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. 2014 Jun 12;6(7):1652–1668. doi: 10.1093/gbe/evu126

The Genome of the Fungal-Interactive Soil Bacterium Burkholderia terrae BS001—A Plethora of Outstanding Interactive Capabilities Unveiled

Irshad Ul Haq 1,*, Katharina Graupner 2, Rashid Nazir 3, Jan Dirk van Elsas 1,*
PMCID: PMC4122924  PMID: 24923325

Abstract

Burkholderia terrae strain BS001, obtained as an inhabitant of the mycosphere of Laccaria proxima (a close relative of Lyophyllum sp. strain Karsten), actively interacts with Lyophyllum sp. strain Karsten. We here summarize the remarkable ecological behavior of B. terrae BS001 in the mycosphere and add key data to this. Moreover, we extensively analyze the approximately 11.5-Mb five-replicon genome of B. terrae BS001 and highlight its remarkable features. Seventy-nine regions of genomic plasticity (RGP), that is, 16.48% of the total genome size, were found. One 70.42-kb RGP, RGP76, revealed a typical conjugal element structure, including a full type 4 secretion system. Comparative analyses across 24 related Burkholderia genomes revealed that 95.66% of the total BS001 genome belongs to the variable part, whereas the remaining 4.34% constitutes the core genome. Genes for biofilm formation and several secretion systems, under which a type 3 secretion system (T3SS), were found, which is consistent with the hypothesis that T3SSs play a role in the interaction with Lyophyllum sp. strain Karsten. The high number of predicted metabolic pathways and membrane transporters suggested that strain BS001 can take up and utilize a range of sugars, amino acids and organic acids. In particular, a unique glycerol uptake system was found. The BS001 genome further contains genetic systems for the degradation of complex organic compounds. Moreover, gene clusters encoding nonribosomal peptide synthetases (NRPS) and hybrid polyketide synthases/NRPS were found, highlighting the potential role of secondary metabolites in the ecology of strain BS001. The patchwork of genetic features observed in the genome is consistent with the notion that 1) horizontal gene transfer is a main driver of B. terrae BS001 adaptation and 2) the organism is very flexible in its ecological behavior in soil.

Keywords: Burkholderia, synteny, pan–core genome, regions of genomic plasticity, secretion systems, membrane transporters

Introduction

As a result of the spatial separation in soil between bacterial cells and substrate, as well as the general recalcitrancy of substrate to degradation, soil bacteria most of the time perceive conditions of nutrient scarcity in their habitat and occur in a state of dormancy. However, there are so-called hot spots in soil in which soil bacteria are activated. One such hotspot for bacterial activity is the mycosphere, that is, the narrow zone of influence around the fungal hyphae in soil.

Evolutionary and genome-shaping events in soil bacteria (mutations and horizontal gene transfer and selective processes) are thought to mostly take place in high-activity microenvironments, and hence a thorough investigation of the genomic diversity of organisms dwelling in the mycosphere is warranted. On the basis of two different experimental setups, Burkholderia terrae like organisms were previously found to be key associates with fungal hyphae in soil (Warmink and van Elsas 2009). Indeed, since this initial discovery, a fascinating interaction between the mycosphere-isolated B. terrae strain BS001 and Lyophyllum sp. strain Karsten has been unveiled (Warmink and van Elsas 2009; Nazir, Zhang, et al. 2012).

The draft genome sequence of strain BS001 was recently announced (Nazir, Hansen, et al. 2012), but no detail on key genetic systems and on how the genomic information determines the ecophysiological behavior of the organism was provided. From the previous work, circumstantial ecologically based evidence was obtained for the contention that the presence of type 3 secretion system (T3SS), motility and biofilm formation traits as well as glycerol uptake and metabolism genes might offer selective advantages to the organism in the mycosphere. However, a tight linkage of the ecological data with genome features of strain BS001 has been lacking so far, in spite of the fact that this linkage is needed to build hypotheses that are testable in further experiments. Thus, in this study, we provide an in-depth analysis of the genome of B. terrae BS001, focusing on the plethora of genetic systems that potentially drive the interactions between B. terrae BS001 and the soil fungi it associates with. As the reference fungus, Lyophyllum sp. strain Karsten was used. To achieve our aim of assessing the genome in a broad sense and also addressing the uniqueness of particular features for strain BS001, we then performed comparative analyses of selected salient features of the genome of B. terrae BS001 with those of other (related) Burkholderia strains. From the data, we make inferences about the implications for the behavior of strain BS001 in soil, in an attempt to narrow the gap between (selected) genotype and phenotype.

Results and Discussion

Genome Properties

We first briefly describe the overall genome characteristics, before considering particular genetic systems that might relate to the ecological behavior of B. terrae BS001. The genome has an estimated size of about 11.5 Mb, and was found to consist of five replicons with a G+C content of 61.8% (Nazir, Hansen, et al. 2012). The genome of B. terrae BS001 is extremely large compared with the genomes of other Burkholderia species, that is, B. rhizoxinica HKI454 (3.75 Mb) (Lackner, Moebius, Partida-Martinez, et al. 2011), B. phytofirmans PsJN (8.21 Mb) (Mitter et al. 2013), and B. phymatum STM815 (8.68 Mb). The total number of predicted coding sequences (CDSs) was 12,047, compared with 4,146 in the genome of B. rhizoxinica HKI454, 8,043 in that of B. phytofirmans PsJN, and 8,434 in that of B. phymatum STM815 (table 1). A relatively high number of CDSs, amounting to 38% of the genome, was predicted to encode proteins of unknown function.

Table 1.

Comparison and General Features of Burkholderia Genomes

Bacterial specie B. terrae BS001 B. rhizoxinica HKI454 B. phytofirmans PsJN B. phymatum STM815
Genome size (Mb) 11.5 3.75 8.21 8.68
GC content (%) 61.8 60.7 62.2 62.3
Number of CDSs 12,047 4,146 8,043 8,434
tRNAs 51 59 63 62
rRNA operons 4 9 18 16
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The giant genome size and dense gene coverage suggest that strain BS001 disposes of a suite of lifestyle choices in the soil that are selected as a result of the conditions that may reign locally in the microhabitat it occupies in soil. In previous studies, we found a (one-sided) correlation between fungal-interactivity in soil bacteria and the presence of 1) motility traits and 2) a T3SS. In the following section, we highlight the ecological features of B. terrae BS001 in soil and explore the genome for the presence of these as well as other characteristics that may correlate with the (fungal-interactive and otherwise) lifestyle in soil.

Ecological Features that Presumably Drive the Genome Structure of B. terrae BS001

Salient ecological features of B. terrae BS001 in soil and the mycosphere are summarized in supplementary table S1, Supplementary Material online. Burkholderia terrae was found to be a key inhabitant of the mycosphere of Laccaria proxima in the field, whereas B. terrae BS001 was an avid migrator with growing Lyophyllum sp. strain Karsten mycelium. The rapid migration along the mycelial growth front endowed strain BS001 with the remarkable capacity to successfully reach novel (remote) microhabitats in the soil (Warmink and van Elsas 2009). Moreover, strain BS001 revealed a migration helper effect, as it stimulated the comigration of the nonmigrator Dyella japonica BS003 along with the fungal hyphae (Warmink et al. 2011). The organism further showed avid biofilm formation around hyphae of the fungal host. The extracellular polysaccharides constituting the biofilm are thought to act like a shield against antifungal agents (Warmink and van Elsas 2009), allowing protection of the fungal host. Burkholderia terrae BS001 was further found to be able to comigrate with a range of other selected soil fungi through soil, allowing the notion that it is a “generalist” migrator. The fungal-interactive capacity was suggested to involve bacterial motility as well as the activity of a T3SS (Warmink and van Elsas 2009). Moreover, considering resource utilization, B. terrae BS001—much like Variovorax paradoxus HB44—was found to grow on Lyophyllum sp. strain Karsten released glycerol (tested in liquid microcosms). The organism was even able to trigger the release of glycerol from host cells by an as-yet-unknown mechanism (Boersma et al. 2010; Nazir et al. 2013). Furthermore, it grew avidly on glycerol as the sole carbon source (data not shown). In this sense, B. terrae BS001 is likely to be an excellent competitor in soil in competitive situations where carbon sources such as glycerol become available. Finally, B. terrae BS001 was indicated to affect the physiology of Lyophyllum sp. strain Karsten, including the induction of a secretome that contained high levels of glycerol, next to the inhibition of primordium formation by fungal mycelial mats in the microcosms (Nazir et al. 2013).

Core and Pan Genome Analyses

We used the “MicroScope” platform (Vallenet et al. 2013) to determine the genetic landscape of the B. terrae BS001 genome, taking as references the available (MicroScope) genomes of 23 other Burkholderia species, that is, B. phymatum STM815, B. phytofirmans PsJN, B. rhizoxinica HKI454, B. glumae (strains; AU6208, LMG 2196, BGR1, and 3252-8), Burkholderia sp. (strains; CCGE1002, CCGE1003, and TJ149), B. lata 383, B. kururiensis M130, B. thailandensis E264, B. mallei ATCC 23344, B. pseudomallei K96243, B. cenocepacia AU 1054, B. phenoliruptrix BR3459a, B. ambifaria AMMD, B. gladioli (strains; 3848s-5 and BSR3), B. vietnamiensis G4, B. multivorans ATCC 17616, and B. xenovorans LB400. First, B. terrae BS001 had the largest genome of all (11.5 Mb compared with 9.7 Mb for B. xenovorans LB400), indicating a large accessory gene part. The pan genome across these genomes is defined as the sum of the core genome (genes present in all twenty four organisms), the variable genomes (genes found in some organisms while being absent from the others) and the strain-specific genes (Medini et al. 2005), whereas the core genome is the collective number of shared genes between all genomes. The analysis revealed a total of 180,124 CDSs to be present in the pan genome of the 24 genomes, which were divided over 81,027 MicroScope gene families (MICFAM; see Materials and Methods section). The core genome across all Burkholderia species comprised 472 gene families; however, the exact number of core CDSs was different across the compared genomes, as a result of some core CDSs having more than one copies. Thus, strain BS001 was found to have a core of 523 CDSs as several core genes had paralogs. The 523 CDSs core genome of strain BS001 encompassed only 4.34% of its total genome. The remaining 95.66% (11,522 CDSs) thus constituted the variable or accessory part. Of these, there were 6,099 (50.63%) strain-specific CDSs in strain BS001 whereas the remainder (∼5,000) were volatile, meaning that they were present across a limited number of genomes (supplementary table S2, Supplementary Material online). Clearly, as found by others, we also found that the sizes of the core and pan genomes depended strongly on the number of genomes analyzed, resulting in shrinking core and expanding pan genomes with depth of genome sampling (fig. 1). Also, across the Burkholderia strains that were analyzed, the pan genome should be considered to be open, as, with every new genome sequenced, several hundreds of novel genes are found (data not shown). Our analysis further revealed that the core genome across all 24 Burkholderia genomes consisted of CDSs encoding key cellular functions such as DNA recombination, replication, metabolism, transcription, translation, glycolysis, amino acid activation (tRNAs), chaperoning, RNA modification, transcription regulation, DNA repair, fatty acid biosynthesis, peptidoglycan biosynthesis, posttranslational modification, and cell division. Overall, the level of homology (translated amino acid sequences) of the genome of B. terrae strain BS001 with these genomes was between 55% and 60%.

Fig. 1.—

Fig. 1.—

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Core–pan genome size evolution. (A) Pan genome: Pan genome size is directly proportional to the number of genomes. (B) Core genome: Core genome size is inversely proportional to the number of genomes.

Phylogeny and Synteny Groups

We first constructed a maximum-likelihood tree on the basis of seven concatenated genes for representative Burkholderia species from groups A (plant/soil related, nonpathogens) and B (pathogens) (Estrada-de los Santos et al. 2013) to locate the position of strain BS001. Indeed, strain BS001 belonged to group A, being very closely related to B. phymatum STM815 (fig. 2). In this analysis, we further found that B. rhizoxinica HKI 454 could not be related to any of the other Burkholderia groups, as also observed by Estrada-de los Santos et al. (2013). We then performed an analysis of synteny between the strain BS001 genome versus selected available genomes in the NCBI (National Center for Biotechnology Information) RefSeq (reference sequence) database. The analyses (fig. 3) revealed that the B. phymatum STM815 genome had highest CDSs synteny to the BS001 genome, that is, 76.78% (fig. 2), followed by B. graminis C4D1M, B. phytofirmans PsJN, and B. kururiensis M130 (69.47%, 67.97%, and 64.79%, respectively). These four Burkholderia species are members of Burkholderia group A (Estrada-de los Santos et al. 2013) to which B. terrae BS001 also belongs (Nazir, Zhang, et al. 2012). A trend of decreasing % CDSs synteny was observed as we moved from Burkholderia group A members to those of group B, that is, B. cenocepacia AU 1056 (58.66%), B. pseudomallei K96243 (58.1%), B. mallei ATCC 23344 (55.49%), and B. vietnamiensis G4 (51.34%) (fig. 3). The levels of synteny of the outgroups, that is, Ralstonia, Cupriavidus, and Escherichia, with the genome of the strain BS001, were much lower. These findings suggest that the conservation of synteny was highest among members of the group A Burkholderia and it gradually decreased as the phylogenetic distance increased.

Fig. 2.—

Fig. 2.—

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A maximum-likelihood tree illustrating the relationship of selected Burkholderia species. The tree is based on alignment of seven concatenated core genes (aroE, dnaE, groeL, gyrB, mutL, recA, and rpoB) and was generated using MEGA. Burkholderia terrae BS001 falls within group A and is closely related to B. phymatum STM815. Bootstrap values (more than 50%) are shown at each node. Group A and group B are defined by Estrada-de los Santos et al. 2013.

Fig. 3.—

Fig. 3.—

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Synteny groups. Syntenous CDSs (%) of Burkholderia species from group A and group B, compared with B. terrae BS001, are shown. Ralstonia, Cupriavidus, and Escherichia coli species are included as outgroups.

Secretion Systems and Effector Proteins

Secretion systems such as the T3SS may be important for bacteria when these interact with hosts such as fungi (Lackner, Moebius, Hertweck, et al. 2011). We investigated the genome of B. terrae BS001 for the presence of (protein) secretion systems, which have a potential role in the interaction with Lyophyllum sp. strain Karsten. Basically, we found all known protein secretion systems, that is, the type 1 secretion system (T1SS) through to the type 6 secretion system (T6SS), in the genome of B. terrae BS001. The T1SS is normally composed of three constituents, including ATP-binding cassette (ABC) transporters, membrane fusion proteins, and outer membrane proteins (Delepelaire 2004; Holland et al. 2005). The BS001 genome harbored ABC transporters and related proteins that are part of the T1SS (discussed later in detail).

The type 2 secretion system (T2SS) (constituted of 9–11 genes), which is used to translocate a wide range of proteins, was present as three gene clusters, numbered 1 (AKAUv1_257005–AKAUv1_2570061), 2 (AKAUv1_2570149–AKAUv1_2570159), and 3 (AKAUv1_790112–AKAUv1_790123) across the genome. The latter (11-genes) cluster was completely syntenous and highly homologous (>80% amino acid identity [AAI]) to a similar system in B. phymatum STM815. The BS001 genome also revealed the presence of two copies of a subtype of the T2SS, the Tad (tight adherence) macromolecular transport system. Tad genes encode a machinery that is required for the assembly of adhesive fimbrial low-molecular-weight protein (Flp) pili. The machinery is associated with a so-called widespread colonization island (Tomich et al. 2007). The Flp pili play crucial roles in the formation of biofilms, colonization, and pathogenesis across several bacterial genera (Tomich et al. 2007). The first 14-genes tad cluster (AKAUv1_990039–AKAUv1_990052) was highly syntenous and homologous to similar systems in B. phytofirmans PsJN (∼60–70% AAI) and B. phymatum STM815 (∼70–75% AAI). The second, 13-gene, tad copy (AKAUv1_550027–AKAUv1_550039), located at a different position, also had highest synteny with a second system in B. phymatum STM815 (>70% AAI) and B. phytofirmans PsJN (>55% AAI).

The T3SS has a crucial role in the virulence of several plant and human pathogens (Tseng et al. 2009). It may also be involved in bacterial–fungal interactions, in particular aiding bacteria in their migratory response to extending fungal hyphae in the mycosphere (Warmink and van Elsas 2009). Indeed, the few bacterial types that were able to migrate along with the hyphal front of Lyophyllum sp. strain Karsten in soil microcosms were all positive for the presence of the T3SS (Warmink and van Elsas 2009). The T3SS makes membrane-bound complexes that can secrete effector proteins into eukaryotic host cells. The B. terrae BS001 genome possesses one T3SS gene cluster, containing 22 canonical T3SS genes (AKAUv1_540178–AKAUv1_540199). Out of these, nine were found to be highly conserved, making up the proposed core apparatus (SctSRQVUJNTC), however without a canonical gene for the “needle.” Interestingly, the system was found to be highly syntenous and homologous (60–65% AAI) to the T3SS of the fungal-interactive strain B. rhizoxinica HKI454 as well as that of B. glumae BGR1 (fig. 4). Remarkably, a CDS with unknown function (AKAUv1_540179) was found, of which the location and length compelled us to argue that it encodes a needle protein. Based on PSIPRED predicted α-helix structures, we assume that this CDS is a functional homolog of SctF (B. rhizoxinica HKI454) or its Ralstonia counterpart HrpY (Lackner, Moebius, Hertweck, et al. 2011). To understand the evolutionary position of the T3SS of strain BS001, we constructed a phylogenetic tree (fig. 5) using eight concatenated core genes (SctS, SctR, SctQ, SctV, SctU, SctJ, SctN, and SctT) that were conserved across the tested genomes. The analysis supports the conclusion that the T3SS of B. terrae BS001 belongs to the so-called Hrp2 family, to which most of the Burkholderia and Ralstonia T3SSs belong (Abby and Rocha 2012). The T3SSs of B. terrae BS001 and B. rhizoxinica HKI454 might fulfill similar roles. Both strains interact actively with fungal hosts and there is evidence that T3SSs play a role in the interaction in both cases (Lackner, Moebius, Hertweck, et al. 2011; Warmink and van Elsas 2008).

Fig. 4.—

Fig. 4.—

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Type 3 secretion system (T3SS). Genetic organization of the T3SS gene cluster found in Burkholderia terrae BS001 compared with other Burkholderia species including B. rhizoxinica HKI454, B. phenoliruptrix 3459a, B. kururiensis M130 and B. glumae BGR1.

Fig. 5.—

Fig. 5.—

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A maximum-likelihood phylogenetic tree. The evolutionary relationship and position of the T3SS of Burkholderia terrae BS001 was inferred from concatenated amino acid sequences of eight conserved T3SS genes (SctS, SctR, SctQ, SctV, SctU, SctJ, SctN, and SctT). The T3SS of B. terrae BS001 belongs to the Hrp2 family of T3SS. Hrp2, Myxo, Rhizo, Hrp1, SPI1, and SPI2 are families within T3SSs (Abby and Rocha 2012).

The type 4 secretion system (T4SS) is responsible for secreting proteins and, most importantly, nucleic acids (coupled to proteins; nucleoproteins) across the inner and outer membranes into recipient cells or into the external milieu. It plays a key role in the transfer of plasmids or integrated chromosomal elements, next to being involved in virulence on animals (protein secretion, e.g., in Helicobacter pylori) or plants (plasmid transfer, e.g., in Agrobacterium tumefaciens) (Dale and Moran 2006). Remarkably, the genome of B. terrae BS001 was found to contain three T4SS gene clusters. The first one, T4SS-I (20 CDSs; AKAUv1_3130024–AKAUv1_3130044), with all canonical T4SS functions (fig. 6), is present on the 70.42-kb region of genome plasticity (RGP) RGP76. The predicted virB10 gene of T4SS-I had very high AAIs with similar genes from a Burkholderia sp. BT03 plasmid (94%) and the B. phymatum STM815 genome (71%). Furthermore, non-T4SS open reading frames (ORFs) on RGP76 were predicted to encode a DNA circulation family protein, DNA replication protein tnpB, transposase tnpA, IS5 transposase insH, next to seven integrases, including two phage family integrases. Further, at the downstream boundary of RGP76, four transposases and two integrases were localized. The T4SS was completely syntenous and highly homologous (70–75%) with a B. phymatum STM815 T4SS located on plasmid pBPHY02 (595,108 bp). Moreover, the regions flanking RGP76 also contained CDSs potentially involved in gene mobility, that is, ORFs similar to the typical plasmid replicon genes parA, parB, parD, parE, mobB, mobC, repB, korC, and klcA. RGP76 further carried several accessory genes, that is, those predicted to function as a putative xanthine dehydrogenase/carbon monoxide dehydrogenase acceptor (AKAUv1_3070015, AKAUv1_3070016) and molybdenum binding xanthine dehydrogenase (AKAUv1_3130017). The remaining accessory ORFs were predicted to encode conserved proteins of unknown functions.

Fig. 6.—

Fig. 6.—

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Type 4 secretion system (T4SS). The gene organization of T4SS-I (gene cluster 1) of Burkholderia terrae BS001 compared with the T4SSs B. phymatum STM815, B. phenoliruptrix 3459a, and B. phytofirmans PsJN is shown.

A second system, T4SS-II, containing 13 genes, was found to be present elsewhere in the genome (AKAUv1_1810006–AKAUv1_1810019). This cluster had a gene composition different from the first one, as it contained the virB8, virB9, and virB10 genes but lacked other vir genes. Instead, it contained CDSs for lytic transglycosylase (two CDSs), pilL, toxin coregulated pilus biosynthesis protein Q, pulD, pulE, pulF (T2SS), and conserved hypothetical proteins genes.

The third system, T4SS-III, (AKAUv1_1120025–AKAUv1_1120032) contained only eight T4SS related genes. T4SS-III contained virB4, traG, traL (relaxase) virB6, trbJ, trbD, trbC, and trbB genes. However, T4SS-III lacked the canonical virB10 gene. In order to assess the origin of the former two T4SSs, we performed a phylogenetic analysis of their respective virB10 gene amino acid sequences. The analysis showed that T4SS-I is highly similar (72% AAI) to T4SSs of both B. phymatum STM815 and B. phenoliruptrix BR3459a. The T4SS-II, being very different from T4SS-I (22% homology), was closely related (76% AAI) to a T4SS of Burkholderia sp. SJ98 (supplementary fig. S1, Supplementary Material online).

The B. terrae BS001 genome further revealed the presence of four copies of predicted T6SSs, numbered T6SS-I through T6SS-IV (12–25 genes). T6SS-1 (16 genes; AKAUv1_650058–AKAUv1_650074) had highest synteny and homology (>40%) with a genomic region of B. gladioli BSR3. T6SS-II (19 genes; AKAUv1_1080195–AKAUv1_1080214) was highly syntenous and homologous (85–95%) with a similar cluster of B. phymatum STM815. T6SS-III and T6SS-IV comprised lower numbers of genes, that is, 8 (AKAUv1_1680089–AKAUv1_1680097) and 5 (AKAUv1_920001–AKAUv1_920005), respectively. T6SS-III had highest synteny with clusters in B. phenoliruptrix BR3459a and B. kururiensis M130 (both 55–60% homology). T6SS-IV, found on RGP25, was syntenous with five-genes clusters (40–50% homology) in B. glumae strains BGR1 and AU6208 and Burkholderia sp. CCGE 1002 (supplementary fig. S2, Supplementary Material online).

The B. terrae BS001 genome also contained several genes for signal peptide bearing proteins (supplementary table S3, Supplementary Material online) that may be exported in a sec-dependent fashion. Among these, the so-called type II secreted proteins fall in the category of lytic and toxin-related proteins (Cianciotto 2005). Furthermore, putative ppiA (encoding peptidyl–prolyl cis–trans isomerase A; rotamase A; AKAUv1_720068), next to genes for parvulin-type peptidyl–prolyl cis–trans isomerase (AKAUv1_1270066), and peptidyl–prolyl cis–trans isomerase (AKAUv1_1390107) were found. The latter protein may have pathogenicity-enhancing abilities in Legionella pneumophila and Xanthomonas campestris pv. campestris 8004 (Fischer et al. 1992; Zang et al. 2007). It has also been reported to exist in B. rhizoxinica HKI454 (Lackner, Moebius, Partida-Martinez, et al. 2011). We also found gene iagB (AKAUv1_790111) and its three duplicates (45–65% AAI), which produces an invasion protein and may be involved in the invasion of eukaryotic host cells by Shigella flexneri and Salmonella enterica serovar typhi (Allaoui et al. 1993; Miras et al. 1995). Three genes predicted to encode ankyrin (AKAUv1_540128, AKAUv1_920061, AKAUv1_1270092) and tetratricopeptide (TPR) repeat containing proteins were also present in the BS001 genome. These proteins may be involved in protein–protein interactions as host interactive factors (Edqvist et al. 2006). Both ankyrin and TPR repeat proteins have also been reported in B. rhizoxinica HKI454 (Lackner, Moebius, Partida-Martinez, et al. 2011).

Putative T3SS secreted effector proteins were then searched for using www.effectors.org (last accessed June 26, 2014) (Jehl et al. 2011) (supplementary table S4, Supplementary Material online), which were analyzed with respect to the putative interaction of B. terrae BS001 with Lyophyllum sp. strain Karsten. These effectors need confirmation, as we ignore their functions at this moment. Nevertheless, we found putative phytoene synthase (AKAUv1_920106), of the squalene–hopene cyclase enzyme family and may be involved in the biosynthesis of terpenoids. This is consistent with the presumption that terpenoids mediate the interactions of B. terrae BS001 with its host, like reported for B. rhizoxinica HKI454 in its interaction with the fungal host (Lackner, Moebius, Partida-Martinez, et al. 2011). Among the predicted effectors we also found flagellin proteins (coined pathogen-associated molecular patterns) both in plants and in mammals (Ronald and Beutler 2010; Wei et al. 2013).

Pilus, Biofilm Formation, Motility, and Insecticidal Toxin Complexes

The genome of B. terrae BS001 further carries gene clusters predicted to encode flagellar biosynthesis, biofilm formation, motility, and (seven genes) type 4 pili. The latter pili may play a role at fungal surfaces, as, once attached to the surface, bacteria can “walk” on it by employing the action of such pili (Conrad 2012). Genes that encode such pili, that is, pilMNQ (AKAUv1_1170009, AKAUv1_1170010, AKAUv1_1170012), were present in a cluster. We also found pilin precursor gene PilA (AKAUv1_2170003), signal peptidase PilD (AKAUv1_2940033), NTP-binding protein PilB (AKAUv1_2940037), and PilC (AKAUv1_2940035) in the genome.

Bacterial biofilm formation is dependent on the ability to produce extracellular matrix which may be composed of polymers like poly-beta-1,6-N-acetyl-d-glucosamine (PGA) (Izano et al. 2007; Bosse et al. 2010). We found several ORFs predicted to be involved in PGA production, that is, pgaA (PGA synthesis protein; AKAUv1_1000016), pgaB (PGA synthesis lipoprotein; AKAUv1_1000017), and pgaC (PGA synthesis N-glycosyltransferase; AKAUv1_1000018). In addition, two other biofilm synthesis gene clusters (both representing a Pel operon homolog) were identified. The Pel operon provides a scaffold in the early stage of biofilm formation by Pseudomonas aeruginosa PA14, (Colvin et al. 2011). Pel is thought to encompass a glucose-rich polysaccharide polymer that is encoded by a seven-genes operon (pelA–F) (Friedman and Kolter 2004). Cluster 1 comprised pelABCDEFGA (AKAUv1_2280066–AKAUv1_2280073), and cluster 2 pelGFEDCBA (AKAUv1_100003–AKAUv1_100009). Thus the Pel region may have undergone a recent duplication followed by some reshuffling, as evident from our analysis. Two genes, taken as proxies in the two systems, that is, pelG and pelF, were 61.84% and 64.69% identical, respectively, whereas pelABCDE had lower (43.52%, 36.33%, 47.9%, 40.08%, and 45.55%) AAI. An extensive screening further revealed the presence of ten other CDSs (i.e., 4 epsA, 4 epsB, and 2 epsP) in the genome that are potentially involved in the biosynthesis of other exopolysaccharide (EPS). Overall, the presence of the pel gene clusters reflects another capacity of B. terrae BS001 to form biofilms during its interaction with Lyophyllum sp. strain Karsten (as well as other hosts) in soil.

Moreover, the B. terrae BS001 genome harbors one gene cluster that encodes flagellar biogenesis proteins. The cluster revealed the presence of 46 genes (AKAUv1_120002–AKAUv1_120048) in a region that was highly syntenous with regions of the STM815 (80–90% AAI) and PsJN (75–85% AAI) genomes. Moreover, the BS001 genome contained a gene cluster comprised 11 genes (AKAUv1_790001–AKAUv1_790011) associated with bacterial chemotaxis and motility. Again, this cluster was syntenous with a similar one in strain STM815 (85–95% AAI). Elsewhere in the genome, another nine flagellar genes (flhA, flhB, 2 [flhC], 3 [flhD], fliC, and fliD) were found, next to a seven-genes chemotaxis/motility cluster (AKAUv1_920023–AKAUv1_920030).

Interestingly, the genome of B. terrae BS001 harbored two ORFs (AKAUv1_2130030 and AKAUv1_2130031) coding for a putative insecticidal toxin complex, as also reported to be present on the 822-kb megaplasmid pBRH01 in B. rhizoxinica HKI454 (Lackner, Moebius, Partida-Martinez, et al. 2011). We analyzed the putative horizontal mobility of such ORFs. Indeed, the two ORFs formed part of the largest genomic island, RGP53 (102,873 bp), suggesting that they may have been acquired horizontally. Thus strain BS001 might have insect pathogenicity traits, indicating its putative interaction with soil insects.

Genomic Islands across the Genome of B. terrae BS001

We employed “MicroScope” (Vallenet et al. 2013) to predict the presence of RGPs in the BS001 genome. The analysis predicted the existence of 79 RGPs, which were scattered across the genome (supplementary table S5, Supplementary Material online). The total size of the collective RGPs was 1,896,071 bp, accounting for 16.48% of the genome. Among the RGPs, we found the aforementioned 70.42 kb plasmid-like RGP76 (AKAUv1_3070018–AKAUv1_3130074). An overview of ten important RGPs with plasmid-related and functional genes is given in supplementary table S6, Supplementary Material online.

Briefly, on RGP34, we identified two ORFs (AKAUv1_1280007 and AKAUv1_1280008) encoding putative addiction proteins (toxin and antitoxin). In this region, four ORFs (AKAUv1_1930029, AKAUv1_1930030, AKAUv1_1230100, and AKAUv1_1230101) encoding HigA, HigB (plasmid maintenance protein), StbD and StbE (stabilization proteins) were found just outside of the RGP. Similarly, putative plasmid segregation antitoxin CcdA and CcdB toxin proteins are encoded by ORFs AKAUv1_2410015 and AKAUv1_2410016, both located outside plasticity regions. On the other hand RGP61 harbored two ORFs, AKAUv1_2490012 and AKAUv1_2490013, which encoded CcdB and CcdA. Downstream of these genes, the region harbored an ORF (AKAUv1_2490015) predicted to encode a protein having 39% (coverage 77%) homology with cryptic plasmid protein A from Neisseria gonorrhoeae NCCP11945. Interestingly, it also revealed an ORF (AKAUv1_2490048) that encoded a beta-lactamase (with 55% and 51% similarity to a similar protein in Bradyrhizobium SG-6C and R. etli CFN 42, respectively). Furthermore, we identified putative parE and parD genes (AKAUv1_2860003 and AKAUv1_2860004) in RGP71, whereas another ORF (AKAUv1_330002) encoding “prevent host death” family protein; Phd was present outside the RGPs. We also identified HigB and HigA (AKAUv1_360025 and AKAUv1_360026) as well as MazE and MazF addiction modules (AKAUv1_920172 and AKAUv1_920173), outside the predicted RGPs. Such toxin–antitoxin system proteins play a role in mediating stability of plasmids by imposition of an addiction mechanism to the host (Arcus et al. 2005). They may also play a role in mediating growth arrest during stress situations (Gerdes et al. 2005).

The largest RGP, RGP53, was found to span 102,873 bp (AKAUv1_2120017–AKAUv1_2140005), carrying ORFs for seven transposases and one integrase. A screen for functional genes in this RGP revealed the presence of one gene sip encoding a siderophore interacting protein (SIP) (AKAUv1_2130016) highly similar to a sip from Burkholderia sp. BT03 (99%). The gene had a duplicate downstream (AKAUv1_2130093), having (99%) similarity with sip of Burkholderia sp. BT03. Another ORF (AKAUv1_2130052) was predicted to encode invasion plasmid antigen J (IpaJ); it had 35% homology with IpaJ from S. flexneri 2002017. RGP53 also carried a gene for type III effector protein HopJ (AKAUv1_2130053); it was 53% homologous to similar gene from Salinivibrio costicola. It appears to furnish a wealth of secondary functions to its host, which may be useful in a wide range of ecological conditions. The clear mobility and divergent accessory nature of RGP53 might suggest that it is an ecological flexibility island.

Primary Metabolites and Metabolic Pathways

We used the “MicroCyc” metabolic database (Caspi et al. 2010) to compare the metabolic potential in the genome of B. terrae BS001 with that of other Burkholderia genomes (supplementary table S7, Supplementary Material online).

Based on the values computed for pathway completion, hierarchical cluster analysis was performed for the B. terrae BS001 genome next to 23 other genomes including next-of-kin Burkholderia species (supplementary fig. S3, Supplementary Material online). The analysis revealed that the Burkholderia strains tended to group together, B. terrae BS001 being close to B. kururiensis M130 and B. phymatum STM815, whereas B. phytofirmans PsJN grouped with B. vietnamiensis G4 and B. ambifaria AMMD. Interestingly, B. rhizoxinica HKI454 grouped outside of the main Burkholderia cluster; however, this might have been compromised by its smaller genome size.

Burkholderia terrae BS001 clearly invested in diverse primary metabolisms, as evidenced from the presence of more than 1,200 putative CDSs for carbohydrate metabolism (Nazir, Hansen, et al. 2012). Specifically, we found a putative glycerol kinase, glpK (AKAUv1_1300029), which was preceded by a glycerol-3-phosphate dehydrogenase (glpD, AKAUv1_1300028).

Remarkably, in the B. terrae BS001 genome, we found a gene for a specific glycerol uptake family transporter, denoted GUP. This system was absent from all other Burkholderia genomes and it is actually typical for the yeast Saccharomyces cerevisiae. In this organism, active uptake of glycerol occurs mainly by GUP 1 and its close homolog GUP 2, which both encode multimembrane-spanning proteins (Holst et al. 2000). Interestingly, the gene for GUP (AKAUv1_1930108) was present on RGP47, showing highest AAI (49%) with Magnetospirillum magneticum AMB-1 (fig. 7). The upstream ORF AKAUv1_1930107 encodes protein of unknown function that also shares 25% AAI with a hypothetical protein from M. magneticum AMB-1. The unique presence of this GUP gene in B. terrae BS001 is consistent with the hypothesis that fungal-exuded glycerol (Nazir et al. 2013), which feeds the bacterium, may be taken up through it. The presence of this system on RGP47 may indicate that the bacterium acquired and fixed it in its quest to thrive in the mycosphere at the expense of exuded glycerol.

Fig. 7.—

Fig. 7.—

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A neighbor joining tree of glycerol uptake gene (GUP) of Burkholderia terrae BS001 and close relatives. Based on the amino acid sequence of GUP gene, B. terrae BS001 GUP clustered with a similar gene product of Magnetospirillum magneticum AMB-1. GUP amino acid sequences of Saccharomyces cerevisiae strains s288c, YJM 789, Candida tropicalis and Nakaseomyces delphensis are taken as an outgroup. Bootstrap values (more than 50%) are shown at the nodes.

Genes for glycerol degradation pathways I and V were predicted to be present in the genome of B. terrae BS001. In comparison with the comparator strains, glycerol degradation pathway V was only complete in B. terrae BS001, whereas it was incomplete in strains PsJN and STM815 while strain HKI454 completely lacked it. Pathway V encompasses two reactions, and only the B. terrae BS001 genome carried ORFs (AKAUv1_760319, AKAUv1_2440011, and AKAUv1_1890037) encoding enzymes catalyzing these. The first two ORFs are duplicates of each other, having 42.9% sequence identity.

Strikingly, the BS001 genome further carried ORF AKAUv1_1970009, which is predicted to encode a tyrosinase (involved in l-dopachrome biosynthesis), present on RGP49. We analyzed the potential phylogenetic history of this gene using a maximum-likelihood tree (supplementary fig. S4, Supplementary Material online) with closest hits in the database. Blasting the whole NCBI database only yielded four close hits, with highest AAI (73%) with Anaeromyxobacter dehalogenans 2CP-1. The ORF directly upstream in BS001, AKAUv1_1970008, was predicted to encode a twin-arginine signal translocation protein (Tat). This gene also occurred (55% AAI) directly upstream in A. dehalogenans 2CP-1. However, the region downstream of ORF AKAUv1_1970010 was different. The presence of the Tat gene upstream of the tyrosinase gene is consistent with the fact that the tyrosinase may require a signal peptide containing transactivator for its export and translocation (Berks et al. 2000; Schaerlaekens et al. 2001). This commonality between the rather unique B. terrae system and one in A. dehalogenans is striking. The latter organism belongs to a very different bacterial clade (delta-Proteobacteria) than B. terrae BS001; however, it occurs in the same habitat (soil) and is a very motile (gliding) bacterium (Thomas et al. 2008). Moreover, its mosaic genome has been postulated to be composed of parts coming from diverse proteobacteria. Based on this analysis and the uniqueness of the commonality, we postulate that the gene for tyrosinase may have been acquired from an organism like A. dehalogenans 2CP-1 through horizontal gene transfer. l-Dopachrome is the building block of melanin, which can protect against ultraviolet radiation (Liu et al. 2004). Moreover, melanin in the cell wall of Aspergillus nidulans has been correlated to resistance against digestion by chitinases, glucanases, and proteases (Kuo and Alexander 1967). Bacterial melanin has been shown to interact with double-stranded DNA and its cellular localization may inhibit cell metabolism (Geng et al. 2010). Bacterial tyrosinases may also act on particular phenolic compounds, such as chlorophenols (Marino et al. 2011) and fluorophenols (Battaini et al. 2002). The role of the unique tyrosinase found in the B. terrae BS001 genome is not well understood, but it may serve the organism to cope with stressful environments and detoxify fungal/plant phenolic compounds.

Furthermore, the B. terrae BS001 genome revealed an enormous biosynthetic potential for a wide range of amino acids, see supplementary table S6, Supplementary Material online, indicating its flexibility in synthesizing the essential cellular building blocks of different kinds.

Plant-Interactive Traits

In the B. terrae BS001 genome, we further found biosynthetic potential for plant hormones, that is, genes for ethylene biosynthesis pathway III (two out of three enzymes are present) and indole acetic acid (IAA) biosynthesis IV and V, whereas IAA biosynthesis pathway VI is partially present. This may suggest that B. terrae BS001 spends (part of) its life occupying a niche close to plants like the rhizosphere. The presence of three copies of a 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase—acdS) gene (AKAUv1_150009, AKAUv1_510080, and AKAUv1_780047), denoted acdS-I, acdS-II and acdS-III, gives support to this hypothesis. Maximum-likelihood phylogenetic analyses showed that acdS-III clustered separately from the other two genes and might have been acquired through horizontal gene transfer (HGT). In contrast, acdS-I and acdS-II may be the result of a duplication very early in evolution (supplementary fig. S5, Supplementary Material online).

Similarly, RGP53 was found to carry two CDSs, here denoted as pnodW (putative nodW genes) (AKAUv1_2130064 and AKAUv1_2130080) that may act as two-component response regulators, with 81% AAI to a putative two-component response regulator from P. resinovorans NBRC 106553. Further analyses revealed that up to 18 paralogs of pnodW genes were present across the genome, with similarities of 30–69%. Downstream one ORF (AKAUv1_2130067), we also found a PAS sensor protein (sensor kinase; nodV). This sensor kinase had seven paralogs in the genome with AAI of 35–46%. Both nodV and nodW belong to the two-component regulator family that activates the expression of other nodulation genes at the plant surface and in response to isoflavonoides (Sanjuan et al. 1992; Loh et al. 1997, 2002). We could not find other nodulation genes (such as nodABCD); however, elsewhere in the genome we found CDSs similar to nodN, nodI, nodJ, and nodT. The exact role of the two-component regulators (nodV and nodW) in strain BS001 is not known yet, but the presence of such an array of nod genes allows the hypothesis that B. terrae BS001 might also display a plant-interactive lifestyle. On the other hand nitrogen fixation genes were not present in the genome, excluding a classical nitrogen fixation interaction.

Detoxification Potential

The B. terrae BS001 genome revealed great potential to detoxify arsenate, phenylmercuric acetate, methylglyoxal, cyanate, fluoroacetate, and superoxide radicals. The gene encoding arsenate reductase (arsC) (AKAUv1_1500002) is present on RGP38. Interestingly, two genes (AKAUv1_1500022 and AKAUv1_1500025) involved in cyanate degradation are located downstream on RGP38. Moreover, ORF AKAUv1_3230012, encoding aliphatic nitrilase that catalyzes a reaction in acrylonitrile degradation, was found to be harbored by RGP78. Another putative ORF, AKAUv1_2920114, was harbored by RGP73. It encodes a putative 2-nitropropane dioxygenase that is involved in alkylnitronate degradation. We also found that strain BS001 is capable of degrading salicylate, anthranilate, benzoate, and catechol. Similarly, proteins for degradation of compounds that are released during the degradation of plant lignin in soil, such as protocatechuate, ferulate and vanillate, as described for Ralstonia solanacearum (Genin and Boucher 2004), are represented by ORFs in the genome of B. terrae BS001.

The afore analyses indicate that B. terrae BS001 has invested considerable genetic luggage in detoxification systems, which may relate to its persistence in ecological niches that are affluent in such compounds and may explain its versatile ecological behavior.

Membrane Transporter Landscape

As gatekeepers to the outside, membrane transporters are important for metabolic activities. Lifestyle is thought to be a key driver of the evolution in numbers and types of transporters (Gelfand and Rodionov 2007). Transporters can be classified into three different classes based on their structure and mechanism of action, that is, energy-dependent (ATP dependent) membrane pumps, ion channels, and secondary transporters (Nagata et al. 2008). We used TransAPP (Transporter Automatic Annotation Pipeline) in the search for membrane transporters. A total of 1,338 transporter-like ORFs were found (supplementary table S8, Supplementary Material online), and so the membrane transporter landscape of B. terrae BS001 was different from that of the other Burkholderia strains. Burkholderia phymatum STM815 had 935 transporters, B. rhizoxinica HKI454 267 while according to Mitter et al. (2013), B. phytofirmans PsJN has 1,196 membrane transporters. As the transporter database lists 997 transporters for B. phytofirmans PsJN, we used this figure for our analysis. Across these Burkholderia species, a relatively constant percentage of the total genomic space is thus dedicated to transporters, and so the larger genomes such as BS001 (11.1%), STM815 (10.8%), and PsJN (12.1%) have more transporters compared with smaller ones such as HKI454 (7.1%). Overall, the ATP-dependent systems represented the bulk of transporters (648), followed by major facilitator family (MFS) (237) membrane transporters. A detailed account of the transporters across all four Burkholderia genomes analyzed is given in supplementary table S9, Supplementary Material online. A total of 81 ATP binding component CDSs of ABC transporters as well as 55 MFS CDSs (supplementary table S10, Supplementary Material online) were randomly picked and analyzed using SplitsTree4 (Huson and Bryant 2006). Thus, for both classes of transporters, a neighbor-net splits network was generated in order to depict different splits indicating potential evolutionary events. The ATP binding components of the ABC transporters revealed varying splits and radiations, indicating duplications to serve different functions in the genome (fig. 8A). Transporters of branched-chain amino acids clustered together, as opposed to those involved in glycerol-3-phosphate and sugars. Similarly, another split indicated that phosphate, Zn/Mn, Fe3+, and nod factor transporters clustered together and are duplicates. Thus, a range of duplication events followed by mutational drift and selection is at the basis of the current diversity of membrane transporters in strain BS001. Similarly, MFS transporters having similar roles clustered together, indicating another suite of duplication events (fig. 8B). Thus, expansion of the genome of B. terrae BS001 may have been driven mainly by nutritional needs in the light of locally fluctuating nutrient availability, for which a diversity of transporter systems is essential. Ren and Paulsen (2005) indicated that gene duplications might be at the basis of expansion, diversification, and selection of distinct transporters families.

Fig. 8.—

Fig. 8.—

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Neighbor-net splits graphs depicting the membrane transporters landscape of B. terrae BS001. (A) NNet splits graph inferred from alignment of 81 ATP binding component CDSs (amino acid sequences) of ABC (ATP binding cassette) transporters. (B) NNet splits graph derived from alignment of 55 MFS (major facilitator superfamily) CDSs (amino acid sequences). Alignments were manually curated and the resulting “nexus” files were analyzed using ProteinMLdist and WAG model (Gamma 4.0) within SplitsTree4 software. The splits indicate that membrane transporters (both ABC and MFS) split out and cluster by their respective functions and that gene duplications are at the basis of membrane transporters diversity. The list of CDS analyzed is given in the supplementary table S10, Supplementary Material online.

Secondary Metabolite Biosynthetic Potential

Many Burkholderia species exhibit a high potential for secondary metabolite production (Kang et al. 1998; Partida-Martinez and Hertweck 2005; Partida-Martinez et al. 2007; Knappe et al. 2008; Schmidt et al. 2009; Rohm et al. 2010; Seyedsayamdost et al. 2010; Tawfik et al. 2010; Mahenthiralingam et al. 2011; Franke et al. 2012). As B. terrae BS001 possesses an exceptionally large genome, one might predict the ample presence of novel biosynthesis gene clusters. Analyses with antiSMASH (Medema et al. 2011; Blin et al. 2013) revealed the existence of a locus (14,592 bp) that likely encodes a nonribosomal peptide synthetase (NRPS) (AKAUv1_1360004–AKAUv1_1360005) and another one (12,975 bp) coding for a hybrid polyketide synthase (PKS)/NRPS enzyme (AKAUv1_1710055, AKAUv1_1710056, and AKAUv1_1710057) (fig. 8A). The NRPS locus (AKAUv1_1360004–AKAUv1_1360005) displays similarity (63% and 60%, respectively) to an NRPS locus of B. cenocepacia that is known to encode the siderophore ornibactin (Agnoli et al. 2006). As a mycosphere-inhabiting bacterium, B. terrae BS001 is postulated to depend on iron uptake systems under conditions of low iron. However, in the predicted ornibactin-like NRPS locus, four genes (pvdF [N5-hydroxyornithine transformylase], orbS [sigma factor], orbH [unknown function], orbG [α-ketoglutarate-dependent hydroxylases]) were missing. Moreover, several transporter genes (orbB, orbC, orbD, and orbF) involved in transport and utilization, differed from those of the ornibactin locus of B. cenocepacia (fig. 9B).

Fig. 9.—

Fig. 9.—

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Modular organization of the NRPS (nonribosomal peptide synthetase) and the PKS (polyketide synthase)/NRPS enzymes encoded in the genome of Burkholderia terrae BS001. (A) Predicted substrates that get activated by the A-domains, are shown. Orn (Ornithine), Asp (Aspartic acid), Ser (Serine), Val (Valine) and Gly (Glycine). A, adenylation domain; T, thiolation domain; C, condensation domain; E, epimerization domain; KS, ketosynthase domain; AT, acetyl transferase domain; ACP, acyl carrier protein; AAT, aminotransferase; TE, thioesterase. (B) Comparison of the ornibactin (orb) biosynthesis gene cluster of B. cenocepacia and the NRPS gene cluster of B. terrae BS001. orb (S, H, G, C, D, F, B, E, I, J, K, A, and L) and pvd (pyoverdine) A and F, are shown.

The modular organization of another cluster—a hybrid PKS/NRPS—is shown in figure 9A. Homologs of such hybrid PKS/NRPS gene clusters have been found in other bacterial genomes by BLASTP analyses (Altschul et al. 1990), notably in Burkholderia sp. BT03 and B. phymatum STM815. However, no secondary metabolite has been described for such gene clusters yet. It would mean a great step forward if the products synthesized by these two clusters could be identified.

The BLAST search for homologs of enzymes of the ribosomally synthesized and posttranslationally modified peptides (RiPPs) pathway revealed no hits, suggesting that B. terrae BS001 is not able to produce any such RiPPs.

Conclusion

We analyzed the genome of the fungal-interactive B. terrae BS001, which was recently sequenced (Nazir, Hansen, et al. 2012). The genome is the largest among all hitherto available Burkholderia genomes, and it is equipped with a repertoire of genetic systems that encode predicted fungal- as well as plant-interactive traits. We thus identified systems potentially involved in physical interactions (biofilm formation and twitching motility) with fungi, next to protein (as well as DNA) secretion systems (in particular T3SS) that may modulate fungal host physiology and unique genes for glycerol uptake and metabolism. The genome further contains extensive RGPs (16.48% of the genome space), which carry functional genes as well as plasmid-related traits, indicating the presumed great role of horizontal gene transfer in shaping the genome. Moreover, B. terrae BS001 apparently invested considerable energy in primary (carbohydrate and amino acid) metabolism, membrane transporters, and even insect-interactive traits, indicating a versatile life style in soil. Such life style may have included (fungal/plant) host-interactive phases interspersed with free-living phases. Concerning the latter, the BS001 genome also contained genes that potentially encode secondary metabolite biosynthesis, which is useful for highly competitive situations that may occur in the soil upon exploration for colonization space. Overall, our analysis unveiled a plethora of ecological features in the genome that provide a firm basis for further studies based on transcriptomics of ecological situations, including interactions with (fungal) hosts.

Materials and Methods

Genome Analysis and Annotation

The genome of B. terrae BS001 was submitted to MicroScope platform that is hosted at Genoscope for analysis. The genome data was integrated to PkGDB database of MicroScope and we have used the gene locus tags from the same database in this study. The genome sequencing data had been submitted to NCBI under accession number PRJNA157903/AKAU00000000.

Pan–Core Genome Computation

The pan–core genome analysis was computed based on single-linkage clustering algorithm in the software package siLix (SIngle LInkage Clustering of Sequences), hosted by MicroScope. The algorithm implemented in the siLix is based on a principle where a sequence A if considered homologous to sequence B and sequence B is homologous to C, then all the sequences (A, B, and C) are clustered in a same family, irrespective of the similarity between sequences A and C (Miele et al. 2011). Therefore if a gene X is homologous to gene Y, they are clustered together and if Y is a homolog of Z, then all three of them are grouped into the same MicroScope gene family (MICFAM).

Synteny Calculations

We employed MicroScope platform to compute synteny of B. terrae BS001 using data from PkGDB and NCBI RefSeq databases. Genes that satisfied BLASTP alignment threshold (35% sequence identity over 80% length of the smallest protein) or bidirectional best hit criteria, were defined as putative orthologs. Based on these relationships, synteny groups or syntons among other bacterial genomes were searched. The approach allows chromosomal rearrangements including insertions, deletions, and inversions. Gap parameter was set to five genes, representing maximum number of consecutive genes not part of synteny group.

Bioinformatics and Comparative Genome Analysis

For comparative analysis of genomes, their metabolic profile prediction and pan–core genome analysis and genomic islands predictions, we used MicroScope platform of the Genoscope (http://www.genoscope.cns.fr/agc/microscope/home/index.php, last accessed June 26, 2014). The RGP finder was used to detect more than 5-kb synteny breaks across a query genome and closely related (comparator) ones, followed by screening of the identified region for HGT features, such as the presence of mobility genes, tRNA hot spots and deviation of the G+C% from the main value. It then employed “SIGI-HMM” (based on the use of Hidden Markov Models, using codon usage to characterize the origin of genes) to measure codon usage aberrations (Waack et al. 2006) and “AlienHunter,” which is an “Interpolated Variable Order Motif” that uses variable-order motif distributions (2–8) to exploit compositional biases (Vernikos and Parkhill 2006).

Membrane transporters prediction and annotation were performed using TransAPP (Transporter Automatic Annotation Pipeline) of TransportDB (www.membranetransport.org, last accessed June 26, 2014). Potential type III effectors were predicted using www.effectors.org (last accessed June 26, 2014) with parameters (classification: [standard set] cutoff: [0.9999; selective]), whereas signal peptides were predicted using online server SignalP 4.1 (Petersen et al. 2011). Antibiotics and secondary metabolites analysis shell (antiSMASH) (Blin et al. 2013) online server was used for prediction and analysis of secondary metabolites.

Metabolic Pathways Comparison

The metabolic profiles of B. terrae BS001 and other genomes in this study were analyzed using MicroCyc based on MetaCyc (Caspi et al. 2010) of the MicroScope platform, as a reference pathway database. For each genome, comparative analysis of metabolic pathways was accomplished using MicroScope platform (https://www.genoscope.cns.fr/agc/microscope/metabolism, last accessed June 26, 2014). Each pathway has its own completion value (completion value = the number of enzymatic reactions in a particular pathway in a given organism/the total number of enzymatic reactions in the same pathway in MetaCyc). Hierarchical cluster analysis of the MicroCyc predicted metabolic pathways for B. terrae BS001 was performed using MeV tool integrated in the MicroScope, taking result comparisons as input and using pathway completion values. The analysis was performed taking into consideration other Burkholderia and a number of additional bacterial genomes integrated in the PkGDB database.

Phylogenetic Analysis

Phylogenetic trees were made with MEGA 6 (Tamura et al. 2013). Poorly aligned regions and gaps were manually eliminated. Maximum-likelihood method based on the JTT matrix-based model (Jones et al. 1992) was used to infer the evolutionary history for the T3SS, T4SS, ACC-deaminase, and tyrosinase genes. For modeling of evolutionary rate differences among sites discrete Gamma distribution was used (4 categories [+G, parameter = 3.8674]). For network analysis of membrane transporters, amino acid sequences of random ORFs (ATP binding component of ABC and MFS transporters) were aligned using ClustalW in MEGA. The matrix was then analyzed in SplitsTree4 software using WAG model with Gamma 4.0 and ProteinMLdist (characters).

Supplementary Material

Supplementary figures S1–S5 and tables S1–S10 are available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).

Supplementary Data
supp_6_7_1652__index.html (1.6KB, html)

Acknowledgments

The authors thank LABGeM (Laboratoire d’Analyses Bioinformatiques pour la Génomique et le Métabolisme) team of the MicroScope platform for their technical assistance and services provision. This research was supported by the European Marie Curie Initial Training Networks (ITN) TRAINBIODIVERSE (project number 289949).

Literature Cited

  1. Abby SS, Rocha EPC. The non-flagellar Type III Secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems. PLoS Genet. 2012;8:e1002983. doi: 10.1371/journal.pgen.1002983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agnoli K, Lowe CA, Farmer KL, Husnain SI, Thomas MS. The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma factor which is a part of the Fur regulon. J Bacteriol. 2006;188:3631–3644. doi: 10.1128/JB.188.10.3631-3644.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allaoui A, Ménard R, Sansonetti PJ, Parsot C. Characterization of the Shigella flexneri ipgD and ipgF genes, which are located in the proximal part of the mxi locus. Infect Immun. 1993;61:1707–1714. doi: 10.1128/iai.61.5.1707-1714.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  5. Arcus VL, Rainey PB, Turner SJ. The PIN-domain toxin-antitoxin array in mycobacteria. Trends Microbiol. 2005;13:360–365. doi: 10.1016/j.tim.2005.06.008. [DOI] [PubMed] [Google Scholar]
  6. Battaini G, et al. Tyrosinase-catalyzed oxidation of fluorophenols. J Biol Chem. 2002;277:44606–44612. doi: 10.1074/jbc.M207829200. [DOI] [PubMed] [Google Scholar]
  7. Berks BC, Sargent F, Palmer T. The Tat protein export pathway. Mol Microbiol. 2000;35:260–274. doi: 10.1046/j.1365-2958.2000.01719.x. [DOI] [PubMed] [Google Scholar]
  8. Blin K, et al. antiSMASH 2.0—a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 2013;41:W204–W12. doi: 10.1093/nar/gkt449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Boersma FGH, Otten R, Warmink JA, Nazir R, van Elsas JD. Selection of Variovorax paradoxus-like bacteria in the mycosphere and the role of fungal-released compounds. Soil Biol Biochem. 2010;42:2137–2145. [Google Scholar]
  10. Bosse JT, et al. Regulation of pga operon expression and biofilm formation in Actinobacillus pleuropneumoniae by sigmaE and H-NS. J Bacteriol. 2010;192:2414–2423. doi: 10.1128/JB.01513-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caspi R, et al. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res. 2010;38:D473–D479. doi: 10.1093/nar/gkp875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cianciotto NP. Type II secretion: a protein secretion system for all seasons. Trends Microbiol. 2005;13:581–588. doi: 10.1016/j.tim.2005.09.005. [DOI] [PubMed] [Google Scholar]
  13. Colvin KM, et al. The Pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog. 2011;27:7–e1001264. doi: 10.1371/journal.ppat.1001264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Conrad JC. Physics of bacterial near-surface motility using flagella and type IV pili: implications for biofilm formation. Res Microbiol. 2012;163:619–629. doi: 10.1016/j.resmic.2012.10.016. [DOI] [PubMed] [Google Scholar]
  15. Dale C, Moran NA. Molecular interactions between bacterial symbionts and their hosts. Cell. 2006;126:453–465. doi: 10.1016/j.cell.2006.07.014. [DOI] [PubMed] [Google Scholar]
  16. Delepelaire P. Type I secretion in gram-negative bacteria. Biochim Biophys Acta. 2004;1694(1–3):149–161. doi: 10.1016/j.bbamcr.2004.05.001. [DOI] [PubMed] [Google Scholar]
  17. Edqvist PJ, et al. Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion. Mol Microbiol. 2006;59:31–44. doi: 10.1111/j.1365-2958.2005.04923.x. [DOI] [PubMed] [Google Scholar]
  18. Estrada-de los Santos P, Vinuesa P, Martínez-Aguilar L, Hirsch AM, Caballero-Mellado J. Phylogenetic analysis of Burkholderia species by multilocus sequence analysis. Curr Microbiol. 2013;67:51–60. doi: 10.1007/s00284-013-0330-9. [DOI] [PubMed] [Google Scholar]
  19. Fischer G, Bang H, Ludwig B, Mann K, Hacker J. Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity. Mol Microbiol. 1992;6:1375–1383. doi: 10.1111/j.1365-2958.1992.tb00858.x. [DOI] [PubMed] [Google Scholar]
  20. Franke J, Ishida K, Hertweck C. Genomics-driven discovery of burkholderic acid, a noncanonical, cryptic polyketide from human pathogenic Burkholderia species. Angew Chem Int Ed Engl. 2012;51:11611–11615. doi: 10.1002/anie.201205566. [DOI] [PubMed] [Google Scholar]
  21. Friedman L, Kolter R. Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol Microbiol. 2004;51:675–690. doi: 10.1046/j.1365-2958.2003.03877.x. [DOI] [PubMed] [Google Scholar]
  22. Gelfand MS, Rodionov DA. Comparative genomics and functional annotation of bacterial transporters. Phys Life Rev. 2007;5:22–49. [Google Scholar]
  23. Geng J, et al. Bacterial melanin interacts with double-stranded DNA with high affinity and may inhibit cell metabolism in vivo. Arch Microbiol. 2010;192(5):321–329. doi: 10.1007/s00203-010-0560-1. [DOI] [PubMed] [Google Scholar]
  24. Genin S, Boucher C. Lessons learned from the genome analysis of Ralstonia solanacearum. Annu Rev Phytopathol. 2004;42:107–134. doi: 10.1146/annurev.phyto.42.011204.104301. [DOI] [PubMed] [Google Scholar]
  25. Gerdes K, Christensen SK, Lobner-Olesen A. Prokaryotic toxin-antitoxin stress response loci. Nat Rev Microbiol. 2005;3:371–382. doi: 10.1038/nrmicro1147. [DOI] [PubMed] [Google Scholar]
  26. Holland IB, Schmitt L, Young J. Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review) Mol Membr Biol. 2005;22: 29–39. doi: 10.1080/09687860500042013. [DOI] [PubMed] [Google Scholar]
  27. Holst B, et al. GUP1 and its close homologue GUP2, encoding multimembrane-spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae. Mol Microbiol. 2000;37:108–124. doi: 10.1046/j.1365-2958.2000.01968.x. [DOI] [PubMed] [Google Scholar]
  28. Huson DH, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006;23(2):254–267. doi: 10.1093/molbev/msj030. [DOI] [PubMed] [Google Scholar]
  29. Izano EA, et al. Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae. Microb Pathog. 2007;43:1–9. doi: 10.1016/j.micpath.2007.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jehl MA, Arnold R, Rattei T. Effective-a database of predicted secreted bacterial proteins. Nucleic Acids Res. 2011:D591–D595. doi: 10.1093/nar/gkq1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8:275–282. doi: 10.1093/bioinformatics/8.3.275. [DOI] [PubMed] [Google Scholar]
  32. Kang Y, Carlson R, Tharpe W, Schell MA. Characterization of genes involved in biosynthesis of a novel antibiotic from Burkholderia cepacia BC11 and their role in biological control of Rhizoctonia solani. Appl Environ Microbiol. 1998;64:3939–3947. doi: 10.1128/aem.64.10.3939-3947.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Knappe TA, et al. Isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of Burkholderia thailandensis E264. J Am Chem Soc. 2008;130:11446–11454. doi: 10.1021/ja802966g. [DOI] [PubMed] [Google Scholar]
  34. Kuo MJ, Alexander M. Inhibition of the lysis of fungi by melanins. J Bacteriol. 1967;94:624–629. doi: 10.1128/jb.94.3.624-629.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lackner G, Moebius N, Hertweck C. Endofungal bacterium controls its host by an hrp type III secretion system. ISME J. 2011;5:252–261. doi: 10.1038/ismej.2010.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lackner G, Moebius N, Partida-Martinez L, Boland S, Hertweck C. Evolution of an endofungal lifestyle: deductions from the Burkholderia rhizoxinica genome. BMC Genomics. 2011;12:210. doi: 10.1186/1471-2164-12-210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liu N, et al. A heat inducible tyrosinase with distinct properties from Bacillus thuringiensis. Lett Appl Microbiol. 2004;39:407–412. doi: 10.1111/j.1472-765X.2004.01599.x. [DOI] [PubMed] [Google Scholar]
  38. Loh J, Garcia M, Stacey G. NodV and NodW, a second flavonoid recognition system regulating nod gene expression in Bradyrhizobium japonicum. J Bacteriol. 1997;179:3013–3020. doi: 10.1128/jb.179.9.3013-3020.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Loh J, Lohar DP, Andersen B, Stacey G. A two-component regulator mediates population-density-dependent expression of the Bradyrhizobium japonicum nodulation genes. J Bacteriol. 2002;184:1759–1766. doi: 10.1128/JB.184.6.1759-1766.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mahenthiralingam E, et al. Enacyloxins are products of an unusual hybrid modular polyketide synthase encoded by a cryptic Burkholderia ambifaria Genomic Island. Chem Biol. 2011;18:665–677. doi: 10.1016/j.chembiol.2011.01.020. [DOI] [PubMed] [Google Scholar]
  41. Marino SM, et al. Investigation of Streptomyces antibioticus tyrosinase reactivity toward chlorophenols. Arch Biochem Biophys. 2011;505:67–74. doi: 10.1016/j.abb.2010.09.019. [DOI] [PubMed] [Google Scholar]
  42. Medema MH, et al. antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 2011;39:W339–W346. doi: 10.1093/nar/gkr466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Medini D, Donati C, Tettelin H, Masignani V, Rappuoli R. The microbial pan-genome. Curr Opin Genet Dev. 2005;15:589–594. doi: 10.1016/j.gde.2005.09.006. [DOI] [PubMed] [Google Scholar]
  44. Miele V, et al. Ultra-fast sequence clustering from similarity networks with SiLiX. BMC Bioinformatics. 2011;12:116. doi: 10.1186/1471-2105-12-116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Miras I, Hermant D, Arricau N, Popoff MY. Nucleotide sequence of iagA and iagB genes involved in invasion of HeLa cells by Salmonella enterica subsp. enterica ser. Typhi. Res Microbiol. 1995;146:17–20. doi: 10.1016/0923-2508(96)80267-1. [DOI] [PubMed] [Google Scholar]
  46. Mitter B, et al. Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants. Front Plant Sci. 2013;4:120. doi: 10.3389/fpls.2013.00120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nagata T, Iizumi S, Satoh K, Kikuchi S. Comparative molecular biological analysis of membrane transport genes in organisms. Plant Mol Biol. 2008;66:565–585. doi: 10.1007/s11103-007-9287-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Nazir R, Hansen MA, Sørensen S, van Elsas JD. Draft genome sequence of the soil bacterium Burkholderia terrae strain BS001, which interacts with fungal surface structures. J Bacteriol. 2012;194:4480–4481. doi: 10.1128/JB.00725-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nazir R, Warmink JA, Voordes DC, van de Bovenkamp HH, van Elsas JD. Inhibition of mushroom formation and induction of glycerol release-ecological strategies of Burkholderia terrae BS001 to create a hospitable niche at the fungus Lyophyllum sp. strain Karsten. Microb Ecol. 2013;65:245–254. doi: 10.1007/s00248-012-0100-4. [DOI] [PubMed] [Google Scholar]
  50. Nazir R, Zhang M, de Boer W, van Elsas JD. The capacity to comigrate with Lyophyllum sp. strain Karsten through different soils is spread among several phylogenetic groups within the genus Burkholderia. Soil Biol Biochem. 2012;50:221–233. [Google Scholar]
  51. Partida-Martinez LP, et al. Rhizonin, the first mycotoxin isolated from the zygomycota, is not a fungal metabolite but is produced by bacterial endosymbionts. Appl Environ Microbiol. 2007;73:793–797. doi: 10.1128/AEM.01784-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Partida-Martinez LP, Hertweck C. Pathogenic fungus harbors endosymbiotic bacteria for toxin production. Nature. 2005;437:884–888. doi: 10.1038/nature03997. [DOI] [PubMed] [Google Scholar]
  53. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8:785–786. doi: 10.1038/nmeth.1701. [DOI] [PubMed] [Google Scholar]
  54. Ren Q, Paulsen IT. Comparative analyses of fundamental differences in membrane transport capabilities in prokaryotes and eukaryotes. PLoS Comput Biol. 2005;1:e27. doi: 10.1371/journal.pcbi.0010027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rohm B, Scherlach K, Möbius N, Partida-Martinez LP, Hertweck C. Toxin production by bacterial endosymbionts of a Rhizopus microsporus strain used for tempe/sufu processing. Int J Food Microbiol. 2010;136:368–371. doi: 10.1016/j.ijfoodmicro.2009.10.010. [DOI] [PubMed] [Google Scholar]
  56. Ronald PC, Beutler B. Plant and animal sensors of conserved microbial signatures. Science. 2010;330:1061–1064. doi: 10.1126/science.1189468. [DOI] [PubMed] [Google Scholar]
  57. Sanjuan J, Grob P, Göttfert M, Hennecke H, Stacey G. NodW is essential for the full expression of the common nodulation genes in Bradyrhizobium japonicum. Mol Plant Microbe Interact. 1992;7:364–369. [Google Scholar]
  58. Schaerlaekens K, et al. Twin-arginine translocation pathway in Streptomyces lividans. J Bacteriol. 2001;183:6727–6732. doi: 10.1128/JB.183.23.6727-6732.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Schmidt S, et al. Production of the antifungal compound pyrrolnitrin is quorum sensing-regulated in members of the Burkholderia cepacia complex. Environ Microbiol. 2009;11:1422–1437. doi: 10.1111/j.1462-2920.2009.01870.x. [DOI] [PubMed] [Google Scholar]
  60. Seyedsayamdost MR, et al. Quorum-sensing regulated bactobolin production by Burkholderia thailandensis E264. Org Lett. 2010;12:716–719. doi: 10.1021/ol902751x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30:2725–2729. doi: 10.1093/molbev/mst197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tawfik KA, et al. Burkholdines 1097 and 1229, potent antifungal peptides from Burkholderia ambifaria 2.2N. Org Lett. 2010;12:664–666. doi: 10.1021/ol9029269. [DOI] [PubMed] [Google Scholar]
  63. Thomas SH, et al. The mosaic genome of Anaeromyxobacter dehalogenans strain 2CP-C suggests an aerobic common ancestor to the delta-Proteobacteria. PLoS One. 2008;3:e2103. doi: 10.1371/journal.pone.0002103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Tomich M, Planet PJ, Figurski DH. The tad locus: postcards from the widespread colonization island. Nat Rev Microbiol. 2007;5(5):363–375. doi: 10.1038/nrmicro1636. [DOI] [PubMed] [Google Scholar]
  65. Tseng T, Tyler BM, Setubal JC. Protein secretion systems in bacterial-host associations, and their description in the gene ontology. BMC Microbiol. 2009;9(Suppl. 1):S2. doi: 10.1186/1471-2180-9-S1-S2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Vallenet D, et al. MicroScope—an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data. Nucleic Acids Res. 2013;41:D636–D647. doi: 10.1093/nar/gks1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Vernikos GS, Parkhill J. Interpolated variable order motifs for identification of horizontally acquired DNA: revisiting the Salmonella pathogenicity islands. Bioinformatics. 2006;22:2196–2203. doi: 10.1093/bioinformatics/btl369. [DOI] [PubMed] [Google Scholar]
  68. Waack S, et al. Score-based prediction of genomic islands in prokaryotic genomes using hidden Markov models. BMC Bioinformatics. 2006;7:142. doi: 10.1186/1471-2105-7-142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Warmink JA, Nazir R, Corten B, van Elsas JD. Hitchhikers on the fungal highway: the helper effect for bacterial migration via fungal hyphae. Soil Biol Biochem. 2011;43:760–765. [Google Scholar]
  70. Warmink JA, van Elsas JD. Selection of bacterial populations in the mycosphere of Laccaria proxima: is type III secretion involved? ISME J. 2008;2:887–900. doi: 10.1038/ismej.2008.41. [DOI] [PubMed] [Google Scholar]
  71. Warmink JA, van Elsas JD. Migratory response of soil bacteria to Lyophyllum sp. strain Karsten in soil microcosms. Appl Environ Microbiol. 2009;75:2820–2830. doi: 10.1128/AEM.02110-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Wei HL, Chakravarthy S, Worley JN, Collmer A. Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana. Cell Microbiol. 2013;15:601–618. doi: 10.1111/cmi.12059. [DOI] [PubMed] [Google Scholar]
  73. Zang N, et al. Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris. Mol Plant Microbe Interact. 2007;20:21–30. doi: 10.1094/MPMI-20-0021. [DOI] [PubMed] [Google Scholar]

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