Received date: February 23, 2014; Accepted date: March 30, 2014; Published date: March 25, 2014
Citation: Prasad Talluri VSSL, Bhavana M, Siva Kumar K, Anil Kumar P, Rajagopal SV (2014) Myroides gitamensis sp.nov., L-asparaginase Producing Bacteria Isolated from Slaughter House Soil Sample in Visakhapatnam, India. J Microb Biochem Technol 6:144-147. doi: 10.4172/1948-5948.1000135
Copyright: © 2014 Prasad Talluri VSSL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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A novel strain designated as BSH-3T was isolated from the slaughter house soil sample in Visakhapatnam, India. Cells of this strain are Gram-negative, aerobic, yellowish, rod shaped, non-motile and non-spore forming bacteria. This isolate was observed to grow optimally at 30°C and pH 7.0. It requires 1- 6% (w/v) NaCl for its growth. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strain BSH-3T belongs to the genus Myroides and is closely related to Myroides odoratimimus (98.4 %). However, DNA-DNA hybridization with M. odoratimimus JCM 7460T showed a relatedness of 48.2% with respect to strain BSH-3T. The major cellular fatty acids identified were iso-C15:0 and iso-C17:0 3-OH and summed feature 3 (comprising iso-C15:0 2OH and/or C16:1ω7c).The polar lipids of the strain BSH-3T were phospholipids, aminolipids and phosphatidyl ethanolamine. The G + C content of the genomic DNA was determined to be 33.8 mol%. The enzyme production was carried out by submerged fermentation and the maximum enzyme activity was 85.7 IU/gds showed by 24 hrs BSH-3T culture at 37°C. Based on the morphological, phenotypic characteristics, phylogenetic inference and chemotaxonomic studies, strain BSH- 3T is proposed as a novel species Myroides gitamensis sp.nov. in the genus Myroides. The type strain is BSH-3T (= MTCC 11601T = BCC 64301T = MCC 2182T). The GenBank/ EMBL / DDBJ accession number for the 16S rRNA gene sequence of strain BSH-3T is HF571338.
BSH-3T; Myroides gitamensis; 16S rRNA gene; L-asparaginase
In the course of screening L-asparaginase producing bacteria from different soil samples in Visakhapatnam, India, many bacterial strains have been isolated and characterized taxonomically. In the present investigation, one of the isolates was found to be novel and designated as BSH-3T. 16S rRNA gene sequence analysis indicated that the novel strain BSH-3T belongs to family Flavobacteriaceae and genus Myroides. This family was first mentioned by Reichmbach in the first edition of Berger’s manual of systematic bacteriology in 1989. At present, the classification of Flavobacteriaceae is still in development stage. The genus Myroides was established by the reclassification of Flavobacterium odoratum as Myroides odoratus, the type species, on the basis of phenotypic, genomic and chemotaxonomic studies . M. odaratum, M. odoratimimus , M. pelagicus , M. profoundi , M. marinus  are the species present in the Myroides family. The main source for Myroides is clinical specimens, wet environment , insect gut , and marine sample . Myroides strains show susceptibility to β-lactam antibiotics . Nabeel et al.  have reported on the production of L-asparaginase from different microbial species. This is the first available report of L-asparaginase production from the Myroides species.
In this study, taxonomic characteristics of novel strain BSH-3T isolated from the slaughter house soil sample in Visakhapatnam, India is being reported. On the basis of phylogenetic analysis, phenotypic characteristics, chemotaxonomic data, the isolate is considered to represent a novel species in the genus Myroides, for which the name Myroides gitamensis sp.nov is proposed.
Isolation and screening
Strain BSH-3T was isolated from slaughter house (17°.68’N 83°.21’E) soil sample of Visakhapatnam, India. The collected soil sample was serially diluted upto 10-1 to 10-6 and plated on modified Czapek Dox’s agar medium which has the composition of glucose-2.0, L-asparagine-10.0, KH2PO4-1.52, KCl-0.52, MgSO4.7H2O-0.52, CuNO3.3H2O-trace, ZnSO4.7H2O-trace, FeSO4.7H2O-trace (grams per liter of distilled water) at pH 6.2 and 0.09 %(v/v) phenol red was added as an indicator to the medium . The plates were incubated at 37°C for 48 hr. To avoid the fungal contamination, nystatin (50 μg/mL) was added to the medium. Control plates were without L-asparagine (NaNO3 was added as nitrogen source instead of L-asparagine) and phenol red dye. Strains with pink color zones around the colonies were considered as L-asparaginase producing strains (Figure 4).
Estimation of L-Asparaginase activity
L-asparaginase activity was estimated according to Mashburn and Wriston . In this assay, the reaction mixture consisting of 100 μL enzyme extract, 200 μL 0.05 M Tris-Hcl buffer (pH 8.6) and 1.7 mL of 0.01 M L-asparagine was incubated for 10 min at 37°C. The rate of hydrolysis of L-asparagine was determined by measuring the released ammonia using Nessler’s reagent.
The reaction was stopped by the addition of 500 μL of 1.5 M TCA. Then the reaction mixture was centrifuged at 3000 rpm at 4°C for 10 min. The supernatant of 0.5 mL was diluted to 7 mL with distilled water and 1 mL of Nessler’s reagent was added. The reaction mixture was incubated on boiling water bath for 10 min and then cooled. The absorbance (A500) was recorded using UV-Vis spectrophotometer (Shimazdu U-800). One Unit (U) of L-asparaginase is defined as the amount of enzyme required to liberate one μmol of ammonia per min at 37°C
Morphological, phenotypic and biochemical characterization
Cell morphology was observed under Nikon inverted microscope (Eclipse TE200-U) and Scanning electron microscope (JEOL JM – 5600). Fully grown 24 hr culture of BSH-3T was taken and fixed for 2hr in 2% formalin . After washing with saline solution, the culture was dehydrated in 30–100% water - ethanol series. The air-dried bacterium was coated with thin layer of platinum in Gatancryostage (Hitachi S-900 FESEM) and was observed using JEOL JM – 5600 electron microscope at 20 kV accelerating voltage with a 5–6 nm electron beam.
Growth of strain BSH-3T was assessed (i) on nutrient agar (NA: HIMEDIA); (ii) at 5, 10, 16, 25, 30, 37, 42, 46, 52, 57°C in Nutrient Broth (NB); (iii) at pH 3, 4, 5, 6, 7, 7. 5, 8, 8, 5, 9, 10, 11 in NB using 0.1 N HCl and 0.1 N NaOH and (iv) in the presence of 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15 and 18% (w/v) NaCl in NB initially devoid of NaCl. Endospore formation was determined by malachite green staining . Gram staining was done according to Gram . Motility was observed by hanging drop method using 18 hr BSH-3T culture . Different biochemical tests namely Indole test, MR-VP test, Simmons citrate, Starch hydrolysis, H2S production, Catalase, Oxidase, Urease, Nitrate reduction test, Gelatin hydrolysis test were performed . Effects of Tween 20, 40, 60 and 80 (1% w/v; Sigma) on BSH-3T were determined .
16S rRNA sequencing
Genomic DNA of the BSH-3T was isolated as described by Sambrook et al. . Gene specific for 16S rRNA coding regions were amplified by PCR  using universal primers, forward (5'–AGTTTGATCCTGGCTCAG–3') and reverse (5'–GGCT/ TACCTTGTTACGACTT–3'). The numbering of positions in the 16S rRNA gene fragments was based on the E.coli numbering system . Amplified 16S rRNA gene products were purified and sequenced with an ABI PRISM big dye terminator cycle sequencing Ready Reaction kit on an ABI PRISM model 310 genetic analyzer. 16S rRNA gene sequence from the strain BSH-3T was subjected to BLAST sequence for similarity search to identify the nearest taxa. The 16S rRNA gene sequence of the nearest taxa, belongs to family Flavobacteriaceae, were downloaded . Multiple alignment of the sequence was aligned using the CLUSTAL_W in MEGA5 . Phylogenetic trees were constructed using the neighbor-joining  as well as maximum parsimony algorithms and maximum likelihood algorithms . Bootstrap analysis was performed to assess the confidence limits of the branching.
Genomic DNA was isolated by using the method of Marmur  and the mol% G + C content was determined from melting point (Tm) curves  using Lambda 35; Perkin Elmer spectrophotometer equipped with Templab 2.0 software package.
For cellular fatty acids determination, strain BSH-3T was grown on Nutrient agar plates at 37°C for 2 days. Cellular fatty acid methyl ester (FAME’s) was isolated from cells by methylation, saponification and extraction following the standard protocol of Sherlock microbial identification system (MIDI).Separated cellular FAME’s was identified by using Gas chromatography (Agilent 6890 series GC system) . Polar lipids were extracted and analyzed from 36 hrs culture freeze dried cells. They were analyzed by two dimensional (2D) TLC followed by spraying with proper detection reagents . Respiratory quinines were established and determined by HPLC (Agilent 1200 series) by using solvents acetonitrile: isopropanol (65:35), C18 column at 240nm wavelengths and the flow rate was maintained at 1mL min-1 .
DNA- DNA hybridization was carried out to assess the DNA relatedness level between strains BSH-3T and JCM 7460T in triplicates using the DIG high prime DNA labeling kit (Roche Applied science) according to the manufacturer’s instruction . The consequential hybridization signals were captured and analyzed with Bio-Rad Quantity one software (version 4.62). The signals formed by hybridization of the probe to the homologous target DNA were calculated to be 100% and signal intensities by the self-hybridization of serial DNA dilutions were used for the calculation of the levels of DNA relatedness between strain BSH-3T and JCM 7460T. The hybridization experiments were always incorrigible by cross-hybridization.
Cells of the strain BSH-3T were found to be Gram-negative, rod shaped, 0.3-0.4×1.4-1.6 μm in width and length (Figure 1) and nonmotile. The diameter of M. odoratus is relatively very high (11-12 μm) compared to remaining species. All the cells of Myroides sps are having rod shaped morphology. Growth of strain BSH-3T was found to occur in the pH range 5.0-9.0, with optimum growth at pH 7.5. The temperature range for growth was determined to be 10-40°C, with an optimum temperature being 37°C. No growth occurred at 50°C. The identified strain was found to be slightly halophilic depending on the extent of its salt tolerance. The optimum growth occurred between salinity of 2-4% (NaCl, w/v) and upto 6% NaCl (w/v) is tolerated.
Colonies were found to be circular, flat, slimy and yellowish color. Strain BSH-3T was determined to be positive for catalase, oxidase, urease, voges- proskauer, gelatin hydrolysis, citrate and negative for methyl red, casein hydrolysis, arginine dihydrolase, starch hydrolysis, esculin hydrolysis. Almost all strains of Myroides sps exhibits negative for hydrolysis of casein except M. profoundi. The type strain BSH-3T is sensitive to Vancomycin (30 μg), Ciproflaxacin (5 μg), Ofloxacin (5 μg), Sparfloxacin (5 μg), Gatifloxacin (5 μg), Cefradaine (30 μg), Erythromycin (10 μg), Linomycin (10μg), Cloxacillin (15 μg), but resistant to Aztreonam (30 μg), Azithromycin (15 μg), Dobyxy cycline hydrochloride (30 μg), Cefuroxime (30 μg), Penicillin-G (2 Units), Tetracycline (10 μg) and Co-trimoxazole (25 μg). Except M. odoratus, remaining five species showed positive for Leucinearyl amidase. The fatty acid profile of strain BSH-3T found to be dominated by branched and hydroxy fatty acids, notably iso-C15:0 (48.6 %), aneteiso-C15:0 (5.8%), iso-C13:0 (9.7%) and iso-C15:0 3-OH (7.5%). The iso-C15:0 value of BSH-3T is high (48.6) compared to other five Myroides species i.e., M. odoratus (43.7), M. odoratimimus (44.7), M. profoundi (27.6), M. pelgicus (29.3), M. marinus (24.2). The polar lipids of the strainBSH- 3T were one unidentified lipid (UL), five identified aminolipids (AL1 -AL5) and one identified phosphatidyl ethanolamine (PE) and three phospholipids (PL1,PL2,PL3) identified by two dimensional thin layer chromatography (Figure 3). DNA-DNA hybridization of strain BSH-3T with M. odoratimimus JCM 7460T showed a relatedness of only 48.2 %, which is lower than the proposed threshold value of 70%  .The G+C content of strain BSH-3T was determined to be 33.8 mol% (Tm), which is in the accordance with the levels (33.8 to 36.7 mol%) for the genus Myroides . The strain BSH-3T shows maximum enzyme activity was 85.7 IU/gds by 24 hr culture at 37°C under solid state fermentation.
Phylogenetic analysis based on 16S rRNA gene sequence (1449 base pairs) of strain BSH-3T (GenBank/ EMBL / DDBJ accession number HF571338) and other closely related to M. odoratimimus with pairwise sequence similarity of 98.4%. Phylogenetic tree constructed with neighbor-joining, maximum-likelihood and maximum- parsinomy Figure 2.
Therefore, on the basis of phenotypic and phylogenetic differences with the most closely related type strain (Table 1), BSH-3T is considered as a representative of novel species in the genus Myroides, for which the name Myroides gitamensis sp. nov.is proposed
|Summed Feature 3||11.7||16.8||9.5||6.7||1.9||1.3|
Strains: (1) BSH-3T, (2) M. marinus JS-08T, (3) M. odoratimimus JCM7460T, (4) M. profoundi DSM19823T, (5) M. pelagicus KCTC12661T, (6) M. odoratus JCM7458T Data were obtained in this study are taken from Cho et al.  and are percentages of total fatty acids, ‘tr’ indicates trace amount less than 1.0% of the total. Prefix esi, OH and Cre presents iso-branched, hydroxyl fatty acids and cis isomer respectively
Table 1: Cellular fatty acid contents of strain BSH-3T (Myroides gitamensis) and closest related strains of genus Myroides.
Description of Myroides gitamensis sp. Nov.
Myroides gitamensis (gi.tam.en’sis. L. masc. adj. GITAM is the university place in Visakhapatnam, where the type strain was isolated).
The strain BSH-3T were determined to be Gram-negative, rod shaped, 0.3-0.4×1.4-1.6 μm (Figure 1) and non-motile. Colonies were circular, flat, slimy and yellowish color (Table 2). The strain was observed to grow between 10 to 40°C and from pH 5–9, with optimum growth at pH 7.5. Optimum growth was observed to occur between salinity 0.5 to 6.0 % (NaCl, w/v). Strain BSH-3T was determined to be positive for catalase, oxidase, urease, voges- proskauer, gelatin hydrolysis, citrate and negative for methyl red, casein hydrolysis, arginine dihydrolase, starch hydrolysis, esculin hydrolysis. The acid production form is negative for adinotol, sorbitol, dextrose, fructose, inositol, lactose, maltose, mannitol, raffinose and rhamnose. Hydrolysis of tween 40, 60, 80 compounds shows positive. The major fatty acids are iso-C15:0, iso-C17:0 3-OH. The polar lipids of the strain BSH-3T were three identified phospholipids, one unidentified lipids, five identified aminolipids and one identified phosphatidyl ethanolamine by two dimensional thin layer chromatography and summed feature 3 contains iso-C15:0 2OH and/or C16:1 ω7c.The major quinine system is menaquinone-7 (MK-7). The DNA G + C content of this type strain is 33.8 mol%.
|Habitat||Slaughter house soil||Seawater||Wound swab||Deep Sea sediment||Seawater||Feaces|
|Pigmentation||Yellow||Yellow to orange||Pale yellow||Pale yellow||Yellow to orange||Yellow|
|Rods||Short rods||Rods||Rods||Spindle-shaped rods||Short rods||Rods|
|Growth temperature (°C)||10-37||10-37||10-37||10-42||10-37||20-37|
|NaCl range (%, w/v)||0.5-6||0-5||0-6||0-8||0-9||0-5|
|DNA G+C content (mol%)||33.8||34.2||31.7||33||33.6||34.7|
Strains: (1) BSH-3T, (2) M. marinus JS-08T, (3) M. odoratimimus JCM7460T, (4) M. profoundi DSM19823T, (5) M. pelagicus KCTC12661T, (6) M. odoratus JCM7458T [2-4]
Table 2: Features that distinguish Myroides gitamensis sp. nov. from the related Myroides species.
From the present investigation, we conclude that the type strain, BSH-3T (=MTCC 11601T = BCC 64301T = MCC 2182T), isolated from slaughter house soil sample in Visakhapatnam, India was found to be novel. The strain was deposited with the GenBank/ EMBL / DDBJ with accession number for the 16S rRNA gene sequence of strain BSH-3T is HF571338.
The authors are thankful to GITAM university management for providing research facilities for carry out this work. The authors also acknowledge the help of MTCC (Chandigarh), MCC (Pune) and BCC (Thailand) for deposition of this strain as a type strain and providing accession number.