Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.Endophytic bacteria reside within the living tissue of plants without substantively harming them. They are of high interest for agrobiotechnological applications, such as the improvement of plant growth and health, phytoremediation 1 or even as biofertilizer 2 . Supply of nitrogen derived from fixation of atmospheric N 2 by grass endophytes, such as Gluconacetobacter diazotrophicus and Azoarcus sp. strain BH72, which has been shown to occur in sugarcane 3 and Kallar grass 2 , is a process of potential agronomical and ecological importance.Although the lifestyle of these endophytes is relatively well documented, the molecular mechanisms by which they interact beneficially with plants have only been poorly elucidated. A combination of features makes Azoarcus sp. strain BH72 an excellent model grassendophyte 4 . (i) It supplies nitrogen derived from N 2 fixation to its host, Kallar grass (Leptochloa fusca (L.) Kunth); in planta it is usually not culturable, but can be detected by culture-independent methods based on nifH-encoding nitrogenase reductase, the key enzyme for N 2 fixation 2 . (ii) It colonizes nondiseased plants in remarkably high numbers: estimates range from 10 8 cells (culturable cells per gram root dry weight (RDW) of field-grown Kallar grass 5 ) to 10 10 cells (estimated on the basis of abundance of bacterial nifH-mRNA in roots) 2 . (iii) It is the only cultured grass endophyte shown by molecular methods to be the most actively N 2 -fixing bacterium of the natural population in roots 2 . (iv) It also colonizes the roots of rice, a cereal of global importance, in high numbers (10 9 cells per g RDW) in the laboratory, and spreads systemically into shoots 6 . Plant stress response is only very limited in a compatible, that is, well-colonized rice cultivar 7 . Notably, Azoarcus sp. strain BH72 is capable of endophytic N 2 -fixation inside the roots of rice 8 .For a wider application in agriculture, more knowledge is required on mechanisms o...

Azoarcus sp. strain BH72, as an endophyte of grasses, depends on successful host colonization. Type IV pili are essential for mediating the initial interaction with rice roots. In the genome sequence analysis, the pilT gene was identified, which encodes for a putative type IV pilus retraction protein. PilT of Azoarcus sp. BH72 shares high similarity to PilT of the human pathogen Pseudomonas aeruginosa PAO1 (77% amino acid sequence identity) and contains a predicted nucleotide-binding motif. To gain more insights into the role of the type IV pili in the colonization process of Azoarcus spp., we constructed an insertional mutant of pilT and a deletion mutant of pilA, the major structural component of the pilus structure. The pilT mutant, as the pilin deletion mutant deltapilA, was abolished in twitching motility. Western blot analyses and electron microscopy studies demonstrated an enhanced piliation of the Azoarcus pilT mutant strain compared with the wild type, indicating that, indeed, PilT has a role in pilus retraction. Studies on rice root colonization in gnotobiotic cultures revealed that the establishment of microcolonies on the root surface was strongly reduced in the deltapilA mutant, whereas the surface colonization was reduced by only 50% in the nontwitching pilT mutant. However, endophytic colonization of rice roots was strongly reduced in both mutants. These results demonstrate that the retractile force mediated by PilT is not essential for the bacterial colonization of the plant surface, but that twitching motility is necessary for invasion of and establishment inside the plant. Thus, a novel determinant for endophytic interactions with grasses was identified.

BackgroundBacterial communication is involved in regulation of cellular mechanisms such as metabolic processes, microbe-host interactions or biofilm formation. In the nitrogen-fixing model endophyte of grasses Azoarcus sp. strain BH72, known cell-cell signaling systems have not been identified; however, the pilA gene encoding the structural protein of type IV pili that are essential for plant colonization appears to be regulated in a population density-dependent manner.Methodology/Principal FindingsOur data suggest that pilAB expression is affected by population density, independent of autoinducers typical for Gram-negative bacteria, likely depending on unknown secreted molecule(s) that can be produced by different bacterial species. We used transcriptomic and proteomic approaches to identify target genes and proteins differentially regulated in conditioned supernatants in comparison to standard growth conditions. Around 8% of the 3992 protein-coding genes of Azoarcus sp. and 18% of the detected proteins were differentially regulated. Regulatory proteins and transcription factors among the regulated proteins indicated a complex hierarchy. Differentially regulated genes and proteins were involved in processes such as type IV pili formation and regulation, metal and nutrient transport, energy metabolism, and unknown functions mediated by hypothetical proteins. Four of the newly discovered target genes were further analyzed and in general they showed regulation patterns similar to pilAB. The expression of one of them was shown to be induced in plant roots.Conclusion/SignificanceThis study is the first global approach to initiate characterization of cell density-dependent gene regulation mediated by soluble molecule(s) in the model endophyte Azoarcus sp. strain BH72. Our data suggest that the putative signaling molecule(s) are also produced by other Proteobacteria and might thus be used for interspecies communication. This study provides the foundation for the development of robust reporter systems for Azoarcus sp. to analyze mechanisms and molecules involved in the population-dependent gene expression in this endophyte in future.

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BackgroundTurbulence intensity, or hydromechanical stress, is a parameter that influences a broad range of processes in the fields of chemical engineering and biotechnology. Fermentation processes are often characterized by high agitation and aeration intensity resulting in high gas void fractions of up to 20% in large scale reactors. Very little experimental data on hydromechanical stress for such operating conditions exists because of the problems associated with measuring hydromechanical stress under aeration and intense agitation.ResultsAn indirect method to quantify hydromechanical stress for aerated operating conditions by the measurement of maximum stable drop size in a break-up controlled dispersion was applied to characterize hydromechanical stress in reactor scales of 50 L, 3 m3 and 40 m3 volume with a broad range of operating conditions and impeller geometries (Rushton turbines). Results for impellers within each scale for the ratio of maximum to specific energy dissipation rate ϕ based on measured values of maximum stable drop size for aerated operating conditions are qualitatively in agreement with results from literature correlations for unaerated operating conditions. Comparison of data in the different scales shows that there is a scale effect that results in higher values for ϕ in larger reactors. This behavior is not covered by the classic theory of turbulent drop dispersion but is in good agreement with the theory of turbulence intermittency. The data for all impeller configurations and all aeration rates for the three scales can be correlated within ±20% when calculated values for ϕ based on the measured values for dmax are used to calculate the maximum local energy dissipation rate. A correlation of the data for all scales and all impeller configurations in the form ϕ = 2.3∙(ϕunaerated)0.34∙(DR)0.543 is suggested that successfully models the influence of scale and impeller geometry on ϕ for aerated operating conditions.ConclusionsThe results show that besides the impeller geometry, also aeration and scale strongly influence hydromechanical stress. Incorporating these effects is beneficial for a successful scale up or scale down of this parameter. This can be done by applying the suggested correlation or by measuring hydromechanical stress with the experimental method used in this study.