Peter H. Janssen

Identifying the Dominant Soil Bacterial Taxa in Libraries of 16S rRNA and 16S rRNA Genes

From near to far , from here to there , funny things are everywhere. —Dr. Seuss, One Fish Two Fish Red Fish Blue Fish In 1909, H. Joel Conn ([25][1]) expressed the hope that methods would soon be at hand by which the significance of the different bacteria present in any soil could be

Three Genomes from the Phylum Acidobacteria Provide Insight into the Lifestyles of These Microorganisms in Soils

ABSTRACT The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria, the Cyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N2 fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

Laboratory Cultivation of Widespread and Previously Uncultured Soil Bacteria

ABSTRACT Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria. In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.

Structure of the Archaeal Community of the Rumen

Members of the domain Archaea contribute about 0.3 to 3.3% of the microbial small subunit (16S and 18S) rRNA in the rumen (22, 39, 60). Archaea have a range of different metabolisms and are found in many habitats (6), but those known to exist in the rumen are strictly anaerobic methanogens. Yanagita et al. (59) observed that 2.8 to 4.0% of ruminal microorganisms displayed autofluorescence characteristic of F420, a methanogen cofactor, able to be seen under UV illumination during microscopy. Taken together with the small subunit rRNA abundance data, this suggests that a large part of the archaeal population is made up of methanogens. Most species of methanogens can grow using H2 and often formate as their energy sources and use the electrons derived from H2 (or formate) to reduce CO2 to CH4. Some species can grow with methyl groups, oxidizing some to CO2 to produce electrons that are used to reduce further methyl groups to methane. A few species can grow with acetate, effectively dissimilating acetate to CH4 and CO2. However, acetate is not metabolized to CH4 to any significant extent in the rumen (13). This is probably because the rate of passage of rumen contents through the rumen is greater than the growth rate of acetate-utilizing methanogens (53). In a normally functioning rumen, proteins and polymeric carbohydrates, which usually make up the largest part of the incoming feed, are fermented by a mixed microbial community to volatile fatty acids (VFAs), NH4+, CO2, and H2. The hydrogen is metabolized by the methanogens. The VFAs are taken up by the animal across the rumen wall and serve as major carbon and energy sources for the ruminant. A part of the VFAs, undigested feed components, and microbial cells leave the rumen and enter the rest of the animals digestive tract. The central role of H2 in the rumen fermentation (12) means that, although methanogenic archaea make up only a small part of the rumen microbial biomass, they play an important role in rumen function and animal nutrition. Efficient H2 removal leads to a nutritionally more favorable pattern of VFA formation and to an increased rate of fermentation by eliminating the inhibitory effect of H2 on the microbial fermentation (26, 53). The rumen can be simplistically described as an open system with discontinuous solid (feed) and liquid (saliva and drinking water) inputs and multiple fractions that have different turnover rates (53). The methanogens in the rumen are found free in the rumen fluid, attached to particulate material and rumen protozoa, associated as endosymbionts within rumen protozoa, and attached to the rumen epithelium. The methanogens associated with these different fractions can be expected to have different growth rates since they will be removed from the rumen at different rates. In addition, the animal itself and the feed also influence the rate of passage of digesta through the rumen system (25). These different habitats may allow niche division among the methanogens and may explain some of the observed phylogenetic diversity of rumen archaea.

Effects of Growth Medium, Inoculum Size, and Incubation Time on Culturability and Isolation of Soil Bacteria

ABSTRACT Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes (including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.

Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range.

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific.

The Genome Sequence of the Rumen Methanogen Methanobrevibacter ruminantium Reveals New Possibilities for Controlling Ruminant Methane Emissions

Background Methane (CH4) is a potent greenhouse gas (GHG), having a global warming potential 21 times that of carbon dioxide (CO2). Methane emissions from agriculture represent around 40% of the emissions produced by human-related activities, the single largest source being enteric fermentation, mainly in ruminant livestock. Technologies to reduce these emissions are lacking. Ruminant methane is formed by the action of methanogenic archaea typified by Methanobrevibacter ruminantium, which is present in ruminants fed a wide variety of diets worldwide. To gain more insight into the lifestyle of a rumen methanogen, and to identify genes and proteins that can be targeted to reduce methane production, we have sequenced the 2.93 Mb genome of M. ruminantium M1, the first rumen methanogen genome to be completed. Methodology/Principal Findings The M1 genome was sequenced, annotated and subjected to comparative genomic and metabolic pathway analyses. Conserved and methanogen-specific gene sets suitable as targets for vaccine development or chemogenomic-based inhibition of rumen methanogens were identified. The feasibility of using a synthetic peptide-directed vaccinology approach to target epitopes of methanogen surface proteins was demonstrated. A prophage genome was described and its lytic enzyme, endoisopeptidase PeiR, was shown to lyse M1 cells in pure culture. A predicted stimulation of M1 growth by alcohols was demonstrated and microarray analyses indicated up-regulation of methanogenesis genes during co-culture with a hydrogen (H2) producing rumen bacterium. We also report the discovery of non-ribosomal peptide synthetases in M. ruminantium M1, the first reported in archaeal species. Conclusions/Significance The M1 genome sequence provides new insights into the lifestyle and cellular processes of this important rumen methanogen. It also defines vaccine and chemogenomic targets for broad inhibition of rumen methanogens and represents a significant contribution to worldwide efforts to mitigate ruminant methane emissions and reduce production of anthropogenic greenhouse gases.

Clostridium paradoxum DSM 7308T contains multiple 16S rRNA genes with heterogeneous intervening sequences

Sequence analysis of the cloned 16S rRNA genes of Clostridium paradoxum DSM 7308T revealed the presence of 15 different sequences in variable region I (Escherichia coli position 73-97) of the 16S rRNA. The majority of the cloned genes contained intervening sequences (IVSs), which varied in length from 120-131 nt, and were present in the DNA obtained from single colonies of C. paradoxum. The absence of IVSs in the mature rRNA was demonstrated by Northern hybridization and sequence analysis of the 16S rRNA reverse transcriptase (RT)-PCR product. This finding was supported by the failure of oligonucleotide probes specific for certain IVSs to hybridize to the RT-PCR product obtained from C. paradoxum. Alterations in culture conditions (temperature, pH, salt) or culture age did not lead to expression of RNA containing IVSs, as indicated by the size of RT-PCR products. Hybridization of the restriction-enzyme-digested genomic DNA of C. paradoxum with probes derived from the IVSs demonstrated that the 16S rRNA genes containing different IVSs are located at different sites on the chromosome.

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable.

ABSTRACT Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (106 CFU/ml) were obtained on solid media. Long incubation times (≥8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the communitys bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.