Miaozhi Zhang

The mycosphere constitutes an arena for horizontal gene transfer with strong evolutionary implications for bacterial-fungal interactions

In the microhabitat that surrounds fungal hyphae in soil, coined the mycosphere, carbonaceous compounds that are released from the hyphae stimulate the growth of heterotrophic bacteria, and thus activate organism-to-organism contacts through genetic interactions. Therefore, the mycosphere is postulated to constitute a gene transfer arena, in which a plethora of genes, including locally adaptive ones, are swapped across the resident microbial communities. Such genetic transfers may have plasmids, in particular ones with broad host ranges, as the basis. Indeed, evidence is increasing for the contention that plasmids play crucial roles as accelerators of evolution in the mycosphere, serving as a horizontal gene pool and, therefore, providing competence factors to local bacteria as well as fungi. The evidence so far points at mycosphere roles for two major plasmid classes, the IncP-1 and PromA groups. Moreover, recent data indicate that bacterium-to-fungus gene transfers are detectable and have been evolutionarily important. The large gene pool present in the mycosphere, coupled with the chances for cell-to-cell contact between mycosphere dwellers allows enhanced recombination frequencies, and as such, organisms are selected locally for enhanced fitness.

The Interactions of Bacteria with Fungi in Soil: Emerging Concepts

In this chapter, we review the existing literature on bacterial-fungal interactions in soil, exploring the role fungi may play for soil bacteria as providers of hospitable niches. A focus is placed on the mycosphere, i.e., the narrow zone of influence of fungal hyphae on the external soil milieu, in which hypha-associated bacterial cells dwell. Evidence is brought forward for the contention that the hyphae of both mycorrhizal and saprotrophic fungi serve as providers of ecological opportunities in a grossly carbon-limited soil, as a result of their release of carbonaceous compounds next to the provision of a colonizable surface. Soil bacteria of particular nature are postulated to have adapted to such selection pressures, evolving to the extent that they acquired capabilities that allow them to thrive in the novel habitat created by the emerging fungal hyphae. The mechanisms involved in the interactions and the modes of genetic adaptation of the mycosphere dwellers are discussed, with an emphasis on one key mycosphere-adapted bacterium, Burkholderia terrae BS001. In this discussion, we interrogate the positive interactions between soil fungi and bacteria, and refrain from considering negative interactions.

The type three secretion system facilitates migration of Burkholderia terrae BS001 in the mycosphere of two soil-borne fungi

The type three secretion system (T3SS) is known to play a critical role in several bacterial-eukaryotic cell interactions. Recent indirect evidence has also pointed to a role of this system in bacterial-fungal interactions in soil. In the current study, we examine if the T3SS of the fungal-interactive Burkholderia terrae strain BS001 can aid in the interaction of this bacterium with two soil fungi, i.e., Lyophyllum sp. strain Karsten and Trichoderma asperellum 302. We first analyzed the T3SS of strain BS001 and then constructed a knockout mutant of the essential sctD gene. The selected sctD mutant strain did not show any differences to the wild-type strain with respect to its growth and nutrient utilization behavior, excluding polar effects of the mutation. Then, the migration ability of the sctD mutant strain along with the hyphae of Lyophyllum sp. strain Karsten growing through presterilized soil was tested, revealing hampered comigration as compared to the wild-type strain. The effect was also observed with T. asperellum 302. However, the migration impairment was only noticed in mixed-inoculation experiments, whereas it remained unnoticed when the two strains were inoculated in separate. These data demonstrate that the T3SS assists B. terrae BS001 in its interaction with two soil fungi, without being essential for these interactions. As far as we know, this is the first time that the role of a T3SS in the comigration of bacteria along with soil-exploring fungi is verified directly.

Role of flagella and type four pili in the co-migration of Burkholderia terrae BS001 with fungal hyphae through soil

Burkholderia terrae BS001 has previously been found to be able to disperse along with growing fungal hyphae in soil, with the type-3 secretion system having a supportive role in this movement. In this study, we focus on the role of two motility- and adherence-associated appendages, i.e. type-4 pili (T4P) and flagella. Electron microcopy and motility testing revealed that strain BS001 produces polar flagella and can swim on semi-solid R2A agar. Flagellum- and T4P-negative mutants were then constructed to examine the ecological roles of the respective systems. Both in liquid media and on swimming agar, the mutant strains showed similar fitness to the wild-type strain in mixed culture. The flagellar mutant had completely lost its flagella, as well as its swimming capacity. It also lost its co-migration ability with two soil-exploring fungi, Lyophyllum sp. strain Karsten and Trichoderma asperellum 302, in soil microcosms. In contrast, the T4P mutant showed reduced surface twitching motility, whereas its co-migration ability in competition with the wild-type strain was slightly reduced. We conclude that the co-migration of strain BS001 with fungal hyphae through soil is dependent on the presence of functional flagella conferring swimming motility, with the T4P system having a minor effect.

The Complete Sequences and Ecological Roles of Two IncP-1β Plasmids, pHB44 and pBS64, Isolated from the Mycosphere of Laccaria proxima

Two novel plasmids, coined pHB44 and pBS64, were recently found in Variovorax paradoxus strains HB44 and BS64 isolated from the mycosphere of Laccaria proxima, on two different sampling occasions. We here describe the full sequences of pHB44 and pBS64 and establish their evolutionary placement and ecological function. Both plasmids, unique for mycospheric V. paradoxus, were around 58 kb in size. They possessed, in a very similar fashion, three main plasmid backbone regions, which were predicted to be involved in plasmid replication, central control of maintenance, and conjugational transfer. Phylogenetic inference on the basis of seven selected and concatenated plasmid backbone genes provided solid evidence for the placement of the two plasmids in the IncP-1β1 group, with the recently isolated IncP-1β1 plasmid pMBUI8 as the closest relative. A comparative analysis of the sequences present in each of the recombinational hot spots (RHS) I to III across plasmids pHB44, pBS64, and pMBUI8 revealed the insertions found in plasmids pHB44 and pBS64 to be different from those of pMBUI8. Whereas, in the former two plasmids, RHS I and III were devoid of any major inserts, their RHS II regions contained inserts of 15,043 (pHB44) and 16,406 kb (pBS64), against about 9,3 kb for pMBUI8. Interestingly, these regions were highly similar across plasmids pHB44 and pBS64, and differed from that of pMBUI8. Closer inspection revealed the insert in the former plasmids to contain, next to transposases, an “mmf” gene cassette previously reported to encode metal “responsiveness” in the PromA plasmid pMOL98. Whereas the plasmid pHB44 RHS II contained the canonical mmf sequence, that in pBS64 contained, in addition, a “two-gene duplicated region” flanking the mmf C2 gene. In vitro experiments on the growth and survival of strains with or without plasmid pHB44 suggested this plasmid was involved in the binding and import of Fe3+ as well as V3+ ions into the host cells, thus yielding a growth advantage under “metal ion-limiting” conditions. In addition, pHB44 was found to confer a bacitracin resistance phenotype to its host strain HB44. The metal import and bacitracin resistance traits were tentatively attributed to specific genes present in the RHS II inserts.

Advances in Applied Microbiology

In this chapter, we review the existing literature on bacterial-fungal interactions in soil, exploring the role fungi may play for soil bacteria as providers of hospitable niches. A focus is placed on the mycosphere, i.e., the narrow zone of influence of fungal hyphae on the external soil milieu, in which hypha-associated bacterial cells dwell. Evidence is brought forward for the contention that the hyphae of both mycorrhizal and saprotrophic fungi serve as providers of ecological opportunities in a grossly carbon-limited soil, as a result of their release of carbonaceous compounds next to the provision of a colonizable surface. Soil bacteria of particular nature are postulated to have adapted to such selection pressures, evolving to the extent that they acquired capabilities that allow them to thrive in the novel habitat created by the emerging fungal hyphae. The mechanisms involved in the interactions and the modes of genetic adaptation of the mycosphere dwellers are discussed, with an emphasis on one key mycosphere-adapted bacterium, Burkholderia terrae BS001. In this discussion, we interrogate the positive interactions between soil fungi and bacteria, and refrain from considering negative interactions.