cysteine arylamidase + + w+ + + + w+
Acid phosphatase + w+ + ++ ++ + +++Napthol-As-BI-phosphohydrolase +++ – w+ + ++ + ++
Proline arylamidase + + + – + + +Glutamyl glutamic acid arylamidase + – – – + – +
HIG HER EDUC ATION COMMISSION
Acinetobacter soli B1T (EU290155)Acinetobacter baylyi DSM 14961T (APPT01000006)
Acinetobacter boissieri SAP 284.1T (JQ771141)
Acinetobacter towneri DSM 14962T (APPY01000064)
Acinetobacter indicus A648T (HM047743)
Acinetobacter puyangensis BQ4-1T (JN664255)
Acinetobacter baumannii ATCC 19606T (ACQB01000091)
Acinetobacter junii CIP 64.5T (APPX01000010)
Acinetobacter gerneri DSM 14967T (APPN01000041)
Acinetobacter marinus SW-3T (AY633607)
Acinetobacter rudis G30T (EF204258)
Acinetobacter guillouiae CIP 63.46T (APOS01000028)
Acinetobacter bereziniae LMG 1003T (AIEI01000248)
Acinetobacter schindleri CIP 107287T (APPQ01000011)
Acinetobacter haemolyticus CIP 64.3T (APQQ01000002)
Acinetobacter brisouii 5YN5-8T (DQ832256)
Acinetobacter oleivorans DR1T (CP002080)
Acinetobacter pittii CIP 70.29T (APQP01000001)
Acinetobacter calcoaceticus DSM 30006T (AIEC01000170)
Acinetobacter nosocomialis NIPH 2119T (APOP01000014)
Acinetobacter pakistanensis NCCP-644T (AB916465)“Acinetobacter kyonggiensis” KSL5401-037T (FJ527818)
Acinetobacter johnsonii CIP 64.6T (APON01000005)Acinetobacter oryzae B23T (GU954428)
Acinetobacter bouvetii DSM 14964T (APQD01000004)
Acinetobacter gyllenbergii 1271T (AJ293694)
Acinetobacter tjernbergiae DSM 14971T (ARFU01000016)Acinetobacter parvus DSM 16617T (AIEB01000124)
Acinetobacter kookii 11-0202T (JX137279)
Acinetobacter beijerinckii 58aT (AJ626712)Acinetobacter venetianus RAG-1T (AKIQ01000085)
Acinetobacter radioresistens DSM 6976T (BAGY01000082)
Acinetobacter harbinensis HITLi 7T (KC843488)Prolinoborus fasciculus CIP 103579T (JN175353)Acinetobacter lwoffii NCTC 5866T (AIEL01000120)
Acinetobacter ursingii DSM 16037T (AIEA01000080)Alkanindiges illinoisensis MVAB Hex1T (AF513979)
82
55
70
72
87
84
98
78
94
89
81
10082
0.5%
97.7 %
10.3%
97.5 %
97.5 %
97.5 %
98.3 %
Symbols: +++, Very strongly positive; ++, strongly positive; +, Positive; w+, weakly positive; –, negative;
Mark
er (1
00 b
p)
a - N
CC
P-6
44
a - J
CM
17071
a –
JC
M18990
a - J
CM
118991
b - N
CC
P-6
44
b - J
CM
17071
b –
JC
M18990
b - J
CM
118991
c - N
CC
P-6
44
c - J
CM
17071
c– J
CM
18990
c - J
CM
118991
d - N
CC
P-6
44
d - J
CM
17071
d –
JC
M18990
d - J
CM
118991
Mark
er (1
-kb
)
Ma
rker
E -
NC
CP
-644
E -
JC
M1
70
71
F-
NC
CP
-644
F -
JC
M1
70
71
G -
NC
CP
-644
G -
JC
M1
70
71
Neg
ativ
e c
on
trol
Targeted Housekeeping Gene(s)
gyrB rpoB atpD infB rpoB rpoB rpoBPrimer pair for
amplification
gyrB 01-F
(forward)
gyrB 02-R
(reverse)
rpoB CM7- F
(forward)rpoB CM31b-R
(reverse)
atpD 01-F
(forward)
atpD 02-R
(reverse)
infB 01-F
(forward)
infB 02-R
(reverse)
Ac696F
(forward)
Ac1093R
(reverse)
Ac1055F
(forward)
Ac1598R
(reverse)
Ac696F
(forward)
Ac1598R
(reverse)
Expected
amplicon size 742 bp 558 bp 657 bp 615 bp 350 bp 450 bp 700 bp
Reference Coenye et al., 1999 La Scola et al., 2006
Strain Name
Name of housekeeping
Gene
Number of nucleotides
housekeeping Gene
Accession number of
housekeeping Gene
Closely related validly published
taxa as per respective gene
sequence similarity
Similarity %age of housekeeping gene sequence
with closely related species
Similarity %age range of
housekeeping gene sequence with all other species of
the genus
Acinetobacter pakistanensis NCCP-644T
gyrB 876 AB924048 Acinetobacter kyonggiensis (AB924049)
92.3 82.7 - 71.1
Acinetobacter pakistanensis NCCP-644T
rpoB 873 AB938199
Acinetobacter kyonggiensis (AB938200 )
95.3 85.3 - 76.7
Acinetobacter pakistanensis NCCP-644T
atpD 756 AB924051 Acinetobacter kyonggiensis (AB924052)
96.6 ---
12.3%
19.6%
Related Documents
