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*
,
Takuji Kudo3, Toshiya Iida3, Toru Fujiwara2 and Moriya Ohkuma3
11
Institute of Microbial Culture Collection of Pakistan (IMCCP), National Agricultural Research Centre, Islamabad-45500, PAKISTAN.
22
Laboratory of Plant Nutrition and Fertilizers, 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-644 TT
was isolated from a textile dyeing wastewater treatment pond, Kohnoor Mills, Islamabad.
Objective: To investigate the taxonomic position of the strain NCCP-644TT as a novel species by polyphasic characterisation.
Materials & Methods: To meet the minimum standards for delineation of any novel species, the strain NCCP-644 TT 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 TT represents a new
species of the genus Acinetobacter and named as Acinetobacter pakistanensis sp. nov.

82 Acinetobacter soli B1T (EU290155)

Acinetobacter baylyi DSM 14961T (APPT01000006) Analysis of Housekeeping gene(s) in
55
0.5% Acinetobacter boissieri SAP 284.1T (JQ771141) Acinetobacter pakistanensis strain NCCP-644T
Acinetobacter towneri DSM 14962T (APPY01000064) in comparison to the reference species and their accession numbers published in DNA database
Acinetobacter indicus A648T (HM047743)
Acinetobacter puyangensis BQ4-1T (JN664255)
Targeted Housekeeping Gene(s)
Acinetobacter baumannii ATCC 19606T (ACQB01000091) gyrB rpoB atpD infB rpoB rpoB rpoB
72 Acinetobacter junii CIP 64.5T (APPX01000010)
Primer pair for gyrB 01-F rpoB CM7- F atpD 01-F infB 01-F Ac696F Ac1055F Ac696F
70 Acinetobacter gerneri DSM 14967T (APPN01000041) amplification (forward) (forward) (forward) (forward) (forward) (forward) (forward)
Acinetobacter marinus SW-3T (AY633607) Ac1093R Ac1598R Ac1598R
gyrB 02-R rpoB CM31b-R atpD 02-R infB 02-R (reverse) (reverse) (reverse)
Acinetobacter rudis G30T (EF204258) (reverse) (reverse) (reverse) (reverse)
Acinetobacter guillouiae CIP 63.46T (APOS01000028) Expected
87 Acinetobacter bereziniae LMG 1003T (AIEI01000248) amplicon size 742 bp 558 bp 657 bp 615 bp 350 bp 450 bp 700 bp
Acinetobacter schindleri CIP 107287T (APPQ01000011)
Acinetobacter haemolyticus CIP 64.3T (APQQ01000002) Reference Coenye et al., 1999 La Scola et al., 2006

Negative control
Acinetobacter brisouii 5YN5-8T (DQ832256)
Marker (100 bp)

b - JCM118991

d - JCM118991
a - JCM118991

c - JCM118991
b – JCM18990

d – JCM18990
a – JCM18990

G - JCM17071
G - NCCP-644
b - JCM17071

d - JCM17071

E - JCM17071
16S rRNA gene

a - JCM17071

b - NCCP-644

c - JCM17071

d - NCCP-644

E - NCCP-644

F - JCM17071
a - NCCP-644

c - NCCP-644

c– JCM18990

Marker (1-kb)
Acinetobacter oleivorans DR1 (CP002080)

F- NCCP-644
T
78
Acinetobacter pittii CIP 70.29T (APQP01000001) sequence DNA- DNA
98
hybrid

Marker
Acinetobacter calcoaceticus DSM 30006T (AIEC01000170) similarity
84
Acinetobacter nosocomialis NIPH 2119 (APOP01000014)
T

94 Acinetobacter pakistanensis NCCP-644T (AB916465)

“Acinetobacter kyonggiensis” KSL5401-037T (FJ527818) 98.3 % 54.7%
Acinetobacter johnsonii CIP 64.6T (APON01000005) 97.5 % 20.6%
89 Acinetobacter oryzae B23T (GU954428)
Acinetobacter bouvetii DSM 14964T (APQD01000004) 97.5 % 10.3%
Acinetobacter gyllenbergii 1271T (AJ293694)
Acinetobacter tjernbergiae DSM 14971T (ARFU01000016)
81 Acinetobacter parvus DSM 16617T (AIEB01000124)
Acinetobacter kookii 11-0202T (JX137279)
Acinetobacter beijerinckii 58aT (AJ626712) 97.5 % 12.3%
Acinetobacter venetianus RAG-1T (AKIQ01000085) Strain Name Name of Number of Accession Closely related Similarity %age of Similarity %age
housekeeping nucleotides number of validly published housekeeping range of
Acinetobacter radioresistens DSM 6976T (BAGY01000082) Gene housekeeping housekeeping taxa as per gene sequence housekeeping gene
Acinetobacter harbinensis HITLi 7T (KC843488) 97.7 % 19.6% Gene Gene respective gene with closely sequence with all
sequence related species other species of
82 Prolinoborus fasciculus CIP 103579T (JN175353) the genus
similarity
100 Acinetobacter lwoffii NCTC 5866T (AIEL01000120)
Acinetobacter gyrB 876 AB924048 Acinetobacter 92.3 82.7 - 71.1
Acinetobacter ursingii DSM 16037T (AIEA01000080) pakistanensis kyonggiensis
Alkanindiges illinoisensis MVAB Hex1T (AF513979) NCCP-644T (AB924049)
Acinetobacter rpoB 873 AB938199 Acinetobacter 95.3 85.3 - 76.7
pakistanensis kyonggiensis
Neighbor-joining phylogenetic tree showing inter-relationship of strain NCCP-644 T NCCP-644T (AB938200 )
Acinetobacter atpD 756 AB924051 Acinetobacter 96.6 ---
with the closely related type species of genera Acinetobacter inferred from sequences of the 16S rRNA pakistanensis kyonggiensis
gene. Nodes indicated by empty circles were recovered by at least two algorithms, whereas nodes with NCCP-644T (AB924052)

solid circles were recovered by three algorithms.

Description of Acinetobacter pakistanensis sp. nov.
Major differentiating characteristics of strain NCCP-644T from the closely related species Etymology: Acinetobacter pakistanensis
(pak’is.tan’en.sis. N.L. masc. adj. pakistanensis from
Characteristics "A. A. A. A. A. A.
NCCP- kyonggiensis" harbinensis bouvetii beijerinckii johnsonii calcoaceticus Pakistan, where the organism was isolated).
644T JCM 17071T KCTC32411T JCM 18991T JCM 18990 T JCM 20194 T JCM 6842T Cell morphology: Gram-stain negative, cocci to short rod (coccobacillus),
Temperature (ºC) range, 3-37 5-30 2-35 5-37 5-40 10-33 5-37
(optimum) (20-25) (20-25) (8-20) † (20-30) (25-33) (20-30) (20-30) occur primarily in pairs and sometimes triplet form, which
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 are non-motile and strictly aerobic.
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: Colonies morphology: Moist, raised, off-white in colour with entire margins and are
Chromium (ppm) 2700 1200 nd 600 1800 300 300 1–2 mm in size after 1–2 days when grown on TSA (Difco)
Arsenic (ppm) 3000 2400 3000 3000 3000 900 1200 medium at 25°C.
Lead (ppm) 2100 900 900 1500 1500 300 900
Copper (ppm) 2700 1200 900 1200 2100 300 300 Cellular Fatty Acids: Summed feature 3 (iso-C15
15 :0
:0
2OH / C16
16 :1
:1
ω7c), followed by
Utilisation of citrate – – – – + – –
C18:1 ω9c, C16:0 , C12:0 3-OH, C12:0 and C10:0 .
Nitrate reduction to N2 + + – + + + + 18:1 16:0 12:0 12:0 10:0

Voges-Proskauer reaction + – + + + + + Major menaquinone: Q-9

Acid from N-acetyl glucosamine – + – – – – –
Assimilation of: G+C (mol%): 40.6 (analysed on HPLC)
Capric acid + – + + + + –
Adipic acid – – – – – – + Cell physiology: Tolerant to Heavy metals (Cr,
Malate + + – + + + + As, Pb, Cu)
Trisodium citrate – – – w+ + + –
Phenyl acetic acid – – – – – – w+ Major polar lipids: Phosphatidyl monomethyl ethanol (PME), phosphatidyl
Enzyme activity (API-Zym and Rapid 32 ID) inositol (PI), diphosphatidyl glycerol (DPG), phosphatidyl
Alkaline phosphatase +++ ++ – ++ w+ w+ +++
ethanolamine (PE), phosphatidyl serine (PS), phosphatidyl
Lipase (C 14) + + w+ ++ +++ ++ w+
Cysteine arylamidase + + w+ + + + w+
glycerol (PG) and one unknown phospholipid.
Acid phosphatase + w+ + ++ ++ + +++ Source of isolation: Textile dyeing wastewater treatment pond, Kohnoor
Napthol-As-BI-phosphohydrolase +++ – w+ + ++ + ++
Proline arylamidase + + + – + + + Mills, Islamabad, Pakistan
+ – – – + – +
Glutamyl glutamic acid arylamidase
Type strain NCCP-644TT = JCM18977TT = LMG 28467TT = KCTC 42081TT
Symbols: +++, Very strongly positive; ++, strongly positive; +, Positive; w+, weakly positive; –, negative;
Species validated: Validation List No. 161 (2015). IJSEM 65(1): 1-4