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Description of Lysinibacillus pakistanensis.

Byline: Iftikhar Ahmed, Yeseul Sin, Jayoung Paek, Muhammad Ehsan, Rifat Hayat, Muhammad Iqbal and Young Hyo Chang


The purpose of this addendum is to provide the additional information for validation of Lysinibacillus pakistanensis sp. nov. as a new name under the procedure described in the Bacteriological Code (1990 Revision). The strain NCCP-54T recently published, however it does not meet the basic requirement as it lacks species description according to the rules of International Code of Nomenclature of Prokaryotes. Additionally, the data on DNA-DNA hybridization was required with all the validly recognized species having more than 97% similarity of 16S rRNA gene sequence. The results of this study showed that DNA- DNA relatedness of strain NCCP-54T is below 70% with all the validly recognized species to date. The diagnostic amino acids in cell wall peptidoglycans were re-analyzed and contained Lys-Asp (type A4a). This addendum also provides the formal description of Lysinibacillus pakistanensis sp. nov. Copyright 2014 Friends Science Publishers

Keywords: Lysinibacillus pakistanensis; Cell wall peptidoglycans type A4a; DNA-DNA hybridizationIntroduction

Recently, strain NCCP-54T has been characterized to describe a novel species, Lysinibacillus pakistanensis (Hayat et al., 2013). To validate the name, Lysinibacillus pakistanensis, by International Committee of Systematics in Prokaryotes (ICSP), it must have to meet the basic requirements of the International Code of Nomenclature of Prokaryotes (Bacteriological Code, 1990 Revision). Unfortunately, the name published by the authors (Hayat et al., 2013) did not meet the basic requirement as described in Rule 27 of the International Code of Nomenclature of Prokaryotes; and thus, it has been declined for validation as a new species by ICSP. Additionally, few other species, Lysisnibacillus mangiferihumi (Yang et al., 2012), L. macrolides (Coorevits et al., 2012) and L. contaminans (KAmpfer et al., 2013) have been published and described in the genus Lysinibacillus, during or after the process of publication of Lysinibacillus pakistanensis (Hayat et al.,2013). These species had 16S rRNA gene sequence similarity more than 97% with strain NCCP-54T, which made it mandatory to provide further evidence to establish the novelty of strain NCCP-54T by DNA-DNA hybridization (Stackebrandt and Goebel, 1994) and to describe strain NCCP-54T as Lysinibacillus pakistanensis sp. nov. Previously, Hayat et al. (2013) have reported meso- diaminopimelic acid as a diagnostic amino acid in cell wall peptidoglycans, which is contradictory to the description of genus Lysinibacillus. Thus, the peptidoglycans analysis of strain NCCP-54T is re-performed. This addendum is submitted to meet such requirements for the validation of Lysinibacillus pakistanensis sp. nov.

Materials and Methods

Phylogenetic Analyses

To understand the exact taxonomic position of strain NCCP-54T, an updated phylogenetic analyses were performed with all the validly recognized species of genus Lysinibacillus as described previously (Roohi et al., 2012). Sequences of closely related validly published type strains were retrieved from the database of EzTaxon Server ( MEGA 5.10 was used to assess the molecular evolutionary distances following the Kimura 2-parameter model and phylogenetic trees were constructed using three algorithms: neighbor joining (NJ), maximum likelihood (ML) and maximum parsimony (MP). The bootstrap analysis was performed to assess the stability of relationship by performing 1,000 re-sampling for the tree topology.

DNA-DNA Hybridization and Cell Wall PeptidoglycanAnalysis

In addition to previously described reference strains (Hayatet al., 2013), the additional strains used as reference species

in this study were Lysinibacillus mangiferahumi KCTC33154T, L. macroides KCTC13806 T and L. contaminans KCTC33155T. DNA of NCCP-54T and the reference strains were extracted using the procedure described previously (Ahmed et al., 2007a). DNADNA hybridization was performed with photobiotin-labelled DNA with five replications of each sample at 45C according to the procedure of Ezaki et al. (1989), using Fluoroskan Ascent Plate Reader (Thermo Labsystems, USA) for fluorescence measurements.To determine the peptidoglycan structure of strain NCCP-54T, cell walls were purified from 2 g wet cells of strain NCCP-54T grown in tryptic soy broth (Difco) for 24 h at 30C according to the protocol described earlier (Kawamoto et al., 1981). The cells were disrupted by sonication for more than 45 min in presence of phosphate buffer (0.05 M Na2HPO4/0.05 M KH2PO4; pH 7.2). The broken cells were reacted with 25% SDS solution at 100C for 40 min and after washing with phosphate buffer (pH 7.2), subsequently suspended in Pronase E solution, and incubated at 37C for 2 h. The broken cells were washed using phosphate buffer (pH 7.6) and treated with 5% trichloroacetic acid (TCA) solution at 100C for 20 min. The precipitated cell walls were washed with Milli-Q water, ethanol and diethyl ether and dried in vacuum. The purified cell walls were hydrolyzed with 6N HCl at 100C for 18 h. The amino acids in the cell wall peptidoglycans were analyzed quantitatively using automatic Amino Acid Analyzer (Hitachi, Japan).


Based on comparison of 16S rRNA gene sequence (1484 bp) with closely related type strains, at least seven species showed the similarity values above 97% with strain NCCP-54T. The results indicated that NCCP-54T was most closely related to L. macroides (99.12%), L. xylanilyticus (99.11%), L. fusiformis (98.57%), L. boronitolerans (98.40%), L. sphaericus (98.38%). L. contaminans (97.63%) and L. mangiferahumi (97.52%). DNA-DNA hybridization was performed with the species having greater than 97% similarity of 16SrRNA gene sequence (Table 1), which have not beenincluded in comparison studies previously (Hayat et al.,2013) to establish the novelty of NCCP-54T. The results revealed that DNA-DNA hybridization values of strainNCCP-54T with the reference species were less than 31%(Table 1). Strain NCCP-54T contained alanine, glutamic acid, lysine and aspartic acid as diagnostic amino acids incell wall peptidoglycans. An estimate of the molar ratio of these amino acids (alanine: glutamic acid: lysine: aspartic acid) in the peptidoglycan was obtained as 1.62: 1.00: 0.64:0.62.


An updated taxonomic position of strain NCCP-54T was revealed by the phylogenetic analysis and the identification command on EzTaxon Server (http://eztaxon- Phylogenetic relationship of NCCP-54T with closely related species of Lysinibacillus showed close association with L. macroides, L. xylanilyticus, and L. boronitolerans at high bootstrap values of greater than 80% and formed a coherent cluster in neighbor-joining phylogenetic tree (Fig. 1). This association was also confirmed by MLH and MP algorithms.Among the closely related taxa with more than 97% similarity of 16S rRNA gene sequence, three species, Lysinibacillus macroides (Coorevits et al., 2012), L. contaminans (KAmpfer et al., 2013) and L. mangiferahumi (Yang et al., 2012), were described in the genus Lysinibacillus, during or after the process of publication of L.pakistanensis (Hayat et al., 2013), whereas the others have been studied in comparison to strain NCCP-54T previously (Hayat et al., 2013). Thus, to establish the novelty of NCCP-54T, the DNA-DNA hybridization was performed with the species having greater than 97% similarity of 16S rRNA gene sequence, which have not been included in comparison studies previously (Hayat et al., 2013). The results revealed that DNA-DNA hybridization values between strain NCCP-54T and the reference strains were less than 31% (Table 1). These values were less than the 70% threshold indicating that NCCP-54T still represents a separate species in thegenus Lysinibacillus (Stackebrandt and Goebel, 1994).Contradictory to the results of peptidoglycans reported by Hayat et al. (2013), strain NCCP-54T contained Lys-Asp as a diagnostic amino acids in cell wall peptidoglycans, which corresponds to variation type A4a. The genus Lysinibacillus have also been reported with variation type A4a peptidoglycan (Ahmed et al., 2007b) and thus, these results are in agreement with the genus description.To meet the basic requirements for validation of a new name, Lysinibacillus pakistanensis sp. nov. according to the Rules of Bacteriological Code of Nomenclature of Prokaryotes, the description of species, Lysinibacillus pakistanensis sp. nov. has been compiled here mainly from the data reported by Hayat et al. (2013) and included as follows:

Description of Lysinibacillus pakistanensis sp. nov

Lysinibacillus pakistanensis (pa.kis.tan.en'sis. N.L. masc. adj. pakistanensis from Pakistan, where the organism was isolated).Cells are rod-shaped, aerobic, motile and Gram- positive. Spherical or ellipsoidal endospores are produced terminally or sub-terminally in slightly bulging sporangia. The colonies are rough having dull surface and are transparent. Texture is butyrous (butter like) and elevation is flat. Diameter of colony is 14 mm and margins are lubatein the older colonies. The color of colony is off white initially and turns to light yellow in older colonies. The form of colony is punctiform and on 2nd to 3rd day spreads on thewhole surface of the plate. Cells can grow at 1045?C (optimum at 28?C), at pH ranges of 6.09.0 (optimum at pH7.0) and can survive in medium with 06% (w/v) NaCl. It can also be able to tolerate 0150 mM boron (optimum growth without boron) in the media. Positive for catalase and can reduce nitrate to nitrogen but negative for oxidase. No acid production observed from sugar contained in API-50CH and API-20E galleries (bioMACopyrightrieux, France). Strong enzyme activity for acid phosphatase and Esterase Lipase (C8), but weak activity for alkaline phosphatase and a-

Table 1: 16S rRNA gene sequence similarity and DNA-DNA relatedness between strain NCCP-54T and the closely related

type species of genus Lysinibacillus [additional to data presented by Hayat et al. (2013)]

Strains###Lysinibacillus pakistanensis NCCP-54T (AB558495)

###16S rRNA gene sequence similarity (%)###DNADNA Hybridization value (%)

Lysinibacillus pakistanensis NCCP-54T (AB558495)###100.0###100.0

Lysinibacillus macroides LMG18474T (AJ628749)###99.12###25.1

Lysinibacillus contaminans KCTC33155T (KC254732)###97.63###21.6

Lysinibacillus mangiferahumi M-GX18T (JF731238)###97.52###22.2

Lysinibacillus composti NCCP-36T (AB547124)###96.90###27.0

Lysinibacillus sinduriensis KCTC13296T (FJ169465)###96.38###31.0

Lysinibacillus odyssey KCTC 3961T (AF526913)###95.81###22.0

Lysinibacillus massiliensis KCTC13178T (AY677116)###95.77###19.0

Lysinibacillus [additional to data presented by Hayat et al. (2013)]

chymotrypsin, whereas negative for all other enzymes of API-Zym (bioMACopyrightrieux, France). Positive for L-alaninamide, a-ketovaleric acid, dextrin and acetic acid; whereas negative for pyruvic acid, alanine, pyruvatic acid methyl ester, thymidine, glutamic acid, AY-hydroxybutyric acid, adenosine, thymidine-5'-Monophosphate, succinic acid mono-methyl ester and D-lactic acid methyl ester (Biolog GP2 MicroPlate system; BIOLOG, USA). Major polar lipids are phosphatidylglycerol diphosphatidylglycerol and phosphatidylethanolamine. The predominant cellular fatty acids are iso-C15:0, iso-C16:0, iso-C16:1?7c alcohol, iso-C17:0, iso-C17:1 ?10c, iso-C16:1 ?11c, C16:0, anteiso-C15:0, anteiso- C17:0, and anteiso-C14:0. Diagnostic amino acids of cell wall peptidoglycans were Lys-Asp (which corresponds to peptidoglycan variation type A4a). MK-7 is the major menaquinone. The DNA G+C contents of the type strain is37 mol%.Strain NCCP-54T (=DSMZ 24784T = KCTC13795T = JCM 18776T) is the type strain, isolated from rhizosphericsoil of legume (Glycine max L.) sampled from the experimental area of Soil Science Department located at Research Farm of Pir Mehr Ali Shah Arid AgricultureUniversity, Rawalpindi, Pakistan.The DDBJ/EMBL/GenBank accession number of the16S rRNA gene sequence of the novel strain NCCP-54T isAB558495.


We gratefully acknowledge the kind help of Dr. Takuji Kudo from JCM, Tsukuba, Japan in analysis of cell wall peptidoglycans. References

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Author:Ahmed, Iftikhar; Sin, Yeseul; Paek, Jayoung; Ehsan, Muhammad; Hayat, Rifat; Iqbal, Muhammad; Chang,
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Apr 30, 2014
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