Biological nitrogen fixation plays an important role in terrestrial ecosystems, reaching approximately the same amount of N2 fixed which is industrially reduced as fertilizer. Our research focuses on diazotrophic bacteria which are endophytes of grasses, especially rice. We are particularly interested in elucidation of (1) the molecular cross-talk between the bacteria and their host, (2) the complex signal-transduction cascades allowing adaptation of bacterial reactions towards changes in the environment, and (3) of the population structure and activities of these bacteria in the natural environment.
Endophytic bacteria are capable of systemic infection and colonization of inner tissues of plants, without causing symptoms of plant disease. In grasses, nitrogen-fixing bacteria such as Azoarcus spp. or e.g. Herbaspirillum seropedicae colonize the root cortex and, to a lesser extent, the vessels. In most cases they cannot be isolated from root-free soil, indicating that they are ecologically dependent on the host plant. Our model system consists of a pioneer plant, Kallar grass, which grows luxouriantly on low-fertility soils in the Punjab of Pakistan, and Azoarcus spp., diazotrophs abundant inside its roots and expressing nitrogenase genes in there. In gnotobiotic culture, these bacteria are also capable of infecting rice seedlings and expressing nitrogenase genes endophytically.
The genome sequence of the diazotrophic model endophyte Azoarcus sp. strain BH72 has been elucidated! (Krause, A. et al. (2006) Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72. Nat. Biotechnol. 24:1385-1391)
This allows us to carry our functional genomic analysis on both partners, the bacterial colonizer and the host rice, in order to learn more about the molecular interplay of the endophytic association. We apply microarrays for transcriptome analysis, proteome analysis, mutational analyses and RNASeq. These techniques will greatly facilitate the solution of many questions addressed below.
(Methods: Bioinformatics, microarras, proteome analysis by 2D-PAGE and mass spectrometry, insertional mutagenesis, RNASeq).
The life style of endophytic bacteria raises many questions about their functions inside the plant, their host specificity and compatibility. Which metabolites are exchanged between bacteria and the host? What is necessary for successful establishment inside a plant such as rice? Which bacterial genes are specifically expressed inside the roots? Some of these questions are addressed by us by reporter gene studies. An example is the transcriptional fusion of nifH to gfp or gus, which allowed us to detect and visualize bacterial expression of nitrogenase genes in the apoplast of rice roots (see below).
How do endophytes establish in roots? The first crucial event in the infectious process is the attachment of the bacteria to the host surface. In many human and mammal pathogens, type IV pili are virulence factors mediating bacterial adherence to host cell epithelia as a prerequesite for invasion. Type IV pili play a role also in plant-microbe and bacteria-fungus interactions: An unusually short type IV pilin gene (pilA) and a second gene in the same operon (pilB) are both required for pilus formation in Azoarcus sp. Both genes are also necessary for establishment of microcolonies on root achsils of rice seedlings and for attachment to the mycelium of a rhizospheric ascomycete, indicating there are common traits in the initial steps of interaction of bacteria with different eukaryotic hosts. Twitching motility appears to be specifically required for endophytic spreading in rice. Moreover, a cell-surface-bound cellulase (endoglucanase) is required for ingress into plant cells. Cyclic-di-GMP signalling, flagella, the ability to utilize ethanol as a plant-derived carbon source, and type VI secretion systems also affect the interaction.
(Methods: light-, fluorescence- and electron microscopy, cloning techniques, quantification of gene expression e.g. by fluoroimager, qRT-PCR).
How the plant responds to the endophytic interaction and which plant genes affect the endophytic interaction is now also in focus.
Azoarcus sp. strain BH72 has some unusual features with respect to nitrogen fixation. N2 fixation, which occurs only under microaerobic conditions, may become more active and efficient in empirically optimized batch cultures at extremely low O2 concentrations. Under these conditions, intracytoplasmic membrane stacks, "diazosomes", are formed which are likely to be involved in efficient nitrogen fixation.
Therefore, we were unraveling the signal transduction cascade regulating fixation and assimilation of nitrogen. Azoarcus sp. is again unusual in this respect, e.g. expressing three different paralogous copies of gln genes encoding PII-like proteins, and using protein interactions with the Rnf membrane complex for regulatuion of "switch off" of nitrogenase activity.
An important aspect of the endophytic lifestyle is the regulation of bacterial genes during the association with plants. Endophytic gene expression and signal transduction cascades e.g. those related to quorum sensing or expression of the type VI secretion system, are studied.
(Methods: functional genomics e.g. by proteome/transcriptome analysis, cloning techniques and mutant construction, site-directed mutagenesis, immunological methods, protein overexpression, qRT-PCR, RNAseq).
The predominant diazotrophic endophytes of Kallar grass are members of the beta subgroup of the Proteobacteria, Azoarcus sensu lato spp. Due to a polyphasic taxonomic study, this genus was splitted, resulting in description of three additional genera and species, Azovibrio restrictus, Azonexus fungiphilus and Azospira oryzae. The latter occurs also in association with rice, especially wild rice species. Improvment of isolation techniques now allow us to cultivate a large porportion of the rice microbiome.
(Methods: classical and novel high-throughput isolation techniques, polyphasic taxonomy including 16S rDNA sequencing, genomic fingerprints)
In order to study the population structure in roots in the natural ecosystem, we are using a molecular approach which can also detect strains that are up to now uncultured. It is based on detection by PCR and molecular phylogenetic analysis of bacterial genes in root DNA, e.g. 16S rRNA genes or structural genes of nitrogenase (nifH). These studies can give an insight into the diversity of root-associated diazotrophs: most strains which we detected are up to now uncultured and therefore of unknown capacities! We developed methods to detect bacterial gene expression and thus bacterial functions in the natural niche, by analysis of bacterial mRNA. Nitrogen-cycling in soils and in association with plants, e.g. in the context of shifting cultivation regimes for rice from flooded to non-flooded, is analyzed by these molecular techniques. In order to learn more about functions of non-cultures endophytes, we analyzed the rice endophyte metagenome, and have extended these studies by metatranscriptomic analyses.
(Methods: PCR, RT-PCR, cloning and sequence analysis, molecular phylogeny, DGGE, T-RFLP, Next generation sequencing of amplicons and metatranscriptomes/genomes).
In the framework of cooperation projects The future Okavango (TFO) and SASSCAL, we are focusing on improvement of yields of smallholder farmers in Namibia and Botswana. Local pulses are often not well nodulated in poor Kavango soils. Thus we isolate adapted rhizobia from this region to develop inoculants for local pulses such as cowpea and Bambara groundnut.