Hitchhikers on the fungal highway: The helper effect for bacterial migration via fungal hyphae

Abstract

Previous work in our laboratory showed that several bacterial strains, either singly or in association with other bacteria (community migration), were capable of migrating together with the saprotrophic fungus Lyophyllum sp. strain Karsten through soil microcosms. A possible involvement of the type III secretion system (TTSS) in migration was indicated. In this study, we addressed the basis of the community migration, which might lie in a migration helper effect exerted by particular single-strain migrators on other members of the community. Different culturing (plating) as well as culture-independent (PCR-DGGE) methods were applied to assess the effects of putative bacterial helpers in the migration. We used, as a model, the migration-proficient Burkholderia terrae BS001 as the canonical helper strain. PCR-DGGE analysis of the soil system with or without added strain BS001, revealed that the latter consistently stimulated the migration of different bacterial species through the soil. This was observed both following introduction of the organism to a bacterial community from soil and on the basis of a similar organism that was naturally present. One strain, Dyella japonica BS003, was identified as an avid comigrator with B. terrae BS001, although it appeared to lag behind the latter strain in its migration speed. Further examination of the B. terrae BS001/D. japonica BS003 interaction at Lyophyllum sp. strain Karsten hyphae showed that the presence of the D. japonica strain did not negatively affect the growth and migration of B. terrae BS001 with the fungus. A biofilm of B. terrae BS001 was formed on the fungal hyphal front, and we postulate a role for this biofilm in the migration helper effect.

Introduction

Soil is a highly structured environment in which conditions fluctuate, leading to both spatial and temporal heterogeneities in local conditions (Standing and Killham, 2007). The living soil is the “house” of a multitude of micro- and macro-organisms that show a wide array of interactions (Marschner et al., 2001, Waterfield et al., 2004). Darwinian theory dictates that each organism established in soil has its own specific niche which is defined by nutritional and spatial aspects of the microhabitat (Van Elsas et al., 2007). Our understanding of the living soil further indicates that novel soil microhabitats may be constantly formed via the movement, growth and death of organisms such as soil fungi (De Boer et al., 2005, Frey-Klett et al., 2007), filamentous or other bacteria, other organisms and plant roots (Marschner et al., 2001). This dynamism of local conditions, next to that caused by higher organisms such as earthworms (Lee, 1985) and insect larvae, strongly affects the soil microbiota. Bacteria that colonize the newly-formed microhabitats may be essential for soil functioning. For instance, they may play major roles in the degradation of organic material (Torsvik et al., 2002) as well as the breakdown of pollutants in soil (Harms and Wick, 2006). Furthermore, bacteria involved in plant growth promotion may show responses to emerging mycorrhizal fungi and thus colonize novel sites in soil (Frey-Klett et al., 2007, Garbaye, 1994, Johansson et al., 2004). Bacteria can interact with fungi in soil using different mechanisms (Nazir et al., 2010a). For instance, Pseudomonas fluorescens may use its polar flagella to anchor to mycelial surfaces (Sen et al., 1996). Recently, Toljander et al. (2006) reported that bacteria in the mycosphere differ in their ability to colonize vital and non-vital hyphae and also that bacterial attachment is influenced by the fungal host. Warmink and van Elsas (2009) provided evidence for biofilm formation and an involvement of TTSS in the interaction.

Given the fact that bacterial movement in soil is often severely restricted, reaching new microhabitats in soil via migration with soil fungi (Warmink and van Elsas, 2009) is a key phenomenon that may determine the ecological success of particular soil bacteria. For instance, the air gaps between soil particles, which pose unsurmountable barriers to the movement of (non-filamentous) soil bacteria, may be passed (Schafer et al., 1998). The use of fungal hyphae as “highways” to move to novel microhabitats has thus been hypothesized to offer a mechanism for, e.g., pollutant-degrading bacteria to reach soil areas where pollutant concentrations are elevated and they can become active (Kohlmeier et al., 2005, Wick et al., 2007), especially in unsaturated soil (Furuno et al., 2009). In a previous study by our group, migration of particular bacteria with hyphae of the saprotrophic soil fungus Lyophyllum sp. strain Karsten growing in soil microcosms was found (Warmink and van Elsas, 2009). Ten different bacterial types were selected from a total soil bacterial community, and of these, only four (Burkholderia terrae BS001, Dyella japonica BS018 and BS021 and Ralstonia basilensis BS017) could migrate as single species with the fungus (single-strain migrators). The remainder, when present as single strains, did not migrate with fungal hyphae. It was thus hypothesized that these strains depended on a so-called migration helper effect exerted by any one of the single-strain migrators for their movement.

In the current study, we investigated the influence of a selected single-strain migrator, B. terrae strain BS001, with Lyophyllum sp. strain Karsten hyphae on the bacterial community during selection for migration. We then characterized the putative migration helper effect exerted by this strain on the non-migrating strain D. japonica BS003.