Micromonospora: an Important Microbe for Biomedicine and Potentially for Biocontrol and Biofuels

ARTICLE IN PRESS

Soil Biology & Biochemistry xxx (2009) 1e7

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Review Micromonospora: An important microbe for biomedicine and potentially for biocontrol and biofuels

Ann M. Hirsch a,b,*, Maria Valdés c a Department of Molecular, Cell and Developmental Biology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90095-1606, USA b Molecular Biology Institute, University of California, 405 Hilgard Avenue, Los Angeles, CA 90095-1606, USA c Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, I. P. N., Plan de Ayala y Carpio, 11340, Mexico article info abstract

Article history: Micromonospora species have long been recognized as important sources of antibiotics and also for their Received 2 September 2009 unusual spores. However, their involvement in plant-microbe associations is poorly understood although Received in revised form several studies demonstrate that Micromonospora species function in biocontrol, plant growth promo- 17 November 2009 tion, root ecology, and in the breakdown of plant cell wall material. Our knowledge of this generally Accepted 20 November 2009 understudied group of actinomycetes has been greatly advanced by the increasing number of reports of Available online xxx their associations with plants, by the deployment of DNA cloning and molecular systematics techniques, and by the recent application of whole genome sequencing. Efforts to annotate the genomes of several Keywords: Actinomycetes Micromonospora species are underway. This information will greatly augment our knowledge of these Biocontrol versatile microorganisms. Hydrolytic enzymes Ó 2009 Elsevier Ltd. All rights reserved. Secondary metabolites

1. Introduction species also produce anti-tumor antibiotics (lomaiviticins A and B, tetrocarcin A, LL-E33288 complex, etc.) and anthracycline antibi- Although the genus Micromonospora has long been recognized otics. Furthermore, the endophyte, Micromonospora lupini,produces as a significant source of secondary metabolites for biomedicine the anthraquinones, lupinacidins A and B, which have anti-tumor and some data have accrued concerning the importance of these activity (Igarashi et al., 2007). Other biologically active molecules bacteria for soil ecology, surprisingly, it is only recently that the synthesized by Micromonospora species are vitamin B12 (Wagman influence of Micromonospora species on plant growth and devel- et al., 1969) and anti-fungal compounds (Nolan and Cross, 1988; opment has been acknowledged. This review focuses more on this Ismet et al., 2004). Marine Micromonospora species have recently newly uncovered impact rather than on other aspects of this been reviewed with respect to their broad distribution and their understudied group of microbes, and also on how Micromonospora potential for use as probiotics (Das et al., 2008). The antimicrobial might be employed for biocontrol and breakdown of cellulosic alkaloid, diazepinomicin, was identified from a marine species biomass for biofuels. (Charan et al., 2004). Since 2008, the genomes of three micromonosporas have been sequenced and are currently being annotated. Several more micro- 2. Definition of the genus, life style, and habitat monosporas are in the pipeline for sequencing. Micromonospora species are best known for synthesizing antibiotics, especially ami- On the basis of molecular and chemical composition data, noglycoside, enediyne, and oligosaccharide antibiotics. Thus, their Stackebrandt et al. (2007) proposed the class Actinobacteria, impact on medicine is considerable, and indeed Streptomyces and which consists of five different subclasses. The family Micro- Micromonospora species produce many of the best-known antibi- monosporaceae in the suborder Micromonosporineae (order, otics. For Micromonospora, this includes the aminoglycoside antibi- Actinomycetales) contains several genera in addition to Micro- otics, gentamicin and netamicin (Bérdy, 2005). Micromonospora monospora, namely, Actinoplanes, Dactylosporangium, Pilimelia, Catenuloplanes, and Couchioplanes (see references in Koch et al., 1996). The genus Micromonospora Ørskov 1923 is Gram-positive, * Corresponding author. Department of Molecular, Cell and Developmental chemo-organotrophic, aerobic, and like the rest of the actinomy- Biology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90095-1606, USA. Tel.: þ1 310 206 8673; fax: þ 1 310 206 5413. cetes, characterized by a high mol% G þ C content genomic content E-mail address: [email protected] (A.M. Hirsch). (Vobis, 1992).

Please cite this article in press as: Hirsch, A.M., Valdés, M., Micromonospora: An important microbe for biomedicine and..., Soil Biology & Biochemistry (2009), doi:10.1016/j.soilbio.2009.11.023 ARTICLE IN PRESS

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Although Micromonospora exhibits considerable physiological wheat roots (Combs and Franco, 2003), from nitrogen-fixing root and biochemical diversity, the genus forms a well-defined group in nodules of the actinorhizal plants Casuarina equisetifolia (Valdés et al., terms of morphology, phylogeny, and chemotaxonomy. Certain 2005) and Coriaria myrtifolia (Trujillo et al., 2006), and from root criteria can be used to differentiate at least 27 described species, nodules of the leguminous plants Lupinus angustifolius (Trujillo et al., including the carbon sources utilized (pentoses and hexoses as well 2007) and Pisum sativum (Carro et al., in press). These microbes as organic acids), growth on different media, mycelial pigments, also grow in complex microbial communities in soil as well as in the and so on (Kawamoto, 1989). Micromonospora peptidoglycan rhizosphere. contains meso-DAP, and/or its 3-hydroxy derivative, and glycine. However, species of the genus Micromonospora are not always Cell wall hydrolysates are also characterized as containing arabi- easy to differentiate on the basis of morphology alone. Phyloge- nose and xylose, in this way differing from other actinomycete nies and species identifications are now more commonly derived genera (Holt et al., 2000). either from 16S rRNA DNA or from gyrB (encoding DNA top- Micromonospora exhibits a complex life cycle, differentiating oisomerase) sequence analyses (Kasai et al., 2000)(Fig. 2). The into both substrate mycelia and spores, but no aerial mycelia are advantage of using gyrB is that it is more likely to differentiate produced (Vobis, 1992). The species often form indistinct colonies species of Micromonospora than a 16S rRNA DNA comparison that can be confused with other actinomycetes. Indeed, their because it has a higher molecular evolutionary rate than that of microscopic cell morphology is similar to other genera in the rDNA (Kasai et al., 2000; de Menezes et al., 2008). A potentially Micromonosporaceae in producing branched, septate hyphae of more exact way to determine species or strain identity is to 0.25e0.6 mm in diameter (Fig.1A, B). The colonies can be a variety of employ multilocus sequence typing (MLST) or multilocus colors, including white, orange, rose, or brown (Fig. 1C, D); the sequence analysis (MLSA) (Gevers et al., 2005; Hanage et al., pigments may or may not diffuse into the medium. When colonies 2006). MLST utilizes allelic mismatch of a small number of sporulate, the single asexual spores of almost all species have spiny housekeeping genes for recognizing distinct strains within named projections. The spores are formed on both short and long side species, whereas MLSA uses sequences from multiple protein branches (Vobis, 1992)(Fig. 1A, B). genes to characterize a more diverse group of microbes. To our Micromonospora are widely distributed in nature, inhabiting such knowledge, so far no one has attempted either MLST or MLSA disparate environments as coastal sediments (Zhao et al., 2004), with Micromonospora, but as more Micromonospora genomes are marine sediments (Mincer et al., 2002; Maldonado et al., 2009), peat sequenced and annotated, this is likely to change. swamp forests (Thawai et al., 2005), floodplain meadows (Zenova and Zviagintsev, 2002), and plant rhizospheres (Merzaeva and Shir- 3. Production of hydrolytic enzymes okikh, 2006). Some members of this genus are also known to form intimate associations with plants. For example, Micromonospora The Actinomycetales have been known for some time as potent bacteria have been isolated from roots of rice (Tian et al., 2007), from degraders of cellulose, chitin, lignin, and other complex

Fig. 1. Microscopic and macroscopic views of Micromonospora aurantiaca ATCC 27029 (A, C) and strain Micromonospora L5 (B, D). Although branched hyphae are observed, the magnification is too low to see septa. (A) Phase contrast microscopy of M. aurantiaca ATCC 27029 grown in a defined minimal medium. The arrows point to the large single spores. Bar, 500 mm. (B) Phase contrast microscopy of Micromonospora L5 grown in a defined minimal medium. Single spores are present (arrows) but some (*) appear to cluster and to be phase bright. Bar, 500 mm. (C) Colonies of M. aurantiaca ATCC 27029 grown on agar-solidified ISP Medium 2. Older colonies burst upon drying. See also Fig. 6D in Vobis (1992). Bar 5 mm. (D) Multi-colored colonies of Micromonospora L5 grown on agar-solidified ISP Medium 2. Bar 5 mm.

A.M. Hirsch, M. Valdés / Soil Biology & Biochemistry xxx (2009) 1e7 3

Fig. 2. Phylogenetic tree constructed by the method of Felsenstein (1993) of 16S RNA DNA sequences from various actinomycetes curated in IMG-ER (Markowitz et al., 2009). Micromonospora aurantiaca ATCC 27029 clusters with Salinospora tropica CNB-440 and both are in the same clade as Frankia CcI3 and Acidothermus cellulolyticus 11B. polysaccharides (Erikson, 1941; Malfait et al., 1984; McCarthy and a large number of alpha- and beta-glucosidases, xylanases, and Broda, 1984; Wilson, 1992; Gacto et al., 2000). Many species pectin-degrading enzymes. degrade polysaccharides in compost heaps, decaying plant mate- Chitin is the second most abundant polysaccharide in nature rial, manure piles, straw, and stover (Lacey, 1997; Godden and and many actinomycetes are able to degrade this polymer. Micro- Penninckx, 1984; Abdulla and El-Shatoury, 2007). Some species monosporas are known to produce chitinase although Micro- even inhabit termite guts or the rumens of cattle, where they monospora chalcea was reported to exhibit low activity (Gacto et al., degrade plant cell walls and fibers thereby providing carbon to 2000). So far, two different methods have been used to detect their host (Hungate, 1946; Maluszynska and Janota-Bassalik, 1974). chitinases in Micromonospora. One study used a chitin-degrading Others even degrade natural rubber (Jendrossek et al., 1997; Rose assay to analyze bacteria from various environments in 20 different and Steinbüchel, 2005). The production of hydrolytic enzymes locations. The most potent chitinase-producing actinomycetes allows Micromonospora species to play an active role in the found were: Streptomyces, Kitasatosporia, Saccharopolyspora, degradation of organic matter in their natural habitats. Nocardioides, Herbidospora, Micromonospora, Microbispora, and Rivas et al. (2003) described a new actinomycete species, Xyla- Actinoplanes, with Micromonospora more frequently detected in nimonas cellulosilytica XIL07T, isolated from a decayed Ulmus nigra extreme and polluted environments (Nawani and Kapadnis, 2003). tree as an abundant producer of cellulases and xylanases, enzymes In a second study, Williamson et al. (2000), using PCR primers that are important for the conversion of plant material to biofuels designed to identify a wide range of chitinase genes, found that and for biomass breakdown (Carere et al., 2008). Using the same group A chitinase sequences were present in six different species of methodology, but in this case, to uncover xylanase-producing Micromonospora.AsMicromonospora genomes go online, more and micromonosporas, Trujillo et al. (2006) isolated a strain, named more hydrolytic enzymes, including chitinases, will be detected. Micromonospora coriaria, from nodules of the actinorhizal plant, Our preliminary investigation of the genome of M. aurantiaca ATCC C. myrtifolia, that strongly resembled the nitrogen-fixing strains 27029 shows that at least four different chitinases are present (data isolated from nodules of C. equisetifolia by Valdés et al. (2005). This not shown). strain was capable of using xylan as a sole carbon source (Trujillo Although terrestrial actinomycetes including species of Micro- et al., 2006). Our preliminary data show that Micromonospora strain monospora are well known as xylanase- and cellulase-producers, L5, isolated from C. equisetifolia nitrogen-fixing nodules not has fresh-water micromonosporas have also been shown to have only cellulase and xylanase activity, but also secretes enzymes that cellulose-degrading activity (de Menezes et al., 2008). Using cotton breakdown pectin and chitin (data not shown). We are currently baits placed at various depths, a number of lake-dwelling Micro- annotating the Micromonospora aurantiaca genome (closely related monospora strains, several closely related to M. chalcea, with to Micromonospora L5; Valdés et al., 2005) to determine how many cellulolytic activity were identified. Under laboratory conditions, genes encoding hydrolytic enzymes are present and also whether M. chalcea has been shown to exhibit strong hydrolytic activity in they may be secreted via the twin arginine translocation (Tat) degrading lignocelluose of rice straw strips (Abdulla and pathway as observed for the thermophile Thermobifida fusca XY. El-Shatoury, 2007). The T. fusca XY genome has 45 genes that potentially encode The ability to produce hydrolytic enzymes may allow these hydrolytic enzymes, including six cellulases (Lykidis et al., 2007). bacteria to grow as saprophytes as well as endophytes within plant We anticipate finding similar numbers of cell wall-degrading organs. Recently, Kirby and Meyers (in press) reported the isolation enzymes in Micromonospora species. We already have annotated of a newly named species, Micromonospora tulbaghiae, from

4 A.M. Hirsch, M. Valdés / Soil Biology & Biochemistry xxx (2009) 1e7 surface-sterilized leaves of the South African wild garlic, Tulbaghia preliminary evidence suggests that the supernatant contains violaceae, strongly suggesting an endophytic origin for these indole-acetic acid and gibberellic acid (Solans et al., 2006). If these microbes. The ability of Micromonospora to enter plant tissues may phytohormones are indeed produced by Micromonospora strain make it possible for various species to act as Plant Growth MM18, this supports the hypothesis of Solans et al. (2009) that Promoting Bacteria (PGPB). these actinomycetes are acting at the level of autoregulation of nodulation. 4. Biocontrol agents and PGPR 5. Micromonospora and nitrogen fixation A major factor influencing plant growth and health is the microbial population living on the plant not only as epiphytes, but In the course of isolating Frankia from surface-sterilized nodules also as endophytes (Taechowisan et al., 2003). A number of studies of C. equisetifolia collected in Mexico, different actinomycete colo- have already implicated Micromonospora and other actinomycetes nies were recovered on liquid Defined Propionate Medium (DPM) as biocontrol agents in the protection of carrot (El-Tarabily et al., (Guillén et al., 1993). The filaments of these bacteria were found to 1997), wheat (Combs and Franco, 2003) Chinese cabbage (Lee et al., be smaller in diameter (<0.5 mm) than Frankia hyphae and did not 2008) and cucumber (El-Tarabily et al., 2009). develop nitrogen-fixing vesicles typically found in Frankia. More- Recently, Conn et al. (2008) demonstrated that Micromonospora over, the strains were unable to reinfect Casuarina or other tested and other endophytic actinobacteria were able to suppress actinorhizal plants upon inoculation (Niner et al., 1996). However, a number of pathogens, both in vitro and in planta, through the these actinobacteria were found to grow better on glucose than on activation of key genes in the systemic acquired resistance (SAR) or propionate and also exhibited nitrogenase activity (evaluated by the jasmonate/ethylene (JA/ET) pathways in Arabidopsis thaliana. acetylene reduction) ex planta (Villegas et al., 1997). Based on PCR- Micromonospora sp. strain EN43 barely induced the SAR or JA/ET amplifying an internal segment of the 16S RNA DNA gene, we pathways in the absence of a pathogen. However, upon diverse proposed that these actinobacteria were in a clade separate from pathogen challenge, either, the SAR or JA/ET pathway was triggered Frankia (Niner et al., 1996). in the Micromonospora-inoculated roots leading to greater host Later, we showed that these actinobacteria are diazotrophs by resistance. Moreover, culture filtrates obtained from cells of PCR-amplifying a nifH gene and also by using an isotope dilution Micromonospora sp. strain EN43 grown in either minimal or rich assay to demonstrate that the cells incorporated 15N. The separa- medium activated the SAR and JA/ET pathways, respectively, indi- tion of these actinomycetes from Frankia was supported by the very cating that two different sets of metabolites are synthesized by this low DNAeDNA homology between them and by an analysis of the actinomycete for eliciting the plant defense pathway. Whether or complete 16S rRNA sequence (Valdés et al., 2005). Together, these not these metabolites are antibiotics or other molecules is not studies showed that Micromonospora strain L5 is more closely known at this time. In any case, these studies show that Micro- related to M. aurantiaca than it is to Frankia. monospora is a very effective biocontrol agent. Similarly, Trujillo et al. (2006) isolated from root nodules of the Micromonospora species also produce antimicrobial and anti- actinorhizal plant C. myrtifolia an actinobacteria producing an fungal compounds that act to protect plants from pathogens. For intense orange color and showing a high capacity to degrade xylan example, Shomura et al. (1983) found that Micromonospora sp. SF- as the sole carbon source (see earlier section). Neither aerial hyphae 1917 produces the antibiotic dapiramicin, which inhibited the nor diffusible pigments were produced. Based on genotypic and growth of Rhizoctonia solani on rice roots. An isolate from mangrove phenotypic data, the actinobacterium was clearly differentiated rhizosphere soil in West Malaysia, Micromonospora sp. M39, from previously described Micromonospora species and was produces metabolites that are effective against the rice blast proposed as a novel species, Micromonospora coriariae. From pathogen Pyricularia oryza MPO 292 (Ismet et al., 2004). A number further investigations, the same research group proposed the of compounds were detected in the crude extract, including 2,3- names of M. lupini and Micromonospora saeliciesensis for two acti- dihydroxybenzoic acid, phenylacetic acid, and the antibiotics, cer- nomycete strains isolated from nitrogen-fixing root nodules of vinomycin A1 and A2. However, it is not known yet whether L. angustifolius (Trujillo et al., 2007). Similar to the situation for the a single compound or a mixture is needed for anti-fungal activity. strains isolated by Valdés et al. (2005), DNA sequences for a nifH In natural environments, it is very likely that a mixture of gene, which are very similar to those from nifH of Frankia alni secondary products is required for suppressing a pathogen. An AC14a, were detected in these legume-isolated micromonosporas earlier study shows that an Micromonospora carbonaceae isolate (M.E. Trujillo, personal communication). can lyse, via cellulase production, the cell walls of Phytophthora A new diazotrophic taxon isolated from actinorhizal nodules of cinnamomi, a pathogen that causes root rot. Moreover, a synergism Elaeagnus plants growing in Tunisia has also been reported. It is an is observed when the cellulase-producing M. carbonaceae is co- actinobacterium that occupies an intermediate taxonomic position inoculated with the antibiotic producing Streptomyces violascens in between Frankia and Micromonospora according to the RFLP and that more of the fungal hyphae are degraded (El-Tarabily et al., sequences of the nifH gene (Gtari et al., 2007). 1996). Inoculating plants with both actinomycetes, M. carbonaceae However, so far genes other than nifH have not been detected and S. violascens, had a greater positive effect on plant growth than even though we used numerous PCR variations to look for nifD and the singly inoculated or uninoculated control. The exact mecha- nifK, the two other structural genes for nitrogenase (Milani, De Hoff, nism of how this positive effect is mediated is not completely and Hirsch, unpubl. results). At this time, we do not understand the understood at this time. reasons for the failure to detect the other nitrogenase genes. Micromonospora also acts as a PGPB through its ability to Analysis of the Micromonospora genomes from which nifH was PCR- promote both the Discaria trinervis-Frankia (Solans, 2007) and the amplified should provide answers to this conundrum. Medicago sativa-Sinorhizobium meliloti nitrogen-fixing symbioses The only other case we know of where nitrogen fixation takes (Solans et al., 2009). Other actinomycetes in addition to Micro- place, based on 15N analysis and growth in N-free medium, but monospora also stimulate alfalfa nodule formation, even in the where no nif genes were detected, is that of Streptomyces ther- presence of nitrogen, but so far little is known about them. In moautotrophicus UBT1, which inhabits the covering of burning contrast, the supernatant of Micromonospora strain MM18 was charcoal piles (Gadkari et al., 1992). This actinomycete has an shown to have a definite positive effect on plant growth, and unusual nitrogen reduction system in that it is coupled to carbon

A.M. Hirsch, M. Valdés / Soil Biology & Biochemistry xxx (2009) 1e7 5 monoxide reduction and is dependent on oxygen (Ribbe et al., interesting questions about how these actinomycetes acquired this 1997). Like dinitrogen, carbon monoxide is held together by a very gene and whether or not this is a recent event. The upcoming stable triple bond. Thus, it is not surprising that S. thermoauto- availability of the genomes of some of these putative diazotrophic trophicus UBT1 is able to use its CO-adapted enzymes to breakdown micromonosporas should help us learn whether all or only part of nitrogen gas. The three enzymes employed for fixing nitrogen are: the nitrogenase operon is present, how the sequences differ from St3, a CO dehydrogenase that generates superoxide radicals; St2, Frankia and other nitrogen-fixing microbes, and whether the genes a superoxide oxidoreductase that scavenges oxygen radicals and were likely to be acquired by horizontal gene transfer. acts as a dinitrogenase reductase, generating electrons to transfer We also still have little understanding of metabolic diversity in to St1, a MoFeS protein that functions as a dinitrogenase (Ribbe this genus and how it applies to the concept of species in Micro- et al., 1997). Several genes encoding the various subunits of CO monospora. Currently, Micromonospora consists of a hodge-podge of dehydrogenase are present in the M. aurantiaca genome, but no species and strains, many of which have no assigned species name. evidence has been found for either a superoxide oxidoreductase Species in Micromonospora are usually described by morphological like St2 or a MoFeS, St1-like protein with dinitrogenase functions. traits such as spore color and shape, hyphal color, or by their site of origin be it plant or environment. The recent explosion in the 6. Genetics and genomics number of prokaryotic genomes sequenced gives us many more tools to demarcate taxa and to understand variability within Although Micromonospora species are well known for secondary a genus. Understanding this variability will be important for us to metabolite production, they are orphan children with regard to the select for micromonosporas that not only impact human health, but study of their genomes and the development of tools for genetic also crop fitness and biofuel production. analyses. During the 1980's and 1990's, cloning procedures for several Micromonospora species were published. Indeed, a number Acknowledgments of genes were cloned, but most of these represent antibiotic production or hydrolytic enzyme activity (Keleman et al., 1991; Lin We express our gratitude to Dr. Nicola Stanley-Wall (University et al., 1994). A number of cloning vectors have been described for of Dundee, formerly of UCLA) for her help with the colony photo- various Micromonospora species (Hasegawa et al., 1991; Li et al., graphs shown in Fig. 1. We thank Drs. Stefan J. Kirchanski, Nancy 2003, 2004), but so far little evidence in the literature has been A. Fujishige, and Annette A. Angus for helpful comments on the provided to show that this actinomycete is easily transformable or manuscript. Former and current UCLA and ENCB/IPN undergrad- mutable. Although several techniques for transformation using uate students, Nima Milani, Johana Alvarado, Natalie Ma, and protoplast isolation have been published (Matsushima and Baltz, Claudia Martínez Catalán, are also thanked for their contributions 1988; Keleman et al., 1989; Li et al., 2003), to our knowledge, these to the Micromonospora project. procedures have not been developed further or routinely applied to This research was funded in part by a grant from UC-MEXUS/ Micromonospora transformation. Conacyt to AMH and MV. 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Please cite this article in press as: Hirsch, A.M., Valdés, M., Micromonospora: An important microbe for biomedicine and..., Soil Biology & Biochemistry (2009), doi:10.1016/j.soilbio.2009.11.023