Research Article Characterization of Antibiotic Producing

Research ArticleCharacterization of Antibiotic Producing RareActinomycete Nonomuraea sp JAJ18 Derived froman Indian Coastal Solar Saltern

Polpass Arul Jose Kunjukrishnan Kamalakshi SivakalaPandiyan Rajeswari and Solomon Robinson David Jebakumar

Department of Molecular Microbiology School of Biotechnology Madurai Kamaraj University Madurai 625 021 India

Correspondence should be addressed to Solomon Robinson David Jebakumar jsolomon mrnayahoocom

Received 31 July 2014 Revised 10 November 2014 Accepted 28 November 2014 Published 18 December 2014

Academic Editor Wen-Jun Li

Copyright copy 2014 Polpass Arul Jose et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Rare actinomycete genera are accepted as a promising source of novel metabolites having pharmaceutical importance One suchgenus of rare actinomycete is Nonomuraea The present study was aimed at characterizing the antibiotic producing Nonomuraeastrain JAJ18 whichwas previously isolated from coastal solar saltern Strain JAJ18 was recognized as amember of genusNonomuraeabased on its almost complete 16S rRNA gene sequence and phenotypic characteristics The strain JAJ18 was found to beclosely related to Nonomuraea maheshkhaliensis 16-5-14T (9890) Nonomuraea candida HMC10T (9858) and Nonomuraeajabiensis A4036T (9843) From cell-free culture broth of strain JAJ18 an antibiotic was extracted and purified by silica columnchromatography The obtained antibiotic was found to be active against a range of Gram-positive and Gram-negative bacteriaincluding drug-resistant Staphylococcus with minimal inhibitory concentration (MIC) ranging from 05 to 160120583gmLminus1 Thestructural characteristics of antibiotic were determined by FTIR and NMR spectroscopy The antibiotic was identified to be analiphatic rich compound with significant dissimilarity to known antibiotics reported frommembers of the genus Nonomuraea Asthe trends to discover novel metabolites fromNonomuraea are vibrant further studies are needed to understand the structural andbiotechnological significance of antibiotic compound produced by Nonomuraea sp JAJ18

1 Introduction

Enduring infectious diseases and rapidlymounting antibioticresistance have intensified the search for new antibioticsin order to maintain a pool of effective antibiotics againstthe pathogenic microorganisms In recent years rare acti-nomycetes are considered as potential producers of novelbioactive compounds [1 2] The rare actinomycetes that areoften very difficult to isolate and cultivate might representa unique source of novel biologically active compounds[3] Some genera of this group are Actinomadura Actino-alloteichus Actinoplanes Amycolatopsis ActinokineosporaAcrocarpospora Actinosynnema Catenuloplanes Cryptospo-rangium Dactylosporangium Kibdelosporangium Kineospo-riaKutzneriaMicrobisporaMicrotetrasporaNocardiaNon-omuraea Planomonospora Planobispora Pseudonocardia

Saccharomonospora Saccharopolyspora Saccharothrix Sali-nispora Streptosporangium SpirilliplanesThermomonosporaThermobifida and Virgosporangium [4]

Nonomuraea is less known among the rare actinomycetegenera as its taxonomic positionwas revised several times [5]The genus Nonomuraea was originally proposed by Zhanget al [6] as a member of the family Streptosporangiaceaewhich forms extensively branched substrate and aerial myce-lia On the basis of detailed polyphasic taxonomical analy-sis the genus currently (November 2014) comprises around36 species and 2 subspecies (httpwwwbacterionetnono-muraeahtmlmaheshkhaliensis) Themembers of this genushave been isolated fromvarious soil andplant samples includ-ing mangrove rhizosphere mud [7] cave soil [8] arid soil [9]acidic soil [10] coastal sediments [11] and medicinal plants[12] Members of genus Nonomuraea have been recognized

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 456070 7 pageshttpdxdoiorg1011552014456070

2 The Scientific World Journal

as a valuable source of novel bioactive metabolites showingantimicrobial anticancer anthelmintic and antipsychoticactivities To this date some antibiotic compounds derivedfrom different species of genus Nonomuraea are actinotiocin[13] maduramycin [14] glycopeptide antibiotic A40926 [15]and pyralomicins [16] The trend to discover novel metabo-lites from the genusNonomuraea has been augmented whichis a challenging signal for further exploration of this naturalcompounds producing resource [5]

The Nonomuraea sp JAJ18 taken for this study waspreviously isolated from a coastal solar saltern [17] whichshows antimicrobial activity against a range of bacteriaThe objective of this paper was to describe strain JAJ18 togenus level based on its complete 16S rRNA gene sequenceand phenotypic characteristics purify the antibiotic fromits culture broth and disclose structural characteristics andantibacterial potential of the purified compound

2 Materials and Methods

21 Strains andTheir Maintenance Antagonistic rare actino-mycete strain Nonomuraea sp JAJ18 was previously isolatedfrom a hypersaline coastal solar saltern [17] Pure culture ofthis strain was maintained over modified inorganic salt agarslants which contained starch 100 g yeast extract 40 g NaCl200 g NH

4

SO4

20 g MgSO4

sdot7H2

O 10 g K2

HPO4

10 g and220 g of agar in 10 liter of distilled water The bacterial teststrains methicillin-resistant Staphylococcus aureus (MRSA)Bacillus subtilisMTCC 441 Klebsiella pneumoniaMTCC 109Salmonella typhi MTCC 733 and Proteus vulgaris MTCC426 used in this study for antimicrobial assays were culturedin Mueller Hinton agar slants The MRSA was obtainedfrom Kovai Medical Center and Hospital Coimbatore IndiaOther bacterial strains were obtained from Microbial TypeCulture Collection (MTCC) Institute of Microbial Technol-ogy Chandigarh India

22 Colonial Cultural and Physiological CharacterizationCultural characteristics of JAJ18 were observed in twelvedifferent agar media starch casein agar [20] potato dextroseagar glycerol nitrate agar [21] asparagine vitamin agarsucrose nitrate agar glucose asparagine agar [22] and seriesof ISP media such as yeast extract maltose agar ISP2 oatmealagar ISP3 inorganic salt starch agar ISP4 glycerol asparagineagar ISP5 peptone yeast extract iron agar ISP 6 and tyrosineagar ISP 7 [23]The agar plates were inoculated and incubatedat 30∘C for 7 d The plates were then visually examinedto determine aerial spore-mass colour substrate mycelialpigmentation and the colour of diffusible pigments

The growth temperature range (15 20 25 30 37 45 or50∘C) and pH range (pH 45 5 6 68 72 9 and 10) forthe growth of strain JAJ18 were determined on modifiedISP-4 after culturing for 2 weeks in shake flasks (250 rpm)for 10 days Salt tolerance of the actinomycete isolate JAJ18was determined on starch nitrate medium prepared withseries of NaCl concentrations between 0 and 30 (0 to 5M)according to Kushner [24] Reduction of nitrate degradationof gelatin and casein andproduction of catalase andH

2

Swere

examined as described by Gordon et al [25] Utilization ofa range of different sole carbon sources was tested on ISP9medium [23]

23 16S rRNA Gene Amplification and Cloning For DNAisolation biomasswas obtained by growing the strain JAJ18 inTrypticase soy broth for 7 d at 30∘C with agitation in 500mLflasks containing 100mL of the medium From the biomassDNA was isolated using standard phenol-chloroform extrac-tion procedure [26] The 16S rRNA gene was amplified fromthe isolated DNA using universal eubacterial primer set 27F51015840 AGTTTGATCCTGGCTCAG 31015840 and 1492R 51015840ACGGCTACC TTG TTA CGA CTT 31015840 [27] PCR amplification wascarried out in a 50 120583L reaction mixture according to Thineshet al [28] The PCR amplified 16S rRNA gene was purifiedusing PCR product purification spin kit (HiPurA HiMediaIndia) Purified 16S rRNA gene amplicon was cloned usingpGEM-T Easy vector (Promega) with E coli DH10B cellsThe plasmid DNA of the recombinant clone was isolatedand sequenced by an automated sequencer (Genetic Analyzer3130 Applied Biosystems USA) using the vector-specificprimers SP6 and T7 promoter

24 Phylogenetic Analysis Almost complete 16S rRNA genesequences of strain JAJ18 and those of other related speciesderived from GenBank were aligned using ClustalW Phy-logenetic tree was inferred by using suitable programs ofthe PHYLIP (Phylogeny Inference Package) version 368Nucleotide distances were calculated according to the algo-rithm of Jukes and Cantor by using DNADIST and aneighbor-joining tree was constructed using NEIGHBORfrom the PHYLIP program package [29] The robustnessof tree topology was assessed by bootstrap analysis of theneighbor-joining datasets on 1000 resamplings using theSEQBOOT and CONSENSE options from the same PHYLIPpackage

25 Production and Extraction of Antibiotic For preparationof seed culture spore suspensions of JAJ18 were inoculatedin modified ISP-4 broth and incubated for 3 d at 28∘C ona rotary shaker (220 rpm) A total of 2mL seed culturewas transferred into 200 mL production medium containingstarch 100 g yeast extract 40 g NaCl 50 g NH

4

SO4

20 gMgSO

4

sdot7H2

O20 g K2

HPO4

10 g CaCO3

10 g FeSO4

sdot7H2

O0010 g ZnSO

4

sdot7H2

O 0001 g MnCl2

sdot4H2

O 0001 g andCuSO

4

sdot5H2

O 0001 g in 1 litre of distilled water for antibioticproduction The flask was incubated for 10 d at 30∘C on arotary shaker with 220 rpm After the incubation period theculture broth was centrifuged at 10000 rpm for 10min toremove the mycelial biomass Ethyl acetate was added to thesupernatants in 1 1 proportion and the mixture was agitatedfor 45minThe solvent layer was separated and centrifuged at5000 rpm for 15min to remove traces of fermentation brothThe ethyl acetate fraction was evaporated and the resultantcrude antibiotic was suspended in 50 120583L of methanol whichwas then checked for antibiotic activity

The Scientific World Journal 3

Table 1 Growth characteristics of strain JAJ18

Agar media Growth Substrate mycelium Aerial mycelium PigmentISP2 Poor Yellow None Golden yellowISP3 Good Brick red White Brick redISP4 Good White White NoneISP5 Good Light wheat Wheat NoneISP6 Good Yellowish red None Yellowish redISP7 Good Mint cream Wheat NoneStarch casein agar Good Wheat Dark wheat NonePotato dextrose agar Poor Red None NoneGlycerol nitrate agar Good Light yellow Mint cream NoneAsparagine vitamin agar Good Light yellow Mint cream NoneSucrose nitrate agar Poor Transparent Colorless NoneGlucose asparagine agar Good Wheat Light wheat None

26 Purification of Antibiotic The purification of active frac-tion was initiated with fractionation of crude extract bysilica column chromatography using gradient solvent system(methanol ethyl acetate gradient 10ndash100) monitored bythin layer chromatography (ethyl acetate methanol 92 08)The purified fractions were screened for antimicrobial activ-ity by disc diffusion method Mueller Hinton agar (HiMediaIndia) plates were inoculated with the test microorganismsby spreading the microbial inoculums on the surface of themedia Purified fractions were loaded on 6 mm sterile discsdried and placed on the surface of the medium inoculatedwith test microorganism Plates were incubated at 37∘C for24 h and observed for zone of inhibition around the discs

27 Spectral Analysis of Purified Compound Purified activecompound was subjected to partial characterization using aseries of spectroscopic methods like FT-IR NMR and MSThe FT-IR (KBr) spectrum of the compounds was recordedon Shimadzu FTIR-8400S spectrophotometer Band posi-tions are reported in reciprocal centimeters (cmminus1) Inter-pretation of FTIR spectrum was done according to infraredfrequencies described by Coates [30] The 1H and 13C NMRspectra of the compounds in DMSOCDCl

3

were measuredat 300MHz and 75MHz respectively onAVANCE 300NMRspectrometer (Bruker USA) Tetramethylsilane (TMS) wasused as internal standard for calibration of chemical shifts of1H and 13C NMR spectra Chemical shifts were reported inparts per million (120575)

28 Determination of Antimicrobial Potential Purified com-pound with antibiotic activity was subjected to determina-tion of minimal inhibitory concentration (MIC) and min-imum bactericidal concentration (MBC) to disclose theirantimicrobial potential The MIC values were determinedby microdilution broth method as per the guidelines ofClinical and Laboratory Standards Institute [31] Minimumconcentration of compound that showedge999 reduction ofthe original bacterial inoculums was determined as the MBC[32] MIC and MBC values were compared with those of anideal broad-spectrum antibiotic erythromycin

3 Results and Discussion

31 Colonial Cultural and Physiological Characteristics ofJAJ18 The JAJ18 had pink coloured aerial mycelium andcherry red coloured substrate mycelium without diffusiblepigments on modified ISP4 The growth characteristics ofNonomuraea sp JAJ18 on twelve different agar media aresummarized in Table 1 The strain JAJ18 grew well on most ofthe tested agar media except on sucrose nitrate agar potatodextrose agar and yeast extract maltose agar Vegetativemycelium was initially white yellow or yellowish red ondifferent media and it was gradually darkened in old cultureLikewise aerial mycelia colour was white mint cream orwheat colour depending on the medium Mild diffusibleyellow and brick red coloured pigments were observed inISP2 and ISP3 agar plates respectively

The strain JAJ18 was studied for its range of biochemical(Table 2) and physiological (Table 3) characteristics Thestrain JAJ18 utilized dextrose cellobiose fructose maltoserhamnose salicin sucrose trehalose and sodium citrate Itwas negative for hydrolysis of casein and gelatin growthin Sabouraud dextrose agar and MacConkey agar and thereduction of nitrate Growth of JAJ18 occurred in the rangeof pH (6 to 9) and temperature (25 and 45∘C) Good growthwas shown in the absence as well as in the presence of NaClup to 2 (wv)

Phenotypic properties (growth and biochemical andphysiological characteristics) of strain JAJ18 were comparedwith the nearest valid species Nonomuraea maheshkhaliensis16-5-14T Nonomuraea candida HMC10T and NonomuraeajabiensisA4036T (Table 4) Strain JAJ18 differs from its neigh-bours in its growth and pigmentation on ISP 3 medium salttolerance and carbon source utilization The JAJ18 producedbrick red colour diffusible pigment on ISP3 which has notbeen observed in closely related strains In the case of carbonsource utilization disparity was found in its inability to utilizeinositol and mannitol

32 Molecular Phylogeny of Strain JAJ18 An almost com-plete 16S rRNA gene sequence of strain JAJ18 was obtained


Table 2 Biochemical characteristics of strain JAJ18

Characteristics ResultGrowth on

Sabouraud dextrose broth minus

MacConkey agar minus

Hydrolysis ofCasein minus

Gelatin minus

Production of H2S minus

Reduction of nitrate minus

Production of catalase +Utilization of

Adonitol plusmn

Arabinose plusmn

Cellobiose +Dextrose +Fructose +Inositol minus

Maltose +Melibiose minus

Mannose +Mannitol minus

Rhamnose +Salicin +Sucrose +Sorbitol minus

Xylose plusmn

Trehalose ++ positive plusmn doubtfulpoor minus negative

(1484 bp) and submitted to GenBank with accession num-ber JN8590052 The 16S rRNA-based phylogenetic analysisshowed that strain JAJ18 was closely related to Nonomuraeamaheshkhaliensis 16-5-14T (9890) Nonomuraea candidaHMC10T (9858) Nonomuraea jabiensis A4036T (9843)Nonomuraea kuesteri GW 14-1925T (9836) and Nonomu-raea salmonea DSM 43678T (9827) In neighbour-joiningphylogenetic tree (Figure 1) strain JAJ18 formed a separateclade with Nonomuraea jabiensis A4036T

33 TLC Profile of Ethyl Acetate Extract from JAJ18 Thinlayer chromatograph of ethyl acetate extract of cell-freefermentation broth indicated the presence of five compoundswith different Rf values The chromatograph was developedusing iodine vapor and visualized under UV light Thecompounds were denoted as 18a 18b 18c 18d and 18e whichhad Rf values of 0067 0581 0527 05 and 045 respectively

34 Purification of Antibiotic Produced by Nonomuraea spJAJ18 An antibiotic produced by Nonomuraea sp JAJ18was purified using the silica column monitored with TLCPurification step yielded 13 fractions numbered from J18-1to J18-13 All the fractions were screened for antibacterialactivity against Bacillus subtilis by disc diffusion method

Table 3 Physiological characteristics of strain JAJ18

Characteristics ResultGrowth at initial pH5 plusmn

6 ++68 ++72 ++9 +

Growth at temperature (∘C)15 minus

25 ++37 ++45 +50 minus

Growth in NaCl ()0 ++1 ++2 ++3 plusmn

4 minus

++ positive + moderate plusmn doubtfulpoor minus negative

Purified fraction J18-04 that showed antibiotic activity wasgolden yellowish in colour with a greasy appearance Rf valueof the purified fraction was found to be 058 in TLC Thepurified antibiotic was designated as J18-04

35 Spectral Characteristics of Purified Antibiotic J18-04Infrared spectra of J18-04 in KBr showed alcohol func-tion (346427 cmminus1) CndashH stretch of saturated hydrocarbon(298591 and 293768 cmminus1) carbonyl function (174178 cmminus1)conjugated C=O (165498 cmminus1) aliphatic nitro function(137529 cmminus1) CndashO bends (115540ndash104738 cmminus1) and =CndashH bends (1000ndash650 cmminus1) 1H-NMR spectrum of antibioticJ18-04 in DMSOCDCl

3

exhibited signals at 120575 H 199 215218 220 250 535 576 724 727 736 738 741 and 769The signals shown in the 05-2 and 2-25 regions indicate thepresence of alkyl protons Similarly signals at 535 and 575clearly showed presence of alkenes 13C-NMR spectrum ofJ18-04 exhibited some common main signals at 120575 C 13761877 2204 2240 2850 2895 2906 3122 3364 and 17433The signals between 1376 and 3364 indicate carbons ofmethyl andmethylene moietiesThe signal at 17433 indicatesthe presence of carbonyl carbon

The spectral data confirmed that purified compoundcontains more aliphatic units and range of functional moi-eties such as carbonyl function aliphatic nitro function andaliphatic halogen groups It shared significant dissimilaritywith the structural nature antibiotics such as actinotiocin1500 [13] maduramycin 937 [14] glycopeptide antibioticA40926 1300 [15] and pyralomicins derived from genusNonomuraea However deeper studies will be needed toestablish chemical structure of this active compound


Table 4 Comparison of the phenotypic properties of Nonomuraea sp JAJ18 with Nonomuraea maheshkhaliensis 16-5-14T NonomuraeacandidaHMC10T and Nonomuraea jabiensis A4036T

Characteristics JAJ18 HMC10T [18] 16-5-14T [7] A4036T [19]Growth on ISP 3 medium

Aerial mycelium White White White NoneSubstrate mycelium Orangered Cream Light wheat Brownish orange

Growth on sole carbon sources (1 wv)Adonitol plusmn ND ND +Arabinose plusmn plusmn + NDCellobiose + plusmn + minus

Dextrose + ND + NDFructose + + + +Inositol minus + plusmn NDMaltose + ND ND +Mannose + + + +Mannitol minus ND + +Rhamnose + + + NDSucrose + + + +Sorbitol minus ND ND minus

Xylose plusmn + + +Growth temperature range 23ndash37∘C 30ndash45∘C 20ndash37∘C 20ndash37∘CGrowth at 3 NaCl minus + + minus

+ positive plusmn doubtfulweak minus negative ND not determined

Nonomuraea jiangxiensis FXJ1102 (FJ418910)

Nonomuraea sp JAJ18 (JN8590052)

Nonomuraea maritima FXJ7203 (GU002054)

Nonomuraea solani NEAU-Z6 (JQ073731)

75

58

91

96

88

74

64

92

70

100

001

Nonomuraea fuscirosea NEAU-dht8T (KC417350)

Nonomuraea maheshkhaliensis 16-5-14T (AB290014)

Nonomuraea kuesteri GW14-1925T (AJ746362)

Nonomuraea coxensis DSM 45129T (ARBV01000098)

Nonomuraea bangladeshensis 5-10-10T (AB274966)

Nonomuraea angiospora IFO 13155T

T

(U48843)

Nonomuraea candida HMC10T (DQ285421)

Nonomuraea jabiensis A4036T (HQ157186)

Nonomuraea salmonea DSM 43678TT (X97892)

Nonomuraea endophytica YIM 65601T

T

T

(GU367158)

Actinoallomurus acaciae GMKU 931T (EU429322)

Figure 1 Neighbour-joining tree based on almost complete 16S rRNA gene sequences of JAJ18The phylogenetic tree shows the relationshipsbetweenNonomuraea sp JAJ18 andmembers of the genusNonomuraeaActinoallomurus acaciaGMKU931T was used as out-group Numbersat nodes indicate the levels of bootstrap support () based on a neighbour-joining analysis of 1000 resampled datasets Score bar represents1 nucleotide substitution per 100 nucleotides


Table 5 Antimicrobial potential of antibiotic J18-04 against diff-erent bacteria

Test organisms MIC (MBC)lowast (120583gmLminus1)Compound Erythromycin

Bacillus subtilisMTCC 441 05 to 2 (gt2) 05 to 1 (gt2)Klebsiella pneumoniaeMTCC 109 4 to 8 2 to 3MRSA (clinical strain) 4 to 16 (gt16) gt128 (gt134)Salmonella typhiMTCC 733 2 to 3 (gt4) 2 to 4 (gt4)Proteus vulgarisMTCC 426 2 to 4 (gt4) 1 to 4 (gt2)lowastValues in the parenthesis are MBCs

36 In Vitro Antimicrobial Potential of J18-04 The in vitroantimicrobial potential of the purified antibiotic from JAJ18was evaluated against a list of bacteriaThe compound J18-04showed potent inhibitory activity against both of the Gram-positive and Gram-negative bacteria (Table 5) The minimalinhibitory concentrations (MICs) of J18-04 observed fortested bacterial strains were in the range between 05 and16 120583gmLminus1 Similarly MBC values were in the range between2 and gt16 120583gmLminus1 Moreover it was found to be comparablewith MICs and MBCs of the broad-spectrum antibioticerythromycin

4 Conclusion

In summary the strain JAJ18 was characterized to be amember of rare actinomycete genus Nonomuraea and wasdesignated as Nonomuraea sp JAJ18 It is clear that theNonomuraea sp JAJ18 produces extracellular antibioticwhichis effective against a range of bacterial strains includingMRSA The antibiotic purified from Nonomuraea sp JAJ18was designated as J18-04 Spectral characteristics of purifiedcompound showed presence of a range of functional moietiesdissimilar to previously reported antibiotics of Nonomuraeaspecies The results reveal the importance and need ofcomplete structural and in vitro pharmacological studies touncover the industrial importance of identified antibioticJ18-04

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors acknowledge the financial supports fromthe Council of Scientific and Industrial Research-HumanResource Development Group (CSIR-HRDG) India for thefunding through Senior Research Fellowship

References

[1] K Tiwari and R K Gupta ldquoRare actinomycetes a potentialstorehouse for novel antibioticsrdquo Critical Reviews in Biotechnol-ogy vol 32 no 2 pp 108ndash132 2012

[2] P A Jose and S R D Jebakumar ldquoNon-streptomycete actino-mycetes nourish the current microbial antibiotic drug discov-eryrdquo Frontiers in Microbiology vol 4 article 240 2013

[3] R H Baltz ldquoMarcel Faber Roundtable is our antibiotic pipelineunproductive because of starvation constipation or lack ofinspirationrdquo Journal of Industrial Microbiology and Biotechnol-ogy vol 33 no 7 pp 507ndash513 2006

[4] K Tiwari and R K Gupta ldquoDiversity and isolation of rareactinomycetes an overviewrdquo Critical Reviews in Microbiologyvol 39 no 3 pp 256ndash294 2013

[5] R Sungthong and N Nakaew ldquoThe genus Nonomuraea areview of a rare actinomycete taxon for novel metabolitesrdquoJournal of Basic Microbiology 2014

[6] Z Zhang Y Wang and J Ruan ldquoReclassification of Ther-momonospora and Microtetrasporardquo International Journal ofSystematic Bacteriology vol 48 no 2 pp 411ndash422 1998

[7] I Ara T Kudo A Matsumoto Y Takahashi and S OmuraldquoNonomuraea maheshkhaliensis sp nov a novel actinomyceteisolated from mangrove rhizosphere mudrdquo Journal of Generaland Applied Microbiology vol 53 no 3 pp 159ndash166 2007

[8] N Nakaew R Sungthong A Yokota and S Lumyong ldquoNono-muraea monospora sp nov an actinomycete isolated from cavesoil in Thailand and emended description of the genus Nono-muraeardquo International Journal of Systematic and EvolutionaryMicrobiology vol 62 no 12 pp 3007ndash3012 2012

[9] M Camas A Sazak C Sproer et al ldquoNonomuraea jabiensis spnov isolated from arid soilrdquo International Journal of Systematicand Evolutionary Microbiology vol 63 no 1 pp 212ndash218 2013

[10] X Li L Zhang Y Ding Y Gao J Ruan and Y HuangldquoNonomuraea jiangxiensis sp nov isolated from acidic soilrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 62 no 6 pp 1409ndash1413 2012

[11] L Xi L Zhang J Ruan and Y Huang ldquoNonomuraea maritimasp nov isolated from coastal sedimentrdquo International Journalof Systematic and Evolutionary Microbiology vol 61 no 11 pp2740ndash2744 2011

[12] J Li G-Z Zhao H-Y Huang et al ldquoNonomuraea endophyticasp nov an endophytic actinomycete isolated from Artemisiaannua Lrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 61 no 4 pp 757ndash761 2011

[13] A Tamura R Furuta S Naruto and H Ishii ldquoActinotiocina new sulfur containing peptide antibiotic from Actinomadurapusillardquo The Journal of Antibiotics vol 26 no 6 pp 343ndash3501973

[14] W F Fleck D G Strauss J Meyer and G Porstendorfer ldquoFer-mentation isolation and biological activity of maduramycina new antibiotic from Actinomadura rubrardquo Zeitschrift furAllgemeine Mikrobiologie vol 18 no 6 pp 389ndash398 1978

[15] N Gunnarsson P Bruheim and J Nielsen ldquoProduction ofthe glycopeptide antibiotic A40926 by Nonomuraea sp ATCC39727 influence of medium composition in batch fermenta-tionrdquo Journal of Industrial Microbiology and Biotechnology vol30 no 3 pp 150ndash156 2003

[16] P M Flatt X Wu S Perry and T Mahmud ldquoGenetic insightsinto pyralomicin biosynthesis in Nonomuraea spiralis IMC A-0156rdquo Journal of Natural Products vol 76 no 5 pp 939ndash9462013

[17] P A Jose and S R D Jebakumar ldquoPhylogenetic diversity ofactinomycetes cultured from coastal multipond solar saltern inTuticorin Indiardquo Aquatic Biosystems vol 8 no 13 article 232012


[18] M le Roes and P R Meyers ldquoNonomuraea candida sp nov anew species fromSouthAfrican soilrdquoAntonie van Leeuwenhoekvol 93 no 1-2 pp 133ndash139 2008


[20] E M H Wellington and T Cross ldquoTaxonomy of antibioticproducing Actinomycetes and new approaches to their selectiveisolationrdquo in Progress in Industrial Microbiology M E BushellEd Elsevier Amsterdam The Netherlands 1983

[21] Y Okami S Arima and M Suzuki ldquoInfluence of agar on themorphology and pigmentation of Streptomycetesrdquo Applied andEnvironmental Microbiology vol 11 pp 493ndash497 1963

[22] R E Gordon and M M Smith ldquoProposed group of charactersfor the separation of Streptomyces and Nocardiardquo Journal ofBacteriology vol 69 no 2 pp 147ndash150 1955

[23] E B Shirling and D Gottlieb ldquoMethods for characterizationof Streptomyces speciesrdquo International Journal of SystematicBacteriology vol 16 pp 313ndash340 1966

[24] D J Kushner ldquoGrowth and nutrition of halophilic bacteriardquoin The Biology of Halophilic Bacteria R H Vreeland and L IHochstein Eds pp 87ndash104 CRC Press Boca Raton Fla USA1993

[25] R E Gordon D A Barnett J E Handerhan and C HorNay Pang ldquoNocardia coeliaca Nocardia autotrophica and thenocardin strainrdquo International Journal of Systematic Bacteriol-ogy vol 24 no 1 pp 54ndash63 1974

[26] D A Hopwood M J Bibb K F Chater et al GeneticManipulation of Streptomyces A Laboratory Manual The JohnInnes Foundation Norwich UK 1985

[27] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic acid Tech-niques in Bacterial Systematic E Stackebrandt andM Goodfel-low Eds pp 115ndash175 John Wiley amp Sons New York NY USA1991

[28] T Thinesh P A Jose and E J K Patterson ldquoPredominantbacterial candidates associated with diseased corals from GulfofMannar Indiardquo Journal of Pure andAppliedMicrobiology vol7 no 3 pp 2397ndash2403 2013

[29] J Felsenstein PHYLIP (Phylogeny Inference Package) Version368 Department of Genome Sciences University of Washing-ton Seattle Wash USA 2008

[30] J Coates ldquoInterpretation of infrared spectra a practicalapproachrdquo in Encyclopedia of Analytical Chemistry R A Mey-ers Ed pp 10815ndash10837 John Wiley amp Sons Chichester UK2000

[31] NCCLS Methods for Antimicrobial Susceptibility Testing ofAnaerobic Bacteria Approved Standard NCCLSdocumentM11-A5 National Committee for Clinical Laboratory StandardsWayne Pa USA 5th edition 2001

[32] G M Eliopoulus and R C J Moellering ldquoAntimicrobialcombinationsrdquo in Antibiotics in Laboratory Medicine V LorianEd pp 52ndash111 Williams ampWilkins Baltimore Md USA 1996

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Microbiology


as a valuable source of novel bioactive metabolites showingantimicrobial anticancer anthelmintic and antipsychoticactivities To this date some antibiotic compounds derivedfrom different species of genus Nonomuraea are actinotiocin[13] maduramycin [14] glycopeptide antibiotic A40926 [15]and pyralomicins [16] The trend to discover novel metabo-lites from the genusNonomuraea has been augmented whichis a challenging signal for further exploration of this naturalcompounds producing resource [5]

The Nonomuraea sp JAJ18 taken for this study waspreviously isolated from a coastal solar saltern [17] whichshows antimicrobial activity against a range of bacteriaThe objective of this paper was to describe strain JAJ18 togenus level based on its complete 16S rRNA gene sequenceand phenotypic characteristics purify the antibiotic fromits culture broth and disclose structural characteristics andantibacterial potential of the purified compound

2 Materials and Methods

21 Strains andTheir Maintenance Antagonistic rare actino-mycete strain Nonomuraea sp JAJ18 was previously isolatedfrom a hypersaline coastal solar saltern [17] Pure culture ofthis strain was maintained over modified inorganic salt agarslants which contained starch 100 g yeast extract 40 g NaCl200 g NH

4

SO4

20 g MgSO4

sdot7H2

O 10 g K2

HPO4

10 g and220 g of agar in 10 liter of distilled water The bacterial teststrains methicillin-resistant Staphylococcus aureus (MRSA)Bacillus subtilisMTCC 441 Klebsiella pneumoniaMTCC 109Salmonella typhi MTCC 733 and Proteus vulgaris MTCC426 used in this study for antimicrobial assays were culturedin Mueller Hinton agar slants The MRSA was obtainedfrom Kovai Medical Center and Hospital Coimbatore IndiaOther bacterial strains were obtained from Microbial TypeCulture Collection (MTCC) Institute of Microbial Technol-ogy Chandigarh India

22 Colonial Cultural and Physiological CharacterizationCultural characteristics of JAJ18 were observed in twelvedifferent agar media starch casein agar [20] potato dextroseagar glycerol nitrate agar [21] asparagine vitamin agarsucrose nitrate agar glucose asparagine agar [22] and seriesof ISP media such as yeast extract maltose agar ISP2 oatmealagar ISP3 inorganic salt starch agar ISP4 glycerol asparagineagar ISP5 peptone yeast extract iron agar ISP 6 and tyrosineagar ISP 7 [23]The agar plates were inoculated and incubatedat 30∘C for 7 d The plates were then visually examinedto determine aerial spore-mass colour substrate mycelialpigmentation and the colour of diffusible pigments

The growth temperature range (15 20 25 30 37 45 or50∘C) and pH range (pH 45 5 6 68 72 9 and 10) forthe growth of strain JAJ18 were determined on modifiedISP-4 after culturing for 2 weeks in shake flasks (250 rpm)for 10 days Salt tolerance of the actinomycete isolate JAJ18was determined on starch nitrate medium prepared withseries of NaCl concentrations between 0 and 30 (0 to 5M)according to Kushner [24] Reduction of nitrate degradationof gelatin and casein andproduction of catalase andH

2

Swere

examined as described by Gordon et al [25] Utilization ofa range of different sole carbon sources was tested on ISP9medium [23]

23 16S rRNA Gene Amplification and Cloning For DNAisolation biomasswas obtained by growing the strain JAJ18 inTrypticase soy broth for 7 d at 30∘C with agitation in 500mLflasks containing 100mL of the medium From the biomassDNA was isolated using standard phenol-chloroform extrac-tion procedure [26] The 16S rRNA gene was amplified fromthe isolated DNA using universal eubacterial primer set 27F51015840 AGTTTGATCCTGGCTCAG 31015840 and 1492R 51015840ACGGCTACC TTG TTA CGA CTT 31015840 [27] PCR amplification wascarried out in a 50 120583L reaction mixture according to Thineshet al [28] The PCR amplified 16S rRNA gene was purifiedusing PCR product purification spin kit (HiPurA HiMediaIndia) Purified 16S rRNA gene amplicon was cloned usingpGEM-T Easy vector (Promega) with E coli DH10B cellsThe plasmid DNA of the recombinant clone was isolatedand sequenced by an automated sequencer (Genetic Analyzer3130 Applied Biosystems USA) using the vector-specificprimers SP6 and T7 promoter

24 Phylogenetic Analysis Almost complete 16S rRNA genesequences of strain JAJ18 and those of other related speciesderived from GenBank were aligned using ClustalW Phy-logenetic tree was inferred by using suitable programs ofthe PHYLIP (Phylogeny Inference Package) version 368Nucleotide distances were calculated according to the algo-rithm of Jukes and Cantor by using DNADIST and aneighbor-joining tree was constructed using NEIGHBORfrom the PHYLIP program package [29] The robustnessof tree topology was assessed by bootstrap analysis of theneighbor-joining datasets on 1000 resamplings using theSEQBOOT and CONSENSE options from the same PHYLIPpackage

25 Production and Extraction of Antibiotic For preparationof seed culture spore suspensions of JAJ18 were inoculatedin modified ISP-4 broth and incubated for 3 d at 28∘C ona rotary shaker (220 rpm) A total of 2mL seed culturewas transferred into 200 mL production medium containingstarch 100 g yeast extract 40 g NaCl 50 g NH

4

SO4

20 gMgSO

4

sdot7H2

O20 g K2

HPO4

10 g CaCO3

10 g FeSO4

sdot7H2

O0010 g ZnSO

4

sdot7H2

O 0001 g MnCl2

sdot4H2

O 0001 g andCuSO

4

sdot5H2

O 0001 g in 1 litre of distilled water for antibioticproduction The flask was incubated for 10 d at 30∘C on arotary shaker with 220 rpm After the incubation period theculture broth was centrifuged at 10000 rpm for 10min toremove the mycelial biomass Ethyl acetate was added to thesupernatants in 1 1 proportion and the mixture was agitatedfor 45minThe solvent layer was separated and centrifuged at5000 rpm for 15min to remove traces of fermentation brothThe ethyl acetate fraction was evaporated and the resultantcrude antibiotic was suspended in 50 120583L of methanol whichwas then checked for antibiotic activity






3














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4 Conclusion




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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3














Gelatin minus




Adonitol plusmn

Arabinose plusmn





Xylose plusmn







6 ++68 ++72 ++9 +


25 ++37 ++45 +50 minus


4 minus




3















75

58

91

96

88

74

64

92

70

100

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T

(U48843)





T

T

(GU367158)








4 Conclusion




Acknowledgment


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Gelatin minus




Adonitol plusmn

Arabinose plusmn





Xylose plusmn







6 ++68 ++72 ++9 +


25 ++37 ++45 +50 minus


4 minus




3















75

58

91

96

88

74

64

92

70

100

001







T

(U48843)





T

T

(GU367158)








4 Conclusion




Acknowledgment


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













75

58

91

96

88

74

64

92

70

100

001







T

(U48843)





T

T

(GU367158)








4 Conclusion




Acknowledgment


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Conclusion




Acknowledgment


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Characterization of Antibiotic Producing