Kings without crowns: Analysis of abundant bacilli in different soil ecosystems Vesela A. Tzeneva Promotor: Prof. dr. W. M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Co-promotoren: Dr. H. Smidt Universitair Docent, Laboratorium voor Microbiologie Wageningen Universiteit Dr. A. D. L. Akkermans Universitair Hoofddocent, Laboratorium voor Microbiologie Wageningen Universiteit Promotiecommissie: Prof. Dr. P. de Vos Universiteit Gent, Belgiё Prof. Dr. J. D. van Elsas Rijksuniversiteit Groningen Prof. Dr. A. H. C. van Bruggen Wageningen Universiteit Dr. Ir. J. Dolfing Globalviewmanagement, Deventer Dit onderzoek is uitgevoerd binnen de onderzoekschool SENSE 2 Kings without Crowns: Analysis of abundant bacilli in different soil ecosystems Vesela A. Tzeneva Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M.J. Kropff, in het openbaar te verdedigen op maandag 9 oktober 2006 des namiddags te 16.00 uur in de Aula. 3 Vesela A. Tzeneva – Kings without crowns: Analysis of abundant bacilli in different soil ecosystems – 2006 Thesis Wageningen University, Wageningen, The Netherlands – With summary in Dutch ISBN – 90-9020989-1 4 Посвещавам на Татко Dedicated to my Father 5 6 Content Abstract 9 Chapter 1 Introduction 11 Chapter 2 Isolation and biodiversity of hitherto undescribed soil bacteria related to Bacillus niacini 35 Chapter 3 Development and application of a selective PCR DGGE approach to detect a recently cultivated Bacillus-group predominant in the soil 55 Chapter 4 Riding the ecological time machine: succession of abundant soil bacteria in reclaimed land 79 Chapter 5 Eukaryotic diversity in historical soil samples 99 Chapter 6 Effect of soil drying and storage on the bacterial and eukaryotic diversity monitored by DGGE 117 Chapter 7 Comparative genomics of Bacillus benzoevorans related strains using a Bacillus subtilis microarray 141 Chapter 8 Discussion 155 Summary 171 Samenvatting 175 Acknowledgement 179 List of Publications 183 About the Author 185 7 8 Abstract This study describes culture-dependent as well as culture-independent strategies to monitor diversity and abundance of Bacillus benzoevorans-related soil bacteria. These bacteria are wide spread around the world, inhabiting a variety of terrestrial environments. A distinguishing feature of the family Bacillaceae is their ability to form endospores. This capacity makes the genus Bacillus amenable to retrospective and biogeographical studies, as their endospores provide the means for survival under environmental conditions of stress, allowing their detection later on. For the rapid detection of B. benzoevorans-related populations in soil samples selective cultivation media and group-specific primers and probes were developed. Using these techniques the global distribution of this group was demonstrated and indicated their adaptive capacity to diverse soil ecosystems. A unique soil sample archive provided insight in the microbiological impact of land reclamation and flooding. Using multivariate statistical approaches were used to assess the microbial community dynamics over time and in response to the changing environmental conditions. Found in a variety of soil ecosystems, microorganisms related to B. benzoevorans seem to be able to populate a broad range of niches, which indicates a high degree of metabolic versatility and strong adaptive capability. Moreover, they account for a significant part of the total bacterial community (up to 30 %). Based on our exploratory study the importance of their role is just indicated, but not acknowledged yet. Therefore, it is proposed to regard B. benzoevorans relatives as ‘kings without crowns’; as this group of bacteria deserves more scientific attention in future studies aiming to unravel their eco-physiology and functionality as major players of the soil microbiota. 9 10 CHAPTER 1 INTRODUCTION Chapter 1 General introduction A single shovel full of rich garden soil contains more species of organisms than can be found seen aboveground in the entire Amazon rain forest. The soil ecosystem is known to provide ecological niches for an extremely high number of microorganisms (94). The relationship between the diversity of living organisms and the ecosystem functions has become an important issue of modern microbial ecology. Microbial communities in the soil are involved in major processes, essential for the existence and sustainability of any terrestrial ecosystem, i.e. decomposition of organic matter (27) and mineralization of nutrients in soil (8). Alongside with this important ecosystem-defining function, soil microorganisms, mainly bacteria and fungi, provide an essential contribution to a wealth of other processes, including nitrogen fixation, solubilization of parent rock minerals, synthesis of plant-growth-promoting and other physiologically active substances, etc. Moreover, microorganisms take part in the decomposition of toxic inorganic and organic pollutants in the soil (13, 25, 82, 83, 85). When excluding plants, soil organisms can be largely divided into bacteria, archaea and eukarya (fungi, protozoa, nematodes, arthropods and earthworms). Among these, bacteria, archaea and fungi comprise the majority of the soil microbiota and are functionally important communities for soil fertility. In agricultural and grassland soils bacteria often are the predominant group (42). Soil microbiota The genus Bacillus The genus Bacillus, belonging to the family Bacillaceae within the Firmicutes, was first discovered by Ferdinand Cohn in 1872 with Bacillus subtilis as the first member. A distinguishing feature of the family is their ability to form endospores. Endospores are structures formed within bacterial cells that enable the cell to resist environmental conditions of stress. The members of the genus Bacillus are furthermore characterized as Gram-positive, rod-shaped, and aerobic or facultatively anaerobic (70). In Bergey's Manual of Systematic Bacteriology (1st ed. 1986), the G+C content of known species of Bacillus is listed to range from 32 to 69% (46), illustrating the genomic heterogeneity of the genus. Variations are observed from species to species, and sometimes 12 Introduction profound differences in G+C content are even found among strains within a species. For example, the G+C content of the B. megaterium group ranges from 36 to 45%. According to the “Bacterial Nomenclature up-to-date” (DSMZ -Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, http://www.dsmz.de/bactnom/bactname.htm), there are currently 188 species and 6 subspecies recognized in the genus Bacillus (available data in May 2006). A large number of valid species have been designated and novel species are discovered regularly, as evidenced by the fact that only since 2005, 29 novel species have been described. The genus Bacillus is both taxonomically and metabolically diverse. The primary habitat is the soil and associated plants or rivers, although some species are pathogenic for mammals (e.g. B. anthracis) and insects (e.g. B. sphaericus, B. thuringensis) (103). Bacilli form an important part of the microbiota in many soils, and many species are of considerable practical importance. Some Bacillus spp., including those that are thermophilic, are crucial for biotechnological applications Figure 1. Electron micrograph of B. cereus ATCC 10987 (T. Lindbäck, as they are sources of enzymes and other products of http://mlstoslo.uio.no/bacillus.html, industrial interest (7). Other Bacillus spp., such as B. accession date: 13 June 2006). thuringensis, B. subtilis, B. cereus and B. sphaericus, play important roles as biological control agents of different phytopathogenic organisms (24, 75, 86). Some members of the genus Bacillus are able to produce antibiotics. The majority of these antibiotics are low-molecular-weight peptides with different biological activities, including antimicrobial, antiviral, and antitumor activities (41), and several Bacillus isolates obtained from soil and plants have recently been reported to be capable of enzymatic inactivation of N-acyl homoserine lactone quorum sensing signals (19). The ability of Bacillus to form highly resistant endospores imparts an enormous competitive advantage in environments such as soil, where long periods of drought and nutrient deprivation are common (103). At least 4% of the 4.2 Mbp large B. subtilis genome, comprising 4100 protein-encoding genes (51), is dedicated to the processes of sporulation, germination and out-growth. Bacteria in temperate soils must be able to adapt rapidly to changes in ambient temperatures. For this purpose the B. subtilis genome encodes numerous stress proteins, including the chaperones (44). The ability of Bacillus spp. to secrete a wide variety of extracellular depolymerases is a major factor contributing to their colonisation in soil. Genes encoding secreted amylases, arabinases, chitonases, mannanases, cellulases and xylanases are 13 Chapter 1 evident in the genome sequence. Proteases are also frequently encountered, both intracellular and extracellular, the latter allowing for the use of protein as sources of both carbon and nitrogen (103). To date, the complete genomes of 12 strains of Bacillus have been elucidated, which all showed to be relatively large, ranging from 4.2 Mbp (B. halodurans C-125) to 5.6 Mbp (B anthracis AMES 0581) (http://genomesonline.org, accession date: 18 March 2006). This might reflect the ability of bacilli to survive and adapt to frequent