Gene mobility in microbiomes of the mycosphere and mycorrhizosphere - role of plasmids and bacteriophages

Microbial activity in soil, including horizontal gene transfer (HGT), occurs in soil hot spots and at "hot moments". Given their capacities to explore soil for nutrients, soil fungi (associated or not with plant roots) can act as (1) selectors of myco(rrhizo) sphere-adapted organisms and (2) accelerators of HGT processes across the cell populations that are locally present. This minireview critically examines our current understanding of the drivers of gene mobility in the myco(rrhizo) sphere. We place a special focus on the role of two major groups of gene mobility agents, i.e. plasmids and bacteriophages. With respect to plasmids, there is mounting evidence that broad-host-range (IncP-1 beta and PromA group) plasmids are prominent drivers of gene mobility across mycosphere inhabitants. A role of IncP-1 beta plasmids in Fe uptake processes has been revealed. Moreover, a screening of typical mycosphere-inhabiting Paraburkholderia spp. revealed carriage of integrated plasmids, next to prophages, that presumably confer fitness enhancements. In particular, functions involved in biofilm formation and nutrient uptake were thus identified. The potential of the respective gene mobility agents to promote the movement of such genes is critically examined

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

Effect of culture conditions on the performance of lignocellulose-degrading synthetic microbial consortia

In this study, we examined a synthetic microbial consortium, composed of two selected bacteria, i.e., Citrobacter freundii so4 and Sphingobacterium multivorum w15, next to the fungus Coniochaeta sp. 2T2.1, with respect to their fate and roles in the degradation of wheat straw (WS). A special focus was placed on the effects of pH (7.2, 6.2, or 5.2), temperature (25 versus 28 °C), and shaking speed (60 versus 180 rpm). Coniochaeta sp. 2T2.1 consistently had a key role in the degradation process, with the two bacteria having additional roles. Whereas temperature exerted only minor effects on the degradation, pH and shaking speed were key determinants of both organismal growth and WS degradation levels. In detail, the three-partner degrader consortium showed significantly higher WS degradation values at pH 6.2 and 5.2 than at pH 7.2. Moreover, the two bacteria revealed up to tenfold enhanced final cell densities (ranging from log8.0 to log9.0 colony forming unit (CFU)/mL) in the presence of Coniochaeta sp. 2T2.1 than when growing alone or in a bacterial bi-culture, regardless of pH range or shaking speed. Conversely, at 180 rpm, fungal growth was clearly suppressed by the presence of the bacteria at pH 5.2 and pH 6.2, but not at pH 7.2. In contrast, at 60 rpm, the presence of the bacteria fostered fungal growth. In these latter cultures, oxygen levels were significantly lowered as compared to the maximal levels found at 180 rpm (about 5.67 mg/L, ~ 62% of saturation). Conspicuous effects on biomass appearance pointed to a fungal biofilm–modulating role of the bacteria

Ralstonia solanacearum Delta PGI-1 Strain KZR-5 Is Affected in Growth, Response to Cold Stress and Invasion of Tomato

The survival and persistence of Ralstonia solanacearum biovar 2 in temperate climates is still poorly understood. To assess whether genomic variants of the organism show adaptation to local conditions, we compared the behaviour of environmental strain KZR-5, which underwent a deletion of the 17.6 kb genomic island PGI-1, with that of environmental strain KZR-1 and potato-derived strains 1609 and 715. PGI-1 harbours two genes of potential ecological relevance, i.e. one encoding a hypothetical protein with a RelA/SpoT domain and one a putative cellobiohydrolase. We thus assessed bacterial fate under conditions of amino acid starvation, during growth, upon incubation at low temperature and invasion of tomato plants. In contrast to the other strains, environmental strain KZR-5 did not grow on media that induce amino acid starvation. In addition, its maximum growth rate at 28°C in rich medium was significantly reduced. On the other hand, long-term survival at 4°C was significantly enhanced as compared to that of strains 1609, 715 and KZR-1. Although strain KZR-5 showed growth rates (at 28°C) in two different media, which were similar to those of strains 1609 and 715, its ability to compete with these strains under these conditions was reduced. In singly inoculated tomato plants, no significant differences in invasiveness were observed among strains KZR-5, KZR-1, 1609 and 715. However, reduced competitiveness of strain KZR-5 was found in experiments on tomato plant colonisation and wilting when using 1:1 or 5:1 mixtures of strains. The potential role of PGI-1 in plant invasion, response to stress and growth in competition at high and moderate temperatures is discussed

Metasecretome analysis of a lignocellulolytic microbial consortium grown on wheat straw, xylan and xylose

Background: Synergistic action of different enzymes is required to complete the degradation of plant biomass in order to release sugars which are useful for biorefining. However, the use of single strains is often not efficient, as crucial parts of the required enzymatic machinery can be absent. The use of microbial consortia bred on plant biomass is a way to overcome this hurdle. In these, secreted proteins constitute sources of relevant enzyme cocktails. Extensive analyses of the proteins secreted by effective microbial consortia will contribute to a better understanding of the mechanism of lignocellulose degradation. Results: Here, we report an analysis of the proteins secreted by a microbial consortium (metasecretome) that was grown on either wheat straw (RWS), xylose or xylan as the carbon sources. Liquid chromatography-tandem mass spectrometry was used to analyze the proteins in the supernatants. Totals of 768 (RWS), 477 (xylose) and 103 (xylan) proteins were identified and taxonomically and functionally classified. In RWS, the proteins were mostly affiliated with Sphingobacterium-like consortium members (similar to 50 %). Specific abundant protein clusters were predicted to be involved in polysaccharide transport and/or sensing (TonB-dependent receptors). In addition, proteins predicted to degrade plant biomass, i.e. endo-1,4-beta-xylanases, alpha-l-arabinofuranosidases and alpha-l-fucosidases, were prominent. In the xylose-driven consortium, most secreted proteins were affiliated with those from Enterobacteriales (mostly Klebsiella species), whereas in the xylan-driven one, they were related to Flavobacterium-like ones. Notably, the metasecretomes of the consortia growing on xylose and xylan contained proteins involved in diverse metabolic functions (e.g. membrane proteins, isomerases, dehydrogenases and oxidoreductases). Conclusions: An analysis of the metasecretomes of microbial consortia originating from the same source consortium and subsequently bred on three different carbon sources indicated that the major active microorganisms in the three final consortia differed. Importantly, diverse glycosyl hydrolases, predicted to be involved in (hemi) cellulose degradation (e.g. of CAZy families GH3, GH10, GH43, GH51, GH67 and GH95), were identified in the RWS metasecretome. Based on these results, we catalogued the RWS consortium as a true microbial enzyme factory that constitute an excellent source for the production of an efficient enzyme cocktail for the pretreatment of plant biomass

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-10 group, with the recently isolated IncP-10 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 pM0L98. Whereas the plasmid pHB44 RHS II contained the canonical mmf sequence, that in pBS64 contained, in addition, a "two-gene duplicated region" flanking the mmfC2 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