Cent. Eur. J. Biol.• 6(6) • 2011 • 997-1005 DOI: 10.2478/s11535-011-0072-2

Central European Journal of Biology

Pathogenicity and biotechnological applications of the genus

Mini-Review

Patrizia Paganin, Silvia Tabacchioni, Luigi Chiarini*

ENEA, C.R. Casaccia, Technical Unit for Sustainable Development and Innovation of Agro-industrial System, 00123 Rome, Italy

Received 06 May 2011; Accepted 20 August 2011

Abstract: belonging to the genus Burkholderia are well known for their adaptability to habitats as diverse as freshwater sediments, lungs of patients and plant tissues. This genus includes also plant, animal and human pathogenic species, such asBurkholderia glumae, Burkholderia pseudomallei and the Burkholderia cepacia complex. Over the past few years, several newly discovered non-pathogenic plant associated Burkholderia species have raised particular interest for their potential use in plant growth promotion, biocontrol of plant pathogens, phytoremediation and xenobiotics degradation. Highlights from recent studies on the , ecology and pathogenicity of different species of the Burkholderia genus are presented with the aim to evaluate their potential use in biotechnology.

Keywords: Burkholderia • Burkholderia cepacia complex • Pathogenicity • Biotechnology • Diazotrophs • Genomics • Transcriptomics © Versita Sp. z o.o.

1. Introduction complex [1,3]. The latter is a group of 17 species, widely distributed in the environment, able to promote For the past two decades, research on species of the plant-growth and to antagonize plant pathogens, but genus Burkholderia has been steadily expanding, so it is also reported to contain opportunistic human that the genus Burkholderia currently comprises more pathogens, so that any biotechnological application is, than 60 species, isolated from remarkably diverse at least temporarily, not allowed. Nevertheless, in recent ecological niches such as river water, soil, freshwater years, a growing number of new Burkholderia species sediments and plant rhizosphere, and including also have been discovered and reported as nitrogen fixing, human, animal and plant pathogens [1]. The natural non pathogenic plant-growth promoting bacteria, thus diversity of members of this genus indicates that the being proposed as candidates for biotechnological range of interactions between these bacteria and their applications [4]. hosts is very complex, diverse, and, often, contradictory. Over the past few years, sequencing of more than The ecological and metabolic versatility of this genus 35 Burkholderia genomes has made available to the ranges from the ability to proliferate in lungs of cystic scientific community a huge amount of data that can fibrosis (CF) patients causing often mortal infections significantly contribute to better define the pathogenicity to that of growing inside plant tissues as obligate and the biotechnological potential of the different endosymbionts, from the ability to degrade recalcitrant Burkholderia species and help clarify the genetic basis xenobiotic compounds to that to protect crop plants by of the great ability of members of this genus to adapt synthesizing antifungal compounds. to extremely diverse habitats. This article presents Since the establishment of this genus [2], research highlights from recent studies on the taxonomy, ecology has been focused primarily on two highly pathogenic and pathogenicity of different species of the Burkholderia species such as B. mallei and B. pseudomallei and genus with the aim to evaluate their potential use for on species belonging to the so-called B. cepacia biotechnological applications.

* E-mail: [email protected] 997 Pathogenicity and biotechnological applications of the genus Burkholderia

2. Taxonomy and ecology due to their epidemiological and sanitary features. B. thailandensis, the third and last member of this Burkholderia is a phylogenetically coherent genus subgroup, is non pathogenic for humans and was within the β-proteobacterial subphylum, which was previously described as a non pathogenic variant of first described by Yabuuchi and colleagues (1992) [2] B. pseudomallei [8]. by transferring seven species from the Pseudomonas The so-called phytopathogenic subgroup includes genus, with Burkholderia cepacia as the type species. some well known and wide spread plant pathogens Since then, more than 50 validly named species and such as B. andropogonis, B. glumae, and B. plantarii. candidate species have been added to this genus. So B. glumae is an emerging seed-borne rice pathogen far, the most comprehensive phylogenetic analysis of in many areas around the world and is known to be Burkholderia species are based on 16S rRNA, recA and the etiologic agent of the so-called panicle blight, which acdS genes [5]. They all point out that there are two can cause severe crop yield losses as high as 40% [9]. main lineages in this genus: one is composed largely B. andropogonis has been long known as the causal of pathogens, whereas the other includes mainly agent of several plant diseases such as stripe disease environmental isolates that may form non-pathogenic of sorghum and leaf spot of velvet bean, and more associations with plants. recently it has been described as the cause of brown The first lineage includes human, animal and plant leaf spot of citrus [10]. It is remarkable that B. rhizoxinica pathogens (Burkholderia cepacia complex (BCC) and B. endofungorum, endosymbiotic bacteria, which species, B. mallei, B. pseudomallei, B. plantarii, live within the cells of the fungus Rhizopus microsporus B. gladioli, B. glumae, etc.). Three minor subgroups and produce the causal agent of rice seedling blight, are also recognizable, corresponding to BCC, rhizoxin, and the toxic cyclopeptide rhizonin, do not B. pseudomallei group and plant-pathogens group. group with the other endosymbiotic species of the The BCC is a collection of genetically distinct but non-pathogenic cluster, but are very close to the phenotypically similar bacteria that belong to at least pathogenic species B. mallei and B. pseudomallei as 17 species [1]. BCC species have been isolated well as to B. caryophylli, a parasitic endophytic species from a variety of natural habitats such as plant infecting vascular plants [11]. It is worth noting that rhizosphere, soil and river water and from several recently B. rhizoxinica and B. endofungorum have urban environments such as playgrounds and athletic also been recovered from clinical specimens, probably fields, and although first described as the causal agent as a consequence of patient infection by the fungus of onion rot, they have generally useful properties for Rhizopus [12]. biocontrol, bioremediation and plant growth promotion The second and more recently established [6]. However, BCC bacteria are also recognized as lineage in the genus Burkholderia comprises more important opportunistic pathogens that can cause than 25 related environmental species, which in most variable lung infections in cystic fibrosis patients, which cases are non-pathogenic and have been found to result in asymptomatic carriage, chronic infection be associated with plants [4]. Several interesting or ‘cepacia syndrome’, which is characterized by a properties found in members of this cluster include rapid decline in lung function and can include invasive (i) the strong catabolic potential to degrade aromatic disease and death. compounds, with B. xenovorans being the best known B. pseudomallei and B. mallei are two highly example; (ii) the potential to increase plant nutrient , responsible for melioidosis and availability via nitrogen fixation and/or phosphate glanders, respectively [3,7]. Melioidosis, which affects solubilisation, as demonstrated for B. unamae, humans, is endemic in Southern Asia and northern B. tropica, and B. silvatlantica; (iii) the ability to Australia, and its etiological agent, B. pseudomallei, colonize the rhizosphere or the internal intercellular is an environmental saprophyte that is commonly spaces in several plants and promote plant growth, as found in wet soils and stagnant waters throughout the is the case for B. kururiensis and B. phytofirmans; and endemic regions [3]. B. mallei, the etiological agent of (iv) the ability to form symbiotic interactions with plants glanders, cannot persist in the environment outside as occurs, for example, with B. tuberum, B. phymatum, its equine host. Horses are the primary carriers of B. mimosarum, B. nodosa, and B. sabiae, and disease and are largely responsible for transmitting with mosses, as reported for B. megapolitana and the infection to healthy animals and humans [3]. These B. bryophila. Although root-nodule symbionts, like two species are phylogenetically very similar and have pathogens, form an intimate association with their nearly identical 16S ribosomal DNA sequences. They eukaryotic hosts, all the symbiotic Burkholderia isolates have been defined as separate species essentially belong to the non-pathogenic lineage.

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3. Pathogenicity of Burkholderia infections, than with mounting a potent acute infection species [15]. All BCC species can be isolated from clinical The most important Burkholderia species in terms of samples as well as from the natural environment, which pathogenic potential are B. mallei, B. pseudomallei and could be acting as a constant “reservoir” for infectious the BCC species [13]. B. mallei and B. pseudomallei BCC pathogens [16]. Furthermore, most BCC species have been long known to be the causative agents of include isolates of both clinical and environmental origin the highly infectious equine disease known as glanders showing the same sequence type (ST) by multi locus and of melioidosis, potentially fatal infections of equines sequence typing (MLST), i.e. belonging to the same and humans, respectively. Both bacterial species are strain. In particular, all species whose isolates have facultative intracellular pathogens that can invade and been proven to have biotechnological potential, such replicate inside epithelial and phagocytic cells and as B. cepacia, B. cenocepacia, B. vietnamiensis and possess an arsenal of recently identified virulence B. ambifaria, include STs shared by both clinical and determinants Indeed, capsule, type III secretion system environmental isolates. These findings suggest that (T3SS), and type VI secretion system (T6SS) have the conservation of intrinsic determinants necessary to emerged as key virulence factors of both B. mallei and thrive in environmental niches may confer the ability to B. pseudomallei. However, knowledge on the T3SS- colonize susceptible humans. In fact, in a successive and T6SS-secreted effectors remains limited [3]. study, B. cenocepacia strain POPR8, isolated from a By the 1980s, the BCC had emerged as a significant radish in Mexico and sharing the same ST with other problem in CF clinics around the world, with some CF isolates from CF sputum, was shown to persist in centers reporting prevalence rates as high as 40%, due high numbers in the lungs of infected mice, with high also to the spread of highly transmissible strains [6]. infection rate, confirming the pathogenic potential of To date, prevalence rates have dropped dramatically environmental strains [17]. Vial and colleagues [18] worldwide also because of the introduction of efficient suggest that B. ambifaria uses phase variation, an segregation measures. Of the 17 species included in the adaptive process conferring to bacteria the capacity BCC, a clear clinical impact on CF patients has been to adapt to fluctuating environmental conditions and so far proven only for B. cenocepacia, B. multivorans, to drastically different environments, such as the lungs and B. dolosa. Some anecdotal evidence of a link to of patients with CF or the plant roots environment, worsening of health status of CF patients has been again reinforcing the idea that differentiation between reported for B. stabilis and B. vietnamiensis [6]. For all environmental and clinical isolates of Bcc, as far as other BCC species, no strong causal association has health hazard is concerned, is unrealistic. so far been established between the presence of such So far, most members of the so-called non- species in pulmonary secretions and the health decline pathogenic lineage have not been associated to human in CF patients. As far as non-CF patients are concerned, disease. Perin and colleagues [19] observed that some BCC detection is uncommon and the relevance for these putative virulence and/or transmission markers that patients is unclear, although recently a major outbreak are widely spread among clinical and environmental of B. contaminans was reported in a non-CF clinical isolates of BCC species, are completely absent among setting, affecting more than sixty patients [14]. plant-associated nitrogen-fixing Burkholderia species. As far as BCC pathogenicity is concerned, research However, the rhizosphere, where species of the has focused mainly on B. cenocepacia because of its pathogenic and non-pathogenic Burkholderia clusters high prevalence among CF patients. According to can be recovered in high numbers, is supposed to Loutet and Valvano [15], most studies point out that function as a hot spot for high-rate transfer of genes determinants mediating host niche adaptation are more between related rhizobacteria (e.g. Burkholderia, involved in the establishment of chronic infections by Pseudomonas and Stenotrephomonas species) [20]. B. cenocepacia than those involved directly in host cell This raises concerns about the possibility of DNA and tissue damage. Indeed, the best characterized exchange among strains that belong to different virulence determinants in B. cenocepacia include Burkholderia species. This would, in principle, enable molecules required for resistance to host antimicrobial an apparently harmless Burkholderia strain to acquire compounds, secretion systems, extracellular and cell pathogenicity traits from a virulent neighbour. Stringent surface polysaccharides, systems that regulate gene assessment of risk to human health associated with the expression, etc. Such factors have more to do with release into the environment of harmless nitrogen-fixing adaptation for survival under changing conditions, Burkholderia strains should evaluate such possibility, which is likely necessary for establishing chronic but, unfortunately, no experimental data are available

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so far. Recently, a few species belonging to the non- from 6 to 9 Mbp, and hence encode over 7000 genes. pathogenic cluster have been linked with human It is also clear from their genomes that 10% or more disease. B. tropica and B. fungorum have been reported of these 7000 genes appear to have been acquired to be the causative agents in two cases of septicaemia through horizontal gene transfer; indeed, they all [21,22], while strains belonging to B. caledonica and contain elements of foreign DNA such as genomic B. xenovorans have been isolated from human clinical islands, integrated phages or plasmids and an array of samples [23,24]. At present it is difficult to interpret these insertion sequences, which promote genomic plasticity findings in terms of broader clinical significance because and general adaptability [26]. All these features are of the limited number of cases; in particular, for the latter claimed to be the basis for the very high ecological two species, no cases have been reported where a and metabolical versatility of the members of this causal association has been established between the genus. In fact, large genomes have been described as presence of such species and human disease. disproportionately enriched in regulation and secondary metabolism genes and depleted in protein translation, DNA replication, cell division and nucleotide metabolism 4. Genomics and transcriptomics genes in comparison with small-sized genomes. This may explain why species containing large genomes The number of whole-genome sequences is dominate in environments, such as soils and plants, rapidly increasing; so far, over 35 Burkholderia where resources are scarce but diverse. For example, genome-sequencing projects have been initiated, the species B. xenovorans shows considerable intra- and draft/finished genomes are available for species genomic variation and the size of its genome members of the B. cepacia complex such as may vary up to 2.3 Mbp (from 7.4 to 9.6 Mbp). This can B. multivorans, B. cenocepacia, B. vietnamiensis, be largely explained by more than 20% of sequences B. ambifaria, B. dolosa, and for B. glumae, acquired by means of lateral gene transfer. In general, B. pseudomallei, B. mallei strains, B. phymatum, a large fraction of the differences in gene content B. rhizoxinica (http://www.burkholderia.com). within species is associated with bacteriophage There is a growing interest in using these whole-genome and transposase elements, revealing an important sequences not only for taxonomic and evolutionary role of these elements during bacterial speciation. studies, but also for investigation of pathogenicity Environmental behaviours of strains and species of the mechanisms and adaptation ability to very diverse genus Burkholderia may have modulated the genome habitats of the different Burkholderia species. sizes and content of these , which are Although this genus is phylogenetically coherent, thus capable of flexibility, plasticity and versatility and as mentioned above, Burkholderia species occupy make them adaptable to various microbial niches. extremely diverse ecological niches. Whole-genome The extensive genomic datasets are very useful sequencing of strains belonging to species thriving in in allowing the genetic basis for the pathogenesis and different habitats can give information to explain the environmental versatility of Burkholderia species to high adaptability to different environmental conditions be studied in more detail. For example, as mentioned of members of this genus, i.e. to find the genetic above, many BCC genome sequences are publicly determinants which favour bacterial growth in so available (http://www.genomesonline.org/); in particular, different niches. This is particularly true in the case the genomes of five strains of B. cenocepacia, one of genetic determinants involved in human and plant of the most virulent and problematic CF pathogens, pathogenicity, which is one of the main reasons for have been completed so far: J2315, AU1054, PC-184 genome sequencing of Burkholderia species. Recently, (representing the three major lineages based on recA Lim and colleagues [25] have published the complete gene polymorphism, i.e. IIIA, IIIB and IIID), HI2424 genome sequence of B. glumae; this species was (a soil isolate), and MC0-3 (a rhizosphere isolate) [27]. chosen because it is the etiologic agent of rice panicle Through comparative genomics it will be possible to blight, a disease which is spreading quickly in most identify and select novel vaccine targets and also to Asian rice-growing countries, and because B. glumae detect genes that are shared among species of related can represent a good model to understand how plant- pathogens or are distinct in pathogenic attenuated or pathogenic bacteria infect rice panicles and flowers and, naturally non-pathogenic strains or species. However, more in general, how pathogenic bacteria and plants such sequence-based approaches applied to BCC interact. bacteria cannot predict those genes that will be Most Burkholderia species have a minimum of two expressed preferentially in the lung of CF patients or in chromosomal replicons and a genome size ranging the rhizosphere of crop plants so that recent research

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on the molecular pathogenesis of these bacteria has genome sequence of B. rhizoxinica, completed earlier focused on global gene expression studies. this year, represents an excellent basis to investigate In particular, transcriptomic approaches, based on bacterium-fungus interactions [37]. Recently, another microarray and RNA-seq techniques, have been used whole-genome sequence of an endosymbiontic to examine mechanisms of virulence in B. cenocepacia species has been completed, i.e. that of B. phymatum [28,29]. By using a microarray technology or cDNA (http://www.burkholderia.com). This species is capable sequencing, several global gene expression studies of of fixing nitrogen from the atmosphere and of nodulating B. cenocepacia that model environmental and infection- roots of plants, such as Phaseolus vulgaris, Mimosa related interactions have been made available. Using invisa and M. pudica. Comparison of the two genomes direct high-throughput Illumina sequencing of cDNAs, will certainly shed light on commonalities and difference Yoder-Himes and colleagues [28] investigated the in their genetic equipment necessary to establish transcriptomes of two related strains of B. cenocepacia symbiosis with their hosts. isolated from two very disparate habitats, a CF patient and soil from an agricultural field. All these studies allowed the identification of many genes induced under 5. Biotechnology CF conditions that may constitute, at least some of them, particularly attractive therapeutic targets due to In the last two decades, most Burkholderia candidates their signal sequence, predicted cellular location, and for biotechnology application, particularly as biofertilizers homology to known therapeutic targets. Moreover, in agriculture, belonged to the BCC. Unfortunately, all by means of these approaches, a deep insight into BCC species have been recovered from both clinical adaptation strategies of environmental and clinical and environmental samples and their biotechnological strains of B. cenocepacia is gained through the analysis use is currently not permitted [6]. As mentioned above, of differences in gene expression on a global scale. the clinical significance, except for B. cenocepacia, Burkholderia xenovorans strain LB400 is one of the B. multivorans, B. dolosa and few other species, most important aerobic polychlorinated biphenyl (PCB) is still unclear. However, the distinction between degraders yet discovered (see also the ‘Biotechnology’ environmental and clinical strains of BCC cannot be the section). The pathways for degradation of PCBs by basis to distinguish safe from unsafe strains, because strain LB400 have been extensively characterized the environment is a source of pathogenic strains. at both the genetic and the molecular level [30,31] Therefore, the risk to human health associated with and the complete sequencing of the LB400 genome the release into the environment of BCC isolates which (http://genome.ornl.gov/microbial/bfun) [32], one of have been proven to be good plant-growth promoting the largest ones among prokaryotes (9.7 Mb), greatly agents and plant pathogen antagonizers, as reported by contributed to the investigation of the biology of many authors, is too high. Thus, it is hardly foreseeable PCB degradation. By use of genomic and proteomic that the ban on biotechnological use of BCC bacteria will approaches, an outline of genome-wide responses ever be lifted. to a range of carbon and cell development conditions By contrast, the newly described nitrogen-fixing has been reported [33,34]. Putative detoxification Burkholderia species, associated with the rhizosphere mechanisms were also revealed during analysis of the of different crop plants and belonging to the non- transcriptome of B. xenovorans LB400 [35] and recent pathogenic Burkholderia lineage, could have a great studies, aimed at exploring the effect of environmental potential for biotechnological applications in plant conditions (e.g., carbon source and growth phase) on growth promotion, biological control of plant pathogens, the variation in PCB degradation profiles ofBurkholderia and, concomitantly, in rhizoremediation and/or for xenovorans LB400, revealed 25 potential genes improvement of phytoremediation, and, finally, in involved in the activation of mechanisms beyond the biomining [4]. biphenyl pathway that provide stability and improve Among the Burkholderia species belonging to the non- PCB degradation [36]. pathogenic lineage, B. tropica shows the highest mineral It is remarkable that within the Burkholderia genus, phosphate-dissolving capability [38]. Its remarkable beside human pathogenic species, there are several ability to convert insoluble mineral phosphorus to an species that are endosymbionts with different hosts, available form to plants is an important trait for allowing such as fungi, plants and mosses. Thus, this genus this nitrogen-fixing species to be defined as a plant can represent a model system to understand how growth-promoting rhizobacterium. Seed inoculation symbiosis and pathogenesis have emerged, spread with efficient phosphate-solubilizing bacteria is known and evolved within a single bacterial genus. The whole- to increase solubilisation of fixed soil phosphorus and

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immobilized phosphates in the soil after application century as congener mixtures and used in hydraulic of mineral fertilizers, resulting in higher crop yields. and heat-transfer systems, inks, lubricants, paints, and B. tropica has also been suggested as a candidate for adhesives. Due to remarkable chemical and thermal biological control of pathogenic nematodes since they stability, their persistence in the environment is very are strongly associated with less pathogenic nematodes high and there is still a scarcity of treatment options and weakly associated with highly pathogenic ones [39]. for contaminated soils and sediments, with very Efficient phosphate-solubilizing bacteria could also few alternatives to in situ treatment. A large number be economically useful in emerging industrial activities of PCB degrading organisms have been isolated; such as biomining [40]; in particular, manufacturing among these, B. xenovorans has raised much interest of metallic iron and steel from iron ore resources among being one of the best aerobic PCB degrader requires the previous removal of phosphorus. The discovered so far. Its capacity to oxidize more than 20 use of the novel species B. ferrariae, isolated from a PCB congeners, with up to six chlorine substitutions on high phosphorus iron ore and able to solubilise highly the biphenyl rings [43] as well as its ability to use some insoluble phosphatic minerals, could contribute to solve congeners as sources of carbon and energy [44,45] this problem in a cost-effective and environment-friendly made it the model organism for PCB biodegradation way. studies. The ability of B. unamae to grow using aromatic hydrocarbons, in particular benzene and phenol, as well as its widespread association with different 6. Conclusions plant species, suggests that it could be suitable for applications in rhizosphere remediation of The Burkholderia genus includes species whose common soil pollutants [38]. In addition, taking into ecological properties can differ enormously from each consideration the host plant range of B. unamae and other, and new species colonizing amazingly different its endophytic character, it would be of great interest habitats are continuously discovered. Over the past few to know the natural ability of this species to improve years, a wealth of new information about the taxonomy, phytoremediation of volatile organic pollutants, as has ecology, epidemiology and pathogenicity of Burkholderia been suggested with the application of engineered species, in particular of BCC, has been gathered thanks endophytic BCC bacteria, which, unfortunately, will to the extensive implementation of MLST and genomic not be possible to use because of their pathogenicity. sequencing and the recent application of transcriptomics Indeed, phytoremediation of highly water soluble and proteomics. Although differences in the pathogenic and volatile xenobiotics is often inefficient because and ecological properties among BCC species have plants do not completely degrade these compounds been confirmed, such differences do not warrant through their rhizospheres. This results in phytotoxicity a reconsideration of the possibility of using these and/or volatilization of chemicals through the leaves, organisms in biotechnology. However, the pioneering which can cause additional environmental problems. work carried out in the laboratory of the late Professor According to Barac and colleagues [41] and Weyens Jesus Caballero-Mellado on nitrogen-fixing bacteria and colleagues [42], endophytic bacteria equipped (see references [4,5,19,38]) greatly helped to bring to with the appropriate degradation pathway improve light an entire group of new non-pathogenic nitrogen- the in planta degradation of toluene. For example, fixing Burkholderia species that are serious candidates B. cepacia L.S.2.4, a natural endophyte of yellow for biotechnological applications, in particular in the field lupine, equipped with the pTOM toluene-degradation of plant-growth promotion and phytoremediation. plasmid of Burkholderia vietnamiensis G4 strongly degraded toluene, resulting in a marked decrease in its phytotoxicity, and a 50–70% reduction of its Acknowledgements evapotranspiration through the leaves [41]. This strategy promises to improve the efficiency of This paper is dedicated to the late Prof. Jesus Caballero phytoremediating volatile organic contaminants. Mellado (Centro de Ciencias Genomicas, Universidad Polychlorinated biphenyls (PCBs) are toxic Nacional Autonoma de Mexico, Cuernavaca, Morelos, xenobiotics, manufactured during the mid-twentieth Mexico).

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