Actinomycetes: Role in Biotechnology and Medicine

BioMed Research International

Guest Editors: Neelu Nawani, Bertrand Aigle, Abul Mandal, Manish Bodas, Sofiane Ghorbel, and Divya Prakash Actinomycetes: Role in Biotechnology and Medicine BioMed Research International Actinomycetes: Role in Biotechnology and Medicine

This is a special issue published in “BioMed Research International.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents

Actinomycetes: Role in Biotechnology and Medicine, Neelu Nawani, Bertrand Aigle, Abul Mandal, Manish Bodas, Soﬁane Ghorbel, and Divya Prakash Volume 2013, Article ID 687190, 1 page

Actinomycetes: A Repertory of Green Catalysts with a Potential Revenue Resource, Divya Prakash, Neelu Nawani, Mansi Prakash, Manish Bodas, Abul Mandal, Madhukar Khetmalas, and Balasaheb Kapadnis Volume 2013, Article ID 264020, 8 pages

Streptomyces misionensis PESB-25 Produces a Thermoacidophilic Endoglucanase Using Sugarcane Bagasse and Corn Steep Liquor as the Sole Organic Substrates, Marcella Novaes Franco-Cirigliano, Raquel de Carvalho Rezende, Monicaˆ Pires Gravina-Oliveira, Pedro Henrique Freitas Pereira, Rodrigo Pires do Nascimento, Elba Pinto da Silva Bon, Andrew Macrae, and Rosalie Reed Rodrigues Coelho Volume 2013, Article ID 584207, 9 pages

Anti-Candida Properties of Urauchimycins from Actinobacteria Associated with Trachymyrmex Ants, Thais D. Mendes, Warley S. Borges, Andre Rodrigues, Scott E. Solomon, Paulo C. Vieira, MartaC.T.Duarte,andFernandoC.Pagnocca Volume 2013, Article ID 835081, 8 pages

Identiﬁcation and Biotechnological Application of Novel Regulatory Genes Involved in Streptomyces Polyketide Overproduction through Reverse Engineering Strategy,Ji-HyeNah,Hye-JinKim,Han-NaLee, Mi-Jin Lee, Si-Sun Choi, and Eung-Soo Kim Volume 2013, Article ID 549737, 10 pages

Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes, Surang Chankhamhaengdecha, Suphatra Hongvijit, Akkaraphol Srichaisupakit, Pattra Charnchai, and Watanalai Panbangred Volume 2013, Article ID 782847, 8 pages

Streptomyces lunalinharesii Strain 235 Shows the Potential to Inhibit Bacteria Involved in Biocorrosion Processes, Juliana Pacheco da Rosa, Elisa Korenblum, Marcella Novaes Franco-Cirigliano, Fernanda Abreu, Ulysses Lins, Rosangelaˆ M. A. Soares, Andrew Macrae, Lucy Seldin, and Rosalie R. R. Coelho Volume 2013, Article ID 309769, 10 pages Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 687190, 1 page http://dx.doi.org/10.1155/2013/687190

Editorial Actinomycetes: Role in Biotechnology and Medicine

Neelu Nawani,1 Bertrand Aigle,2,3 Abul Mandal,4 Manish Bodas,1 Sofiane Ghorbel,5 and Divya Prakash1

1 Dr.D.Y.PatilBiotechnologyandBioinformaticsInstitute,Dr.D.Y.PatilVidyapeeth,Pune411033,India 2 Universite´ de Lorraine, UMR 1128, Dynamique des Genomes´ et Adaptation Microbienne, 54506 Vandœuvre-les-Nancy,` France 3 INRA, Dynamique des Genomes´ et Adaptation Microbienne, UMR 1128, 54506 Vandœuvre-les-Nancy,` France 4 School of Life Sciences, System Biology Research Center, University of Skovde,¨ Box 408, 541-28 Skovde,¨ Sweden 5 LaboratoiredeGenie´ Enzymatique et de Microbiologie, Ecole Nationale D’Ingenieurs´ de Sfax, Sfax, Tunisia

Correspondence should be addressed to Neelu Nawani; [email protected]

Received 16 May 2013; Accepted 16 May 2013

Copyright © 2013 Neelu Nawani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Actinomycetes, one of the most diverse groups of filamentous is on par with commercial antimicrobials, clearly manifest- bacteria, are well recognized for their metabolic versatility. ing a new explorable niche “actinobacterial symbionts of The bioactive potential of these bacteria facilitates their plants and animals.” The term “antimicrobials” often leads survival even in distress and unfavourable ecological con- our thoughts to “medicine-related” but it’s “environment- ditions. This special issue is dedicated to the importance of related” applications are less contrived. Streptomyces lunal- multitudeofprimaryandsecondarymetabolitesproducedby inharesii produces antimicrobial substances against sulfate- actinomycetes. The six articles published in this issue balance reducing bacteria commonly responsible for corrosion in the the biocatalytic and biocidal potential of actinomycetes. petroleum industry, with an ability to replace the existing The importance of large repertory of enzymes from biocides. Making the best out of the already good can be actinomycetes and their potential in replacing chemical achieved for actinomycetes by strain improvement. Advanced catalysts is discussed. Successful commercialization of these microarray-driven reverse engineering strategies for the enzymesisanimportantsteptowardsrevolutionizing“green understanding and modulation of independently functioning technology.” Reduction in the cost of enzyme production is regulatory pathways can allow these microfactories to over- demonstrated by production of endoglucanases from Strep- produce important antibiotics. tomyces sp. on low-cost substrates. Such low-cost production In a nutshell, actinomycetes offer the most promising initiatives can be extended to other enzymes and metabolites. synthesizers of many industrially and commercially mean- Novel properties like thermal and ionic stabilities and a better ingful metabolites. Novel and unexplored habitats may offer turnover make these systems infallible and regenerative. bacterial assemblages not reached hitherto. An integration The activity of enzymes from actinomycetes is not confined of newer habitats, screening, and improvement technologies to substrate conversion alone but broadened to biocontrol can offer promising candidates for biotechnology and health- of quorum-sensing-dependent phytopathogens, as mediated related applications. by acyl-homoserine-lactone-degrading enzymes from endophytic actinomycetes. Neelu Nawani Unexplored environments often appeal to researchers Bertrand Aigle inthehopeofaccruingnovelbacteria,acontinuousquest Abul Mandal which has actually led to discovery of unusually industrious Manish Bodas microbes. Antimicrobial potential of actinobacteria isolated Sofiane Ghorbel from the integument of Trachymyrmex fungus-growing ants Divya Prakash Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 264020, 8 pages http://dx.doi.org/10.1155/2013/264020

Review Article Actinomycetes: A Repertory of Green Catalysts with a Potential Revenue Resource

Divya Prakash,1 Neelu Nawani,1 Mansi Prakash,1 Manish Bodas,1 Abul Mandal,2 Madhukar Khetmalas,1 and Balasaheb Kapadnis3

1 Dr. D. Y Patil Biotechnology & Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune 411 033, India 2 System Biology Research Center, School of Life Sciences, University of Skovde,¨ P.O. Box 408, 541 28 Skovde,¨ Sweden 3 Department of Microbiology, University of Pune, Pune 411 007, India

Correspondence should be addressed to Neelu Nawani; [email protected]

Received 25 December 2012; Revised 27 March 2013; Accepted 28 March 2013

Academic Editor: Bertrand Aigle

Copyright © 2013 Divya Prakash et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Biocatalysis, one of the oldest technologies, is becoming a favorable alternative to chemical processes and a vital part of green technology. It is an important revenue generating industry due to a global market projected at $7 billion in 2013 with a growth of 6.7% for enzymes alone. Some microbes are important sources of enzymes and are preferred over sources of plant and animal origin. As a result, more than 50% of the industrial enzymes are obtained from bacteria. The constant search for novel enzymes with robust characteristics has led to improvisations in the industrial processes, which is the key for profit growth. Actinomycetes constitute a significant component of the microbial population in most soils and can produce extracellular enzymes which can decompose various materials. Their enzymes are more attractive than enzymes from other sources because of their high stability and unusual substrate specificity. Actinomycetes found in extreme habitats produce novel enzymes with huge commercial potential. This review attempts to highlight the global importance of enzymes and extends to signify actinomycetes as promising harbingers of green technology.

1. Introduction Commercially available enzymes are derived from plants, animals, and microorganisms. The enzymes derived from Biocatalysis offers green and clean solutions to chemical plants include papain, bromelain, ficin, lipooxygenase, processes and is emerging as a challenging and reverred among several others [2], and those derived from animal alternative to chemical technology. The chemical processes sources include pepsin and renin. However, a major fraction are now carried out biologically by biocatalysts (enzymes) of commercially available enzymes are derived from microbes which are integral components of any biological system. due to their ease of growth, nutritional requirements, and However, the utility of enzymes is not na¨ıve to us, as they downstream processing. To meet the increasing demand have been an integral part of our lives from immemorial of robust, high turnover, economical and easily available times. Their utility dates back to 1914, when they were used in biocatalysts,researchisalwayschannelizedfornovelityin detergents even before their protein nature was determined enzyme or its source or for improvement of existing enzymes in 1960 [1]. Their use in fermentation processes like wine and by engineering at gene and protein level [1]. Search for beer manufacture, vinegar production, and bread making has novelenzymesfromunusualecologicalnichesisoftenmore been practised for several decades. However, a commercial attractive option leading to development of high-throughput breakthrough happened in the later half of 20th century with screening programs. Enzymes with new physical and phys- first commercial protease production in 1957 by Novozymes iological characteristics like high productivity, specificity, [1]. Since then, due to the advent of newer industries, the stability at extreme temperature, pH or other physiological enzyme industry has not only seen an enormous growth but conditions, low cost of production, and tolerance to inhbitors has also matured with a technology-oriented perspective. arealwaysmostsoughtafterpropertiesfromanindustrial 2 BioMed Research International

Table 1: Commercially relevant enzymes produced by actino- This review touches the global and Indian enzyme market mycetes. scenario and further highlights the potential of actinomycetes as sources of important industrial enzymes like cellulases, Industry of Enzyme Use pectinases, proteases, and chitinases which can be employed application for the recovery of numerous value added products with Detergents Detergent applications in biomedicine and waste management. Cheese making Food Protease Clarification- low calorie beer Brewing Dehiding Leather 2. Enzyme Market: Global and Indian Scenario Treatment of blood clot Medicine The global market is expected to experience a growth of Removal of stains Detergent 6% in enzyme requirement with an estimated market of $7 Cellulase Denim finishing, softening of Textile billion in 2013 [28]. North America and Western Europe are cotton predicted to show an increased growth, while the highest Deinking, modification of fibers Paper and pulp growth is likely in developing countries of Asian, African Removal of stains Detergent and Mideast regions, along with Latin America and Eastern Stability of dough and Europe. China is emerging as an important base and market Lipase Baking conditioning for industrial enzymes due to various R&D activites set up by Cheese flavoring Dairy many industrial giants which accounts for 10% of the global Deinking, cleaning Textile scenario [29]. The demand of diagnostic and therapeutic enzymes is expected to increase owing to improvement in Conditioning of dough Baking medical care facilities in developing countries and global Xylanase Digestibility Animal feed health care reforms. Few enzyme manufacturing industries Bleach boosting Paper and pulp in the world include AB Enzymes GmbH, Advanced Enzyme Clarification, mashing Beverage Technologies Ltd., Amano Enzyme Inc., Asahi Kasei Pharma Pectinase Scouring Textile Corporation, Cargill Texturizing Solutions, Genencor Inter- Removal of stains Detergent national Inc., DSM Food Specialties, Hayashibara Company, Nexgen Biotechnologies Inc., Novozymes A/S, and Maps Softness of bread softness and Baking Enzymes Ltd. Amylase volume The global industrial enzyme market has evolved con- Deinking, drainage Paper and pulp improvement tinounsly due to numerous mergers and acquisitions. In the year 2011, enzyme industry giants like Novozymes and Production of glucose and Starch industry fructose syrups DuPont occupied market shares of 47% and 21%, respectively [30]. Technical enzymes were valued at $1.2 billion in 2011, Removal of starch from woven Textile fabrics andthisisexpectedtoriseto$2.2billionin2016withthe highest sales predicted in the leather and bioethanol markets Glucose Strengthening of dough Baking [31]. Similarly, food and beverage enzyme sector is expected oxidase to achieve about $2.1 billion by 2016, from a value of $1.3 Lipoxygenase Bread whitening Baking billionin2011asshowninFigure 1. This is well correlated with Phytase Phytate digestibility Animal feed thenumerouspatentswhichhavebeenfiledoveraperiod Peroxidase Removal of excess dye Textile of years which indicate an increasing trend. From Figure 2, it can be inferred that due to the lack of information on intellectual property rights (IPRs) in the 1970s, there were hardly any patents on any of the industrial enzymes [32]. But standpoint. An important criterion for enzymes derived from from the year 2000 till date, there has been a tremendous microbes remains that the source microbe should have a increase in the number of patents filed or obtained for generally regarded as safe (GRAS) status [3]. various enzymes. The maximum number of patents is for Many microbes particularly bacteria and fungi are cur- proteasesfollowedbyamylasesandcellulasesperhapsdue rently employed for the production of various industrial to maximum utility of these enzymes. The application and enzymes [4]. Hydrolases cover more than 75% of commer- issuing of patents for various enzymes is expected to grow in cially used enzymes and are often in great demand. These the future due to green technologies. are however used in a crude form to make the process Speciality enzymes due to their unique properties like economically viable and also to meet the demand of enzyme extreme thermostability, specific activity, and activity over at a large scale [5]. Amongst the hydrolases, proteases occupy a wide range of pH are expected to occupy an important an important platform, as they are extensively used in categoryinfutureduetotheirrobustness.Thesespeciality detergent industry, followed by starch industry which is the enzymes also include those useful in medicine and biotech- second largest user of enzymes and textiles, baking, food, and nology, for example, kinases, polymerases, and nucleases [33]. animal feed industries. The applications of few commercially Besides this, their utility in wide range of personal care significant enzymes are enlisted in Table 1. products is revolutionalizing the cosmetic industry too. The BioMed Research International 3

Technical enzymes Other enzymes Technical enzymes Other enzymes 1.2 2.2 1.5 2.2

Food and beverage Food and beverage enzymes enzymes 1.3 2.1

(2011) (2016) (a) (b)

Figure 1: Global enzyme industry market in the years 2011 and 2016.

5000 3. Actinomycetes as a Source for 4500 Amylase Industrial Enzymes 4000 Xylanase Amylase 3500 Cellulase Actinomycetes are one of the ubiquitous dominant groups of 3000 Xylanase gram positive bacteria. Actinomycetes have been commer- 2500 cially exploited for the production of pharmaceuticals, neu- 2000 Amylase Cellulase Protease traceuticals, enzymes, antitumor agents, enzyme inhibitors, 1500 Xylanase

Number of patents Cellulase 1000 Amylase and so forth [36]. These bioactive compounds are of high Protease 500 Xylanase Protease commercial value, and hence actinomycetes are regularly Protease 0 screened for the production of novel bioactive compounds. 1970–79 1980–89 1990–99 2000–09 2010–13 A wide array of enzymes and their products applied in Years biotechnological industries and biomedical fields has been Figure 2: Growth in number of patents issued for important reported from various genera of actinomycetes. Since there industrial enzymes over past few decades. is vital information available due to the advent of genome and protein sequencing data, actinomycetes have been continuously employed of the production of proteases, cellulases, chitinases, amylases, xylanases, and others. Representative examples of industrially important enzymes from actino- speciality sector is expected to reach $4.3 billion by 2015, mycetes are discussed below, and their enzymatic properties and industrial enzyme segment is worth $80 million, accord- are enlisted in Table 2. ingtoreportsbyreputedmarketresearchersandindustry analysts [34]. This growth would be mainly accelerated by the pharmaceutical and diagnostics industry, the largest end- 3.1. Cellulases. Cellulases convert cellulose to fermentable users of these enzymes which will continue to grow due to the sugars fit for human consumption and the largest known emergence of enzyme replacement therapies and innovations producers are from genus Streptomyces [37]. Cellulases from in thrombolytics. Streptomyces sp. are reported to have an alkaline pH opti- India imports 70% of the total enzyme consumed by its mum and high thermostability. Subsequently, the enzyme market which indicates need of indigenous manufacturers was used as a supplement in detergents to clean, soften, and technologies. The most important enzymes in demand and restore the color of the fabrics. It was also tested for are of the pharmaceutical sector consuming more than 50% the treatment of textiles, processing of paper and pulp, of the total enzymes. This is followed by detergent enzymes and as an animal feed additive [6]. Besides Streptomyces, (20%), textile enzymes (20%), and the rest comprises of food several other genera like Thermobifida and Micromonospora enzymes [35]. Novozymes is one of the leading industries produce recombinant cellulases that can be commercially in the enzyme market in India. The need of the hour is a exploited [7]. A recombinant cellulase with thermal and strong R&D in terms of investment. There is necessity for pH stability is reported from Streptomyces thermoviolaceus; strong legislation and IPR regulations which would make this enzyme retains its activity in the presence of commer- India withstand the stiff competition on the global front. In cial detergents highlighting its superiority to the existing addition, the enzyme market is risky and has high captial commercial cellulases [8]. Cellulase from Thermomonospora costs. India offers excellent human resource power which can fusca has been used for degradation of cotton and avi- lower production costs compared with many other countries cel [9]. These enzymes not only hold a biotechnological making this country an attractive location for investment. promise but can be economical due to their low cost Besides, the biodiversity in India is valuable for the screening of production. Their production can be carried out on of novel enzymes and metabolites which can be produced and cheap substrates like rice and wheat straw [10] and fruit utilized at the industrial level. peels [38]. 4 BioMed Research International

Table 2: List of industrially viable enzymes from actinomycetes and their characteristics.

Enzyme Producing Strain pH stability Thermal stability Substrate specificity Reference ∘ Recombinant Streptomyces sp. 5.0–12.0 40–50 CCMC[6] ∘ Thermobifida halotolerans 6.0–8.0 40–50 CCMC[7] ∘ Cellulase Recombinant Streptomyces sp. 10.0 40 CCMC[8] ∘ Thermomonospora sp. 7.0–10.0 50 CCMC[9] ∘ Streptomyces ruber 5.5–7.0 35–40 CCMC[10] ∘ Actinomadura sp. 4.0 70 CXylan[11] ∘ Recombinant strain 5.0–7.0 70–80 CXylan[12] Xylanase ∘ Recombinant strain 5.0–7.0 60–70 CBirchxylan[13] ∘ Streptomyces spp. 8.0–11.0 45–60 CXylan[14] ∘ Streptomyces sp. 5.0–7.0 45–50 CStarch[15] ∘ Streptomyces erumpens 9.0-10.0 40–50 CStarch[16] ∘ Amylase Nocardiopsis sp. 8.6 70–80 CStarch[17] ∘ Thermobifida fusca 5.0–7.0 60 CStarch[18] ∘ Nocardiopsis sp. 5.0–10.0 35–45 CStarch[19] ∘ Pectinase Streptomyces lydicus 4.0–7.0 45 C Polygalacturonic acid [20] ∘ Thermoactinomyces sp. 4.0 50 CNA[21] ∘ Nocardiopsis sp. 10.0 40–50 CCasein[22] ∘ Protease Streptomyces pactum 7. 5 4 0 CCasein[23] ∘ Streptomyces thermoviolaceus 6.5 65 C Keratin [24] ∘ Streptomyces sp. 4.0–11.0 30–60 C Keratin azure [25] ∘ Nocardiopsis prasina 7. 0 5 0 – 6 0 C Colloidal chitin [21] ∘ Chitinase Streptomyces thermoviolaceus 6.0 60 C Colloidal chitin [26] ∘ Microbispora sp. 3.0–11.0 30–50 C Colloidal chitin [27] CMC: Carboxymethyl cellulose; NA: not available.

3.2. Xylanases. Streptomyces spp. are prolific producers 3.3. Amylases. Another important group of enzymes is the of another commercially important enzyme, xylanase. amylases which are employed in the starch processing indus- Xylanases from Streptomyces sp. are preferred in treatment tryfortheconversionofstarchtohighfructosesyrups[15]. of rice straw pulp to improve the pulp bleachability. This One of the focus areas with respect to starch industry is preference is due to absence of cellulase contamination in the production of tailor length maltooligosaccharides which thexylanaseandalsoduetoreducedusageofchemicals can be produced with amylases with a very specific mode during bleaching and pulping [39]. Further, xylanases from of action. Thermophilic and acidophilic amylases which can actinomycetes are stable on kraft pulps and can be used in the findapplicationsinbakery,brewing,andalcoholindus- crude form thereby making the process economical. High tries have been studied from Streptomyces erumpens [16]. thermostability and specific activity, two desirable properties Thermostable amylases are reported from Nocardiopsis sp. of enzymes to be employed in industrial processes, are which have important applications in bakery and paper reported in xylanases from Actinomadura sp. FC7 and industries [17]. The amylase from Thermobifida sp. produced Nonomuraea flexuosa [11, 12]. Similarly, fused xylanases from maltotriose as the major end product from refined starch and fungi and actinomycetes have been employed in paper and raw sago starch [18]. Such amylases are lucrative catalysts pulp industries, due to high thermal and pH stability [13]. in nutrition and healthcare [18]. Besides this, end-product In many higher plants and agricultural wastes, the content specific amylases can be used for the production of mal- of xylan is almost 20–40% of the dry weight. Xylan with tooligosaccharides from low cost starch substrates [40]. Many actinomycetes have also been reported for the production hemicelluloses is the second most renewable biopolymer 𝛼 [14]. Since pure xylan is expensive, alternate cheap substrates of cold-active -amylases which can be employed in tex- like these can serve as potential substrates at industrial level. tile industries, detergents, bioethanol producing industries Streptomyces spp. were able to produce high levels of xylanase [19]. when untreated rice straw was utilized which resulted in significant biobleaching [39]. Similarly, Streptomyces sp. 3.4. Pectinases. Food industry uses pectinases particularly was able to hydrolyze various agricultural residues like oil in clarification of fruit juices, in degumming of fibres, wine cake and straw waste which resulted in increased biogas making, and retting of bast fibres. Pectinases from Strepto- production [14]. myces sp. are reported [20]; however, reports of pectinases BioMed Research International 5 from other genera of actinomycetes are scanty. The demand 3.7. Other Enzymes from Actinomycetes. An array of other of cold-active pectate lyases is increasing due to their ability enzymes with industrial potential reported from actino- to retain the palatability and nutritional characteristics of mycetes includes lignin peroxidases, laccases, and tyrosi- food products. Occurrence of pectin degrading genes in few nases which are effective in the treatment of textile dyes actinomycetes suggests their characterization could perhaps [43] promising their application in waste treatment plants. yield pectinases with novel properties. Esterases and amidases from Nocardia sp. have been used to increase the hydrophilicity of polyethylene terephthalate and polyamide fibers. This can be an ecofriendly and cost- 3.5. Proteases. The quest for novel proteases and their effective method in textile industries [43]. To get enzymes formulations for industries like detergents, animal feed, and with novel properties or functionalities, high throughput breweries is observed from several decades. Most of the screening (HTS) programs are adopted for choosing rare proteases reported from Streptomyces spp. are alkali-tolerant, actinomycetes which are a source of novel compounds. Suc- andsomeofthemaresalttolerantandbelongtogeneraother cess examples include therapeutic enzymes like thrombinase than the genus Streptomyces [21]. Proteases from Nocardiopsis and L-asparaginase from marine Streptomyces sp. which are spp. are employed as detergents additives [22]andforthe used in the treatment of myocardial infarction and leukemia. depilation of hides and skins in the leather industry. AspongeassociatedStreptomyces sp. produced phytoene, a Dehairing of goat skin by proteases from Streptomyces carotenoid with enhanced antioxidant activity making it a sp. makes the process economically and environmentally promising food additive [44]. feasible [23]. Keratin-rich wastes like feathers, hair, nails, Access to advanced technologies has made it possible to and horn are waste products of agroindustrial processes. obtain untapped microbes with novel properties, and actino- Keratinolytic Streptomyces spp. capable of degrading keratin ∘ mycetes have been natural reservoirs of excellent enzymes. at temperatures higher than 50 C are reported [24]. Some The existing HTS methods are used for choosing industrially Streptomyces hydrolyze keratin by pronases as seen for Strep- important bacteria and have not been actively extended to tomyces griseus [24]. Proteases from other sources are used in actinomycetes. Several HTS methods which can be used for conjunction with enzymes from actinomycetes for recovery exploring novel enzymes from actinomycetes are discussed. of antioxidants from shellfish waste. Protease production Fluorescence activated cell sorting (FACS) is successfully was also carried by growing Microbispora sp. on the shellfish employed for sorting of desired clones from a genomic waste [25]. End products of protein hydrolysis rich in amino library, where fluorescent substrate specific for a particular acidsandpeptidesserveasalowcostanimalfeed. enzyme is used. The positive fluorescence indicates biocatalytic activity of the clone [45]. Gel MicroDrop technology 3.6. Chitinases. Chitinases are another class of hydrolases detects clones positive for specific enzymes by capturing which have gained tremendous importance in the past the fluorescence emitted due to catalytic breakdown of two decades. They are glycosyl hydrolases that catalyze the biotinylated substrate by the clone [46]. degradation of chitin, which is an insoluble linear 𝛽-1,4- Metagenomics has offered rapid screening methods linked polymer of N-acetylglucosamine (GlcNAc). Chitin is where the bioactive potential of unculturable microbes can be a major constituent of the shells of crustaceans, exoskeletons explored. A clonal library is prepared using the metagenome of insects, and cell walls of a variety of fungi [26]. Chiti- obtained from extreme habitats like arid regions, ocean nases are useful in protoplast preparation from fungi, as beds, stratosphere, and others without expecting the actual biocontrol agents against plant pathogenic fungi, nematodes, microbes to grow under laboratory conditions which usually andsoforthandarerecentlyusedfortheextractionof limits the exploitation of the bioactive potential of these chitin oligomers which are important biomedical products. nonculturable microbes [47]. These technologies also help Chitinases occur in several actinomycetes and possess unique to determine the functional aspects of a microenvironment properties in terms of thermostability and activity in wide pH [48]. Although metagenomic approach has many advantages, range which makes them suitable for industrial applications it suffers from a common disadvantage like low or no expres- [21, 26, 27]. One of their most resourceful applications is sion of desired gene(s). Multiple displacement amplification the production of chitin oligosaccharides. Chitin oligosac- has allowed researchers to overcome the problem of low or no charides (COS) have anticoagulant, antimicrobial, antic- expression. Here whole genome amplification is carried out holesteremic, anticancer, wound-healing, antitumor, and from single cells, which unlocks entire biochemical potential antioxidant activities which make them bright candidates for of an uncultured microbe from a complex habitat. Other biomedical applications [26]. COS can be recovered from innovative approaches like substrate-induced gene expres- lowcostsubstrateslikeshrimp,crab,andsquidpenwaste sion screening (SIGEX), preamplification inverse-PCR (PAI- [41, 42]. Chitinase from Microbispora sp. was employed for PCR), and metagenomic DNA shuffling provide insights on the recovery of chitobiose, a potential antioxidant which the functional metagenomics of a particular habitat [48]. can be used as a food additive and for other biomedical A smart and diversified technique which accelerates applications [25].Thisrenewableresourcecanbeutilizedfor evolution is the “directed evolution” approach, where a library the growth of many chitinolytic organisms as well as for the of genetic variants is created and the mutants are screened for effective recovery of COS at the industrial level. The disposal desired enzymatic properties. The best variants are shortlisted of the waste is also carried out effectively by the biological and reorganized for another round of library creation which utilization by actinomycetes. is repeated a number of times to possibly get the best variants, 6 BioMed Research International a process which follows the rule of natural evolution but is Acknowledgments performed at a pace that gives the result of evolution in a short time [49]. This method is a significant driving force The authors are grateful to the Department of Science and for the discovery of next generation biocatalysts [50]. An Technology, Government of India, Swedish International added advantage to this method is the inclusion of ultrahigh- Development Cooperation Agency, Sweden, and Dr. D. Y. throughput FACS-based screening which aids in the rapid Patil Vidyapeeth, Pune, for support. screening of the variant library [51]. Recent improvements in screening technologies which can be very useful for enzymes References from actinomycetes are the Drop-based microfluidics which was successfully used to screen mutants with ten times potent [1]M.Leisola,J.Jokela,O.Pastinen,O.Turunen,andH.E.Schoe- horseperoxidaseactivitythanwildtype[52]. HTS in drop- maker, “Industrial use of enzymes,” in In Encyclopedia of Life based microfluid platform carries a small foot print chip Support Systems (EOLSS),vol.2,UNESCO,EolssPublishers, with an array of insoluble substrates specific for the enzyme Oxford, UK, 2004. of interest; this gives rapidity, parallel execution, and cost [2] http://naturebiochem.com/downloads/Enzymes Pharma economics to the screening protocol [53]. applications. Reporter gene technology is another step up in the [3] http://ec.europa.eu/food/fs/sc/scan/out85 en.pdf . screening methods which offer simplicity and sensitivity. [4] P. 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Research Article Streptomyces misionensis PESB-25 Produces a Thermoacidophilic Endoglucanase Using Sugarcane Bagasse and Corn Steep Liquor as the Sole Organic Substrates

Marcella Novaes Franco-Cirigliano,1 Raquel de Carvalho Rezende,1 Mônica Pires Gravina-Oliveira,1 Pedro Henrique Freitas Pereira,1 Rodrigo Pires do Nascimento,2 Elba Pinto da Silva Bon,3 Andrew Macrae,1 and Rosalie Reed Rodrigues Coelho1

1 Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Goes,´ Centro de Cienciasˆ da Saude´ (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Avenida Carlos Chagas Filho 373, Bloco I, Laboratorio´ 055, 21941-902 Rio de Janeiro, RJ, Brazil 2 Departamento de Engenharia Bioqu´ımica, Escola de Qu´ımica, Centro de Tecnologia (CT), Universidade Federal do Rio de Janeiro (UFRJ), Avenida Athos da Silveira Ramos 149, Bloco E, sala 203, 21941-909 Rio de Janeiro, RJ, Brazil 3 Departamento de Bioqu´ımica, Instituto de Qu´ımica, Centro de Cienciasˆ Matematicas´ e Natureza (CCMN), Universidade Federal do Rio de Janeiro (UFRJ), Avenida Athos da Silveira Ramos 149, Bloco A, sala 539, 21941-909 Rio de Janeiro, RJ, Brazil

Correspondence should be addressed to Rodrigo Pires do Nascimento; [email protected]

Received 1 October 2012; Revised 8 January 2013; Accepted 4 February 2013

Academic Editor: Divya Prakash

Copyright © 2013 Marcella Novaes Franco-Cirigliano et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Streptomyces misionensis strain PESB-25 was screened and selected for its ability to secrete cellulases. Cells were grown in a liquid medium containing sugarcane bagasse (SCB) as carbon source and corn steep liquor (CSL) as nitrogen source, whose concentrations −1 were optimized using response surface methodology (RSM). A peak of endoglucanase accumulation (1.01 U⋅mL )wasobservedin a medium with SCB 1.0% (w/v) and CSL 1.2% (w/v) within three days of cultivation. S. misionensis PESB-25 endoglucanase activity ∘ ∘ was thermoacidophilic with optimum pH and temperature range of 3.0 to 3.6 and 62 to 70 C, respectively. In these conditions, −1 2+ values of 1.54 U mL of endoglucanase activity were observed. Moreover, Mn was demonstrated to have a hyperactivating effect −1 2+ on the enzyme. In the presence of MnSO4 (8 mM), the enzyme activity increased threefold, up to 4.34 U⋅mL .Mn also improved ∘ 2+ endoglucanase stability as the catalyst retained almost full activity upon incubation at 50 Cfor4h,whileintheabsenceofMn , enzyme activity decreased by 50% in this same period. Three protein bands with endoglucanase activity and apparent molecular masses of 12, 48.5 and 119.5 kDa were detected by zymogram.

1. Introduction regions. Its hydrolysis is carried out by endo-𝛽-1,4-glucanase (EC 3.2.1.4), which cleaves internal 𝛽-1,4-glycosidic bonds Enzymatic hydrolysis of cellulose is a challenge worldwide, at random positions and forms insoluble reducing sugars, because currently we lack inexpensive and efficient enzymes and by exo-𝛽-1,4-glucanase (EC 3.2.1.91) that hydrolyses to hydrolyse the 1.5 trillion tons of cellulose produced cellulose from its reducing and nonreducing ends releasing annually [1]. Enzyme blends and optimization are required soluble reducing sugars with prevalence of cellobiose. The tospeedupenzymatichydrolysistomaketheprocesscom- enzyme 𝛽-glucosidase (EC 3.2.1.21) converts cellobiose into mercially viable. Cellulose is a homopolymer of 𝛽-1,4 linked glucose monomers [2]. An increase in the formation of free glucose units presenting both amorphous and crystalline reducing and nonreducing ends from endo-acting cellulases 2 BioMed Research International couldspeeduptheactionoftheexoglucanasesandthetotal using ABI Prism dye terminator cycle sequencing reaction cellulose hydrolysis process. A significant amount of research kit (Applied Biosystems) in an automatic sequencer (ABI on new endoglucanases has been done [3, 4]. model 3730; Applied Biosystems). The sequence of rrs gene Cellulolytic organisms are ubiquitous in nature. They obtained was compared with sequences online at the Ribo- are mostly bacteria and fungi, aerobic or anaerobic, and somal Database Project (RDP) release 10 [17]andGenBank mesophilic or thermophilic. Actinomycetes, which are Gram- [18] using the NCBI (The National Center for Biotechnol- positive filamentous soil bacteria, are well known for their ogy Information) basic local alignment search tool, BLAST abilitytodecomposecomplexmolecules,particularlythelig- (http://blast.ncbi.nlm.nih.gov/Blast.cgi)[19]. nocellulose components, which make them important agents in decomposition processes [5]. They have also been shown 2.3. Endoglucanase Production Using Experimental Design. to produce thermostable cellulases, with alkalophilic and Streptomyces misionensis PESB-25 was cultivated in liquid acidophilic characteristics [6, 7]. The work that has been done medium with SCB and CSL as the main carbon and nitrogen in our laboratory with strains from the Streptomyces genus sources, respectively. SCB consists of 43.8% cellulose, 25.8% indicates that endoglucanase activity is predominant in these hemicellulose, 22.1% lignin, 6.1% extractives, and 1.4% ash bacterial cellulases. In previous studies from our laboratory, [20]. It contains, approximately, 45.3% carbon and 0.5% we reported that the culture supernatant of S. malaysiensis nitrogen [21]. CSL is a major by-product of the corn wet- AMT-3, S. drozdowiczii M-7A, and S. viridobrunneus SCPE- milling industry and contains 47% crude protein, 26% lactic 09 presented endoglucanase activity with optimal pH in the ∘ acid, 7.8%phytic acid, 2.5% reducing sugars (as dextrose), and range of 4.0 to 5.0, optimal temperature around 50 Cand 17% ash, total nitrogen being 7.5% [22]. molecular masses, according to zymogram analyses, in the Response surface methodology (RSM) was used as a range of 37 to 178 kDa [8–10]. tool for the optimization of SCB and CSL concentrations This study investigated cellulase production by an acti- (independent variables) in the range indicated in Table 1. −1 nobacterial strain, S. misionensis PESB-25. Experimental Endoglucanase activity (U⋅mL ) was the dependent variable. 𝛽 2 design was performed to optimize endo- -1,4-glucanase A2 central composite rotational design (CCRD) was used to productionusingSCBasthemaincarbonsourceandCSL design experiments. as nitrogen source. As seen before, these low-cost materials Cultivations were carried out in 125 mL Erlenmeyer flasks can be suitable for cellulases production [8–10]. The elec- −1 containing 25 mL of mineral salts solution [23](ing⋅L : trophoretic profiles of extracted enzymes were determined NaCl,2.0;KH2PO4, 3.0; K2HPO4, 6.0; MgSO4⋅7H2O, 0.5; by zymogram analyses. Enzymatic activity was investigated CaCl2,0.05),supplementedwithatraceelementsolution[13] over a range of pH and temperature values in the culture −1 (in g⋅L :CuSO4⋅5H2O, 6.4; ZnSO4⋅7H2O, 1.5; FeSO4⋅7H2O, supernatants (crude enzyme preparation). The effect of metal 2+ 1.1; MnCl2⋅4H2O,7.9),withSCBandCSLattherelevant ions, most importantly Mn , on the endoglucanase activity concentrations. The medium start pH was adjusted to 7.0.The and stability was also evaluated. growth medium was inoculated with 25 𝜇Lofasporesuspen- 9 −1 ∘ sion (10 spores⋅mL )andincubatedat28 C, under agitation 2. Materials and Methods (200 rpm), for 3 days. The cultures were filtered through glass microfiber filter (Millipore), and the culture supernatant 2.1. Microorganism Screening, Preservation, and Cultivation. (crude enzyme preparation) was used for endoglucanase Streptomyces misionensis PESB-25wascollectedfromasugar- activity determination. cane crop soil in the State of Pernambuco, Brazil. The dilution plate technique was used for the isolation of the bacterial 2.4. Standard Endoglucanase Activity Assay. Endoglucanase strain, which was selected as cellulolytic via its cultivation on activity was determined by measuring the release of reducing solid medium containing carboxymethylcellulose low viscos- sugars in a reaction mixture containing 0.5 mL of the crude ity (CMClw) as carbon source followed by the identification enzyme preparation and 0.5 mL of CMClw (SIGMA) 4.0% oftheCMC-degradingzonesusingtheCongoreddye[11]. (w/v) solution in sodium citrate buffer 50 mM (pH 4.8) Spore suspensions were prepared according to Hopwood ∘ ∘ incubated at 50 C for 10 min. Reducing sugars were assayed and colleagues [12] after cultivation at 28 C for 15 days in bythedinitrosalicylicacidmethod[24]. One unit (IU) of yeast extract-malt extract-agar medium [13]. Spores were endoglucanase activity corresponded to the formation of − ∘ maintained in 20% (v/v) glycerol at 20 C. 1 𝜇mol of reducing sugars equivalent per minute under the assay conditions [25]. 2.2. Molecular Identification of Bacterial Strain PESB-25. Genomic DNA was extracted using the method described 2.5. Effect of pH, Temperature, and Ions on the Enzyme Activity by Kurtzman and Robnett [14]. PCR amplification of the and Stability. To study the effect of pH and temperature 2 rrs gene was carried out using the GoTaq Green Master on the supernatants endoglucanase activity, a CCRD 2 was Mix kit (Promega Corporation), with primers 27F [15]and used. In the 12 experiments which were carried out, the ∘ ∘ 1541R [16], in a thermal cycler model Gene Amp PCR temperature ranged from 40 to 70 CandthepHvaluesfrom System 9700 (Applied Biosystems). Amplified fragments 3.0 to 7.0 as shown in Table 3.Citratebuffer(50mM)wasused were purified using the Illustra GFX PCR DNA and Gel for pH 3.0, 3.6 and 5.0 and phosphate (50 mM) for pH 6.4 and Band Purification kit (GE Healthcare) and sequenced directly 7. 0 [ 26]. Statistical analysis of the results was performed using BioMed Research International 3

Table 1: Observed and predicted values of endoglucanase activity for the independent variables SCB and CSL concentrations used in central composite rotational design (CCRD), from the crude enzyme extract of Streptomyces misionensis PESB-25.

Endoglucanase activity Endoglucanase activity Run SCB (%w/v)/Coded level CSL (%w/v)/Coded level −1 ∗ −1 (U⋅mL )Observed (U⋅mL ) Predicted 1 0.65 (−1) 0.77 (−1) 0.95 ± 0.11 0.91 2 1.35 (+1) 0.77 (−1) 0.72 ± 0.005 0.67 3 0.65 (−1) 1.63 (+1) 0.86 ± 0.003 0.85 4 1.35 (+1) 1.63 (+1) 0.98 ± 0.038 0.96 5 0.5 (−1.41) 1.2 (0) 0.93 ± 0.022 0.95 6 1.5 (1.41) 1.2 (0) 0.83 ± 0.032 0.86 7 1.0 (0) 0.6 (−1.41) 0.66 ± 0.003 0.71 8 1.0 (0) 1.8 (1.41) 0.87 ± 0.024 0.88 9 1.0 (0) 1.2 (0) 1.03 ± 0.016 1.01 10 1.0 (0) 1.2 (0) 1.03 ± 0.044 1.01 11 1.0 (0) 1.2 (0) 1.00 ± 0.003 1.01 The statistical analysis of the results was performed using the software Design Expert 7.0 (trial version). ∗ Values are based on Mean ± SD of 3 individual observations. the software Design Expert 7.0 (trial version), and response Table 2: Statistical ANOVA for the model of endoglucanase pro- surface graphics were plotted with STATISTICA 7.0 (trial duction at different levels of concentrations of SCB and CSL. version). Source of Sum of Degrees of Mean 𝑃 value 𝐹 value The influence of sodium, calcium, potassium, and barium variations squares freedom square (prob >𝐹)a ions in the chloride form and copper, magnesium, cobalt, Model 0.13 5 0.03 11.74 0.01 manganese,andironinthesulfateformontheendoglucanase Residual 0.01 5 0.002 activity was done by the addition of the relevant salts at 2 mM final concentration in the enzyme activity assay using Lack of fit 0.01 3 0.003 2.08 0.34 the previously determined optimal conditions for pH and Pure error 0.003 2 0.001 2+ temperature. The effect of Mn was studied using at final Total 0.14 10 2 concentrations of 1, 2, 4, 8, and 10 mM. aStatistically significant at 90% of confidence level; 𝑅 =0.84. ∘ Endoglucanase thermal stability was evaluated at 65 C ∘ and 50 C upon incubation at different time intervals. Stability experiments were also performed in the presence of MnSO4 3. Results and Discussion (8 mM or 16 mM) in mixtures with 1.5 mL of the crude The sequencing of rrs gene resulted in a 1491 base sequence enzyme plus 1.5 mL of MnSO4 solutions. In all cases, residual enzymatic activity was assayed at optimal conditions for pH which was 100% similar to Streptomyces misionensis Type and temperature, taking into account the relevant enzyme Strain NRRL B-3230, and as such PESB-25 was putatively dilutions. identified as a strain belonging to S. misionensis.Thesequence obtained was submitted to the GenBank database (GenBank ID: JN869290). S. misionensis Type Strain NRRL B-3230 was 2.6. Zymogram of Endoglucanase Activity. The culture super- isolated in Misiones, Argentina, and it produces misionin, natants from optimized growth conditions were analyzed an antibiotic active against phytopathogenic fungi, including by electrophoresis on denaturing 10% sodium dodecyl sul- Helminthosporium and Alternaria [28]. Strains from this phate (SDS)-polyacrylamide gel added of copolymerized species have been cited in the literature confirming their CMClw (SIGMA) 0.2% (w/v) as the zymogram substrate. presence in certain soils [29] and their antibiotic production Electrophoresis was performed at constant voltage (100 V) capacity [30]; however, there have been no reports that strains ∘ at 4 C for 3 h followed by incubation with Triton X-100 of this species can be cellulolytic. sodium acetate 1.0% buffer for 30 min in ice bath for SDS TheuseofRSMandCCRDtoolsfortheoptimizationof removal. The detection of protein bands with endoglucanase Streptomyces misionensis endoglucanase production resulted ∘ in enzyme activity accumulation in the range of 0.67 to activity was performed by incubating gels at 50 CandpH −1 4.8 (sodium citrate buffer 50 mM) for 30 min, followed by 1.03 U⋅mL (Table 1). The fitted response surface for the the gel immersion in Congo red 0.1% (w/v) for 10 min and production of endoglucanase is given in Figure 1. Best results washing with NaCl 1M until the visualization of the enzyme were obtained at center-point conditions, with SCB 1.0% bands [27]. The molecular masses of the enzyme bands seen (w/v) and CSL 1.2% (w/v), although results obtained in some in gels were estimated by comparing their position in the other concentrations were not so different (e.g., 1.35% SCB gel with a molecular mass ladder using standard molecular and 1.03% CSL). The interaction effect evident between SCB masses ranging from 12 to 225 kDa (Full-Range Rainbow- andCSLcouldberelatedtotheC:Nproportionnecessary GE Healthcare), which was run along with the sample and for microbial growth, and consequently better enzyme pro- photographed before Congo red staining. duction. The relevant regression equations, resulting from 4 BioMed Research International

Table 3: Observed and predicted values of endoglucanase activity for the independent variables pH and temperature used in CCRD, from the crude enzyme preparation of Streptomyces misionensis PESB-25. 1.2 Endoglucanase Endoglucanase Temperature 1 pH/Coded ∘ activity activity ) Run ( C)/Coded ⋅ −1 ⋅ −1 −1 level (U mL ) (U mL ) level 0.8 Observed Predicted − − 0.6 13.6(1) 44 ( 1) 1.02 0.96 2 6.4 (+1) 44 (−1) 0.77 0.78 Endoglucanase activity (U mL (U activity 0.4 33.6(−1) 66 (+1) 1.53 1.42 0.2 4 6.4 (+1) 66 (+1) 0.81 0.77 53.0(−1.41) 55 (0) 1.21 1.31 1.8 6 7.0 (1.41) 55 (0) 0.72 0.72 1.5 1.5 75.0(0)40(−1.41) 0.78 0.79 1.24 Corn steep liquor1.2 8 5.0 (0) 70 (1.41) 1.02 1.11 1 9 5.0 (0) 55 (0) 1.27 1.23 (% w/v) 0.9 0.76 10 5.0 (0) 55 (0) 1.26 1.23 0.6 0.5 Sugarcane bagasse (% w/v) 11 5.0 (0) 55 (0) 1.18 1.23 12 5.0 (0) 55 (0) 1.20 1.23 1.01 0.7 1 0.6 0.9 0.5 Table 4: Statistical ANOVA for the model of endoglucanase activity 0.8 0.4 at different levels of pH and temperature values.

Figure 1: Response surface on endoglucanase production by Strep- Source of Sum of Degrees of Mean 𝑃 value 𝐹 value b tomyces misionensis PESB-25 using SCB and CSL concentrations as variation squares freedom square (prob >𝐹) the independent variables. Model 0.67 5 0.13 19.67 0.001 Residual 0.04 6 0.01 Lack of Fit 0.03 3 0.012 6.44 0.08 theanalysisofvariance(ANOVA)(Table 2)haveshown endoglucanase production as a function of the codified values Pure Error 0.005 3 0.002 of SCB and CSL. The equation that represented a suitable Total 0.71 11 2 model for endoglucanase production (𝑌)isgivenin: bStatistically significant at 95% of confidence level; 𝑅 =0.89. ∗ ∗ ∗ 𝑌 = 1.01 − 0.03 SCB − 0.06 SCB2 + 0.06 CSL 2 (1) The coefficient of determination obtained (𝑅 = 0.895) − 0.11∗ 2 + 0.09∗ ∗ ± 0.024. CSL SCB CSL indicates that 89.5% of the variability of the responses can be The model 𝐹 value of 11.74 implies that the model is explained by the model. significant at a high confidence level. The probability 𝑃 value The regression equations, obtained after the ANOVA, was also very low (<0.1) indicating the significance of the demonstrated endoglucanase activity as a function of the 𝛼 = 0.1 codified values of pH and temperature. The equation that model. The lack of fit term was insignificant at( ). 𝑌 The validation of the mathematical model used was represented a suitable model for endoglucanase activity ( ) performed in triplicates and confirmed the maximal values is given in: −1 ⋅ ∗ ∗ ∗ for endoglucanase activity obtained, from 0.9 to 1.04 U mL 𝑌 = 1.23 − 0.21 pH −0.11 pH2 + 0.11 T when SCB 1.0% (w/v) + CSL 1.2% (w/v) were used. (2) According to the data presented in Table 3,maximum − 0.14∗ 2 − 0.12∗ ∗ ± 0.04. −1 T pH T endoglucanase accumulation of 1.54 U⋅mL was observed ∘ at 66 C and pH 3.6. As expected, the enzyme levels were The graphic of response surface (Figure 2)suggeststhat influenced by pH and temperature. Acidic conditions as well other ranges should be studied, so a new CCRD was performed using new pH and temperature ranges (from 2.0 to 5.0 as higher temperatures favored endoglucanase activity. The ∘ ∘ analysis of the resulting surface response plots revealed that and 55 Cto85C), but the results were not an improvement. the maximal endoglucanase activity occurred in pH range of In this new matrix, the maximal endoglucanase activity ∘ −1 ∘ 3.0–3.6 and temperature of 62.5–70 C(Figure 2). was 1.30 U⋅mL at pH 4.6 and 66 C. In this case, the best The model was tested for adequacy by ANOVA (Table 4). temperaturewasthesameasbefore,buttheenzymeactivity The model 𝐹 value of 19.67 indicates that the model is signif- had decreased. icant at a high confidence level. The probability 𝑃 value was Based on the first CCRD experiment, a validation of the also very low (<0.1) indicating the significance of the model. model was performed, using the best concentrations of C BioMed Research International 5

Table 5: Effect of metal ions on endoglucanase activity. Enzyme was produced by S. misionensis PESB-25 grown on 1.0% (w/v) SCB and 1.2% (w/v) CSL.

1.6 a Relative activity Endoglucanase activity Ion ∗ − (%) (U⋅mL 1)

) Control (no addition) 100.0 1.72

−1 1.2 NaCl 133.2 ± 2.0 2.23 CuSO 140.6 ± 0.3 2.30 0.8 4 ± MgSO4 126.6 0.9 2.18 Endoglucanase activity (U mL (U activity CoSO 161.2 ± 0.6 2.73 0.4 4 ± MnSO4 201.5 0.1 3.48 ± FeSO4 131.1 0.1 2.34 70 ± CaCl2 137.6 2.3 2.25 7 62.7 KCl 125.3 ± 4.9 2.17 6 Temperature55 ( BaCl 109.3 ± 0.9 1.97 5 2 aThe final concentration in the reaction mixture was 2mM. 47.3 4 pH ∗ ∘ Values are based on Mean ± SD of 3 individual observations. C) 40 3

1.2 Table 6: Effect of different manganese concentrations on endoglu- 0.8 canase activity. 0.4 Mn2+ Relative activity Endoglucanase activity ∗ −1 Figure 2: Response surface for Streptomyces misionensis PESB-25 concentrationa (%) (U⋅mL ) endoglucanase activity by using pH and temperature values as the Control (no addition) 100.0 1.72 independent variables. 1mM 182.2± 1.5 3.08 2mM 201.5± 0.08 3.48 ± 3.28 and N sources for enzymatic production [(SCB 1.0% (w/v) 4mM 185.4 9.0 and CSL 1.2% (w/v)] and one of the pH and 𝑇 conditions 8mM 243.0± 5.7 4.34 ∘ suggested by model, pH 3.0 and 70 C, in triplicate. The 10 mM 233.7 ± 1.6 3.96 −1 results obtained were 1.54 ± 0.01 U⋅mL of endoglucanase aFinal Concentration in the reaction mixture. ∗ activity that represented an increase of 50% in endoglucanase Values are based on Mean ± SD of 3 individual observations. activity in comparison to that observed at pH 4.8 and ∘ 50 C. Based on these results, we can conclude that Strep- tomyces misionensis PESB-25 produces a thermoacidophilic Metal ions may be a requirement for enzymatic activity endoglucanase. and might even be an integral component of the enzyme Cellulases with maximum activity at the acidic pH range complex [34]. Ions may also be required as cofactors for their areoftenobservedforfungalenzymes[31]aswellasforStrep- maximum activity [35]. According to Chauvaux et al. [36], tomyces.Assuch, endoglucanase produced by S. malaysiensis manganese and other metal ions can enhance the substrate AMT-3, S. viridobrunneus SCPE-09, S. drozdowiczii M7A, binding affinity of the enzyme and stabilize the conformation and Streptomyces sp. J2 presented maximal activity in the of the catalytic site. The results for the effect of several metal pH range from 4.0 to 6.0 [8–10, 32]. However, optimum pH ions on endoglucanase activity of S. misionensis PESB-25 are for the Streptomyces misionensis PESB-25 endoglucanase was shown in Table 5. None of the ions studied inhibited the enzyme activity at a concentration of 2 mM. The addition of determined as 3.0, which is noteworthy. 2+ Ba resulted in a small increase in activity (9.3%), which In general, the optimum temperature for endoglucanase ∘ differs from the results reported by Grigorevski-Lima and activity for Streptomyces strains is around 50 C[8–10, 33]. ∘ colleagues [10], who showed that endoglucanase activity of S. Ourstrainshowedmaximumactivityat70 C, a characteristic drozdowiczii M7A greatly increased (86%) in the presence of that differs from most other Streptomyces. Jaradat et al. 2+ 2+ 2+ ∘ [32] described an optimal endoglucanase activity at 60 C, Ba . In these experiments, the addition of Mn and Co to obtained from Streptomyces sp. J2, but as far as we are the S. misionensis PESB-25 supernatant resulted in significant increases in endoglucanase activity (101.5 and 61.2%, resp.). aware, there are no reports in the literature of an endoglu- 2+ canase Streptomyces origin with optimal activity at such a Considering the significant effect of Mn 2mM on high temperature. These unusual results concerning pH and endoglucanase activity, this effect was further evaluated. temperature make our strain a very promising candidate The results are shown in Table 6 and they show the effect 2+ for biotechnological applications, especially when very acidic of Mn in the concentration range of 1 to 10 mM. This and thermophilic conditions will be necessary. ion had a hyperactivating effect on endoglucanase, with 6 BioMed Research International

−1 2+ maximum activity of 4.34 U⋅mL observed with Mn 8mM 100 which corresponded to an increase of 143% in endoglucanase 2+ activity in relation to when no Mn was added. 80 Although studies dealing with the activation of cellulase activity by manganese in Streptomyces strains have not 60 been previously reported, there is a report on the positive effect of this ion on Bacillus subtilis cellulase 5A [37]. Also 40 2+ some fungal cellulases are activated by Mn . Gao et al. Relative activity (%) activity Relative [38]studiedtheinfluenceofseveralmetalionsonactivity 20 of a purified endoglucanases from Aspergillus terreus and 2+ foundanincreaseof43%whenusingMn 2mM. Tao 0 0123456789 et al. [39], studying Aspergillus glaucus, found increments 2+ Time (h) of 30% when the final concentration of Mn 4mM was used for a purified endoglucanases obtained when grow- Figure 3: Thermal stability of Streptomyces misionensis PESB-25 ∘ ∘ ing the fungus in SCB medium. Manganese was also able endoglucanase activity at 65 C(--)and50C (—): crude extract to increase enzymatic activity of other enzymes, such as (filled diamond), crude extract + 4MnSO 8 mM (filled triangle), endonucleases from Penicillium chrysogenum PCL501, where and crude extract + MnSO4 16 mM (filled circle). Residual activity 2+ is expressed as a percentage of the original activity. Error bars an increase of 219.6% in presence of Mn 2mMwasobserved 𝑛=3 [34]. represent one standard deviation of each experimental point ( ). Fewarticleshavebeenpublisheddescribingcellulase production by actinomycetes using agroindustrial residues and over 70% after 9 h of incubation, shows beyond doubt as substrates, and most of them have given very low values 2+ for endoglucanase activity when using wheat straw (WS) the positive effect of Mn 16mMontheenzymestructural stabilization. According to the overall results, incubation of [40, 41] or wheat bran (WB) [10]asthemainsubstrate. 2+ ∘ ⋅ −1 the crude extract with Mn at 50 Cincreasedthehalf-life Our group has obtained values of 0.71 U mL when using 2+ ⋅ −1 oftheenzymefrom4h(noMn addition) to more than 8 h brewer spent grain (BSG) [8], and more recently 2.00 U mL 2+ ⋅ −1 (addition of Mn 8 mM) or even more than 30 h (addition when using wheat bran [9]. Values as high as 4.34 U mL , 2+ obtained in the present research, have not been described yet of Mn 16 mM). Values of half-lives of 8 h have been for endoglucanase production by actinomycetes using low- currently reported in the literature for Streptomyces strains cost residues, especially SCB. [9, 10]. The results of the endoglucanase thermal stability are These are very promising results for the Streptomyces shown in Figure 3. When the enzyme crude extract was misionensis endoglucanase. Its natural thermal stability ∘ incubated at 65 C,theenzymeactivitydecreased70%ofits (which can be significantly enhanced with manganese) indi- initial activity within 15 min of incubation. However, upon cates potential as a biocatalyst for industrial process that ∘ incubation at 50 C, activity decreased to 40% within 30 min, demands long processing times at elevated temperatures, ∘ retaining this activity for 2 h. The enzyme half-life at 50 Cwas such as those in the food, sugar, and fuel ethanol industries 4h. [33]. Also, additional studies for the determination of its It is known that metal ions play an important role in stability at different pH values and different periods of time stabilizing proteins, protecting against thermal denaturation would be interesting for future industrial applications. by binding at specific sites36 [ , 42]. Several studies have The zymogram analysis of the culture supernatant of shown increased enzyme thermal stability in presence of Streptomyces misionensis PESB-25 is shown in Figure 4.Three calcium [27, 42, 43], which is known to regulate the stability protein bands with endoglucanase activity and estimated and reactivity of a wide variety of biological proteins [43]. molecular masses of 12.0, 48.5 and 119.5 kDa are clearly 2+ Given the strong positive effect of Mn on endoglucanase shown. Cellulose degrading microorganisms commonly pro- activity, the effect of this ion on the enzyme stability was duce multienzyme systems [44]. As such, and in accordance further investigated. It was observed that in the presence of to previous reports, Nascimento and colleagues [8]observed 2+ three cellulolytic bands (51, 115, and 178 kDa) in the super- Mn 8 mM, the crude enzyme preparation increased 25% of ∘ natants of S. malaysiensis AMT-3 when BSG 0.5% (w/v) and its initial activity upon incubation for 30 min at 65 C, and 2+ CSL1.2%(w/v)wereused.DaVinhaetal.[9], in their when Mn 16 mM was used, the activity increased to over study, cultured Streptomyces viridobrunneus SCPE-09 in 2.0% 70%. Moreover, at a manganese ion concentration of 16 mM, ∘ wheat bran (w/v) and 0.19% CSL (w/v). In these conditions, theenzymehalf-lifeat65Cwasalmost2hours. ∘ two bands of endoglucanase activity were observed, one Results from enzyme stability at 50 Cwereevenmore 2+ with estimated molecular masses of 37 and the other with promising. The incubation of crude extract with Mn at 119 kDa. final concentration of 16 mM resulted in an increase in Additional studies about these enzymes are required to thermal stability of 40% after 4 hours incubationFigure ( 3), better evaluate their feasibility for further industrial applica- in comparison to the results for the experiments in the tions. Purification would enable kinetics studies and also the absenceoftheion.Activityretentionofover92%for5h, determination of their specific activity. BioMed Research International 7

Abbreviations 150 ANOVA: Analysis of variance 119.5 BLAST: Basic local alignment search tool 102 BSG: Brewer spent grain CCRD: Central composite rotational design CMClw: Carboxymethylcellulose low viscosity CSL: Corn steep liquor 52 NCBI: National center for biotechnology information (kDa) RDP: Ribosomal database project 48.5 RSM: Response surface methodology (kDa) SCB: Sugarcane bagasse SDS: Sodium dodecyl sulphate 38 WB: Wheat bran WS: Wheat straw. 24

12 Acknowledgments This work was supported by grants from the Conselho Figure 4: Zymogram analysis (duplicate experiment) of the culture Nacional de Desenvolvimento Cient´ıfico e Tecnologico´ supernatant of S. misionensis PESB-25 showing three bands with (CNPq), Coordenaçao˜ de Aperfeiçoamento de Pessoal do endoglucanase activity. Cells were grown on SCB 1.0% (w/v) and Ensino Superior (CAPES), and Financiadora de Estudos CSL1.2%(w/v).Thecalculatedmolecularmasses(inkDa)ofthe e Projetos (FINEP). The authors also thank the Postgrad- endoglucanases are indicated on the right side of the figure. The uate Programme in Plant Biotechnology (Programa de molecular masses of the markers Full-Range Rainbow (GE-RPN pos-graduac´ ¸ao˜ em Biotecnologia Vegetal) and Postgraduate 800E) are indicated on the left side of the figure. For details see text. Programme in Science, Microbiology (Programa de pos- graduaçao˜ em Ciencias,ˆ Microbiologia) at Universidade Fed- 4. Conclusions eral do Rio de Janeiro (UFRJ). In this study, S. misionensis PESB-25 was able to grow and References produce endoglucanase in a culture medium containing a salt solution and agroindustrial by-products, specifically [1] J. Kim, S. Yun,and Z. Ounaies, “Discovery of cellulose as a smart sugarcane bagasse and corn steep liquor, as the main car- material,” Macromolecules,vol.39,no.12,pp.4202–4206,2006. bon and nitrogen substrates. Characterization of the crude [2] P. Beguin´ and J. P. Aubert, “The biological degradation of enzyme showed that the endoglucanases produced were cellulose,” FEMS Microbiology Reviews,vol.13,no.1,pp.25–28, acidic, thermophilic, and thermotolerant. An optimum pH 1994. of 3.0 was reported which is rare. An optimum activity [3]J.Liu,W.D.Liu,X.L.Zhao,W.J.Shen,H.Cao,andZ.L.Cui, ∘ temperature at 70 Cwasseenandisnovelforactinobacterial “Cloning and functional characterization of a novel endo-𝛽- strains. The activity of these endoglucanases was also strongly 1,4- glucanase gene from a soil-derived metagenomic library,” increased and more stable in the presence of a number of Applied Microbiology and Biotechnology,vol.89,no.4,pp.1083– 2+ −1 metal ions, especially Mn .Activityof4.34U⋅mL was 1092, 2011. obtained under these conditions. This level of activity places [4] S. L. Liu and M. Xing, “Purification, properties and mass spec- this study amongst the highest described in the literature for trometry analysis of two novel thermotolerant endoglucanases cellulase production by Streptomyces strains using low-cost from Bacillus akibai I-2,” Advanced Materials Research,vol.393– +2 395, pp. 911–915, 2012. residues as substrates. The effect of Mn 16 mM on enzyme [5] J. Lacey, “Actinomycetes in composts,” Annals of Agricultural stability was also important and noteworthy. Manganese at and Environmental Medicine,vol.4,pp.113–121,1997. that concentration increased the enzyme stability half-life ∘ from less than 4 h to greater than 30 h at 50 Candfromless [6] C. S. Park, T. Kawaguchi, J. I. Sumitani, G. Takada, K. Izumori, ∘ and M. Arai, “Cloning and sequencing of an exoglucanase gene than 30 minutes to 2 h at 65 C. from Streptomyces sp.M23,anditsexpressioninStreptomyces The characteristics of thermoacidophiles, thermal sta- lividans TK-24,” Journal of Bioscience and Bioengineering,vol. bility, and induction by manganese suggest that endoglu- 99,no.4,pp.434–436,2005. canases from S. misionensis PESB-25 could be considered [7]F.Alani,W.A.Anderson,andM.Moo-Young,“Newisolate as promising alternatives in biotechnological applications. of Streptomyces sp. with novel thermoalkalotolerant cellulases,” For example they could be used as a complement to fungal Biotechnology Letters,vol.30,no.1,pp.123–126,2008. enzymatic mixtures improving the lignocellulose hydrolysis [8] R. P. Nascimento, N. A. Junior, N. Pereira, E. P. S. Bon, and R. for ethanol production. Combining advantageous enzyme R. R. Coelho, “Brewer’s spent grain and corn steep liquor as characteristics with the use of low-cost residues (SCB and substrates for cellulolytic enzymes production by Streptomyces CSL), we have the potential for a new low-cost enzyme malaysiensis,” Letters in Applied Microbiology,vol.48,no.5,pp. production process. 529–535, 2009. 8 BioMed Research International

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Research Article Anti-Candida Properties of Urauchimycins from Actinobacteria Associated with Trachymyrmex Ants

Thais D. Mendes,1,2 Warley S. Borges,3,4 Andre Rodrigues,1,5 Scott E. Solomon,6 Paulo C. Vieira,4 Marta C. T. Duarte,7 and Fernando C. Pagnocca1,5

1 Center for the Study of Social Insects, Sao˜ Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil 2 EMBRAPA Agroenergy, Parque Estac¸ao˜ Biologica,´ 70770-901 Bras´ılia, DF, Brazil 3 Chemistry Departament, Federal University of Esp´ırito Santo (UFES), 29075-910 Vitoria,´ ES, Brazil 4 Chemistry Department, Federal University of Sao˜ Carlos (UFSCar), 18052-780 Sao˜ Carlos, SP, Brazil 5 Department of Biochemistry and Microbiology, Sao˜ Paulo State University (UNESP), 13506-900 Rio Claro, SP, Brazil 6 Department of Ecology and Evolutionary Biology, Rice University, Houston, TX, USA 7 Division of Microbiology, Center for Chemistry, Biology and Agriculture Research (CPQBA/UNICAMP), 13081-970 Paul´ınia, SP, Brazil

Correspondence should be addressed to Fernando C. Pagnocca; [email protected]

Received 12 November 2012; Revised 29 January 2013; Accepted 2 February 2013

Academic Editor: Manish Bodas

Copyright © 2013 Thais D. Mendes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

After decades of intensive searching for antimicrobial compounds derived from actinobacteria, the frequency of isolation ofnew molecules has decreased. To cope with this concern, studies have focused on the exploitation of actinobacteria from unexplored environments and actinobacteria symbionts of plants and animals. In this study, twenty-four actinobacteria strains isolated from workers of Trachymyrmex ants were evaluated for antifungal activity towards a variety of Candida species. Results revealed that seven strains inhibited the tested Candida species. Streptomyces sp. TD025 presented potent and broad spectrum of inhibition of Candida and was selected for the isolation of bioactive molecules. From liquid shake culture of this bacterium, we isolated the rare antimycin urauchimycins A and B. For the first time, these molecules were evaluated for antifungal activity against medically important Candida species. Both antimycins showed antifungal activity, especially urauchimycin B. This compound inhibited the growth of all Candida species tested, with minimum inhibitory concentration values equivalent to the antifungal nystatin. Our results concur with the predictions that the attine ant-microbe symbiosis may be a source of bioactive metabolites for biotechnology and medical applications.

1. Introduction Actinobacteria are widely known for their ability to produce bioactive secondary metabolites, especially compounds The increased resistance of microorganisms to antibiotics isa with antimicrobial activity. These bacteria are responsible for problem of public health [1]. The increasing number of fungal producing two-thirds of the commercially available antibi- species that can infect humans, particularly immunocompro- otics [2, 3]. Most actinobacteria species explored commer- mised individuals, further reinforces this concern. A limited cially were isolated from the soil. However, after decades of number of antifungal agents are commercially available when bioprospecting actinobacteria from this environment, it is compared to antibacterial drugs. This scenario motivates the becoming more difficult to obtain strains producing novel search for new bioactive compounds in various biological bioactive metabolites [4]. Thus, many companies have turned systems using several approaches, including metagenomics the search for microbial producers of novel antifungal com- and microbial genome-mining. pounds to other environments such as hydrothermal vents, 2 BioMed Research International marine environments, tropical rain forests, and microbial were carefully excavated in order to reach the first fungus symbionts associated with plants and animals hosts [5, 6]. garden chamber. Fungus garden with the tending workers For example, the occurrence of actinobacteria associated with and brood was sampled using an alcohol-flamed spoon and marine sponges and the fact that such strains produce com- stored in sterile plastic containers. All containers were kept poundswithantimicrobialactivityconfirmsthispotential[7– in a cooler during transport to the laboratory where they ∘ 9]. In addition, endophytic actinobacteria are also explored were maintained at 25 C. for their capacity to produce antimicrobial compounds [10– From each colony, we randomly selected four workers 12]. for actinobacteria isolation. Then, the propleural plates Several studies have focused on the association between were scraped with a sterile needle under a low power actinobacteria and insects from an ecological perspective stereomicroscope. All ants used in the present study had a [13–25].Ontheotherhand,fewstudieshavefocusedon visible, whitish covering on the propleural plates. Scrapings −1 the multitude of chemical compounds that are involved were plated on SCN agar (in g ⋅ L : 10.0 starch, 0.3 casein, in such interactions [26]. The best studied example is the 2.0 KNO3, 2.0 NaCl, 2.0 K2HPO4, 0.05 MgSO4 ⋅ 7H2O, symbiosis between actinobacteria and fungus-growing ants 0.02 CaCO3, 0.01 FeSO4 ⋅ 7H2O and 18.0 agar supplemented (Hymenoptera: Formicidae: tribe Attini). In this association, with 0.05 Nystatin) [36]. After scraping, the entire body theactinobacteriaarefoundontheants’integumentand of all workers was inoculated on SCN agar. All plates ∘ produce antimicrobial compounds that help the ants to were incubated at 25 Cfor30days.Fromeachsampled suppress the microfungus Escovopsis sp. [13, 14]. This fungus Trachymyrmex colony, one representative strain was is considered a specialized parasite of the ant cultivar and selected from each morphotype obtained. The strains were −1 causes negative impacts to the ant colony [27]. subcultured in YMA (in g ⋅ L : 3.0 yeast extract, 3.0 malt Actinobacteria isolated from the integument of attine extract, 5.0 peptone, 10.0 glucose, 18.0 agar) and stored at ∘ ants are generally classified in the genus Pseudonocardia and −80 Cin15%glycerol. Streptomyces. Bioactive molecules have already been isolated We used a molecular approach to provide taxonomic and characterized from actinobacteria isolated from several affiliation to actinobacteria strains. Genomic DNA was attine genera [26]. Pseudonocardia isolated from Acromyrmex extracted following the method of Sampaio et al. [37]. We octospinosus and Apterostigma dentigerum are known to pro- carried out 16S rDNA PCR with the universal primers 󸀠 󸀠 󸀠 duce several compounds like (i) dentigerumycin, a complex 27F (5 -AGAGTTTGATCATGGCTCAG-3 )and1492R(5- 󸀠 compound active against Candida albicans and Escovopsis TACGGTTACCTTGTTACGACTT-3 )[38]. Reactions were [28]; (ii) a nystatin-like antifungal [29]; (iii) the novel quinone conducted in a final volume of 25 𝜇L and contained 1 𝜇L pseudonocardones A–C active against the malaria causal of diluted DNA template (1 : 10), 2.0 𝜇L of each primer agent Plasmodium berghei [30]; (iv) the already known antibi- (10 mM), 2.5 𝜇Lof10Xbuffer,1.0𝜇L of MgCl2 (50 mM), otics 6-deoxy-8-O-methylrabelomycin and X-14 881, both 4.0 𝜇L of dNTPs (1.25 mM each), 0.2 𝜇LofTaqpolymerase active against Bacillus subtilis and P. b e rg he i [30]. In addition (5 U/𝜇L), and 12.3 𝜇L of ultrapure water. Amplicons were to Pseudonocardia, actinobacteria in the genus Streptomyces cleaned up with GFXPCRDNAandGelBandPurifica- arealsofoundontheintegumentofAcromyrmex workers tion Kit (GE Healthcare). Forward and reverse sequences and were shown to produce (i) candicidin, active against weregeneratedusingthesameprimers,alongwithan 󸀠 Escovopsis sp. [29, 31–34], (ii) antimycins active against internal primer U519F (5 -CAGCMGCCGCGGTAATWC- 󸀠 Escovopsis sp. [32–34], and (iii) actinomycin D, actinomycin 3 ). Sequences were generated using BigDye Terminator v.3.1 X2 and valinomycin that are active against B. subtilis [32]. Cycle Sequencing Kit (Life Technologies) in an ABI 3130 Poulsen [35] suggested that the attine ant-microbe asso- sequencer and manually edited in BioEdit v. 7.1.3 [39]. Contigs ciation is little explored regarding the search for new were compared with those available in the databases NCBI- antimicrobials. The author highlights the various symbiotic GenBank (http://blast.ncbi.nlm.nih.gov/)andRibossomal associations between attine ants and microorganisms as a Database Project (RDP, http://rdp.cme.msu.edu/). Sequences promising source for drug discovery, especially those with generated in the present study were deposited in NCBI- antimicrobial activity. Here, we explored the antimicrobial GenBank (accessions KC480554-KC480557). potential of actinobacteria isolated from the integument of A phylogenetic analysis was carried out in order to deter- Trachymyrmex fungus-growing ants and demonstrate the mine the taxonomic affiliation of strain TD025. Sequences action against different medically important Candida species. of closest relatives were retrieved from the NCBI-GenBank We also report two previously described urauchimycins from and the RDP Project and aligned in ClustalW. Phylogenetic a Streptomyces strain and emphasize the newly discovered reconstruction was performed using the neighbor-joining anti-Candida activity of these compounds. algorithm implemented in PAUP v. 4.0 [40]. Genetic distances were calculated using the Kimura 2-parameter model of nucleotide substitution [41]. Robustness of the relation- 2. Material and Methods ships was estimated from 1000 bootstrap pseudoreplicates. 2.1. Actinobacteria Isolation and Identification. Twelve Trachymyrmex colonies were collected in different Brazilian 2.2. Organic Extracts and Antifungal Assays. All actinobac- biomes (see Table S1 in Supplementary Material available teria were grown in Erlenmeyer flasks (250 mL) containing −1 online at http://dx.doi.org/10.1155/2013/835081). Colonies 50 mL of modified MPE medium (in g ⋅ L :5.0soyflour, BioMed Research International 3

20.0 glucose, 5.0 of NaCl, 4.0 CaCO3)[42]. Each flask was compounds, they were evaluated for antimicrobial activity inoculated with five mycelium disks (1 cm in diameter) cut following the method described previously. Besides the MIC from a previously grown culture and then incubated at determination, we also evaluated the minimum fungicidal ∘ 28 Cfortwodaysonanorbitalshakerat150rpm.From concentration (MFC). The MFC was determined by inoculat- this culture, 10 mL was inoculated in two Erlenmeyer flasks ing Sabouraud dextrose medium with 10 𝜇Lofthecontents (250 mL) containing 100 mL of the same medium and incu- of each of the wells where there was growth inhibition of bated for five days on the same conditions. After incubation, yeast, the MFC was defined as the lowest concentration of the the fermented broth was separated from the mycelium by substance capable of preventing the onset of colony forming centrifugation and partitioned three times with ethyl acetate units. (EtOAc). The organic solvent was evaporated under vacuum, and the EtOAc extracts were diluted in RPMI-1640 culture broth containing 10% DMSO and used in the antimicrobial 3. Results and Discussion assays. 3.1. Actinobacteria Isolation and Identification. Several acti- The antimicrobial activity of the extracts was evalu- nobacteria colonies were observed after incubation of iso- ated against the yeasts Candida albicans CBS 562, Candida lation plates. We selected just one morphotype of each per dubliniensis CBS 7987, Candida glabrata CBS 138, Candida antcolony,renderingatotalof24strainsoufof12Tra- krusei CBS 573, Candida parapsilosis CBS 604, and Candida chymyrmex spp. nests (Table S1). Four actinobacteria genera tropicalis CBS 94. The minimum inhibitory concentration were identified and Streptomyces was the most abundant (MIC) was determined using the microdilution method taxon (Table 1), corresponding to 66.67% of the strains. It according to the M27-A2 standard of the Clinical and Lab- is assumed that the main actinobacteria associated with the oratory Standards Institute [43]. integument of attine workers is the genus Pseudonocardia [14–16, 20]. However, several authors have demonstrated the 2.3. Isolation and Characterization of the Bioactive Com- isolation of actinobacteria other than Pseudonocardia on the pounds of Strain TD025. The actinobacteria strain that exhib- integument of attine ants [17–19, 21, 22, 29, 31]. The prevalence ited both a broad antifungal spectrum and lower MIC values of Streptomyces and absence of Pseudonocardia among our was strain TD025. In order to identify the compounds isolates may be due to the culture medium used [44]. The responsiblefortheobservedresults,thestrainwasculturedin SCN medium is suitable for the isolation of fast-growing 5 L of modified MPE medium and the extracts were obtained actinobacteria, but according to other authors [13–16], the use as described. of a low-nutrient medium, such as chitin agar, may provide The fermented broth (5 L) was separated from the cells by the recovery of Pseudonocardia strains. centrifugation and portioned three times with ethyl acetate Ineightoutof12antcolonies,weobtainedmorethan (EtOAc). The organic solvent was evaporated under vacuum. one actinobacteria morphotype (Table S1). From colony The crude extract, a dark green oil (1.40 g), was separated CTL080820-02, the two morphotypes isolated from two by means of column chromatography using silica gel 60 different workers were identified as the same actinobacteria eluted with n-hexane/EtOAc as the elution gradient, yielding species (Tables S1 and 1). On the other hand, different 8 fractions. All fractions were submitted to antimicrobial actinobacteria species were isolated in the seven remaining assays against C. albicans following the procedure described colonies. We also observed the occurrence of different acti- above. The most active fraction (67.5 mg) was subjected to nobacteria strains in a single worker (Tables S1 and 1). This preparative TLC (thin layer chromatography) eluted with result demonstrates the diversity of actinobacteria present on n-hexane/EtOAc (7 : 3) two times, yielding 3 subfractions. the integument of these ants (Table S1, nests SES080911-04 The subfractions obtained were submitted to antimicrobial and SES080924-01). assays against C. albicans.Themostactivesubfractionwas The 16S rDNA sequence of strain TD025 showed 99% submitted to semipreparative HPLC separations carried out similarity with sequences of several species of the genus in a Shimadzu (LC-6AD apparatus, Japan) multisolvent Streptomyces deposited in the databases. For a better charac- delivery system, Shimadzu SPD-M10Avp Photodiode Array terization, we performed a phylogenetic analysis (Figure 1). Detector, and an Intel Celeron computer for analytical system The result suggests a differentiated phylogenetic position for control, data collection and processing (software Class-VP). strain TD025 when compared with the remaining sequences. The separation was carried out using VP 250/10 NUCLEOSIL This preliminary analysis allowed us to assign this strain 120-5 C18 column eluted with acetonitrile/water/acetic acid as belonging to the genus Streptomyces,withS. cirratus as −1 (50:50:0.01)ata flowrateof3mL⋅ min , yielding com- the closest relative strain (Figure 1). However, more refined pounds 1 and 2. The isolated molecules were characterized by phylogenetic analyses, along with morphological and physi- 1 13 Hand C NMR spectroscopic experiments recorded on a ological studies, are necessary to ensure the identification of BRUKER DRX-400 spectrometer with CDCl3 as solvent and TD025 to the species level. TMS as internal standard. 3.2. Screening for Antifungal Activity. Our screening for 2.4. Minimum Inhibitory Concentration and Minimum Fungi- antifungal activity revealed that seven out of 24 extracts cide Concentration of Bioactive Compounds. After isola- (29.16%) inhibited the growth of at least one Candida tion and determination of the structure of the targeted species. C. albicans was the most sensitive yeast and was 4 BioMed Research International

Table 1: Actinobacteria identification according to 16S rDNA sequencing.

Isolate Id bp1 NCBI-GenBank closest relative Coverage % Accession # TD016 1251 Nocardia neocaledoniensis DSM 44717 100 100 JF797311 TD017 1248 Nocardia neocaledoniensis DSM 44717 100 100 JF797311 TD018 1184 Streptomyces zaomyceticus NRLL B-2038 99 100 NR044144 TD019 1342 Streptomyces alivochromogenes NBRC 3404 99 100 AB184761 TD020 1167 Streptomyces sannanensis NBRC 14239 99 99 NR041160 TD021 1250 Streptomyces mauvecolor NBRC 13854 100 99 NR041154 TD022 1254 Streptomyces lydicus CGMCC 4.1412 100 100 JN566018 TD023 1246 Streptomyces sp. QZGY-A17 100 100 JQ812074 TD025 1333 Streptomyces sp. QLS92 100 99 JQ838121 Streptomyces cirratus 100 99 JQ222143 TD027 1342 Streptomyces alivochromogenes NBRC 3404 99 100 AB184761 TD028 1353 Actinoplanes ferrugineus 100 99 AB048221 TD030 1255 Streptomyces sp. CA13 100 99 AB622252 TD032 1263 Streptomyces chartreusis NBRC 12753 100 100 NR041216 TD033 1278 Streptomyces griseoplanus 99 100 HQ699516 TD034 1283 Amycolatopsis decaplanina DSM 44594 100 100 NR025562 TD035 1320 Streptomyces atriruber NRLL B-24676 100 99 FJ169330 TD045 1183 Amycolatopsis equina 100 99 HQ021204 TD047 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251 TD049 1260 Streptomyces luteogriseus NBRC 13402 100 99 NR041128 TD050 1261 Streptomyces rubiginosohelvolus NBRC 12912 100 100 NR041093 TD051 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251 TD053 1265 Streptomyces kunmingensis NBRC14463 99 98 AB184597 TD055 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251 TD058 1257 Streptomyces globisporus KCTC 9026 100 100 HQ995504 1 bp: base pair. inhibited by seven extracts with MIC ranging between 10 Compounds 1 and 2 exhibited typical NMR data of −1 and 1000 𝜇g ⋅ mL (Table 2). The yeasts C. glabrata and C. urauchimycins (Figures S1 and S2). Their NMR data are in tropicalis were the most resistant strains, being inhibited by agreement with those previously reported by Imamura et al. one and two actinobacteria extracts, respectively, with MIC [46]. Although these urauchimycins (1 and 2)havealready −1 values of 1000 𝜇g ⋅ mL (Table 2). been isolated, they have never been tested on various species of Candida as carried out in the present study. Except for strain TD034 identified as Amycolatopsis 13 decaplanina (Table 1), the other actinobacteria exhibiting The CNMRspectrumof1 showed 22 carbon signals: antimicrobial activity were identified as belonging to the four carbonyls (𝛿 179.0, 𝛿 170.6, 𝛿 169.8, and 𝛿 158.7), three 2 genus Streptomyces.Thisgenusisrecognizedasthelargest quaternary sp carbons (𝛿 150.6, 𝛿 127.4, and 𝛿 112.8), three producer of antibiotics because from approximately 3,000 methine aromatic carbons (𝛿 124.8, 𝛿 120.6, and 𝛿 119.0), two 3 3 known antibiotics obtained from actinobacteria, the genus sp methylene group (𝛿 35.6 and 𝛿 30.5), three sp methine Streptomyces contributes with 90% of this total [45]. groups (𝛿 54.0, 𝛿 50.1, and 𝛿 32.4), three oxymethinic groups The extracts of Streptomyces sp. TD025 and Streptomyces (𝛿 77.1, 𝛿 76.3, and 𝛿 70.9), and four methyl groups (𝛿 18.4, crystallinus TD027 showed activity against all yeast strains 𝛿 18.4, 𝛿 15.1, and 𝛿 11.4). Two carboxylic carbons 𝛿 170.6 except for C. glabrata. These extracts were effective against and 𝛿 173.9 showed correlations with different hydrogens of C. albicans and C. krusei and showed low activity against the structure, showing a dilactone system of nine members, C. tropicalis (Table 2). More interestingly, both strains were typical of the antimycin class. 1 isolated from the same colony but from independent workers The H NMR spectrum showed a singlet at 𝛿 8.50, (Table 1). Because lower MICs were obtained for the extract assigned to a hydrogen bounded to a carbonyl group and of Streptomyces sp. TD025, this strain was selected to verify three aromatic hydrogens at 𝛿 8.55 (dd, J 8.1 and 1.2 Hz), 𝛿 7. 24 the chemical compounds responsible for the antimicrobial (dd, J 8.1and1.2Hz),andd6.92(t,J 8.1 Hz), suggesting a 1,2,3 activity. trisubstituted aromatic ring. The substance was identified as urauchimycin A by comparison with the literature data [46]. 1 13 3.3. Bioactive Compounds of Streptomyces sp. TD025. Chro- The Hand CNMRspectramof2 were very similar to matographic procedures revealed that EtOAc extract from those observed for compound 1. Differences were observed in TD025 contains two compounds (1 and 2, Figure 2). chemical shifts of the hydrogen of the methyl and methylene BioMed Research International 5

TD025 (KC480562) 100 Streptomyces sp. (JQ838121) 100 73 Streptomyces cirratus (JQ422143)

65 Streptomyces sp. (EF093114)

74 Streptomyces spiroverticillatus (NR041214)

Streptomyces griseobrunneus (AB249912)

Streptomyces albolongus (NR041144)

100

Streptomyces flavogriseus (NR028988) 58

71 Streptomyces griseolus (NR041207)

Streptomyces cinereorectus (NR041173) 86 Streptomyces badius (NR043350) 58

68 Streptomyces rubinosohelvolus (NR041093)

Streptomyces globisporus (NR044145)

Streptomyces crystallinus (NR041177) 0.001

Figure 1: Phylogenetic relationships of strain TD025 (in bold) isolated from the integument of Trachymyrmex sp. The phylogeny was inferred from 16S rDNA sequences retrieved from the NCBI-GenBank using the neighbor-joining algorithm and the Kimura 2-parameter model of nucleotide substitution. Numbers in parentheses correspond to GenBank accessions. Numbers on branches indicate the bootstrap support after 1,000 pseudoreplicates. The scale bar denotes the number of substitutions per site. groups of the side chain. Based on published data [46] Urauchimycins A and B were previously isolated from compound 2 was identified as Urauchimycin B, an isomer of Streptomyces sp. Ni-80 isolated from a marine sponge in compound 1. Urauchicove, Irimore, Japan. These substances were the Urauchimycins belong to the antimycin class, a group first antimycins having an odd number of carbons and a of well-known antifungals. Antimycins act by inhibiting the branching side chain [46]. Imamura et al. [46]suggestedthat electron flow in the mitochondrial respiratory chain [47]. such structures are the result of an evolution of actinobacteria Antimycins have been previously identified in Streptomyces in the marine environment, which could have resulted in a isolated from the integument of attine ants [32–34]. Schoe- change in their secondary metabolism. nian and colleagues [32] detected the well-know antimycins In 2006, two new urauchimycins were described: urau- A1–A4in50%oftheactinobacteriaidentifiedasStreptomyces chimycin C, isolated from Streptomyces sp. B1751 from marine isolated from workers of several Acromyrmex species. These sediment, and urauchimycin D, isolated from Streptomyces data along with the rare antimycins identified in the present sp. AdM21 from soil [48]. In the study by Imamura and studyindicatethatthischemicalclassisoftenproduced coworkers [46], the urauchimycins A and B inhibited the by Streptomyces associated with attine ants. Compounds morphological differentiation of C. albicans up to a con- −1 belonging to this class may have an important role in the centration of 10 𝜇g ⋅ mL .UrauchimycinsCandDshowed attine ant-microbe association. no inhibitory activity against C. albicans, Mucor miehei,and Another antifungal compound widely distributed in bacteria [48]. Streptomyces associated with attine ants is candicidin The study of antimicrobial activity of urauchimycins A [31–34], which was not detected in Streptomyces sp. TD025. It andBwasrestrictedtoC. albicans in the work by Imamura is possible that candicidin was lost in one of the purification and colleagues [46]. The reisolation of these molecules in the steps of the AcOEt extract or it is not produced by this strain. present study allowed a better evaluation of its spectrum of 6 BioMed Research International

− Table 2: Minimum inhibitory concentrations (𝜇g⋅mL 1) of actinobacteria extracts towards different medically important Candida species.

C. albicans C. dubliniensis C. glabrata C. krusei C. parapsilosis C. tropicalis Isolate ID CBS 562 CBS 7987 CBS 138 CBS 573 CBS 604 CBS 94 TD016 ∗∗∗∗∗∗ TD017 ∗∗∗∗∗∗ TD018 ∗∗∗∗∗∗ TD019 60 900 ∗ 40 80 1000 TD020 ∗∗∗∗∗∗ TD021 ∗∗∗∗∗∗ TD022 800 ∗ 1000 ∗∗ ∗ TD023 ∗∗∗∗∗∗ TD025 40 700 ∗ 15 125 1000 TD027 60 1000 ∗ 15 200 ∗ TD028 ∗∗∗∗∗∗ TD030 ∗∗∗∗∗∗ TD032 1000 ∗∗∗ ∗ TD033 10 800 ∗ 125 200 ∗ TD034 500 ∗∗∗ ∗ TD035 ∗∗∗∗∗∗ TD045 ∗∗∗∗∗∗ TD047 ∗∗∗∗∗∗ TD049 ∗∗∗∗∗∗ TD050 ∗∗∗∗∗∗ TD051 ∗∗∗∗∗∗ TD053 ∗∗∗∗∗∗ TD055 ∗∗∗∗∗∗ TD058 ∗∗∗∗∗∗ ∗ − Minimum inhibitory concentration > 1000 𝜇g⋅mL 1.

O O O O H N H N O N H N O H OH H O H OH OH O O O OH O O

1 2

Figure 2: Chemical structures of compounds isolated from Streptomyces sp. TD025. (1) urauchimycin A; (2) urauchimycin B.

activity. The urauchimycins from Streptomyces sp. TD025 pre- antifungal agents that complement or substitute for the scarce sented MIC values equivalent to the reference antifungal nys- products available on the market, it is interesting and nec- tatin for C. albicans and C. glabrata (Table 3). Urauchimycin essary to determine the toxicity presented by urauchimycin B showed inhibitory activity against all Candida strains eval- B, to assess whether it can be used as an antifungal agent for uated, showing MIC similar to those provided by nystatin. humans and animals. In addition, evaluation of the isolated Urauchimycin B showed a broad spectrum of activity compound against Candida species resistant to commercially against Candida spp. with MIC values equivalent to the available antifungal agents should be performed to confirm antifungal nystatin, which indicates the potential for medical the potential of this relatively unexplored antifungal. use. For many years, antimycins were used for the treatment Here we show that Trachymyrmex ants, one attine genus of human infections, but due to its mechanism of action understudied with respect to its microbial symbionts, har- andassociatedsideeffects,itsuseinhumantreatmentwas bor antimicrobial-producing actinobacteria. As observed by discontinued [47]. However, with the pressing need for new other authors [28–33], the present study demonstrates that BioMed Research International 7

Table 3: Minimum inhibitory concentration (MIC) and minimum thank two anonymous referees for helpful comments on this − fungicide concentrations (MFC) (𝜇g⋅mL 1) of Urauchimycins A and paper. BobtainedfromStreptomyces sp. TD025 in comparison with the antifungal Nystatin. References Urauchimycin A Urauchimycin B Nystatin Candida species MIC MFC MIC MFC MIC MFC [1] J. Travis, “Reviving the antibiotic miracle?” Nature,vol.264,no. 5157, pp. 360–362, 1994. C. albicans 1 ∗ 1312 [2] S. Miyadoh, “Research on antibiotic screening in Japan over C. dubliniensis 800 ∗ 2312 the last decade: a producing microorganism approach,” Actino- C. glabrata 215,62 2 11 mycetologica,vol.7,no.2,pp.100–106,1993. C. krusei 15,6 15,6 2 3 2 3 [3]G.L.ChallisandD.A.Hopwood,“Synergyandcontingency ∗∗ C. parapsilosis 2212 as driving forces for the evolution of multiple secondary C. tropicalis ∗∗2244 metabolite production by Streptomyces species,” Proceedings of ∗ − >1000 𝜇g⋅mL 1. the National Academy of Sciences of the United States of America, vol. 100, no. 24, pp. 14555–14561, 2003. [4] M. Goodfellow and H. P. Fiedler, “A guide to successful bio- actinobacteria of attine ants are able to produce antifungal prospecting: informed by actinobacterial systematics,” Antonie compounds active against other fungal species and not only van Leeuwenhoek, vol. 98, no. 2, pp. 119–142, 2010. against the specific fungal parasite Escovopsis. [5]A.C.W.WaughandP.F.Long,“Prospectsforgeneratingnew Moreover, our study corroborates previous work [35]that antibiotics,” Science Progress,vol.85,no.1,pp.73–88,2002. suggests the attine ant-microbe association is a promising [6] J. Clardy, M. A. Fischbach, and C. T. 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Review Article Identification and Biotechnological Application of Novel Regulatory Genes Involved in Streptomyces Polyketide Overproduction through Reverse Engineering Strategy

Ji-Hye Nah, Hye-Jin Kim, Han-Na Lee, Mi-Jin Lee, Si-Sun Choi, and Eung-Soo Kim

Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea

Correspondence should be addressed to Eung-Soo Kim; [email protected]

Received 13 November 2012; Revised 15 December 2012; Accepted 5 January 2013

Academic Editor: Sofiane Ghorbel

Copyright © 2013 Ji-Hye Nah et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Polyketide belongs to a family of abundant natural products typically produced by the filamentous soil bacteria Streptomyces. Similar to the biosynthesis of most secondary metabolites produced in the Streptomyces species, polyketide compounds are synthesized through tight regulatory networks in the cell, and thus extremely low levels of polyketides are typically observed in wild-type strains. Although many Streptomyces polyketides and their derivatives have potential to be used as clinically important pharmaceutical drugs, traditional strain improvement strategies such as random recursive mutagenesis have long been practiced with little understanding of the molecular basis underlying enhanced polyketide production. Recently, identifying, understanding, and applying a novel polyketide regulatory system identified from various Omics approaches, has become an important tool for rational Streptomyces strain improvement. In this paper, DNA microarray-driven reverse engineering efforts for improving titers of polyketides are briefly summarized, primarily focusing on our recent results of identification and application of novel global regulatory genes such as wblA,SCO1712,andSCO5426inStreptomyces species. Sequential targeted gene manipulation involved in polyketide biosynthetic reguation synergistically provided an efficient and rational strategy for Streptomyces strain improvement. Moreover, the engineered regulation-optimized Streptomyces mutant strain was further used as a surrogate host for heterologous expression of polyketide pathway.

1. Introduction: Streptomyces polyketide synthase (PKS), which is typically classified by Polyketide Biosynthesis and 3 different types. Type I PKSs are multifunctional enzymes Pathway-Specific Regulation whose domains are arranged into several modules, each of which controls incorporation of a specific precursor unit into The high G+C Gram-positive filamentous soil bacteria Strep- a growing polyketide backbone during chain elongation, gen- tomyces are well known for their superior characteristics erating macrolide compounds such as erythromycin, tylosin, in producing a variety of secondary metabolites, includ- avermectin, amphotericin, and tautomycetin [5]. Type II ingmanypharmaceuticallyvaluablecompoundssuchas PKSs are multienzyme complexes that perform a single set antibiotics, anticancer agents, and immunosuppressants [1, ofenzymaticactivitiesforiterativebiosynthesisofaromatic 2]. These secondary metabolites are commonly synthesized products including actinorhodin, tetracycline, and doxoru- by biosynthetic enzymes, whose corresponding genes are bicin [6]. Type III PKSs are homodimeric enzymes that typically clustered in the Streptomyces chromosome and catalyze iterative condensation reactions, typically known are proposed to be under tight, complicated regulation at as chalcone synthase-like PKSs [7]. The genes, enzymes, the transcriptional level [3]. Among Streptomyces secondary and the mechanism of Streptomyces polyketide biosynthesis metabolites are polyketides, which belong to one of the have been thoroughly reviewed previously [8]. In general, largest natural product families [4]. Streptomyces polyketide asimplecarboxylicacidstarterunitsuchasacetyl-CoAor biosynthesis is initiated by the key enzyme(s) referred to as propionyl-CoA is transferred to the cysteine active site of 2 BioMed Research International a 𝛽-ketoacyl synthase (KS) in the PKS complex. A specific the recursively and randomly mutagenized overproducing extender unit determined by an acyl transferase (AT) domain industrial mutant (OIM) with the wild-type (WT) strain at in the PKS complex, such as a malonyl-CoA, methylmalonyl- the molecular gene level, eventually leading us to identify CoA, or ethylmalonyl-CoA, is transferred to the thiol group previously unknown primary and/or secondary metabolic of the phosphopantetheine arm of an adjacent acyl carrier genes critical for secondary metabolite overproduction. The protein (ACP). These subunits are joined by a decarboxylative S. coelicolor DNA microarray system was successfully applied condensation catalyzed by KS and remain covalently attached to the erythromycin-overproducing Saccharopolyspora ery- to ACP, followed by no, partial, or full series of keto group thraea OIMaswellastylosin-overproducingS. fradiae OIM processing reactions involving 𝛽-ketoacyl reductase (KR), strains [26]. A similar genome-wide transcriptome analysis dehydratase (DH), and enoyl reductase (ER). The growing also showed that a pleiotropic antibiotic regulator, afsS in chainistransferredtoadownstreammoduleoftypeIor S. coelicolor A3(2), and various AfsS-dependent genes are iteratively recycled in type II and III for further rounds of regulated by various nutritional stress responses [27]. S. elongation and processing to produce a full-length polyketide peucetius DNA microarray analysis also revealed that expres- chain. The completed chain is typically released from the PKS sion patterns of S. peucetius genes involved in doxorubicin by a terminal thioesterase (TE) to form a macrocyclic lactone production change under different culture conditions28 [ ]. ring or an aromatic compound. In addition, genomics-driven approaches were applied to Although a general molecular-level mechanism for stimulate cryptic pathways such as 51-membered glycosylated polyketide biosynthesis has been relatively well characterized, macrolides, stambomycins [29, 30]. Chemical perturbation a comprehensive understanding of the complex polyketide of secondary metabolism also demonstrated important links regulatory networks in Streptomyces species is yet to be to primary metabolism, implying that small molecules could elucidated [3]. The biosynthesis of Streptomyces polyketide is also enhance yields of secondary metabolites for discovery regulated via multiple regulatory pathways induced by both and biochemical characterization [31]. nutritional and environmental stimuli [17, 18]. While various global regulatory systems present in most Streptomyces species are known to control both morphological differen- 3. Identification and Manipulation of a Novel tiation and polyketide production, polyketide biosynthetic Regulatory Gene wblA in S. coelicolor gene sets are subject to pathway-specific regulation by linked regulatory genes [19–21]. Some pathway-associated regula- A putative negative regulatory gene involved in polyketide tory genes encode specialized types of regulatory protein, biosynthesis,SCO3579,wasoriginallyproposedasawhiB- such as “SARPs” (Streptomyces antibiotic regulatory proteins) like putative transcription factor gene referred to as wblA often associated with genes for type II aromatic polyketide in S. coelicolor [33]. whiB is one of five key regulatory biosynthesis [22, 23] and “LALs” (large ATP-binding regu- genes including whiA, whiB, whiG, whiH, and whiI and lators of the LuxR family) associated with some gene sets known to be required for early stages of the transition of for type I macrolide polyketide biosynthesis [16]. Most of aerial hyphae to spores. Moreover, the whiB mutant exhibits these pathway-specific regulatory genes are transcriptionally poorly septated aerial hyphae with long and tightly coiled regulated by various global regulatory networks in most phenotype, and its expression is proposed to be repressed Streptomyces species, but their detailed mechanisms remain by a transcription factor BldD. [33]. Although whiB is a largely unknown. developmental regulatory gene identified and characterized in S. coelicolor as being essential for the sporulation of aerial hyphae, the biological function of wblA with regard to 2. Reverse Engineering Approaches Applied in secondary metabolite regulation has not yet been examined. Streptomyces Strain Improvement Duringthesearchforpreviouslyunknownpolyketideregula- tory genes, wblA was identified as a novel antibiotic downreg- Since all genome sequences of the first model species, ulatory gene in a Streptomyces reverse-engineering approach Streptomyces coelicolor, became publically available around [9]. The recursively mutated doxorubicin-overproducing S. the year 2002, various Omics-guided strategies have been peucetius OIM and the wild type S. peucetius subsp.caesius applied to increase the understanding of Streptomyces global ATCC 27952 were examined for potential transcriptional regulatory networks involved in polyketide biosynthesis [24, differences between the 2 strains. After systematic analyses 25]. Although most pathway-specific regulatory genes have of growth phase-dependent transcription profiles, 20 genes beenidentifiedonthebasisoftheirtypicallocationwithinthe with particularly large transcriptional changes between the biosynthetic pathway gene cluster, global regulatory genes are 2 strains were selected and individually overexpressed in S. much more difficult to identify even in the well-characterized coelicolor under the control of the strong promoter of the S. coelicolor because of the presence of more than 300 Streptomyces expression vector. Among these genes, overex- annotated putative regulatory open reading frames (ORFs) pression of wblA inhibited actinorhodin biosynthesis in S. inthegenomesequences[24, 25]. Recently, Omics-guided coelicolor, and the transcript encoded by an actinorhodin- reverse engineering approaches have emerged as an effective specific activator gene was reduced in wblA-overexpressing tool for investigating gene expression alterations associ- S. coelicolor [9]. These results suggest that wblA is a broadly ated with polyketide overproduction in several Streptomyces functioning downregulatory gene for polyketide biosynthesis industrial strains [26]. This strategy enabled us to compare in Streptomyces species. It was suggested that WblA, which BioMed Research International 3

0.2

Flux optimization 0.15

Downregulator deletion 0.1

Downregulator deletion

0.05 Specific productivity (g/g) productivity Specific

0 24 48 72 96 120 144 168 Time (hr)

M145 M145/Δ𝑤𝑏𝑙𝐴/Δ1712 M145/Δ𝑤𝑏𝑙𝐴 M145/Δ𝑤𝑏𝑙𝐴/Δ1712/Δ5426

Figure 1: Time-dependent ACT volumetric productivities of wild-type S. coelicolor M145 (⧫), S. coelicolor ΔwblA (◼), S. coelicolor ΔwblAΔSCO1712 (󳵳), and S. coelicolor ΔwblAΔSCO1712Δ5426 (∙) cultured in modified R5 media during 9 days in a 2-liter bioreactor16 [ ].

contains 4 conserved cysteine residues, may be sensitive to Moreover, the transcription level of actinorhodin pathway- redox changes, perhaps via disulfide bond formation as has specific actII-ORF4 was significantly increased from the been found for the E. coli OxyR transcription factor [32, 34]. S. coelicolor M145ΔSCO1712, while an opposite transcrip- Recently, transcriptome analysis with S. coelicolor microarray tion pattern was observed in the SCO1712-overexpressing approach in a wblA mutant exhibited that approximately S. coelicolor M145 strain, implying that SCO1712 had a 180 genes involved in primary metabolism and actinorhodin global inhibitory effect on polyketide biosynthesis in S. biosynthesis and 100 genes related to the aerial hyphal growth coelicolor [36]. Interestingly, the expression of not only were overexpressed and underexpressed, respectively [9]. wblA but also SCO1712 complemented the ΔwblA mutant WblA was then proposed to be important in the slow-down phenotype independently. These results suggest that wblA of biomass accumulation, the change from aerial hyphal may not be required for SCO1712 to downregulate polyke- initial cells to the subapical stem, apical compartments that tide biosynthesis. SCO1712 was additionally disrupted in precede sporulation, and oxidative stress response similar to an S. coelicolor M145ΔwblA mutant strain; the S. coeli- that of Corynebacterium glutamicum WhcA [35]. WhcA in color M145ΔwblAΔSCO1712 double knock-out mutant strain; C. glutamicum was proved to physically bind to a specific exhibited considerably higher actinorhodin volumetric pro- protein named SpiA (stress protein interacting with WhcA) ductivity (Figure 1). This implies that SCO1712 is not directly only under the nonoxidative condition. Interestingly, a SpiA related to wblA function and more likely encodes a wblA- ortholog present in S. coelicolor also showed a similar WblA- independent polyketide downregulator [36]. These results SpiA interaction in the oxidative stress response in S. coeli- suggest that sequential targeted gene disruptions of indepen- color, and a global regulatory protein called AdpA (A-factor- dently working downregulatory genes may be an efficient and dependent protein A) might control the wblA expression rational strategy for Streptomyces strain improvement. through direct binding to the promoter region of wblA (Lee et al., unpublished data). Currently, several wblA ortholog genes have been identified in various Streptomyces species, and they 4. Identification and Engineering of wblA are presumably involved in secondary metabolite regulation Ortholog in S. peucetius Industrial Mutant (Table 1). In addition to identifying wblA, comparative microarray Toexamine whether the wblA ortholog is also present in other analysis revealed that SCO1712 expression was considerably Streptomyces strains with similar biological significance in lower in actinorhodin overproducing S. coelicolor M145 than polyketide regulation, a total genomic DNA library from the in the actinorhodin less-producing S. coelicolor J1501 [36]. doxorubicin-producing S. peucetius OIM was constructed. SCO1712 encodes a 205-amino acid protein with an N- Cosmid library construction and screening successfully gen- terminal TetR-family helix-turn-helix (HTH) DNA-binding eratedonepositivecandidatecontainingtheentirewblA domain, whose biological function related to secondary ortholog gene (wblA𝑠𝑝𝑒)[10]. Sequence analysis of 345 bp metabolite regulation has not been reported. A significant ORF containing 𝑤𝑏𝑙𝐴𝑠𝑝𝑒 showed a protein-coding sequence decrease in the blue pigment actinorhodin was observed showing a high degree of amino acid similarity with the trans- in the SCO1712-expressing S. coelicolor in plate culture. lated products of wblA genes from several previously reported 4 BioMed Research International

Table 1: wblA orthologs identified from various Streptomyces species.

Strain Secondary metabolite name Amino acid homology (%) Reference and its structure Polyketide actinorhodin OH O HO O OH CH3 S. coelicolor 100 [9] M145 O O OO CH OH O 3 OH O OH Polyketide doxorubicin OOOH OH OH S. peucetius H 95 [10] OIM OOOH O O

NH2 Polyktide tautomycetin

O OH OOOH OH Streptomyces 96 [11] CK4412 O O O O

Polyketide avermectin R

H O HO O O O O O S. avermitilis O O 95 [12] MA-4680 H O O OH Avermectin B = 1a R CH2CH3 O Avermectin B 1b H OH R = CH3 Aminoglycoside streptomycin O CH3 HO H HO OHO OH O S. griseus HO N 88 [13] NBRC 13350 O O NH2

HO OH NH2 HN CH3 N NH2

H2N Betalactam cephalosporin

2 R H H N S S. clavuligerus 96 [14] ATCC 27064 O N 1 R O O OH BioMed Research International 5

Table 1: Continued.

Strain Secondary metabolite name Amino acid homology (%) Reference and its structure Phosphoglycolipid moenomycin OOH HO HO O OO OH O HO P O H2N O O O OH O O HO O O O S. ghanaensis OH O N 96 [15] ATCC 14672 HOHH O N OH O O OH OO N O H HO OH OH

Streptomyces genomes, including S. coelicolor (95%), S. aver- regions present in both the wblA sequences of S. coelicolor mitilis MA-4680 (92%), S. griseus NBRC 13350 (96%), and S. A3(2) and S. avermitilis ATCC31780 [11, 38]. Streptomyces clavuligerus ATCC 27064 (91%). As expected, approximately sp.CK4412hasbeenknowntoproduceanunusuallinear 35% more doxorubicin and 150% more 14-deoxydoxorubicin polyketide compound named tautomycetin (TMC), which (daunorubicin) were produced by S. peucetius OIMΔwblA𝑠𝑝𝑒 inhibits T cell proliferation at concentrations 100-fold lower compared with the S. peucetius OIM [10]. than those needed to achieve maximal inhibition with Through a second round of microarray analysis between cyclosporinA.SinceTMCisbelievedtospecificallyblock S. peucetius OIM and OIMΔwblA𝑠𝑝𝑒,sixgenesshowing tyrosine phosphorylation of intracellular signal mediators more than 4-fold transcriptional changes between these downstream of Src tyrosine kinases in a T cell-specific man- two strains were identified, followed by expression in S. ner, TMC is a novel potent T cell-specific immunosuppressive coelicolor [10]. Since the only S. coelicolor exconjugant con- agent whose mechanism of action is different from that of taining the SCO4967 construct produced approximately 2- cyclosporin A or FK506. [39–41]. Through Streptomyces sp. fold more actinorhodin than the control, the Streptomyces CK4412 DNA cosmid library screening, one positive cosmid expression vector pSET152 derivative containing SCO4967 containing the entire wblA ortholog gene (named wblA𝑡𝑚𝑐) was reintroduced into S. peucetius OIMΔwblA𝑠𝑝𝑒.SCO4967 was selected. Complete sequence analysis revealed that the is listed to encode a conserved hypothetical protein with 390-bp wblA𝑡𝑚𝑐 gene encodes a 130-amino acid protein with little information of in vivo biological function. SCO4967 a high degree of amino acid similarity with that of the overexpression in S. peucetius OIMΔwblA𝑠𝑝𝑒 resulted in translated wblA gene products from S. coelicolor (96%), S. approximately 1.7-fold more aklavinone (another doxoru- avermitilis MA-4680 (93%), S. griseus NBRC 13350 (89%), bicin precursor polyketide) productivity than that of S. and S. clavuligerus ATCC 27064 (93%), all of which contain peucetius OIMΔwblA𝑠𝑝𝑒 exconjugant harboring an empty 4 conserved cysteine residues and a helix-turn-helix (HTH) vector [10]. Moreover, the SCO4967-containing S. peucetius motif [11]. OIMΔwblA𝑠𝑝𝑒 strain exhibited the highest total volumetric Although in silico sequence analyses of wblA𝑡𝑚𝑐 from production of doxorubicin/daunorubicin/aklavinone, which Streptomyces sp. CK4412 was consistent with its putative was approximately 1.9-fold and 1.3-fold higher than those of regulatory roles in other Streptomyces polyketide biosynthe- S. peucetius OIM and S. peucetius OIMΔwblA𝑠𝑝𝑒,respectively ses, the in vivo function of wblA𝑡𝑚𝑐 was confirmed using a (Figure 2). This implies that sequential genetic manipulation gene disruption approach. Construction of the wblA mutant of target genes identified through interspecies comparative (Streptomyces sp. CK4412-001) was generated using PCR- microarray analysis may be an efficient and rational strategy targeted disruption followed by PCR analysis confirmation. for Streptomyces strain improvement [10, 37]. Culture broths of Streptomyces spp. CK4412, CK4412-001, CK4412-001/wblA𝑡𝑚𝑐, and CK4412/wblA𝑡𝑚𝑐 growninMS media were extracted, these were analyzed using an anti- 5. Identification and Engineering of wblA fungal bioassay, and the presence of TMC was quantified Ortholog in Streptomyces sp. CK4412 using high-pressure liquid chromatography (HPLC). Strep- tomyces CK4412-001 produced approximately 3-fold more To isolate another wblA ortholog gene from a type I TMC compared to WT Streptomyces CK4412 (Figure 3). polyketide-producing Streptomyces species, a total genomic Significantly enhanced antifungal activity against Aspergillus DNA library from Streptomyces sp. CK4412 was screened niger wasalsoobservedintheextractsofStreptomyces sp. using PCR degenerate primers based on the highly conserved CK4412-001underthesamecultureconditions.Moreover,an 6 BioMed Research International

250

200 Upregulator addition

150 Downregulator deletion 100

Concentration (mg/L) Concentration 50 Recursive mutation

0 24 48 72 96 120 144 168 Time (hr)

WT OIM/Δ𝑤𝑏𝑙𝐴 OIM OIM/SCO4967

Figure 2: Time-dependent DXR/DNR/aklavinone volumetric productivities of S. peucetius WT (󳀅), S. peucetius OIM (◼), S. peucetius OIMΔwblA𝑠𝑝𝑒 (󳵳), and S. peucetius OIMΔwblA𝑠𝑝𝑒 /SCO4967 (∙)[32]. integrating conjugative vector, into which the coding region synergistically provide an efficient and rational strategy for of wblA𝑡𝑚𝑐 and its own upstream promoter region were Streptomyces strain improvement. cloned, was constructed (pSETHYGwblA). Both HPLC and a bioassay confirmed that TMC productivity and antifungal activity were reduced in the Streptomyces sp. CK4412-001 7. Potential Application as a Surrogate Host mutant strain carrying pSETHYGwblA back to the WT for Synthetic Biology level, implying that wblA𝑡𝑚𝑐 also plays a global antibiotic downregulatory role in type I polyketide biosynthesis in As a complementary strategy for valuable Streptomyces Streptomyces sp. CK4412 (Figure 3). polyketide production, functional expression of the target polyketide pathway in a Streptomyces heterologous host has been applied. Several secondary metabolite pathways were 6. Synergistic Redesign of Polyketide and expressed in relatively well-characterized and genetically Precursor Flux Regulatory Pathways amenable Streptomyces surrogate hosts including S. albus, S. lividans, S. coelicolor, S. avermitilis, S. ambofaciens, S. Comparative transcriptome analysis between S. coelicolor roseosporus,andS. grisefuscus [44, 45]. Recently, the genomes WT and an S. coelicolor ΔwblAΔSCO1712 double knock-out of these strains have been further engineered to maximize mutant revealed an additional 14 genes that displayed no particular (less than 1.2-fold) transcriptional changes [42]. foreign polyketide production by deleting some endogenous These putative wblA/SCO1712-independent genes include biosynthetic gene clusters and/or preventing diversion of pre- a carbon flux-regulating SCO5426, which is one of the cursors into competing secondary metabolic pathways. Espe- 3 6-phosphofructokinase genes. SCO5426 disruption was cially, a genome-minimized S. avermitilis industrial mutant previously reported to enhance both precursor carbon flux strain was also used successfully for foreign polyketide and NADPH supply for polyketide biosynthesis by activating pathway expression as a heterologous expression host [46], the pentose phosphate pathway, resulting in significantly suggesting a realistic alternative strategy for overproducing enhanced actinorhodin production in S. coelicolor [43]. exogenous natural and unnatural polyketides. Based on the above observations, additional deletion of A polyketide nonproducing S. coelicolor mutant strain SCO5426 in the S. coelicolor ΔwblAΔSCO1712 double knock- was generated by deleting the entire actinorhodin cluster out mutant may further enhance actinorhodin precursor flux from the chromosome of a previously generated S. coelicolor as well as NADPH supply in S. coelicolor. While all mutant ΔwblAΔSCO1712ΔSCO5426 triple knock-out mutant strain, strains exhibited comparable growth patterns, the S. coelicolor which was shown to stimulate actinorhodin biosynthesis ΔwblAΔSCO1712ΔSCO5426 triple knock-out mutant strain through deletion of 2 antibiotic down-regulators as well exhibited the highest actinorhodin productivity, which was as a polyketide precursor flux downregulator [47]. Using 1.7-fold and 1.3-fold higher than those of the single knock- this engineered S. coelicolor mutant strain as a surrogate out S. coelicolor ΔwblA and the double knock-out S. coelicolor host or a cell factory from a synthetic biology perspective, ΔwblAΔSCO1712 mutant strains, respectively [42]. These a model minimal polyketide pathway for aloesaponarin results suggest that sequential targeted gene disruption of II [48] was cloned and functionally expressed in a high- independently working downregulators as well as precursor copy expression plasmid, followed by quantitative polyke- flux downregulators involved in polyketide biosynthesis may tide analysis. As expected, aloesaponarin II production was BioMed Research International 7

30 25 )

4 20

×10 15 10 uV( 5 0 31 32 33 34 35 36 37 38 39 (min) (A) 250

) (a)

4 200

×10 150

uV( 100 50

250 (b) 200 ) 4 150 ×10 100 uV( 50 250 (c) 200 ) 4 150 ×10 100 uV( 50 250 (d) 200 ) 4 150 ×10 100 uV( 50 0 31 32 33 34 35 36 37 38 39 (min) 2.5 (B)

1.5 Downregulator deletion 1

0.5 Concentration (mg/L) Concentration

0 24 72 120 168 Time (hr)

CK4412 CK4412/Δ𝑤𝑏𝑙𝐴 (C)

Figure 3: (A) Authentic TMC standard. (B) TMC volumetric productivities measured by quantitative HPLC analyses of the ethyl acetate- −1 −1 −1 extracted culture broths were 1.69 mg ⋅ L for the wild-type strain CK4412 (a), 5.44 mg ⋅ L for the wblA-tmc disruptant (b), 4.04 mg ⋅ L −1 for CK4412-001/wblA-tmc (c), and 1.41 mg ⋅ L for CK4412/wblA-tmc (d). (C) Time-dependent tautomycetin volumetric productivities of Streptomyces. sp. CK4412WT (⧫) S. sp. CK4412/ΔwblA𝑡𝑚𝑐 (◼)[36]. 8 BioMed Research International

rep ori 25

ori pMMBL004 20 (8.5 kb)

bla 15 tsr aprR oriT 10 Specific productivity (mg/g) productivity Specific 5 ND PstIactIII actI actI actI actII actIV PstI pAloeII-1

0 EcoRI pAloeII-2 PstI actIII actI actI actI actII actIV 12345678 actII-orf4 (a) (c)

OO O OOCH3 O O CH3

CoA-S + S-CoA actI O actIII COOH COOH O HO OOO O O

actVII

OH O CH3 OHOOCH3 OH CH3

R1 OO OH OH COOH actIV COOH OH OH O (b)

Figure 4: (a) Plasmid map of the pMMBL004, pAloeII-1 (pMMBL004 containing PstI fragment), and pAloeII-2 (pAloeII-1 containing actII- orf4); rep: replicon; ori:originofreplication;bla: 𝛽-lactamaseR; tsr:apramycinR; apr:apramycinR; oriT:originoftransfer.(b)Schematic representation of the aloesaponarin II biosynthetic pathway. Solid and dotted arrows represent presumed enzymatic and spontaneous steps, respectively. 3,8-DMAC: 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid. R1 groups of aloesaponarin II and 3,8-DMAC are hydrogen and carboxylic acid, respectively. (c) Productions of aloesaponarin II and 3,8-DMAC in recombinant S. coelicolor strains. 1, S. coelicolor ΔACT/pMMBL004; 2, S. coelicolor ΔACTΔwblAΔSCO1712ΔSCO5426/pMMBL004; 3, S. coelicolor ΔACT/pAloeII-1; 4, S. coelicolor ΔACTΔwblAΔSCO1712ΔSCO5426/pAloeII-1; 5, S. coelicolor ΔACT/pAloeII-2; 6, S. coelicolor ΔACTΔwblAΔSCO1712ΔSCO5426/pAloeII-2; 7, S. coelicolor ΔACT/pAloeII-2; 8, S. coelicolor ΔACTΔwblAΔSCO1712ΔSCO5426/pAloeII-2; 1∼6, liquid culture; 7∼8, solid culture; ND, not detected [29]. observed at the highest level in the actinorhodin cluster- Acknowledgment deleted and downregulators-deleted mutant strain, S. coelicolor ΔACTΔwblAΔSCO1712ΔSCO5426 (Figure 4). These This work was supported by Grant no. D00040 from the results imply that this engineered actinorhodin-free and Global Research Network (GRN) Program of the National regulation-optimized S. coelicolor mutant strain can be used Research Foundation of Korea. as a general surrogate for efficiently expressing foreign polyketide pathways. In conclusion, biotechnological appli- References cations of the independently functioning regulatory pathway [1] R. H. Baltz, “Molecular engineering approaches to peptide, identified through microarray-driven reverse engineering polyketide and other antibiotics,” Nature Biotechnology,vol.24, strategy may be beneficial for Streptomyces strain improve- no.12,pp.1533–1540,2006. ment for polyketide overproduction as well as for efficient [2]D.A.Hopwood,“Soiltogenomics:theStreptomyces chromo- host cell factory construction for synthetic biology. some,” Annual Review of Genetics,vol.40,pp.1–23,2006. BioMed Research International 9

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Research Article Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

Surang Chankhamhaengdecha,1, 2, 3 Suphatra Hongvijit,2, 3, 4, 5 Akkaraphol Srichaisupakit,1, 2, 3 Pattra Charnchai,2, 3, 4 and Watanalai Panbangred2, 3, 4, 5

1 Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand 2 Mahidol University-Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU:CRC), Bangkok, Thailand 3 Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand 4 Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok, Thailand 5 Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

Correspondence should be addressed to Watanalai Panbangred; [email protected]

Received 7 November 2012; Revised 28 December 2012; Accepted 2 January 2013

Academic Editor: Bertrand Aigle

Copyright © 2013 Surang Chankhamhaengdecha et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30±3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain inHSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6-toC12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is thefirstreportofHSL-acylaseactivityderivedfromanendophyticStreptomyces.

1. Introduction monitoring population density which is known as quorum sensing (QS) mechanism [3–5]. In Gram-negative bacteria, Bacterial pathogens infecting plants, animals, and humans QS is often mediated by N-acyl-L-homoserine lactone (HSL) cause a tremendous economic loss worldwide. Conventional signal molecules [4]. Interference of HSL-dependent QS, treatments, such as application of antibacterial agents, sig- commonly known as quorum quenching (QQ), has been nificantly contribute to selection of resistant microorganisms demonstratedtobeaneffectiveantimicrobialstrategyfor as well as environmental contamination [1]. A number of controlling virulent pathogens [6–9].QQcanbeachievedby concerns on using antibiotics have brought investigators degradation of QS signal molecules by enzymatic digestion, to search for alternative strategies to combat with these significantly decreasing functions of signaling molecules. The pathogens. Attenuation of virulence phenotypes rather than QQ enzymes (QQEs) have been reported from several Gram- killing of causative agents has gained an interest since this positive bacteria including Arthrobacter sp. IBN110, Bacillus strategy does not introduce selective pressure which poten- sp. 240B1, Geobacillus kaustophilus strain HTA426, Mycobac- tially leads to development of antibiotic-resistant bacteria terium avium subsp. paratuberculosis K-10, Solibacillus sil- [2]. Certain bacterial pathogens regulate their virulence by vestris StLB046 [10], and Streptomyces M664 [8]. However, 2 BioMed Research International

there are differences in the catalytic spectrum among QQEs 40 𝜇M N-decanoyl-L-homoserine lactone (C10HSL) in 0.1 M ∘ toward HSLs. In the search for QQE-producing bacteria, acti- Tris-HCl pH 6.8. The mixture was incubated at 28 Cfor nomycetes are of great interest since they possess an ability to 1 h with gentle agitation and the reaction was stopped by ∘ produce and secrete various extracellular hydrolytic enzymes heating at 95 Cfor5min.Then10𝜇L of reaction mix- [11, 12]. A number of actinomycetes have been isolated from ture was used to determine the remaining of C10HSL on several natural sources, including rhizospheric soil and plant a bioassay plate. For determination of HSL-degrading activ- tissues. Biological functions of actinomycetes predominantly ity, Agrobacterium tumefaciens NTL4 (pZLR4) (kindly pro- depend on sources from which the bacteria are isolated. vided by Professor Stephen K. Farrand, Department of Crop Natural resources in megabiodiversity with high selective Sciences and Microbiology, University of Illinois, USA) was pressure and microbial competition in tropical regions are used as a biosensor strain in bioassay to long-chain HSL well recognized as an important resource of new anti-mic- (C8 to C12HSL) [15]. Chromobacterium violaceum CV026 robial agents as well as QQE [13]. (kindly provided by Professor Paul Williams, Depart- To date, the isolation of actinomycetes with high QQE ment of Molecular Microbiology, University of Nottingham, activity from endophytes has never been reported. Screening UK) was used to detect N-butanoyl-L-homoserine lactone for QQE-producing actinomycetes in this study showed the (C4HSL), N-hexanoyl-L-homoserine lactone (C6HSL), and diversity and abundance of HSL-degrading actinomycetes 3-oxohexanoyl-L-homoserine lactone (3-oxo-C6HSL) [16]. from soil and plant tissues for the first time. The QQE from Each reporter strain was cultivated overnight in nutrient thehighactivitystrainwasshowntohydrolyzeHSLwitha broth (NB) and Luria-Bertani broth (LB), respectively. To broad range of chain length from N-hexanoyl-L-homoserine determine HSL-degrading activity qualitatively, an overnight lactone (C6HSL) to N-dodecanoyl-L-homoserine lactone culture of the biosensor strain (2.5 mL) was mixed with (C12HSL);hence,thisenzymecouldbepotentiallyusedto 5 mL of agar. The mixture was overlaid on an AB minimal attenuate virulence of a broad range of bacterial pathogens medium bioassay plate [17] supplemented with 40 𝜇g/mL with different QS signal molecules. of 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) for A. tumefaciens NTL4 (pZLR4) and Luria-Bertani agar (LA) for C. violaceum (CV026). A well on an agar plate was 2. Materials and Methods made by punching with a cork borer (0 = 0.4 cm). Ten microliters of the heat inactivated reaction mixture was dropped in 2.1. Isolation of Rhizospheric and Endophytic Actinomycetes. ∘ each well. All plates were incubated at 30 C overnight to allow Rhizospheric soil samples were collected from various color zone developing. Blue and purple color zones were provinces in Thailand, including Bangkok, Chanthaburi, Nongbualumpoo, Prachinburi, and Rayong provinces. To developed around colonies of A. tumefaciens NTL4 (pZLR4) isolate rhizospheric actinomycetes, 1.0 g of soil was serially and C. violaceum (CV026), respectively, by the induction diluted in 4 mL of 0.85% normal saline solution (NSS). The of the residual HSL in the reaction mixture. The residual appropriate dilution was spread on selective agars, which are amounts of HSL were calculated using relationship equations Pridham’s and water proline supplemented with 25 𝜇g/mL basedonthecolorzonesizeandknownamountsofHSL[6]. nalidixic acid and 50 𝜇g/mL cyclohexamide [14]. Isolates Relative activity of HSL-degrading enzyme was calculated with typical actinomycetes colony morphology were selected by using the following formula: relative HSL-degrading acti- × for screening of HSL-degrading activity. In order to isolate vity = (HSL-degrading activity/initial amount of substrate) endophytic actinomycetes, leaves, fruits, seeds, and stems of 100. For quantitative HSL-degrading activity determination, plant samples were collected from various regions in Thai- bioassay agar medium in the plate was cut into separated land including Bangkok, Salaya botanical garden in Nakhon slices across the plate (1 cm in width). Five microliters of the pathom, and Nam Nao national park in Phetchabun. All dirt reaction mixture was added to one end of an agar slice and was removed by running tap water. Samples were air-dried at then the culture of biosensor strain at an OD600 of 1 was room temperature for one week. Plant samples were cut into progressively spotted (0.6 𝜇L per spot) at further distances small pieces. Surface sterilization was performed by treating from the loaded reaction mixture. The last induced color with 10% sodium hypochlorite (MERCK), 70% ethanol and colony to the origin of the reaction mixture sample in each followed by washing twice with sterile water. The samples agar slice was measured. Amounts of HSL in the reaction were then air-dried and placed on water agar medium mixture were determined based on relationship equation of (containing only 1.5% agar) supplemented with 25 𝜇g/mL adding known amounts of HSL to the bioassay slices and nalidixic acid and 50 𝜇g/mL cyclohexamide. Actinomycetes determining the distance of the color colony from the origin. colonies were selected for further study. HSL-degrading activity reported as nmole/h was calculated from subtraction of the initial amount of HSL substrate with leftoveramountfromtheenzymaticdigestion. 2.2. HSL Inhibition Assay. Actinomycetes isolates were grownonWaskman’sagar(1%glucose,0.5%peptone,0.5% meat extract, 0.3% NaCl, and 1.2% agar) for 3 days, sub- 2.3. Strain Identification. The 16S rRNA gene of isolate cultured in 301 broth (2.4% starch, 0.1% glucose, 0.3% LPC029 was amplified using conserved primers18 [ ]. PCR was ∘ ∘ peptone, 0.5% meat extract, and 0.3% CaCO3), and further performed for 30 cycles at 95 C for 45 seconds, 45 Cfor45 ∘ ∘ incubated at 28 Cwithshakingfor6days.Fortymicroliters seconds, and 72 C from 1.5 minutes. The PCR product was of cell-free supernatant was mixed with an equal volume of sequenced by the dideoxy chain-termination method [19]. BioMed Research International 3

2.4. Preparation of Partially Purified HSL-Degrading Enzyme. each potato tuber were each filled with 0.85% NSS (negative The endophytic isolate LPC029 was harvested after 6 days control), 1000 cells of Pcc (positive control), the mixture of ∘ of culture in 1,000 mL 301 medium by centrifugation at 4 C Pcc and HSL-acylase, and HSL-acylase alone. The inoculated (10,000 g) for 10 min. The supernatant was filtered through potato tubers were further sealed with a sterilized sticker. To ∘ a0.45𝜇mmembraneandstoredat4C. Ammonium sulfate make a moisture condition, the tubers were wrapped with was added to the culture supernatant to achieve 60% satura- aluminum foil and sterile moist towels. The wrapped-potato ∘ ∘ tion and the solution was left at 4 C for 16 h. The precipitate tubers were incubated in a closed box at 30 Cfor3days. was collected by centrifugation, dissolved, in 6 mL of 20 mM sodium phosphate buffer pH 7.0, and dialyzed (dialysis tubing, MW cut-off 10 kDa; Sigma, USA) overnight against 3. Results and Discussion thesamebuffer.Thedialyzedsamplewasthencollectedand ∘ 3.1. Screening of HSL-Degrading Actinomycetes and HSL- stored at 4 C. Degrading Activity. The abundance and diversity of HSL- degrading actinomycetes isolated from soil and plant tissues 2.5. HSL Degradation Assay. C10HSL was used as a substrate were assessed. They were isolated on the basis of their typical in the determination of hydrolytic products of the HSL- morphologies (filamentous growth, spore chain, and several degrading enzyme. Substrates were prepared as a stock solu- types of convex and margin colonies on selective medium) tion at 8 mM in 80% ethanol. The HSL-degrading product according to Bergey’s Manual of Systematic Bacteriology was analyzed by mixing 13 𝜇g of the partially purified HSL- [21]. While 344 actinomycetes could be isolated from 43 degrading enzyme and 350 mM of C10HSL in 700 mL of rhizospheric soil samples, only 132 isolates were obtained 20 mM sodium phosphate buffer pH 7.0. After incubation from 64 plant samples. Among these actinomycetes isolates, ∘ at 30 C for 16 h, the reaction mixture was extracted three the number of isolates with HSL degrading activity was times with equal volumes of ethyl acetate. The organic phase found in higher frequency in endophytic isolates (51.5%) ∘ was evaporated to dryness at 40 C by a rotary evaporator. than in rhizospheric isolates (36.9%) (Table 1). While several Thedriedsamplewasdissolvedin50𝜇LofHPLCgrade previous studies have indicated the HSL-degrading activity methanol. Five microliters of HSL-degraded products was in soil bacteria [8, 22, 23], this study is the first report of introduced onto Shiseido Capcell Pak C18 (4.6 × 250 mm such activity in endophytic actinomycetes. The evolution race I.D.;particlesize5𝜇m) previously equilibrated with 10% for survival in tropical ecosystem reinforces strong compe- acetonitrile. The elution was performed with linear gradient tition among organisms, which may result in a plethora of of 10–90% acetonitrile, at a flow rate of 1 mL/min under the chemical molecules [24], and enzymes [11, 12] with biological following condition: 10% acetonitrile from 0 to 8 min; linear functions. Consequently, there is a high probability that gradient to 90% acetonitrile from 8 to 35 min. Homoserine microorganisms associated with tropical plants might be a lactone (3.5 mM) and 35 mM of C10HSL were used as a source of bioactive compounds and enzymes. Among our standard. HPLC chromatogram of the products was analyzed isolates with HSL-degrading activity, 4 rhizospheric actino- by diode array detector with UV ranging from 200 to 280 nm mycetes (PS032, SWP036, SWP042, and SWP043) and 5 (Agilent 1200 HPLC). To check substrate specificity, HSL- endophytic actinomycetes (LPC026, LPC029, PC005, PC052, acylase was reacted with different HSL-substrates including and PC053) were found to be the potential strains in HSL C4HSL, C6HSL, 3-oxo-C6HSL, 3-oxo-C8HSL, C10HSL, and degrading as they were able to degrade HSL of greater than C12HSL. Reaction mixture was composed of 0.4 mM each of 96.9% relative activity (Table 1). Quantitative measurement HSLsubstancesin20mMsodiumphosphateatpH7.0and of enzymatic activity among these 9 isolates showed that 2 𝜇g of the partially purified HSL-degrading enzyme in 50 𝜇L. isolate LPC029 from Gmelina arborea Roxb. possessed the ∘ The mixture was incubated at 30 Cfor1h.TheHSL-substrate highest HSL-degrading activity at 151.30 ± 3.1 nmole/h/mL that remained in the reaction mixture was determined by the (Table 2). Strain LPC029 was later identified with 16S rRNA bioassay method as described above. gene sequence (GenBank accession number KC153060) to be closely related to S. globisporus at 99% homology. The presence of HSL-degrading activity in both soil and plant 2.6. In Vitro Pathogenicity Assay. The assay was performed isolates may be due to their common need of HSL-degraded as described by Burr et al. [20]. The potatoes’ tubers of about product as carbon and energy sources [25]andforcom- thesamesizewerewashedwithtabwaterandpretreated petition with other microbes to protect their ecological in 5% sodium hypochlorite (Merck) for 10 min then soaked niche [26]. In addition, endophytic bacteria have previously with sterile water. The tubers were dried in a laminar flow been reported to be involved in several biological functions cabinet. P. carotov or um ssp. carotovorum (Pcc)wascultured ∘ such as promoting plant growth, biocontrol agents, and in 50 ml of LB broth at 30 Covernight.Thecultureat phytoremediation [24, 27–29]. These functions stem from OD600 of 1 was further serially diluted with 0.85%NSS to −5 the production of their natural compounds and antipathogen 10 . Ten microliters of 100,000-folded dilution of Pcc (ca. metabolites inside the host plant [29]. 1000 cfu/10 𝜇L) was mixed with 2 𝜇gofpartiallypurifiedHSL- degrading enzyme. The reaction was then further incubated ∘ at 30 C for 1 h and was inoculated into a potato tuber. A 3.2. HPLC Analysis of C10HSL Hydrolysis by LPC029 HSL- two-hundred-microliter pipette tip was used to punch a hole Degrading Enzyme. The products of HSL degradation by of 22 mm in depth, 2 holes per tuber. The two holes in enzyme from LPC029 were analyzed by HPLC after ethyl 4 BioMed Research International

Table 1: Relative HSL-degrading activity of actinomycetes isolated from rhizospheric and endophytic samples.

∗ Relative HSL-degrading activity (%) Location No. of isolates 0 >0–50 >50–75 >75–87.5 >87.5–93.8 >93.8–96.9 >96.9–100 Rhizospheric samples Bangkok 278 182 54 16 11 8 4 3 Chanthaburi 13 6 5 0 0 1 1 0 Nongbualumpoo 3 2 0 0 0 0 0 1 Prachinburi 27 12 9 3 3 0 0 0 Rayong 23 15 8 0 0 0 0 0 Total 344 217 76 19 14 9 5 4 Total (percentage) 217 (63.1%) 127 (36.9%) Endophytic samples Bangkok 43 29 12 0 1 1 0 0 Nakhonpathom 29 20 8 1 0 0 0 0 Phetchabun 60 15 25 0 10 1 4 5 Total 132 64 45 1 11 2 4 5 Total (percentage) 64 (48.5%) 68 (51.5%) ∗ The activity of each isolate was qualitatively determined by comparing with known concentrations ofC10HSL (200, 100, 50, 25, 12.5, 0.625, and 0 pmole).

Table 2: Quantitative HSL-degrading activity of isolates with high efficiency of HSL degradation.

Source Name of isolate HSL-degrading activity (nmole/h/mL) Rhizosphere soil, Nongbualumpoo PS032 143.42 ± 1.5 Rhizosphere soil, Bangkok SWP036 144.67 ± 2.1 Rhizosphere soil, Bangkok SWP042 144.37 ± 1.5 Rhizosphere soil, Bangkok SWP043 144.26 ± 1.3 Leaf of Ta khram (Garuga pinnata Roxb.) LPC026 146.45 ± 2.8 Leaf of So (Gmelina arborea Roxb.) LPC029 151.30 ± 3.1 Leaf of Kling klang dong (Stephania venosa Spreng.) PC005 146.93 ± 1.0 Leaf of Malabar melastome (Melastoma malabathricum L.) PC052 143.45 ± 4.3 Leaf of Grape-leaf Wood Rose (Merremia vitifolia Hallier f.) PC053 147.68 ± 1.8

acetate extraction. Hydrolysis of C10HSL by LPC029 enzyme Although the enzymes were derived from a variety of resulted in releasing HSL as shown by HPLC (Figure 1(c)) microorganisms, most of HSL-acylases were active against compared to standard HSL (Figure 1(a))andC10HSL sub- HSLwiththecarbonchainlessthan8atoms[10]. Testing strate alone (Figure 1(b)). Therefore the HSL-degrading ofthepartialpurifiedenzymefromLPC029withvarious enzymefromLPC029wasproventobeHSL-acylaseaccord- HSL substrates showed the ability to degrade medium-to- ing to the deacylation activity to break the amide bond of long chains of HSL (C6–C12)inwhichC10HSL was the most C10HSL, out of which HSL was released as an end prod- preferred substrate (Figure 2). The activity of this enzyme uct. The substrate (C10HSL) was incompletely hydrolyzed was not dependent on the substitution of oxo-group on the (Figure 1(c)) due to an insufficient amount of enzyme used thirdcarbonatom(Figure 2)whichshowednosignificant in the assay. HSL-acylase has been explored in (i) Gram- difference between two substrates,6 C HSL and 3-oxo-C6HSL negative bacteria including Comamonas strain D1 [9], Pseu- (𝑃 ≥ 0.05). The results suggested the high possibility domonas aeruginosa PAO1 [30], P. ae r ug ino s a PAI-A [31], of LPC029 HSL-acylase in inhibiting QS of medium-chain P. sy r ing ae [32], Ralstonia solanacearum GMI1000 [33], She- HSLs dependent on Pcc (3-oxo-C6HSL, 3-oxo-C8HSL) and wanella sp. strain MIB 015 [7], Tenacibaculum maritimum Burkholderia cepacia (C6HSL, C8HSL) and might provide [34], and Variovorax paradoxus VAI-C [25]; (ii) Gram- the most effective quenching agent against long-chain HSLs positive bacteria including Rhodococcus erythropolis strain produced by Vibrio anguillarum (C10HSL) and P. ae r ug ino s a W2 [35]andStreptomyces sp. M664 [8]; and (iii) cyanobac- (C12HSL). teria such as Anabaena sp. PCC 7120 [36]. Comparison of HSL-acylase activity among these HSL-degrading microbes 3.3. Effect of LPC029 HSL-Degrading Enzyme on Pathogenic- is difficult due to the difference of both quantitative and ity. In vitro efficacy of HSL-degrading enzyme from LPC029 qualitative measurement methods. Nevertheless, substrate to quench QS-regulated functions was determined by Pec- specificity of the enzymes could be compared qualitatively. tobacterium carotovorum ssp. carotovorum (Pcc)mediated BioMed Research International 5

400 1000 C10HSL 300 800 600 mAU mAU 200 HSL 400 100 200 0 0 5 1015202530 5 1015202530 (min) (min) (a) (b) 1000 800 C HSL 600 HSL 10 mAU 400 200 0 5 1015202530 (min) (c)

Figure 1: HPLC analysis of enzymatic hydrolysis product of C10HSL with the HSL-degrading enzyme from LPC029. The initial C10HSL (35 mM) was reacted with partially purified enzyme from LPC029 for 16 h and separated with HPLC. HPLC profiles of (a) HSL standard (3.5 mM); (b) unreacted C10HSL (35 mM); and (c) reaction product of C10HSL with LPC029 HSL-degrading enzyme. The product peak was eluted at 2.8 min. mAU is the abbreviation for milli-absorbance unit. soft-rotting potato tuber assay. In Pcc, the virulence and 1600 pathogenicity are QS dependent, but different strains of this 1400

H 1200 subspecies prefer different signal molecules and they are ঴ 1000 divided into two groups. One group used 3-oxo-C6HSL as 800 a predominant signal molecules, whereas another group 600 exploited mainly 3-oxo-C8HSL as HSL-dependent QS [37]. QNPMFI 400 3 200 Inoculation of Pcc (10 cells per tuber) evidently resulted in activity HSL-degrading 𝜇 0 tissue maceration. The addition of 2 gofpartiallypurified C4HSL C6HSL 3-oxo- 3-oxo- C10HSL C12HSL HSL-acylaseresultedinastatisticallysignificantreductionin C6HSL C8HSL soft-rot weight𝑃 ( < 0.05)asshowninFigure 3(e).Inthe control experiment, neither NSS nor HSL-acylase inoculation Figure 2: Substrate specificity of LPC029 HSL-degrading enzyme. inducedsoftrotinplanttissue(Figures3(a) and 3(d),resp.). Partially purified enzyme was mixed with each of HSLs. The activity 3 Inoculation of Pcc alone (10 cells) caused the highest plant was determined based on HSL leftover on C. violaceum CV026 and A. tumefaciens NTL4 (pZLR4) bioassay plates. Relative activity is tissue necrosis (Figure 3(b)) with the highest soft-rot weight 3 given in parenthesis. Bars indicate SD values of four replicates. (Figure 3(e)). When the same number of Pcc (10 cells) was mixed with 2 𝜇g of HSL-acylase before inoculation, the enzyme attenuated the bacterial pathogenicity by reduction of plant tissue necrosis (Figure 3(c))andcausedreductionof soft-rot weightFigure ( 3(e)). Several studies have shown the to possess this enzyme activity at higher frequency than the effectiveness of QS targeting strategy in reducing virulence soil isolates. It is interesting to note that this study was the of P. ae r ug ino s a [38], Enterobacteriaceae [39], B. cepacia first report on studying the distribution of HSL-degrading [40], P. carotov or um [23, 41, 42], Serratia liquefaciens [43], enzyme in actinomycetes as well as the first report on finding Agrobacterium tumefaciens [23],andsomeaquaticconsortia such an activity in endophytic actinomycetes. Endophytic [44]. These results offer an alternative strategy to alternate Streptomyces LPC029 showed the highest C10HSL-degrading 151.30 ± 3.1 virulence of plant pathogen using QS-degrading enzyme activity at nmole/h/mL and its enzyme showed from endophytic Streptomyces. Due to its ability to degrade broad substrate specificity against medium-to-long-chain long-chain HSL with moderate activity toward short-chain HSLs.TheenzymewasclassifiedasHSL-acylasewhich HSL, the QQE from Streptomyces LPC029 might be of interest hydrolyzes an amide bond between an acyl-side chain and to control certain plant and human pathogens. However, a homoserine lactone and releases a free HSL. The partially more in-depth in vitro and also in vivo investigations are purifiedenzymefromLPC029attenuatedsoft-rotdiseases warranted. caused by Pcc, which made the strain as well as many other QQ actinomycetes in this study potential candidates for bio- 4. Conclusion control against QS-dependent phytopathogens. Further study on growth conditions including nutrients, temperature, pH, The HSL-degrading activity among rhizospheric and endo- and aerations is required to maximize enzyme productivity phytic actinomycetes was assessed and the latter was found in the selected strain(s). 6 BioMed Research International

0.85% NSS P. carotov or um

(a) (b)

P. carotov or um + HSL-acylase HSL-acylase (c) (d) 4

3.5

3 2.8

2.5

1.5 1.3 So-rot weight (g) weight So-rot

0.5 0 0 0 ABCD (e)

Figure 3: Effects of LPC029 HSL-degrading enzyme on pathogenicity of Pcc on potato tubers. Pathogenicity of Pcc was determined by inspection of lesion zones induced upon inoculation of potato tuber at two different sites. (a) Negative control consisting of tuber treated 3 3 with 0.85% NSS, (b) inoculation of Pcc alone at 10 cells per tuber, (c) inoculation of Pcc at 10 cells mixed with 2 𝜇g of partial purified HSL- acylase per tuber, (d) 2 𝜇g of partially purified HSL-acylase, and (e) pathogenicity of Pcc determined by soft-rot weight. Soft-rot tissue from treatment (a)–(d) was measured after inoculation of Pcc for3days;barsindicateSDvaluesoffourreplicates.

Acknowledgments References

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Research Article Streptomyces lunalinharesii Strain 235 Shows the Potential to Inhibit Bacteria Involved in Biocorrosion Processes

Juliana Pacheco da Rosa, Elisa Korenblum, Marcella Novaes Franco-Cirigliano, Fernanda Abreu, Ulysses Lins, Rosângela M. A. Soares, Andrew Macrae, Lucy Seldin, and Rosalie R. R. Coelho Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goes,´ Centro de Cienciasˆ da Saude,´ Universidade Federal do Rio de Janeiro, Bloco I, Ilha do Fundao,˜ 21941-590 Rio de Janeiro, RJ, Brazil Correspondence should be addressed to Juliana Pacheco da Rosa; [email protected] and Rosalie R. R. Coelho; [email protected]

Received 28 September 2012; Accepted 12 December 2012

Academic Editor: Neelu Nawani

Copyright © 2013 Juliana Pacheco da Rosa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Four actinomycete strains previously isolated from Brazilian soils were tested for their antimicrobial activity against Bacillus pumilus LF-4 and Desulfovibrio alaskensis NCIMB 13491, bacteria that are well known to be involved in biofilm formation and biocorrosion. Strain 235, belonging to the species Streptomyces lunalinharesii, inhibited the growth of both bacteria. The antimicrobial activity was seen over a wide range of pH, and after treatment with several chemicals and heat but not with proteinase K and trypsin. The antimicrobial substances present in the concentrated supernatant from growth media were partially characterized by SDS-PAGE and extracellular polypeptides were seen. Bands in the size range of 12 to 14.4 kDa caused antimicrobial activity. Transmission electron microscopy of D. alaskensis cells treated with the concentrated supernatant containing the antimicrobial substances revealed the formation of prominent bubbles, the spherical double-layered structures on the cell membrane, and the periplasmic space completely filled with electron-dense material. This is the first report on the production of antimicrobial substances by actinomycetes against bacteria involved in biocorrosion processes, and these findings may be of great relevance as an alternative source of biocides to those currently employed in the petroleum industry.

1. Introduction architectural features [8]. Jack et al. [9] demonstrated a significant increase in corrosion rates of carbon steel in Corrosion is a leading cause of pipe failure and high main- a continuously flowing freshwater reactor where a biofilm of tenance costs in gas pipelines [1]. Biocorrosion is defined as Bacillus andSRBhadbeenobserved. a corrosive damage initiated or aggravated by the direct or The most common method for controlling microbial indirect activities of microorganisms [2]. A wide range of growth in industrial water systems is the use of biocides bacteria are present in most if not all areas of oil production [10]. Oxidizing (chlorine, ozone) or nonoxidizing com- and have been described from water injection plants, drilling pounds (quaternary ammonium salts, aldehydes, and tetrakis mud, and live reservoir cores [3–6]. Among the aerobic and (hydroxymethyl)phosphonium sulfate—THPS) are com- anaerobic bacteria related to biocorrosion in oil industries, monly applied [11, 12]. However, the environmental impact the sulfate-reducing bacteria (SRB) have been extensively and cost of adding large quantities of these compounds must studied because as well as forming corrosive biofilms they also be considered when such biocides are used [13]. So, the also produce hydrogen sulfide [3], which can result in health use of biocides of a microbial nature and origin offers another risks to workers [7]. A variety of Bacillus species have been option. Indeed, the production of antimicrobial substances shown to form biofilms on metal surfaces and produce elab- (AMSs) able to inhibit SRB growth has already been shown orated multicellular communities that display conspicuous as an attractive alternative to the use of biocides [5, 13]. 2 BioMed Research International

Actinomycetes are well known as potent producers of Table 1: Genus and origin of the actinomycete strains. a variety of secondary metabolites with distinct biological activities [14, 15], including AMSs active against both Strain Genus Origin pathogenic [16–18] and phytopathogenic microorganisms 221 Streptomyces [22] a Soil of the Atlantic Forest [18–20]. However, the production of AMSs active against 224 nd (Vista Chinesa—RJ, Brazil) [23] bacteria involved in biocorrosion process has been poorly 235 Streptomyces [22] described thus far. The exploration of soils and other habitats 606 Streptomyces [22] Soil of the Atlantic Forest for microbes of biotechnological interest has led to the iso- (Mendanha—RJ, Brazil) [22] 𝑎 lation of novel actinomycete strains [21]. Brazilian soils offer nd: not determined. great potential for bioprospection for novel strains and new bioactivecompounds.Inpreviousstudies,wehaveisolated several actinomycete strains from these soils. Therefore, the described by Rosado and Seldin [31]. All strains were spot search among these strains for those with antimicrobial prop- inoculated (5 𝜇L) on the surface of YMA plates, and after ∘ erties against microorganisms that negatively impact on the incubation at 28 C for 7 days, they were killed by exposure to oil industry was likely to reveal a strain with biotechnology chloroform vapors for 15 min. The plates were then flooded potential. with either B. pumilus LF-4 or D. alaskensis and incubated ∘ In this study four different strains of actinomycetes, pre- at 30 C for 24 h or 5 days, respectively. Manipulation of viously isolated from Brazilian tropical soils and previously D. alaskensis was performed in an anaerobic chamber (Plas selected as promising for AMS production, had their ability Labs, Lansing, MI, USA). Clear inhibition zones around to produce AMSs against the Bacillus pumilus LF-4 and the spot inoculum indicated antimicrobial production. The the Desulfovibrio alaskensis NCIMB 13491 evaluated. In this diameters of the inhibition zones were scored as follows: (−) paper, one strain, identified as belonging to the Streptomyces no inhibition, (+) weak inhibition with clear zones <7 mm, lunalinharesii species, is reported and discussed. A prelimi- (++) moderate inhibition with clear zones between 7 and nary characterization of the AMS obtained from the culture 12 mm, and (+++)strong inhibition with clear zones >12 mm supernatant and its effect on SRB cells are also described. [32]. The antimicrobial substance produced by S. lunalinharesii To test the antimicrobial activity of the supernatants, 235 might be an important alternative biocide for use against 20 𝜇L aliquots were spotted on solid LB media containing a undesirable bacteria that colonize pipe and flow lines used in growth lawn of B. pumilus LF-4. Plates were maintained at ∘ theproductionofoilandgas. 30 C/24 h and then inhibition zones were observed and their size recorded. 2. Material and Methods 2.1. Bacterial Strains and Growth Conditions. The actino- 2.3. Influence of Growth Conditions on the AMS Production. mycete strains used in this study were originally isolated from The overlay method was also performed using the actino- Brazilian tropical soils prior to 2001. The genus and the origin mycete strains grown in two chemically defined agar media of these bacterial strains are presented in Table 1.Theywere [32], containing a mineral salt solution and either glucose or glycerol as a carbon source. After incubation in aerobic previously selected as promising for the production of AMSs ∘ against different microbial strains22 [ –25] and now were used conditions at 28 C for 7 days, the antimicrobial activity of to test their capability to produce AMSs with antimicrobial strains was tested against B. pumilus LF-4. activity against aerobic and anaerobic bacteria associated The influence of pH and aeration on AMS production with biocorrosion processes. Unless otherwise stated, these wastestedinliquidmediumusingthechemicallydefined strains were grown in yeast extract-malt extract-agar (YMA) medium containing glucose [32]. Four different pH values ∘ (5.0, 6.0, 7.0,and 8.0) and two aeration conditions (stationary [26] under aerobic conditions at 28 Cfor7days. ∘ The Bacillus pumilus LF-4 and the SRB Desulfovibrio or shaking, 200 rpm) were tested. After incubation at 28 C alaskensis NCIMB 13491 were used as indicator strains for for 7 days, each culture was filtered with filter Whatman the production of AMSs.B.pumilusLF-4 was originally paper no. 1, and the supernatants were lyophilized for further isolated from an oil reservoir in Brazil located in a deep- analysis. The lyophilized supernatants were concentrated water production basin at an off-shore platform in Rio de 100-fold and 300-fold. The antimicrobial activity of each Janeiro [27]. It was grown under aerobic conditions for 24 h concentrated supernatant was tested as described above. ∘ at 30 C, in the Luria-Bertani broth (LB) [28]. The SRB strain was isolated from a soured oil reservoir [29]andgrownin 2.4. Molecular Identification of Streptomyces sp. 235. After ∘ ∘ Postgate C medium [30]at30C for 3 days, in anaerobic growth of Streptomyces sp. 235 in YMA for four days at 28 C conditionsusingsealedserumbottles(10mL).Thebottles with agitation (200 rpm), genomic DNA was extracted as were purged with a N2 flux to achieve anaerobiosis. All the described in earlier reports [33]. PCR amplification of the bacterial strains were maintained in a long-term storage by ∘ rrs gene was performed using a GoTaq Green Master Mix freezing the cells at −20 Cin20%glycerol. Kit (Promega Corporation) according to the manufacturer’s instructions. The amplification was carried out using the 2.2. Antimicrobial Activity Assay. For testing the actino- pair of universal primers 27F [34] and 1541R [35]. The PCR ∘ ∘ mycete strains, the overlay method was performed, as amplification conditions were 35 cycles of 95 C(30s),55C BioMed Research International 3

∘ (30 s), and 72 C (50 s) in a thermal cycler model Gene Table 2: Properties of the antimicrobial substances produced by Amp PCR System 9700 (Applied Biosystems). A hot start Streptomyces lunalinharesii 235. ∘ (5 min at 95 C) was applied to avoid initial mispriming Responses to Sensitiveb and enhance primer specificity. A final extension step was ∘ a run for 7 min at 72 C and the reaction tubes were then Enzymes ∘ cooled to 4 C. Amplified fragments were purified using the Pronase E − Illustra GFX PCR DNA and Gel Band Purification Kit (GE Proteinase K + Healthcare), which was used according to the manufacturer’s Trypsin + instructions. A negative control (without DNA) was run Solvents (10% and 50%) in all amplifications. DNA preparation and PCR products Methanol − were visualized after electrophoresis in 1x TBE buffer Ethanol − on a 1.2% agarose gel [36]. The purification product was Acetone − sequenced by the Center for Human Genome Studies at the Chloroform − University of Sao˜ Paulo, Brazil. All sequences were identified Chemicals using BLAST [37]attheNationalCenterforBiotechnology − Information (http://www.ncbi.nlm.nih.gov/blast). Sequences Urea 6 M − retrieved were aligned with the most similar-type NaOH (0.2 M and 1 M) strains obtained using CLUSTAL X [38]. BioEdit v. 7.0.0 HCl 0.2 M − (http://www.mbio.ncsu.edu/Bioedit/bioedit.html)wasused Heat treatment ∘ for manual editing of the sequences and a phylogenetic tree 40 C for 20, 45 and 60 min − ∘ with 1000 bootstrap replicates was constructed using MEGA 60 C for 20, 45 and 60 min − ∘ 4software(http://www.megasoftware.net/). The sequence 80 C for 20, 45 and 60 min − ∘ obtained was deposited in the GenBank database under 100 C for 20, 45 and 60 min − ∘ accession number GU126551. Autoclavation (121 C for 20 min) + Genomic DNA-DNA hybridization experiments were pHc performed to compare strain 235 with its closest-type strain − relative as determined by 16S rRNA gene sequences. Levels 3.0–6.0 7.0–9.0 − of genomic DNA-DNA similarity were determined at the a Deutsche Sammlung von Mikroorganismen und Zellkul- All enzymes were used at final concentration of 1 mg/mL, and tested with turen GmbH (DSMZ). Cells were disrupted by using a French 300-fold concentrated supernatant containing the AMS. b − pressure cell (Thermo Spectronic) and the DNA in the crude ( ) inhibition zones similar to those observed in control without treatment, (+) no inhibition zone observed. lysate was purified by chromatography on hydroxyapatite cpH of supernatants was adjusted from 3.0 to 9.0 varying 1.0 unit before [39]. DNA-DNA hybridization was carried out as described testing their activity. by De Ley et al. [40] under consideration of the modifications described by Huss et al. [41]usingamodelCary100 Bio UV/VIS spectrophotometer equipped with a Peltier- for 2 hours at room temperature (for organic solvents and thermostatted 6X6 multicell changer and a temperature ∘ chemicals) or at 37 C(forenzymes).Fortheheattreatment, controller with in situ temperature probe (Varian). the concentrated supernatant was incubated during 45 or ∘ ∘ 60min,at40,60,80,and100C, or autoclaved (121 Cfor 2.5. Partial Characterization of AMSs Produced by Strain 235. 20 min). For pH stability, the concentrated supernatants were Partial characterization was performed using a concentrated mixed to the same volume of citric acid-sodium citrate buffer supernatant containing the AMSs, obtained from growth of to achieve different pH values lower than 6.0 and with Tris- the strain 235 in the liquid medium containing glucose at ∘ HCl buffer for pH higher than 7.0. Antimicrobial activities pH 7.0 [32]. After incubation at 28 Cfor7days,theculture against B. pumilus LF-4 were determined before and after all was filtered with Whatman paper no. 1, and the supernatant treatments. lyophilized and 300-fold concentrated in the same chemically defined medium. To estimate the molecular mass of AMSs the concentrated 2.6. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophore- supernatant (500 𝜇L) was submitted to centrifugation at sis (SDS-PAGE). Ten microliters of the same 300-fold con- ∘ 4 C and 3 000 rpm for 1 h in an ultrafiltration membrane centrated supernatant described above were treated with (Millipore Corp., USA) with a 10 000 Da molecular mass cut equal volume of SDS-PAGE sample buffer (125 mM Tris- off. Both fractions, the concentrated one (MM > 10 000 Da) HCl, pH 6.8, 4% SDS, 20% glycerol, 2% mercaptoethanol, and the excluded one (MM < 10 000 Da), were tested for 2% bromophenol blue). Proteins were analyzed in 20–5% antimicrobial activity against B. pumilus LF-4 in LB plates. gradient SDS-PAGE by the method described by Laemmli ∘ The effect of organic solvents, chemicals, proteolytic [42].Followingelectrophoresisconductedat100Vat4C enzymes, heat and pH on activity of AMSs was tested for 140 min, each lane of the gel was cut vertically. One (Table 2)using20𝜇L of the concentrated supernatant. Sys- lane was stained with 0.025% Comassie brilliant blue R- tems containing the concentrated supernatant and equal 250 in methanol-acetic acid-water (40 : 7 : 53) and destained volume of each solvent, chemical, or enzyme were incubated in the same solvent, to determine the secretory protein 4 BioMed Research International profile. The apparent molecular masses of the polypeptides were calculated by comparison with the mobility of Full- Range Rainbow Molecular Weight Markers (GE Healthcare, Buckinghamshire, UK). To localize the in situ antimicrobial activity, a bioautography was performed: the other lane was prewashed with 10 volumes of 1% Triton X-100 in water for 1 h at room temperature, under agitation, to remove the SDS. Then, the gel strips were washed three times (20 min each) with double-distilled water, transferred to a LB agar plate, and overlaid with 4 mL semisolid (0.6%) LB containing 0.4mLoftheindicatorstrain(B. pumilus LF-4). Plates were ∘ then incubated in aerobic conditions at 30 Cfor24hand examinedforthepresenceofaninhibitoryzone.

2.7.InhibitoryEffectofAMSsonD.alaskensis. The inhibitory effect of AMSs on D. alaskensis was assessed by the determi- Figure 1: Inhibition zone of D. alaskensis NCIMB 13491 by S. nation of the minimal inhibitory concentration (MIC) and/or lunalinharesii 235 on agar plate. the minimal bactericide concentration (MBC). The MIC was performed using a microdilution method. The SRB were ∘ growninliquidPostgateCmediumforabout24–48hat30 C in PolyBed 812. Ultrathin sections were obtained using a Leica under anaerobic conditions. Experiments were performed ultramicrotome, stained with uranyl acetate and lead citrate, usinga96-wellplate,whereeachwellcontained100𝜇LofSRB and observed in a FEI Morgagni TEM at 80 kV. 7 suspension (10 cells/mL), and suspensions were exposed to different amounts of AMSs (from 0.1 to 0.003 g protein/mL) 3. Results diluted in Postgate C medium, in order to obtain successive dilutions of AMSs (from 1/1, 1/2, 1/4, 1/8, 1/16, until 1/2032) 3.1. Antimicrobial Activity Assay. All four actinomycete and a final volume of 200 𝜇L. Experimental controls included strains (Table 1)wereabletoinhibitaerobicB. pumilus LF- a medium without AMSs or a medium plus cell suspensions 4 and anaerobic D. alaskensis when tested using the overlay ∘ without AMSs. After incubation at 30 C for 7 days, growth of method in YMA medium. Figure 1 shows the inhibition of D. D. alaskensis was confirmed visually, by the observation of a alaskensis by strain 235, where a 12 mm inhibition zone was blackish precipitate (FeS, from the reaction of H2S, produced observed. by the SRB cells, and Fe, present in the medium), and then measured spectrophotometrically at 630 nm. The MIC was 3.2. Influence of Growth Conditions on the AMS Production defined as the lowest amount of AMSs required to ensure that and Strain Selection. All four strains were also able to inhibit SRB growth was absent. B. pumilus LF-4grownonsolidmediawitheitherglucoseor To perform the MBC test, aliquots of 10 𝜇Lfromthe glycerol as a carbon source. Strains 221 and 235 were more wells where growth was absent in the MIC test were used antagonistic on solid media and were selected for further to inoculate fresh Postgate C medium (90 𝜇L). The same studies in liquid media. Strain 221 supernatants failed to ∘ controls were used, and after incubation for 7 days at 30 C, inhibit B. pumilus LF-4 at less than a 400-fold concentration. the MBC was determined as the lowest concentration where Given that the 300-fold concentrated supernatant from strain growth was absent. All the inoculation procedures and 235 did show inhibitory activity, strain 235 was chosen for incubations in MIC and MBC tests were performed in an identification and further studies. anaerobic chamber (Plas Labs, Lansing, MI, USA) and the experiments conducted five times. Protein determination was 3.3. Molecular Identification of Streptomyces sp. 235. The according to Bradford [43]. identification of strain 235 was carried out by PCR amplification of rrs gene (1498 bp), its sequencing, and then 2.8. Transmission Electron Microscopy (TEM). TEM was genomic DNA-DNA whole-genome hybridization homol- performed to examine the ultrastructural changes in the ogy. According to the phylogenetic tree (Figure 2), the most D. alaskensis cells treated with MIC, sub-MIC, and supra- closely related bacterial type strain was Streptomyces lunal- MIC of AMSs during 7 days. Postgate C medium was also inharesii RCQ1071 (accession number DSM 41876T) [44], tested as a negative control. The mixtures were harvested by which shares 99.0% similarity within its 16S rDNA gene centrifugation at 4 000 ×gfor15minandthepelletwashed sequence. Genomic DNA homology between the Strepto- three times in a reducing solution (0.0124% sodium thiogly- myces lunalinharesii type strain and 235, analyzed by DNA- collate, 0.01% ascorbic acid, and 4 mL of a solution of 0.025% DNA hybridization (DSMZ service), demonstrated that these resazurin). Then cells were fixed for 2 h, at room temperature, strains belong to the same species. with 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2. Postfixation was carried out in1 3.4. Partial Characterization of AMSs Produced by Strepto- % osmium tetroxide in 0.1 M cacodylate buffer for 1 h. Cells myces sp. 235. The AMS that was retained by the ultrafil- were further dehydrated in an acetone series and embedded tration membrane (10 000 Da cutoff) and its characterization BioMed Research International 5

97 IMC S-0802T (ISP5492) 99 NBRC 15452T (AB184678) 85 YIM 41004T (AF346818) sp. 235 (GU126551) 42 100 RCQ1071 DSM 41876T (DQ094838) 62 ATCC 25470T (Y15507) 36 JCM 3399T (AY999753) NRRL B-5408T (DQ026654) NBRC 13452T (AB184414) 98 LMG 20087T (AJ781351) 99 NBRC 13786T (AB184479) DSM 44494T (AJ421018)

0.01

Figure 2: Phylogenetic tree obtained by the neighbor-joining method, based on the alignment of 16S rDNA of strain 235 and other Streptomyces species. Nocardiopsis halophila wasusedasanout-group.Bootstrapanalyseswereperformedwith1000repetitions.Thescale bar corresponds to 0.01 substitutions per nucleotide position.

111

68.9 (kDa)

34.8

14.4 12 (a) (b)

Figure 3: Antimicrobial activity profile presented by strain 235 in SDS-PAGE. The gel strips containing 300-fold concentrated supernatant fluids were stained with Coomassie brilliant blue to reveal the secretory protein profile (a) or overlaid with semisolid LB agar containing the indicator strain (LF-4) to show the inhibition zones (b). For details see text. Numbers on the left indicate relative molecular mass markers (in kDa). canbeseeninTable 2. The antimicrobial activity was resistant region of the gel (from 12 to 14.4 kDa), which exhibited to pronase E, different chemicals (urea 6 M, NaOH 0.2 and inhibitory activity when overlaid with the indicator strain B. 1 M, and HCl 0.2 M) and organic solvents (acetone, ethanol, pumilus LF-4 (Figure 3(b)). methanol,andchloroformat10and50%)butsensitiveto proteinase K and trypsin. It was heat stable after incubation ∘ at 100 C for 1 h but did not maintain its activity after being 3.6. Inhibitory Effect of AMSs against D. alaskensis. Exper- ∘ autoclaved at 121 Cfor20min.Itwasactiveintherangeof iments concerning the type of activity of AMSs against pH values between 3.0 and 9.0. D. alaskensis cells have shown a bacteriostatic effect at a 0.03 g protein/mL (MIC), which corresponded to a 1/4 dilution of the 300-fold concentrated supernatant, whereas 3.5. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophore- a bactericidal effect was observed at a 0.05 g protein/mL sis (SDS-PAGE). Streptomyces sp. 235 was able to secrete (MBC), which corresponded to the 1/2 dilution. polypeptides to the extracellular environment during its growth in a chemically defined medium for 7 days, as demonstrated by SDS-PAGE analysis (Figure 3(a)). The strain 3.7. Transmission Electron Microscopy (TEM). Untreated D. released a large amount of extracellular polypeptides with alaskensis cells in Postgate C medium showed an undamaged molecular masses varying from 12 to 111 kDa, including one structureoftheinnermembraneandanintact,slightly 6 BioMed Research International

(a) (b)

(d)

(f)

(e) (f)

Figure 4: Transmission electron microscopy micrographs of D. alaskensis cells. In untreated cells, the inner and outer membranes are visible as continuous and intact structures (a). After treatment with a sub-MIC of AMSs, spherical double-layered structures inside and outside the cell can be observed ((b), black arrow), and the periplasmic space filled with electron-dense material. MIC of AMSs causes the formation of numerous spherical double-layered structures ((c)-(d), black arrows) and the periplasmic space to be filled with electron-dense material. After treatment with a supra-MIC of AMSs, the periplasmic space is still filled with electron-dense material and prominent bubbles are emerging from the cell surface ((e)-(f), black arrows).

waved outer membrane, and the periplasmic space was thin of prominent bubbles emerging from the cell wall surface with a uniform appearance (Figure 4(a)). D. alaskensis cells was observed (black arrows in Figures 4(e)-4(f)), whereas an treatedwithsub-MIC(0.01gprotein/mL)oftheconcentrated electron-dense material was still visible in the periplasmic supernatant of AMSs presented spherical double-layered space (Figures 4(e)-4(f)). structures between inner and outer membranes and out of the cell (black arrows in Figure 4(b)). After incubation with a MIC (0.03 g protein/mL) of AMSs, numerous spherical 4. Discussion double-layered structures could be observed (black arrow in Figures 4(c)-4(d)). The periplasmic space became completely Although the four actinomycete strains isolated from the filled with electron-dense material (Figures 4(c)-4(d)). For Brazilian soils were able to effectively inhibit the growth of B. a supra-MIC (0.05 g protein/mL) of AMSs, the formation pumilus LF-4 and D. alaskensis NCIMB 13491 in vitro,strain BioMed Research International 7

235 was the only one able to inhibit them on solid and in characterization of AMSs produced by S. lunalinharesii 235 liquid media. This strain was originally isolated from soil of have shown that its antimicrobial activity was sensitive to the Atlantic Forest, Vista Chinesa, RJ [23], and characterized trypsin and proteinase K, indicating its proteic nature. The as belonging to the Streptomyces genus [22]. The molecular SDS-PAGE analysis and the presence of an inhibitory zone characterization by PCR amplification of the rrs gene had between 12 and 14.4 kDa have confirmed this finding. This 99.0% identity with rrs gene sequences of Streptomyces lunal- apparent molecular mass range is in accordance with the inharesii RCQ1071. Within the Streptomyces genus, the rrs ultrafiltration membrane approach. However, the AMS was gene sequences are highly conserved and sequence identities resistanttopronaseEsuggestingthatitisresistanttosome arehighandcanbemorethan99%.Asaresult,itbecomes typesofproteolyticcleavage.Theresistancetoproteolytic very difficult to identify streptomycete species based solely enzymes may indicate the presence of unusual amino acids on 16S rDNA sequencing, even when 99.0% similarity to a in the AMS structure. Moreover, the AMS could also present type strain is observed [45]. Dastager et al. [46] described a compact structure, or the lack of cleavage recognition sites, Streptomyces deccanensis as a new Streptomyces species even making it resistant to proteolytic enzymes. Cyclic peptides though its rrs gene is 99.4% similar to its most closely can be resistant to hydrolysis by proteases because their related type strain. Whole-genome DNA-DNA hybridization cyclic structure renders them relatively inflexible, which may remains the “gold standard” to distinguish bacterial species make cleavage sites inaccessible because of steric hindrance and is necessary to decide whether a strain belongs to a [52]. species. When DNA-DNA homology is less than 70%, then Another interesting characteristic shown by the AMS in the two strains being compared do not belong to the same this study was its resistance to organic solvents, indicating species. Strain 235 shared around 100% homology with S. that its structure should not contain a lipid portion. Also this lunalinharesii RCQ1071, which is far above the 70% thresh- AMS was able to resist different chemicals and continued old recommended for the recognition of separate genomic to inhibit growth over a range of pH values and high species [47]. These results have clearly demonstrated that temperatures. Similar characteristics to those of the AMSs strain 235 indeed belongs to this same species. produced by S. lunalinharesii have been observed for the AMSs produced by other bacterial genera, such as Bacillus S. lunalinharesii-type strain RCQ1071T was described [5, 53–56], Paenibacillus [32, 57], Lactobacillus [58], and Ente- recently by our group [44]. It was isolated from another rococcus [59]. AMS from Paenibacillus peoriae,forinstance, Brazilian soil, in the Central Plateau, under cerrado veg- presented a high stability after treatments with a broad pH etation [48]. A preliminary study has shown it has the ∘ ∘ range (3.2–9.6), heat (100 C for 1 h or 121 Cfor10min), ability to produce antimicrobial substances against human proteolytic enzymes and organic solvents, among other tests pathogenic microorganisms [22]. Its activity against some [32]. Bacillus subtilis, B. firmus,andB. licheniformis,isolated phytopathogenic fungi was assessed and the strain was char- from an oil reservoir in Brazil, have also produced AMSs ∘ acterized as an excellent chitinase producer [48]. In another that are stable at 100 C for 1 h as well as in presence of other studybyourgroup[49], strain 80 from this same species has chemicals. In those studies, however, the AMSs were resistant been shown to be a promising strain for the biological control to different proteolytic enzymes and sensitive to several of Sclerotinia sclerotiorum.Herewereportonapreviously organic solvents, indicating a different chemical nature5 [ ]. undescribed strain of this species which produces bioactive Lactobacillus paracasei strains have produced an AMS stable ∘ compounds able to inhibit bacteria involved in the microbial at 100 C for only 3 min, and its inhibitory activity was also colonization and corrosion of pipe systems in the oil industry. totally lost after treatment with different proteolytic enzymes S. lunalinharesii strain 235 was grown in a mineral salt [58]. Enterococcus faecium was able to produce a bacteriocin ∘ ∘ ∘ solution containing glucose, pH 7.0, at 28 Cfor7dayswithout which has shown stability at 30 Cfor1handat100Cfor shaking. These growth conditions for AMS production are 30mininthepHrange2.0–7.0and2.0–5.0,respectively, very interesting if we think about future biotechnological being also sensitive to different proteolytic enzymes [59]. application: the use of glucose, a simple, cheap, and easy The stability at high temperatures and different pH values, carbon source along with a mineral salt solution would in particular, may be very useful in oil reservoirs, where ∘ be very appropriate for an industrial scale production; the temperatures above 60 C and pH values ranging between 3.0 use of a neutral pH and fermentation without agitation and 7.0 are usually found [4]. would be also be very favorable. Most of the methods for Cellular alterations caused by AMSs in D. alaskensis cells antibiotic production described in the literature for Strepto- observed by TEM were basically related to the appearance myces [50], other bacteria [5], and fungi [51]requiresimilar of an electron-dense material in the periplasmic space and conditions of carbon source, pH, and temperature. However, to membranes, where spherical double-layered structures they require growth under agitation. Von Der Weid et al. appeared and increased from sub-MIC to MIC of AMSs, [32] also observed maximum antimicrobial activity when and prominent bubbles emerge from the cell wall surface at Paenibacillus peoriae was cultivated in a chemically defined ∘ a supra-MIC concentration. Some of these alterations have medium containing glucose at pH 7.0 and incubation at 30 C been observed in other bacterial strains treated with antimi- in stationary conditions. crobial peptides. For instance, Meincken et al. [60]treated Peptidic antibiotics, an abundant class of special Escherichia coli cells with the synthetic peptide peptidyl- metabolites, are commonly produced by many microbial glycyl-leucine-carboxamide (PGLa) and observed numerous species including Streptomyces.Thepreliminarytestsforthe regularly distributed protrusions from cell surface. Latter, 8 BioMed Research International

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