The ecology and evolution of bacteriophages of mycosphere-inhabiting Paraburkholderia spp.
Pratama, Akbar Adjie
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bacteriophages of mycosphere-inhabiting
Paraburkholderia spp.
(GREEN) research group, Groningen Institute for Evolutionary Life Sciences (GELIFES), University of Groningen, The Netherlnads, according to the requirement of the Graduate School of Science (Faculty of Science and Engineering, University of Groningen).
Akbar Adjie Pratama receive a PhD grant from Lembaga Pengelola Dana Pendidikan, Kementrian Keuangan, Republik Indonesia (Indonesian Endowment Fund for Education).
Layout: Nikki Vermeulen | Ridderprint BV Printing: Ridderprint BV | www.ridderprint.nl Cover: Akbar Adjie Pratama
ISBN: 978-94-034-1044-9 (printed version) ISBN: 978-94-034-1043-2 (electronic version)
bacteriophages of mycosphere-inhabiting
Paraburkholderia spp.
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. E. Sterken
and in accordance with the decision by the College of Deans. This thesis will be defended in public on Monday 5 November 2018 at 14.30 hours
by
Akbar Adjie Pratama born on 30 July 1987
Assessment committee Prof. dr. K. Smalla Prof. dr. J. Kok Prof. dr. R. Lavigne
General introduction 9 SECTION I: Genomics-based dissection of mycosphere-inhabiting Paraburkholderia strains, yielding a basis for bioinformatics-assisted searches for prophages
Chapter 2
Draft genome sequences of three fungal-interactive Paraburkholderia terrae
strains BS007, BS110 and BS437 35
Chapter 3
Species boundaries and ecological features among P. terrae DSM 17804T,
P. hospita DSM 17164T and P. caribensis DSM 13236T 57
SECTION II: Conceptualisation and theoretical perspective on (soil) viruses Chapter 4
The “neglected” soil virome – potential role and impact 87 Chapter 5
The significance of mutualistic phages for bacterial ecology and evolution 109 SECTION III: Paraburkholderia viruses and their evolutionary trajectory
Chapter 6
A novel inducible prophage from the mycosphere inhabitant P. terrae BS437 127 Chapter 7
Evolutionary history of bacteriophages in the Genus Paraburkholderia 167 Chapter 8
The dynamics and diversity of the mycosphere viromes 211 Chapter 9 Synthesis 233 References 252 Summary/Samenvatting 276 Acknowledgements 281 Author affiliations 284
General Introduction
Akbar Adjie Pratama and Jan Dirk van Elsas Partly published in Book chapter:
The viruses in soil – potential roles, activities and impacts.
Van Elsas J.D., Trevors J.T., Rosado A.S., and Nannipieri P., Taylor and Francis, 2018. Modern Soil Microbiology III. In press
1
Essentially, all life depends upon the soil. There can be no life without soil and no soil without life; they have evolved together
(Charles E. Kellogg)
The living soil, soil activity hot spots and phages
The living soil is a very heterogeneous and dynamic system that influences microbial contact, attachment, niche differentiation and diversity. Its heterogeneity stems from the diversity of soil physicochemical characteristics and soil environmental conditions (e.g. pH, salinity) that have shaped both macro- and microhabitats across soil space and time (Fierer, 2017). The most dynamic activity in soil can be observed in microhabitats that are nutrient-rich and have been denominated hotspots. The factors that affect the life in soil hotspots include soil density, redox potential and nutrient contents, the latter spurring dynamically active and diverse microbiomes. Key known activity hotspots in the living soil are (i) the rhizosphere, i.e. the soil volume surrounding living roots; (ii) the detritusphere, i.e. organic litter that may consist of plant and/or animal residues, (iii) biopores, i.e. the soil surrounding animal-caused burrows (mainly earthworms), also known as the drillosphere. Other biopores such as the termitosphere (burrow caused by termites) and myrmecosphere (burrow caused by ants) should also be noted, yet the extent to which these affect microbial dynamics is understudied. (iv) aggregate surfaces, i.e. hotspots formed as the consequence of the movement of dissolved organic matter (DOM) through soil pores. Finally (v), the mycosphere, i.e. the region surrounding fungal hypae in soil (for details, see Zhang et al., 2014) stands out as a highly relevant yet hitherto understudied soil activity hot spot. All hotspots are ecologically significant for the functioning of the soil (e.g. decomposition, mineralization as well as build-up of soil organic matter). In generic terms, they increase microbial processes and interactions and consequently accelerate microbial exchange pools and evolution. With respect to the mycosphere, we may safely state that it is a highly dynamic microhabitat in terms of local conditions that are shaped by the microbially-mediated as well as physicochemical processes that shift over time. This is discussed further in the next section. The dynamic conditions are thought to shape the lifestyle of mycosphere inhabitants.
In general, soil microbiomes include bacteria, fungi, archaea, protozoa, as well as their respective viruses. Compared to bacteria, the study of soil viruses is still in its infancy. Adding up to it, the role of soil viruses as shapers of the ecology and the evolution of soil microbiomes is still poorly understood, as compared to marine counterparts. In the study reported in this thesis, I focus on the importance of soil viruses, especially bacteriophages, for the ecology and evolution of mycosphere inhabitants. The phage like or related genes in there lay the basis of this thesis. The importance of horizontal gene transfer (HGT) in soil is undoubtedly great, as evidenced by major studies on soil-derived bacteria. I conclude this introduction by developing concepts on the roles of phages in mycosphere dwellers, which yield hypotheses underlying the work described in each of the chapters.
The mycosphere in soil and the key mycosphere inhabitant
Paraburkholderia terrae
In soil microbiomes, bacteria are often dominant, both in terms of numbers, diversity and activity. Their diversity across soil space and time is immense and has been well documented (Torsvik et al., 1990). Moreover, their role in global ecological processes such as degradation, mineralization and fixation processes is essential. Next to bacteria, fungi also (i) are abundant with respect to cell numbers (expressed as hyphal length), and (ii) play significant roles in soil functioning. Some of the fundamental questions with respect to the ecology of soil microbial communities are therefore: how are bacterial-fungal interactions (BFI) established? What are the key players and roles in these interactions? What are the molecular/ecological mechanisms behind it? And, what are the consequences of such interactions for the ecosystem functions in soil and the benefits between the partners.
The mycosphere is known to provide carbonaceous compounds that are released by fungal cells, including oxalate, glycerol, formate, acetate, fumarate, mannitol, trehalose, erythritol, arabitol, citric acid and amino acids (Boersma et al., 2010; Frey, 1997; Haq et al 2018). These offer ecological opportunities to soil bacteria that are able to utilize, and thrive on, these released compounds.
Here, I briefly describe the system used in this study, the mycosphere inhabitant Paraburkholderia terrae interacting with host fungi in the soil. It was found this organism has intricate strategies to interact with soil fungi (Haq et al., 2014; Nazir et al., 2012; Warmink et al., 2011; Yang et al., 2016). P. terrae has been shown to have “high-affinity” fungal-interactive behavior, with the ectomycorrhizal fungus Laccaria proxima in soil under hazel trees (see Figure 1.1). Thus, assessments based on culture-dependent (isolation and colony-plate counting) and culture-independent
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assays (molecular detection of a type-3 secretion system (T3SS) proxy, the hrcC gene) for three consecutive years on L. proxima mycospheres showed significant increases of P. terrae (exemplified by strain BS110), as compared to the corresponding bulk soil. This suggested the occurrence of positive selection of these organisms presumably by the nutrients released by the fungi (Warmink and van Elsas, 2008). Further studies in soil microcosms revealed a strong profiency of another P. terrae strain, denoted BS001, for migration along the growing hyphae of the soil saprotroph Lyophyllum strain Karsten. This fungal-interactive behavior possibly involved a complex array of mechanisms, including motility, the T3SS and biofilm formation, which was observed surrounding the fungi hyphae (Warmink and Van Elsas, 2009). Strain BS001 was indeed a proficient single-strain migrator along the growing hyphae, next to P. terrae strains BS007, BS110, DSM 17804T and P. hospita DSM 17164T (Nazir et al., 2012). The mechanisms behind the interaction of P. terrae with soil fungi have also been addressed extensively (Haq et al., 2014, 2016, 2017, Yang et al., 2016, 2017, 2018). The involvement of the T3SS was investigated by comparing the knock-out strain P. terrae BS001ΔsctD with the wild-type strain, as regards its growth, nutrient utilization ability and migration profiency. No significant differences were observed in terms of growth, nutrient utilization (BIOLOG GEN III plates assay) and migratory ability in single inoculation experiments. However, mixed (1:1 ratio) inoculation experiments showed the wild-type to outcompete the mutant. Thus the T3SS possibly played a subtle role in the comigration along fungal hyphae, helping P. terrae in attachment to the host cell surface. Interestingly, this behaviour was shown with both L. sp. strain Karsten and Trichoderma asperellum 302 (Yang et al., 2016). The ability of P. terrae to adhere to fungal cell walls was then investigated (using ELISA), with a focus on the glycosphingolipids ceramide monohexosides (CMHs) that possibly play roles in fungal cell envelopes. The results showed adherence of the P. terrae wild-type to CMH of L. sp. strain Karsen but not to that of T. asperellum 302 and to a lesser extent of the ΔsctD mutant. Subsequently, addition of P. terrae BS001 enhanced the total biomass with L. sp. strain Karsten but not with T. asperellum 302 (Haq et al., 2016).
Work with a flagellar mutant (ΔfliP) and a T4P one (ΔpliN) revealed the essential role of functional flagella for migration along with fungal hyphae, next to a minor positive effect of the T4P. In conclusion, the migration of P. terrae BS001 along fungal hyphae was posited to be a strongly flagellar-driven process (Yang et al., 2017). The movement of P. terrae strain BS001 towards the fungal-released compounds glycerol and oxalic acid has also been addressed (Haq et al., 2016, 2018). Both compounds indeed promoted the movement of the cells. A transcriptome study of P. terrae strain BS001 in confrontation with L. sp. strain Karsten on soil extract agar plates supported the data with respect to the key role of chemotaxis. Confrontation
with fungal hyphae upregulated the expression of chemotaxis and flagellar motility genes, as well as of oxalate uptake and utilization gene clusters (i.e. oxalate/ formate antiporter oxalyl-CoA decarboxylase, CoA transferases, formyl-CoA transferase, tartronic semialdehyde reductase and glyoxylate carboligase).
LOG-CFU PER GRAM SOIL
A
Abundance and diversity
BB Biofilm C Mutants generated N.S. N.S. *** SWIMMING WT∆SctD ∆pilN∆fliP D Mobility HO HO HO Glycerol O O HO HO Oxalic acid E Chemotaxis 0.01 7648 97 Five-gene cluster F Genomic sequencing and transcriptome G G
?
C E MycosphereFigure 1.1. Paraburkholderia terrae strains isolated from the mycosphere. (A) Abundance and diversity assessments using culture-dependent and culture-independent methods showed that P. terrae to be mycosphere inhabitant (Warmink and van Elsas, 2008), (B) P. terrae strain BS001 forming biofilms on Lyophyllum sp. Karsten hyphae (Haq et al., 2016; Nazir et al., 2012; Warmink and Van Elsas, 2009), (C) Mutants generated to investigate the mechanisms behind the interactions of P. terrae BS001 and fungal hyphae; ΔSctD, ΔpilN and ΔfliF (Yang et al., 2016, 2017, 2018), (D) Mobility/comigration along L. sp. hyphae is flagellar-driven (swimming mobility) and type 4 pili (T4P) and type 3 secretion system (T3SS) offer an anchoring/ twitching devices (Yang et al., 2017, 2018), (E) P. terrae BS001 moved toward fungal released compounds, including glycerol and oxalic acid (Haq et al., 2016, 2018), (F) Genomic analyses and transcriptome studies enhanced our understanding on the fungal-interactive behaviour of P. terrae BS001, reveling the important of a five-gene cluster for energy generation at the fungus (Haq et al., 2014, 2017). (G) The “enigmatic role” of bacteriophages in shaping P. terrae genomes.
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However, no enhancement of expression was observed for a glycerol uptake (GUP) gene. The latter finding indicated that (i) glycerol might have been at low concentration and (ii) in the current setting, glycerol uptake might have occurred through another mechanism, such as passive diffusion through the membrane. Interestingly, a five-gene cluster was dynamically modulated and significantly upregulated upon physical contact with the fungal mycelium. This five-gene cluster includes genes for (i) alkyl hydroperoxidase AhpD family core domain protein, (ii) a cupin, (iii) a LysR transcriptional regulator, (iv) a putative nucleoside-diphosphate sugar epimerase (nmrR like) and (v) a conserved exported protein: alpha/beta hydrolase fold. To my surprise, this five-gene cluster, presumably involved in aerobic energy generation, was found to be conserved across several other fungal-interactive Paraburkholderia strains. In summary, to this point, it is hard to pinpoint to which extent and in what temporal manner P. terrae employs the plethora of capabilities in its interaction with soil fungi. However, the studies performed so far have certainly broadened the understanding of the interaction of P. terrae with its fungal counterparts in soil. Considering the soil microhabitat from which the P. terrae strains were obtained and in which the organism probably thrives, i.e. the mycosphere, one may posit that P. terrae needs to be phenotypically flexible. The reason is that the mycosphere can be short-lived, and so the organism may – following selection by active fungi – be delivered into the bulk soil, where it will be subjected to the vagaries of that system. It may thus be expected that such a flexible phenotype is supported by flexibility at the genetic level. Considering the latter, HGT was found to have contributed to the shaping of the genome of P. terrae strain BS001 (Haq et al., 2014). The comparative genome analyses performed indicated that only 4% of the P. terrae strain BS001 genome could be identified as the core genome, the remainder belonging to variable parts, including prophage and plasmid regions and genomic islands (Haq et al., 2014). However, the work by Haq et al, with one exception, stopped short of an in-depth analysis of the prophage/island regions. I therefore here placed a focus on these, using both phage culture-dependent (i.e. double-agar layer, spot-assay, induction) and –independent approaches (mining phage signal from bacterial genome and virome community metagenomics - viromics) as the method of choice.
Evolution in the mycosphere: horizontal gene transfer
Bacterial evolution is rarely a simple speciation event. Rather, it is a complex process, in which processes like HGT, duplications, phage host switching and phage/island gains and losses play roles (Conow et al., 2010; Hutchinson et al., 2017). In early work, van Elsas et al., (1988) showed that the rhizosphere of wheat constitutes a habitat
that incites gene (plasmid) transfer among the local bacteria. Fairly recently, Zhang et al., (2014) provided arguments for the notion that the mycosphere also constitutes a soil hotspot that drives HGT.
In general, HGT is facilitated by mobile genetic elements (MGEs), which include, in a broad sense, bacteriophages, plasmids, genomic islands, transposons and integrons (see Figure 1.2). HGT occurs through the three main mechanisms conjugation, transformation and transduction. Transformation is the uptake of naked DNA, whereas conjugation involves the transfer of genetic material from donor to recipient cells by cell-to-cell contact. Transduction (generalized and specialized) is phage-mediated gene transfer in which donor DNA can be incorporated into the genome of the recipient cell (or persist as a plasmid). There is an increased interest in the study of HGT in soil because of the concern about the spread of DNA from genetically-modified plants into microbiomes, next to the key role of HGT as an accelerator of bacterial evolution in soil. The major HGT mechanism transduction or effects of prophage presence on bacterial fitness have, unfortunately, not been extensively studied in soil. A study by Herron and Wellington, (1994) showed that in harst soil conditions Streptomyces lividans TK24 lysogenic for phage KC301 decreased in abundance as a consequence of phage excision/lysis. This indicated a possible strategy of prophage KC301, i.e. active excision, allowing subsequent transduction, in order to survive. However, the ecological significant of this putative phage-mediated HGT is still unclear.
Currently, it is estimated that between 1.6 and 32.6 % of all genes that occur in bacterial genomes have been acquired by HGT (Boto, 2010). HGT clearly provides a venue for the reassortment of newly-introduced (as well as existing) genes in bacterial genomes. Such genomic plasticity is at the basis of diversification and adaptive evolution (see Box 1.1). An early study by Freeman (1951) already demonstrated that Corynebacterium diphtheriae infected with bacteriophage gives origin to enhanced virulence, providing an evolutionary asset for the newly emerged organism. Furthermore, Strauch et al., (2008) showed that phage 2851 of Shigella sonnei transfers (stx) virulence genes to ’naive’ S. sonnei cells, turning these into virulent forms. Thus, major changes in bacterial lifestyle are driven by phage-dependent HGT processes. Clearly, phages play important roles in the evolution of pathogenic bacteria, resulting in the intricate co-evolution of these partners (see review by Brüssow et al., 2004).
Key factors that influence phage fate in soil are the spatial separation between these and recipient. Water availability aids the mobility (and survival) of bacterial cells, within a range for optimal survival of between (roughly) 25 and 70% of WHC – water-holding capacity (Yang and van Elsas, 2018). Excess water in the soil leads to
1
unfavourable anaerobic conditions, while water scarcity leads to stress, forcing cell to become inactive or silent.
With respect to genetic barriers, DNA:DNA homology is a major determinant of recombinational success and hence a lack of homology poses a ‘general’ barrier to recombination-dependent gene ‘fixation’ mechanisms. Other barriers encompass the aforementioned incompatibilities of incoming DNA with available molecular machinaries pertaining to fixation in the recipient bacterium (van Elsas et al., 1986). A review by Baltrus, (2013) defined the ‘cost’ of HGT as a sum of all the inefficiencies in the genotypic/phenotypic changes that determine the evolutionary potential. This includes chromosomal distruption, misfolded protein, distruption in transcription-translation and suboptimal protein function and system-level effects (e.g. loss of phenotype, altered metabolic flux).
A B C D E Bacteriophage Transposon Plasmid Integron Mobile gene cassette Transduction Prophage
Figure 1.2. Transfer of DNA between bacterial cells facilitated by some of the mobile elements. (A) bacteriophage, (B) transposon, (C) conjugative plasmid, (D) integron and (E) mobile gene cassette.
Box 1.1. Proposal on how to assess phage-mediated evolution?
Precise and accurate methods to quickly identify phage-mediated evolution events, for example HGT, are needed to examine the processes that have shaped the genomes of bacteria in soil. The current low-cost and advanced next-generation sequencing technologies and the growth of whole genome sequence availability have enabled large-scale analyses of such events. A review by de Carvalho and Loreto, 2012 divided the methods that allow to identify evolutionary events in complete genomes into so-called ‘surrogate’ or ‘comparative’ methods. The former class does not require the construction of phylogenetic trees to identify evolutionary events. The base composition of bacterial genomes is relatively uniform and it takes time for newly introduced genes to adapt to the recipient host genome. The methods are based on the proposition that newly acquired sequences harbor nucleotide compositions that are different from those in the recipient genome. The method examines the best match of genes in a genome. Such surrogate methods provide quick scans of evolutionary events. However, high rates of false positive results have been obtained and so care is required in interpretation of the results. The second method uses phylogenetically-based analyses to detect the occurrence of evolutionary events. This method can produce more details, as the involved species, time of the evolutionary event and direction of transfer can be estimated. Using phylogenetically based analyses allows to locate the order, absence or loss of genes in closely-related species. Evolutionary events can also be identified using methods based on inferring phylogenetic incongruence. A disagreement between a robust gene tree and the known species phylogeny is often best explained by invoking the occurrence of HGT (Fitzpatrick et al., 2008). Phylogenetic incongruence is often quite successful and has been widely adopted in microbial relatedness studies (Hutchinson et al., 2017). The drawback of phylogenetic tree analysis to identify HGT events lies in the fact that it generates large data sets, is laborious and requires computational skills and resources. Additionally, the donor-recipient unipartite network has been used to characterize the extent of HGT mediated by phahe transduction (Popa et al., 2017). However, in the end, the choice of methods to identify HGT events should be clearly based on the availability of data sets. Surrogate methods may be only applicable for large-scale analyses, quick scans, and no data are suitable for the use of comparative methods. Furthermore, comparative methods using phylogenetic tree analysis should be employed when full-genome data sets are available for the analysis.
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Evolution, diversity and ecological role of phages
Viruses
Viruses are extremely abundant agents (Suttle, 2007) and they are also highly diverse. Viruses are able to infect three domains of life, i.e. Eukarya, Bacteria and Archaea (see Table 1.1). The origin and evolution of viruses are still rather enigmatic. Three major hypotheses have been proposed (see reviews by Forterre, 2006):
(1) The virus-first hypothesis: viruses emerged as first ‘organisms’ in the primitive-soup/precellular world,
(2) The evolutionary reduction hypothesis: viruses originated from a simple cell that lost its replication machinery and became dependent on another cell to survive and (3) The evolutionary escape hypothesis: viruses originated from genes of cellular
organisms that became selfish elements.
A close inspection of the existing theories of viral origin and evolution led Koonin et al., (2006) to propose “an ancient virus world hypothesis” (see Figure 1.3). This hypothesis was based on the notion that the primordial world may have allowed a rampant and extensive flow of genetic information through a variety of selfish elements. The continuation of the “virus world” theory from the primitive soup world may have enabled the evolution of the first primitive selfish genetic elements into fully-fledged viruses (RNA to DNA based) and other elements to the pre-cellular and eukaryogenesis stages (Figure 1.3A). Thus, this hypothesis offers a scenario in which the “ancient virus world” stages, to certain extent viruses, contribute to the evolution of cellular life forms (bacteria, archaea and eukarya) (Figure 1.3B). The ancient virus world may connect the origin of viral lineages and give a perspective of the origin of different types of cells (see details in Koonin et al., 2006; Koonin and Dolja, 2013). Koonin also emphasized the importance of compartmentalization (inorganic to the pre-cellular compartment/membrane) as a prerequisite for successful gene transfers, ensuring a sufficiently high concentration of substrates and genetic elements for effective replication and, in consequence, evolution. This scenario, in an era in which descent is uncertain, blurs the distinction between monophyly and polyphyly origins of viruses, as no genes are shared (there is no universal virus gene, such the small subunit RNA gene in all life forms) among viruses. This appears to rule out any conventional notion of common origin. However, homologies at the structural and mechanistic levels, for example, between viral RNA and DNA helicases and replication mechanisms (i.e. rolling circle replication - RCR), as well as commonalities in viral structures/hallmarkgenes (e.g. capsid gene), have been observed (Krupovic and Koonin, 2017). At present, viruses are seen as the driving force of evolution. Examples may be found across the board, however need to be addressed in the mycosphere- see Figure 1.3C.
Table 1.1.
The main features of soil viruses from three domains of life.
Domain Viruses Main F eatur es Ex amples Refer enc es Bacteria Bact erio -phages
Most studied vir
al type t ermed bact eriophage. Di verse genomes: dsDN A, ssDN A, dsRN A and +ssRN
A, linear and cir
cular
. Di
verse
morphotypes, most ha
ve icosahedr
al capsid, with or without
en
velope. Filament
ous and spherical t
o pleomorphic f
orms also
found. Based on their lif
esty le, phages ar e di vided int o l ytic v ersus ly sogenic ones. Man y e xamples:
Most phages belong t
o the Caudo vir ales (My oviridae, P odo viridae and Sipo viridae ), Ino viridae, Plasmaviridae and Leviviridae
Salmond and Finer
an, 2015 Archaea Ar chaea virus Widespr
ead, as hosts occur acr
oss e
xtr
eme and non-e
xtr eme en vir onments. Ha ve either dsDN A or ssDN A. Morphotypes di vided int o tw o cat egories: ( i) ar
chaea-specific, without structur
al or genetic count erparts in pr ok ary ot e and euk ary ot e domains ( ii)
cosmopolitan, with structur
e and genetic mak
e-up similar t
o
viruses in other domains.
Ar chaeal-specific vir al types: Ampullaviridae, Bic audaviridae, S pir aviridae and Fuse llo viridae Cosmopolitan-Ar chaeal viruses: My oviridae, S ipo viridae, P odo viridae, Sphaer olipo viridae and Turriviridae . Pr angish vili et al., 2017 Eukary a Plant viruses Di verse genomes: ssDN A, dsRN A, ssRN A(+), ssRN A(-), ssRN A (R T) and dsDN A (R T) viruses. Morphotypes di verse: non-en veloped, r ound, twinned ( geminat e) incomplet e icosahedr
al, double-capsid, filament
ous, rigid helical
rods, bacillif
orm. Others ha
ve en
veloped, allant
oid, spherical and
hig hl y fle xuous f orm. Tobacco mosaic t obamo virus (TMV), T omat o spott ed wilt t ospo virus (TSWV), T omat o yello w leaf cur l begomo virus (TYL CV), P otat o
virus Y (potyvirus, PVY), Cauliflo
w
er mosaic
caulimo
virus (CaMV) and P
otat o virus X (pot exvirus, PVX). Ry bicki, 2015 My co viruses Lack e xtr acellular phase, tr ansmitt ed via h yphal anast omosis or v erticall y. Oft en s ympt omless, ma
y change host phenotype,
hypo
virulent
, used in biocontr
ol. Di
verse genome structur
es:
linear dsRN
A, linear ssRN
A(+), linear ssRN
A(-) and cir
cular ssDN A. Totiviridae, Chr yso viridae, P artitiviridae, Meg abirnaviridae, Quadriviridae, R eo viridae, Alphafle xiviridae, Endornaviridae, Barnaviridae, Gamm afle xiviridae, Hypo viridae, Narnaviridae , and My comononeg aviridae Ghabrial et al., 2015 Arthr opod viruses Arthr opods ar e v ect
ors of viruses that inf
ect plants and mammals.
Mostl y RN A viruses (ssRN A(+)/ssRN A (-)). Fla vi virus, Ar bo viruses, T og aviruses, Bun ya viruses, Mesoni viruses, R eo viruses, Rhabdo viruses, Negi
viruses and Birn
viruses
Calisher and Higgs, 2018; Li et al., 2015
Nemat
ode
viruses
Kno
w
ledge still in its inf
ancy . Nemat ode: natur al v ect or f or some
plant viruses. Orsa
y virus is the first kno
wn virus that natur
all y inf ects Caenor habditis e leg ans or other nemat odes. It has icosahedr al vir on and ssRN A(+) genome. Orsa y virus inf ect Caenor habditis e leg ans Guo et al., 2014 Pr ot ozoal viruses The kno w ledge of pr ot
ozoal viruses is still in its inf
ancy . Viruses that inf ect Pr ot ozoa ar e mostl y RN A viruses. F or e xample, Cry spo viruses, dsRN A, icosahedr al viruses. Cry spo virus inf ects Tric homonas v aginalis viruses (TVV), Giar dia lamblia viruses. Mimi virus (
giant virus) inf
ects amoebae Colson et al., 2017; W ang and W ang, 1991
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1 3 4 5 2 3 3 2 2 1 1 5 5 4 4 (i) (ii) prophage genomes: HGTs P.hospita species-clusterBacteria Archaea Eukarya
LUCA
Primoldial “soup” (RNA world)
A
C
Host cell genome
(i) (ii) (iii) (iv) (v) (vi)
Time
Pre-Darwinian Darwinian
B
RNA world The emergence of capsid genes,more complex molecules and
RNA virus RNA to DNA The emergence of DNA virus LUCA Modern virus world
Figure 1.3. Viral evolution. The ancient viral world hypothesis, as proposed by Koonin et al., (2006); (A) Depiction of pre-evolutionary (blue) and evolutionary (pink) time. Only after a “threshold” (LUCA- last universal common ancestor) is positioned, Darwinian evolution takes place. (B) Ancient viral world scenario, in which viruses evolve and contribute to the evolution of cellular life forms. (i) Initially, selfish genetic elements (e.g. group I introns) are formed, as a consequence of rampant gene and RNA flow in the primordial soup; flow related to compartmentalization, e.g. inorganic compounds, (ii) continuation of this event, including emergence of capsid genes and more complex molecules; small genomes, that later evolve into RNA viruses, (iii) RNA viruses using primitive enzymes, transforming RNA into DNA, (iv) Mixed RNA-DNA systems promote retroid elements (i.e. group II introns), later DNA viruses, (v) Viruses (RNA and DNA) involved in “birth” of early life forms (LUCA) (vi) Emergence of three domains of life: bacteria, archaea and eukarya. Viruses as the “effectors” of ecology and evolution. (C) Present-day impact of viruses and phages on evolution, (i) the integration of prophages, rearranging host cell genomes, adding moron genes that spur ecological fitness and (ii) phages as mobile genetic elements (MGEs) in the mycospere.
Bacteriophages
Independent research in 1915 by Frederick Twort, and in 1917 by Félix d’Hérelle on micrococci and Shigella, respectively, discovered viruses of bacteria, denoted as bacteriophages (bacterium-eaters) (D’Herelle, 1917; Twort, 1915). From comparative genomics, we have learned that phages have significantly contributed to bacterial genome over evolutionary time (Touchon et al., 2009). Thus, often-new bacterial traits have emerged, allowing genetic flexibility to the host, which eventually helps
to adapt quickly to conditions in local environments. A case in point is given by E. coli in which a plethora of genomic islands – often including prophages - have been discovered that each confer different ecological behaviour to the host (Touchon et al., 2009).
Since their discovery, phages have significantly revolutionalized our fundamental understanding of biology, i.e. (i) establishing the central dogma of molecular biology, (ii) shown to be essential in ecological functioning and evolutionary of microbes and (iii) reservoir of accessory genes for modern biology techniques (e.g. CRISPR-Cas9) (see review by Salmond and Fineran, 2015). Although studies of soil viruses, including phages, have emerged only as from the early 1970s (see Table 1.2), they are still understudied. It is expected that soil viruses play significant roles in the ecological functioning and evolution of their hosts in soil.
Current understanding of, and new insights in, phage biology
The two known phage lifestyles, lytic action and lysogeny, govern the ecology of host cells. They also significantly contribute to the fate of cell populations in soil. For example, lysis (caused by lytic phages) essentially controls bacterial population densities – the phenomenon fits the “kill-the-winner” theory that stipulates that lytic “predation” suppresses the most abundant cell populations in a microbial community, thus modifying host diversity (see Box 1.2 on the phage-bacteria dynamics in highly dense bacterial populations). Lysis of local cells by phages in a biofilm releases extracellular DNA (eDNA) and polysaccharide that strengthens the structural integrity of the biofilm (see Obeng et al., 2016; Secor et al., 2015) for details on the role of phages in bacterial biofilm). In contrast, integrated prophages can offer beneficial genes that may advance the fitness of host cells.
The abundance of soil viruses has been determined in varying soil systems. A study by Srinivasiah et al., (2015) examined the mechanisms affecting the production and abundance of soil viruses as related to the extant bacterial community. Using microcosms, the authors focused on bacteriophage community dynamics. To avoid the growth of fungi, cycloheximide (100 µg/gdw) was applied. Three different carbon sources were added, i.e. chitin, yeast extract (YE) and methanol. Over time, soil samples were collected to assess both bacterial and viral community abundance and diversity, using fingerprinting assays. The results showed that soil viral titers in yeast extract increased from ~1 × 107 to 3.4 × 108 viruses/gdw on day 7 and then declined to 1.6 × 108 viruses/ gdw on day 30. In parallel, the soil bacterial populations went from about 1 × 106 to 8 × 107 bacterial cells/gdw on day 3, after which these gradually increased to 11 × 107 bacterial cells/gdw on day 30. Similar results were found with chitin, albeit with some delay for both bacterial and viral community increases.
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However, only subtle changes occurred in both communities with the methanol-treated and control microcosms. The fingerprint analyses (16S rRNA-based DGGE and RAPD-PCR) across treatments for both communities showed temporal changes in the community structures. The bacterial community compositions shifted in accordance with the amendments, with regards to 16S rRNA DGGE; the chitin and YE treatments showed greatest changes in 30 days, revealing ~34% similarity with the initial point. The lowest change occurred in the control and methanol treatments, with 56% and 49% similarity between initial and final points (30 days), respectively. These results showed that the ability of particular bacterial populations to utilize certain carbon sources spurs the growth of specific phage hosts, and, as a consequence, specific phage populations.
Box 1.2. Phage-bacterial host dynamics in dense bacterial populations
Lytic viruses regulate bacterial populations in accordance with the classical predator-prey model (called kill-the-winner; KtW). In this model, lytic viruses lyse the most abundant (presumably active) bacterial type in a community, promoting population control and bacterial diversity. However, in situations in which the bacterial host population is highly dense and therefore less actively growing, the rate of lysis by phages is decreased and - consequently - the virus-to-microbe ratio (VMR) lowered. This model is known as the ‘piggy-back-the-winner’ (PtW) model (Knowles et al., 2016). Indeed, in highly-dense bacterial populations, VLPs have been less observed. On the other hand, the abundances of integrases and excisionases were shown to be increased. The PtW strategy has emerged in lysogenic viruses to mitigate the highly-dense host condition. The energy cost to build up resistance in the host through superinfection to viral reproduction is lower than that in mutation ((Knowles et al., 2016). The strategy is universally observed in environments like soil and marine settings, as well as the human gut. We still ignore the extent to which the mechanism operates in the mycosphere. An evolutionary arms race often exists between a bacterial host and its phage. In consequence, a considerable diversity of (both bacterial and phage) defensive and offensive strategies has evolved. Thus, the restriction modification (R-M) system, the abortive bacterial infection (ABI) and clustered regularly interspaced short palindromic repeats and Cas associated proteins (CRISPR-Cas) have emerged. CRISPR array and spacers have been used to study the dynamic interaction and evolutionary history between host/phage partners (Puschnik et al., 2017; Touchon et al., 2016). The heritable immunity conferred by CRISPR-Cas systems offers a major fitness advantage for bacterial cells against specific foreign DNA or, in some cases, RNA.
Table 1.2
. Overview of studies on soil viruses
a.
Soil type / habitat
Vir al abundanc e Vir al di versity Methods used Remar ks Refer enc es Soil de void of v egetation and e xposed t o the sun, New Zealand 1.2 x 10 8 - 3.4 x 10 10 pfu ml -1 suspension Tailed phages DAL on Bacillus c er eus, B. coagulans, B. lic henif ormis, B. meg at erium, B. pumilus, B. sphaericus , B. subtilis, B.st ear ot hermophilus This stud y hig hlig hts that Bacillus phages ar e agents that acceler at e e volution. Reanne y and Marsh, 1973 Nile V alle y, Egypt > l0 6 pfu ml -1 suspension Tailed phages DAL on Az ot obact er chr ooc oc cum C32 This stud y assessed Az ot obact er
lytic phages in soils
Heg
azi and Jensen,
1973 Illinois, US A and tr opical soil, Br azil 10 9 - 10 l0 pfu ml -1 suspension
Tailed phage MJ-1 Sipho
viridae) and phages
MP9 t o MP50 (My oviridae and Sipho viridae) DAL on Bacillus.meg at erium F4 and B. meg at erium QM BI55I (A TC C I2872), respecti vel y
The papers hig
hlig ht the pot ential r ole of B. meg at erium phages in soil
Van Elsas and Penido, 1982; V
ary and Halse y, 1980 Rhizospher e ( Bet a vulg aris v ar . Am ythest , Poa pr at ensis L, Epibolium t etr ag onum L. Griseb. Senecio j ac obaea L., and Car damine fle xuos ) and r especti ve bulk soils, UK 1.5 x 10 7 - 1.5 x 10 8 VLP s g -1 dry soil Tailed phages Dir
ect counting TEM, on
S. quiniv or ans CP6 and P. aeruginosa PU21 De veloped methods f or counting the t otal numbers of bact eriophages in soil Ashelf or d et al. 2003 Agricultur al soil, US A 4.0 x 10 7 - 2.15 x 10 8 VLP s g -1 dry soil Tailed phages
TEM and EFM count (No significant diff
er ence fr om both methods) This stud y sho w
ed that land use
is a significant f
act
or influencing
phages abundance and di
versity in soils Williamson et al. 2005 Rhizospher e ( Triticum ae stivum ), bulk soil, UK 1.1 - 1.2 x 10 9 VLP s g -1 dry soil
Tailed phages (Caudo
vir ales), spherical, bacillif orm, filament ous and r od-shaped viruses Dir
ect counting (TEM)
Report
ed that majority of
virus particles observ
ed ar e indigenous soil viruses Sw anson et al. 2009
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Table 1.2
. Overview of studies on soil viruses
a. (Continued)
Soil type / habitat
Vir al abundanc e Vir al di versity Methods used Remar ks Refer enc es
Hyper-arid desert soil, Antar
ctic 2.3 - 6.4 x 10 8 VLP s g -1 dry soil Dominant-Caudo vir ales; signatur e gene: Adeno viridae, Bic audaviridae, Hytr osaviridae, Retr oviridae , and Rudiviridae
Virus community metagenomics (vir
ome) stud y This stud y hig hlig ht ed the fact
ors that contribut
e t o vir al abundance and di versity in deserts ecos yst em Zablocki et al. 2014
Namib desert soil, Africa
22 Mb vir ome reads Tailed phages Irido viridae, Ph yc odnaviridae , ssDN A viruses
Virus community metagenomics (vir
ome) stud y This stud y e valuat ed the r ole of aridity and x eric gr adient on
the both micr
obial and phages
community structur
e and
function in Namib desert soil,
Scola et al. 2017
Soil metagenomics
10,009 vir
al
contigs identified (IMG/VR)
18,470 vir al gr oups Mining vir al signal fr om bact erial metagenomes This stud y sho w ed vir al distribution acr oss di verse ecos yst ems; str ong habitat specificity f or the v ast majority of viruses, ne xt t o some cosmopolitan viruses. Paez -Espino et al. 2016 For
est soil, pastur
e land, cr opland 10 8 - 10 9 VLP g -1 dry soil Not mentioned Micr oscopic enumer ation,
virus community fingerprinting appr
oach This stud y sho w ed that sampling sit e w as a k ey f act or
for shaping the abundance and community structur
e of soil viruses Narr et al. 2017 My cospher e Up t o 10 8
copies of major capsid gene ml
-1 Tailed phages (ϕ437) Mit om ycin C induction Soil (inducible) pr ophage fr om P. t err ae BS437 Pr atama and v an Elsas 2017 aNotes: DAL:
double agar layer; PFU:
Plaque-forming unit; TEM: transmission
electron microscope;
EFM: Epi-fluorescence
Microscope;
Mitomycin C:
prophage
induction agent; IMG/VR: Integrated- microbial genome/Virus
(https://img.jgi.doe.gov/cgi-bin/vr/main.cgi); VLP:
Virus-like particles; ssDNA:
CRISPR spacers harbor a legacy of past infections by phages and/or other mobile elements. However, according to the current database, CRISPR-Cas systems are only present in about 45% of the bacterial genomes sequenced so far. The reasons for this meager prevalence are currently unknown. It is possible that, given that defense systems other than CRISPR-Cas have evolved, some bacteria have depended on these. Thus, systems like BREX (Goldfarb et al., 2015) and type IV pilus (T4P) glycosylation have recently been discovered (Harvey et al., 2018). A very recent study even described the existence of nine novel antiphage systems, next to one antiplasmid system (for details see Doron et al., 2018). Hence, a full account of all key systems that allow bacteria to deal with phages lies still ahead of us. It is also important to give attention to non-model bacteria and archaea, as it might lead to the discovery of other novel antiphage mechanisms.
In counter-defense, for example, anti-CRISPR proteins have evolved in phages. To date, 21 unique families of such anti-CRISPR proteins have been discovered (Pawluk et al., 2018). These are known as phage “moron” genes (Maxwell, 2017). Key “anti-CRISPRs” were recently discovered in Pseudomonas spp. phages (Maxwell, 2017; Pawluk et al., 2018). The genes for these “anti-CRISPR” proteins, such as AcrF1-5 and AcrE1-4, block CRISPR-Cas types I-F and I-E, respectively. The other “anti-CRISPRs” are encoded by anti-CRISPR associated gene 1 (aca1), giving the distinct proteins denoted AcrIIC1, AcrIIC2 and AcrIIC3 and AcrF6 to AcrF10 (Pawluk et al., 2018). The life cycle of E. coli phage lambda has long served as a simplified model for the lysis/lysogeny decision-making process, known as the “genetic switch”. Upon infection, phage lambda can make its host cell enter one of two distinct pathways, i.e. lysis or lysogeny. However, the trigger that ultimately determines the decision has been unknown. A recent study discovered that the Spβ group phages of Bacillus subtilis rely on the host communication system to decide whether to go through lytic or lysogeny phases upon infecting the bacterial host (Erez et al., 2017). This communication system, named arbitrium, is reminiscent of bacterial quorum sensing, in terms of function (as a cue of bacterial population activity is used) and protein sequence. It is regulated by a six-residue peptide known as arbitrium. High population densities of the bacterial host, leading to low per-cell arbitrium levels, induce the system to the lytic cycle. However, after several infection cycles, when the bacterial cell number is reduced, raised concentrations of arbitrium emerge. This signal leads phages to progressively enter the lysogenic state (for details see Erez et al., 2017). In summary, phage-host interactions are very intricate, and abundance-diversity, arms races and defense systems play key roles with bearing on ecological and evolutionary processes. Especially in soil, we are only at the beginning of exploring this phage-host dynamics and hence soil virome studies need greater attention.
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The effect of bacteriophages on the host
Here I will briefly give some examples of the eco-evolutionary effect of bacteriophages on host bacteria. The current growing interest in phage studies is spurred by our increased understanding of the ’fitness advantages’ that can be provided by lysogenic conversion to the host cell. This includes alteration of pathogenicity, of stress tolerance, of biofilm formation capacity and of metabolism of the host. For example, filamentous phage CTXϕ was found to be able to become integrated in the Vibrio cholerae chromosome in a site-specific manner (Faruque et al., 2002). Acquisition of the CTXϕ element by non-toxigenic V. cholerae was key to the emergence of V. cholerae as a pathogen. V. cholerae and CTXϕ have likely coevolved for mutual benefit. The production of cholera toxin by CTXϕ lysogens results in diarrhea in infected animals and promotes the transmission of V. cholerae. In return, CTXϕ is able to utilize host functions for reproduction without compromising V. cholerae. Integrated phages also offer protection to secondary phage infection, thus benefiting the host (Bondy-Denomy et al., 2016). The costs of the Rex system in E. coli carrying phage λ against E. coli HK97 were assessed in the absence and presence of the phage. As expected, E. coli HK97 populations were completely eradicated within short time, even with a very low phage load. In contrast, E. coli λ populations with the Rex system showed decreasing growth as the phage titer was higher. The benefit of the phage λ Rex system was clearly to strengthen the host defense mechanism, thus protecting the host cell populations. Phages thus mediate survival, diversification and selection of host populations, by a range of mechanisms (Refardt et al., 2013). In summary, the intricate relationship between phages and bacterial hosts is subjected to co-evolution –the reciprocal adaptation between interacting species in specific ecological niches (Obeng et al., 2016). It may shape microbial genotypic and phenotypic diversity. The examination of E.coli K-12 from which cryptic prophages were delected (CP4-6, DLP12, e14, rac, Qin, CP4-44, CPS-53, CPZ-55, CP4-57 and Δ9 – deleted all cryptic prophages) has shown that host fitness in adverse environments can be promoted. For instance, osmotic pressure, oxidative and acid stresses were survived better, and biofilm formation was affected. Furthermore, prophages enhance host resistance to sub-lethal concentrations of quinolone (i.e. CP4-6 and rac) and β-lactam antibiotics (i.e. rac and Qin) (Wang et al., 2010). Also, phages have been found to affect host metabolism, energy and nutrient acquisition (auxiliary metabolic genes – AMGs), as well as increase the early stages of biofilm formation. In summary, the presence of both lytic (i.e. population and diversity control, provide readily available nutrient and enhance hosts’ biofilm formation/structure) and lysogenic (i.e. AMGs, pathogenocity) phages affects the ecological functioning of individual host cells to the community level.
This thesis
General hypotheses of this thesis
Given the current state of knowledge with respect to the lifestyle and genomics of the mycosphere inhabitant Paraburkholderia terrae, I hypothesized that mobile genetic elements, notably phages, have shaped the genomes of P. terrae strains in soil and the mycosphere, and thus modulated the lifestyle of this organism. I also hypothesized that such lifestyle modulation has, to a considerable extent, contributed to the capacity of P. terrae to interact with soil fungi.
Specific hypotheses
In the light of the general hypotheses, I pose the following specific hypotheses for further exploration in this thesis:
(1) Mycosphere inhabitants such as P. terrae share genetic potential that enable them to, for example, interact with fungal mycelium that emerges in a soil setting, (2) Given that fungal mycelium is dynamic in time and space, P. terrae mycosphere inhabitants need to be genetically ‘flexible’; they thus foster the presence of prophages as key contributors to the flexibility. Such prophages occasionally give rise to phage progeny in biofilms, which – to a certain extent – improves the ‘health’ of the biofilm and so benefits the mycosphere P. terrae community, (3) Phage communities have shaped the evolutionary trajectory of Paraburkholderia
spp. with different life styles in different ways. Phage relatedness reflects host relatedness,
(4) The dynamics of gene flow across Paraburkholderia spp in the mycosphere can be followed through CRISPR-Cas system analyses in the respective genomes and (5) The mycosphere Paraburkholderia community contains unexplore viral
communities that, in conjunction with the host communities, differ from those in corresponding bulk soil.
Aim of the thesis and research questions
The aim of this thesis was to assess the putative role of bacteriophages in the ecology and evolution of the mycosphere inhabitant Paraburkholderia terrae. Given that no data on this topic existed prior to this work, I ignored the extent to which lytic phages would be prevalent. Hence, I used both culture-dependent and culture-independent approaches to enable the detection of both lytic and temperate phages in the P. terrae mycosphere communities. The culture-dependent approach consisted of phage isolation on the basis of the classical double agar layer (DAL) method and a spot-assay method. In addition, I investigated the prevalence of prophage sequences
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across a variety of Paraburkholderia genomes using sequencing and molecular tools. Subsequently, I used a combined culturing/metagenomics approach to analyze viromes produced from the mycosphere as well as soil by various bioinformatics tools. This thesis aims to provide a foundation for the integration of Paraburkholderia phages into models that depict host ecology as well as genetic processes in the mycosphere. I focused on the following research questions:
(1) How similar are the genomes of mycosphere inhabitants belonging to Paraburkholderia terrae and related species? I used genomic information obtained from P. terrae strains BS001, BS110, BS007 and BS4367; next to three type strains (P. terrae DSM 17804T, P. hospita DSM 17164T and P. caribensis DSM 13236T). Do these bacteria share the genetic potential that drives the fungal-interactive behaviour in the mycosphere?
(2) What is the state-of-the-art of knowledge on the diversity and role of soil phages? Do phages play a role in the ecology and evolution of Paraburkholderia spp.? (3) Are prophages present in diverse mycosphere/rhizosphere/soil-inhabiting
Paraburkholderia spp.?
(4) How is the interaction between mycosphere dwellers and their phages in the mycosphere?
(5) How diverse is the soil virome of a Paraburkholderia community selected from the mycosphere and to what extent did they contribute to host ecology and evolution?
Scope of this thesis
This thesis is divided into three parts. In the first part, I focus on the system-of-interest in this study, i.e. mycosphere-inhabiting Paraburkholderia strains. In the second part, I examine the conceptual and theoretical perspective on soil viruses. Finally, in the last part, I study the newly discovered inducible prophage from P. terrae strain BS437, ϕ437, and its eco-evolutionary significance in the genus Paraburkholderia. Also,I provide insight into the viral community in the mycosphere. Hereafter, I briefly introduce the aims and findings of each thesis chapter.
Section I: Genomics-based dissection of mycosphere-inhabiting
Paraburkholderia strains, yielding a basis for
bioinformatics-assisted searches for prophages
In chapter 2, in an attempt to give ecological significance to the current classification of soil Burkholderia spp., we reclassified the former B. terrae strains BS007, BS110 and BS437 to Paraburkholderia terrae. This reclassification was based on
our molecular genome analyses and on the fact that these bacteria are excellent mycosphere colonizers. We, thus, report the draft genome sequences of these three fungal-interactive Paraburkholderia terrae strains. We compared the three genomes and showed that all strains possess the genetic repertoire necessary for adaptation to diverse soil niches, including those influenced by soil fungi, as well as plants. Furthermore, all genomes were found to harbor highly diverse metabolic (primary and secondary) systems, mobility, biofilm formation, secretion systems (T1SS, T2SS, T3SS, T4SS and T6SS), glycerol uptake systems and energy generating gene clusters. Finally, our analyses also highlight the presence of prophage-like sequences, complete or incomplete, in the three P. terrae genomes.
In chapter 3, I used whole-genome sequencing to explore the evolutionary relationships and ecological versatilities of P. terrae DSM 17804T, P. hospita DSM 17164T and P. caribensis DSM 13236T, next to selected own P. terrae isolates. The type strains have been used as taxonomical and ecophysiological references for a subgroup within the genus Paraburkholderia. Our study showed that the previously designated P.terrae strains BS001, BS007, BS110 and BS437 were closely related to P.hospita DSM 17164T. They share the recommended threshold for species circumscription (using 95-96% threshold for ANIm and >0.99 for TETRA values) of the taxon. Moreover, the
P. terrae type strain DSM 17804T also adhered to the criterion, but P. caribensis DSM
13236T did not. Through genomic analyses of all type strains we showed that they all share highly similar features with respect to fungal- (and plant-) interactive traits. This includes: secretion systems, biofilm formation, glycerol and oxalate uptake, IAA production, ACC deaminase and nodulation genes. Overall, the results confirm the tight relatedness of the type strains P. terrae DSM 17804T and P. hospita DSM 17164T, together with all own P. terrae isolates. This, next to the more remote species
P. caribensis DSM 13236T.
Section II: Conceptualisation and theoretical perspective on (soil)
viruses
Chapter 4 examines the current huge gap in virome studies between soil and marine ecosystems. We examined the state-of-the-art of the environmental virome studies, with a focus on soil in comparison to marine systems. After a description of the selective forces exerted on organisms in marine versus soil settings, we examine the current understanding of phage abundance, diversity, evolution and putative role in these ecosystems. We also identify the current challenges in this area and suggest potential strategies to boost our knowledge with respect to the relevant questions on the soil virome.
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In chapter 5, together with co-authors, I provide a perspective on the ecological and evolutionary consequences for bacteria interacting with phages, when phage and host interests are aligned. Temperate phages can introduce a plethora of genes that provide functions of different kinds to their hosts. Such lysogenic conversions can take the form of stable phenotypic changes (e.g. auxiliary metabolic genes (AMGs), virulence factors) or increased host plasticity (e.g. stress tolerance, quorum sensing), thereby improving bacterial survival under changing conditions. We argue that phage–host antagonism and mutualism get blurred at the population scale. The negative consequences of infection that an individual may experience may actually lead to population-level benefits or vice versa. Furthermore, we examine the question whether bacterial hosts can lower immune barriers to phage infection, thereby facilitating infection by beneficial phages. Taking recent evidence together, we suggest that in many cases temperate phages are to be considered as being mutualistic as well as parasitic, at the same time.
Section III: Paraburkholderia viruses and their evolutionary
trajectory
In chapter 6, I present the first study on a prophage induced from the mycosphere-isolated P. terrae BS437, denoted ɸ437. Moreover, our analyses showed evidence for the contention that all of the analyzed Paraburkholderia sequences contain substantial amounts of prophage regions. Most of these regions were remnants of prophages that have been subjected to host selective pressure (deletion bias). As a consequence, prophages often lose their abilities to produce progeny. The inducible phage ɸ437 was apparently ‘spontaneously’ released and its particle numbers were raised by successful induction with mitomycin C. The experimental results and genomic analysis of ɸ437 showed evidence for a lysogenic lifestyle of this phage with its host. Importantly, I found one moron gene (orf64) that potentially endows the host with a superinfection defense mechanism against other phage infections, enhancing host fitness and biofilm formation. However, experimental works still need to be done to support this finding.
In chapter 7, using the prophage found in chapter 6 and other prophages identified across the genus Paraburkholderia, we examined to what extent the relationship between Paraburkholderia and their prophages can be taken to have bearing on evolutionary processes. We found the genome structures of the full prophages in the selected Paraburkholderia species to be highly diverse, yet classifiable into five defined groups. Moreover, phage remnants were frequently found. We also found some genes (e.g. DNA adenine methylase) that might be advantageous for the host cell. Cophylogenetic analyses showed complex coevolutionary scenarios between the
Paraburkholderia hosts and their prophages that include cospeciation, duplication, host-switching and phage losses. These coevolutionary scenarios also yielded insight into the interaction between these partners. Analysis of CRISPR-Cas systems observed in the Paraburkholderia genomes showed the record of past phage infections. In summary, our results revealed the importance and extent of the role of phages with respect to the coevolution with their Paraburkholderia host.
In chapter 8, I performed a viral metagenomics (virome) study with focus on the mycosphere. Two fungi collected at a forest floor were selected for the study, next to a forest floor bulk soil. Using the respective microbiomes as inocula, we selectively grow Paraburkholderia/Burkholderia in semi-selective medium (PCAT), followed by growth of the resulting community and identification of the respective virome upon induction. This study thus addressed the dynamic diversity of the Paraburkholderia/ Burkholderia community from two forest habitats, the mycosphere versus the bulk soil, next to the diversity of its virome upon induction.
In chapter 9, I present a synthesis and discussion of all results obtained throughout this thesis. I provide insight in the importance of integration of viruses into considerations of the soil microbial ecosystem. Phages play a significant role in the ecology and evolution of soil microbiomes, especially with respect to mycosphere inhabitants. Future research avenues in, and practical applications of, soil viromes for host cell and soil microbial community-level questions are also discussed.
Draft genome sequences of three
fungal-interactive Paraburkholderia terrae
strains, BS007, BS110 and BS437
Akbar Adjie Pratama, Irshad Ul Haq, Rashid Nazir,Maryam Chaib De Mares and Jan Dirk van Elsas Published in Stand. Genomic Sci. (2017) 12:81 doi: 10.1186/s40793-017-0293-8.
Abstract
Here, we report the draft genome sequences of three fungal-interactive Paraburkholderia terrae strains, denoted BS110, BS007 and BS437. Phylogenetic analyses showed that the three strains belong to clade II of the genus Burkholderia, which was recently renamed Paraburkholderia. This novel genus primarily contains environmental species, encompassing non-pathogenic plant- as well as fungal-interactive species. The genome of strain BS007 consists of 11,025,273 bp, whereas those of strains BS110 and BS437 have 11,178,081 and 11,303,071 bp, respectively. Analyses of the three annotated genomes revealed the presence of (1) a large suite of substrate capture systems, and (2) a suite of genetic systems required for adaptation to microenvironments in soil and the mycosphere. Thus, genes encoding traits that potentially confer fungal interactivity were found, such as type 4 pili, type 1, 2, 3, 4 and 6 secretion systems, and biofilm formation (PGA, alginate and pel) and glycerol uptake systems. Furthermore, the three genomes also revealed the presence of a highly conserved five-gene cluster that had previously been shown to be upregulated upon contact with fungal hyphae. Moreover, a considerable number of prophage-like and CRISPR spacer sequences was found, next to genetic systems responsible for secondary metabolite production. Overall, the three P. terrae strains possess the genetic repertoire necessary for adaptation to diverse soil niches, including those influenced by soil fungi.
Keywords: Paraburkholderia terrae, mycosphere, fungal-interactive, genome sequence
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Introduction
The genus Burkholderia was coined in 1993 by Yabuuchi et al., (1992). Following this, continuing emendation of the genus has occurred, mainly as a result of the addition of new species. Recent molecular and phylogenetic analysis of the genus divided it into two clades, with clade I containing the pathogenic Burkholderia spp. and clade II mainly environmental bacteria. The latter clade was reclassified as a novel genus, named Paraburkholderia (Estrada-De Los Santos et al., 2013; Sawana et al., 2014). This genus encompasses a suite of highly diverse and environmentally adaptable bacteria that are able to occupy various ecological niches, ranging from soil (Nazir et al., 2012b; Salles et al., 2002) to plants and humans (Sahl et al., 2015). Members of the genus Paraburkholderia are also known to harbor some of the largest genomes among all known bacteria (Compant et al., 2008; Haq et al., 2014).
Paraburkholderia terrae strain BS001, which was isolated as a co-migrator in soil with the saprotrophic fungus Lyophyllum sp. strain Karsten (Warmink and van Elsas, 2008), has been extensively described, and it is used here as a reference organism. P. terrae strain BS110 was isolated from the mycosphere of the ecotomycorrhizal fungus Laccaria proxima (Nazir et al., 2012b; Warmink and van Elsas, 2008) and also showed comigration capacity with the aforementioned fungus. The other two Paraburkholderia terrae strains (BS007, BS437) were isolated – similarly – as mycosphere dweller / comigrator, from soils collected in Gieterveen and Wageningen, the Netherlands, respectively (Nazir et al., 2012b; Warmink and van Elsas, 2008). Being avid mycosphere inhabitants, all these Paraburkholderia strains might play essential roles in the ecology of soil fungi and so in (degradative) ecosystem functions. Several studies have been performed to address such interactions and understand the mechanisms involved. An in-depth study of the genome of P. terrae strain BS001 revealed its remarkable genetic potential, including genetic systems that presumably enable it to interact with saprotrophic fungi like Lyophyllum sp. strain Karsten (Haq et al., 2014; Nazir et al., 2012b). Moreover, the strain BS001 genome was found to contain numerous regions of genomic plasticity that are typified by different plasmid- and prophage-like genes (Haq et al., 2014). We took this finding as a token of the remarkable ability of P. terrae to adapt – via horizontal gene transfer - to fluctuating local challenges, including the presence of fungal counterparts. The strategies that are presumably used in this fungal interactivity include (but are not limited to): (i) biofilm formation on fungal surfaces (Haq et al., 2016; Warmink and van Elsas, 2008), (ii) a type-3 secretion system (T3SS) with a subtle role in the cellular migration along fungal hyphae and adherence (Haq et al., 2016; Yang et al., 2016) and (iii) chemotaxis towards growing fungal hyphae and subsequent adherence to fungal surfaces (Haq et al., 2016). In a recent study, it was shown that P. terrae strain BS001 differentially
