Modulation of rosR Expression and Exopolysaccharide Production in Rhizobium leguminosarum bv. trifolii by Phosphate and Clover Root Exudates

The acidic exopolysaccharide (EPS) secreted in large amounts by the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii is required for the establishment of an effective symbiosis with the host plant Trifolium spp. EPS biosynthesis in rhizobia is a very complex process regulated at both transcriptional and post-transcriptional levels and influenced by various nutritional and environmental conditions. The R. leguminosarum bv. trifolii rosR gene encodes a transcriptional regulator with a C2H2 type zinc-finger motif involved in positive regulation of EPS synthesis. In silico sequence analysis of the 450-bp long rosR upstream region revealed the presence of several inverted repeats (IR1 to IR6) and motifs with significant identity to consensus sequences recognized by PhoB and LysR-type proteins associated with phosphate- and flavonoid-dependent gene regulation in R. leguminosarum. Using a set of sequentially truncated rosR-lacZ transcriptional fusions, the role of the individual motifs and the effect of phosphate and clover root exudates on rosR expression were established. In addition, the significance of IR4 inverted repeats in the repression, and P2–10 hexamer in the activation of rosR transcription, respectively, was found. The expression of rosR increased in the presence of phosphate (0.1–20 mM) and clover root exudates (10 μM). PHO boxes and the LysR motif located upstream of the rosR translation start site were engaged in the regulation of rosR transcription. The synthesis of EPS and biofilm formation decreased at high phosphate concentrations, but increased in the presence of clover root exudates, indicating a complex regulation of these processes

Rhizobium leguminosarum bv. trifolii rosR is required for interaction with clover, biofilm formation and adaptation to the environment

<p>Abstract</p> <p>Background</p> <p><it>Rhizobium leguminosarum </it>bv. <it>trifolii </it>is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants <it>Trifolium </it>spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as <it>Trifolium</it>, <it>Pisum</it>, <it>Vicia</it>, and <it>Medicago </it>spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the <it>R. leguminosarum </it>bv. <it>trifolii </it>RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.</p> <p>Results</p> <p>In this work, we extend our earlier studies to the characterization of <it>rosR </it>mutants which exhibit pleiotropic phenotypes. The mutants produce three times less exopolysaccharide than the wild type, and the low-molecular-weight fraction in that polymer is greatly reduced. Mutation in <it>rosR </it>also results in quantitative alterations in the polysaccharide constituent of lipopolysaccharide. The <it>rosR </it>mutants are more sensitive to surface-active detergents, antibiotics of the beta-lactam group and some osmolytes, indicating changes in the bacterial membranes. In addition, the <it>rosR </it>mutants exhibit significant decrease in motility and form a biofilm on plastic surfaces, which differs significantly in depth, architecture, and bacterial viability from that of the wild type. The most striking effect of <it>rosR </it>mutation is the considerably decreased attachment and colonization of root hairs, indicating that the mutation affects the first stage of the invasion process. Infection threads initiate at a drastically reduced rate and frequently abort before they reach the base of root hairs. Although these mutants form nodules on clover, they are unable to fix nitrogen and are outcompeted by the wild type in mixed inoculations, demonstrating that functional <it>rosR </it>is important for competitive nodulation.</p> <p>Conclusions</p> <p>This report demonstrates the significant role RosR regulatory protein plays in bacterial stress adaptation and in the symbiotic relationship between clover and <it>R. leguminosarum </it>bv. <it>trifolii </it>24.2.</p

Intragenomic diversity of Rhizobium leguminosarum bv. trifolii clover nodule isolates

BACKGROUND: Soil bacteria from the genus Rhizobium are characterized by a complex genomic architecture comprising chromosome and large plasmids. Genes responsible for symbiotic interactions with legumes are usually located on one of the plasmids, named the symbiotic plasmid (pSym). The plasmids have a great impact not only on the metabolic potential of rhizobia but also underlie genome rearrangements and plasticity. RESULTS: Here, we analyzed the distribution and sequence variability of markers located on chromosomes and extrachromosomal replicons of Rhizobium leguminosarum bv. trifolii strains originating from nodules of clover grown in the same site in cultivated soil. First, on the basis of sequence similarity of repA and repC replication genes to the respective counterparts of chromids reported in R. leguminosarum bv. viciae 3841 and R. etli CFN42, chromid-like replicons were distinguished from the pool of plasmids of the nodule isolates studied. Next, variability of the gene content was analyzed in the different genome compartments, i.e., the chromosome, chromid-like and 'other plasmids'. The stable and unstable chromosomal and plasmid genes were detected on the basis of hybridization data. Displacement of a few unstable genes between the chromosome, chromid-like and 'other plasmids', as well as loss of some markers was observed in the sampled strains. Analyses of chosen gene sequences allowed estimation of the degree of their adaptation to the three genome compartments as well as to the host. CONCLUSIONS: Our results showed that differences in distribution and sequence divergence of plasmid and chromosomal genes can be detected even within a small group of clover nodule isolates recovered from clovers grown at the same site. Substantial divergence of genome organization could be detected especially taking into account the content of extrachromosomal DNA. Despite the high variability concerning the number and size of plasmids among the studied strains, conservation of the location as well as dynamic distribution of the individual genes (especially replication genes) of a particular genome compartment were demonstrated. The sequence divergence of particular genes may be affected by their location in the given genome compartment. The 'other plasmid' genes are less adapted to the host genome than the chromosome and chromid-like genes

Older Adults and Voice Interaction: A Pilot Study with Google Home

In this paper we present the results of an exploratory study examining the potential of voice assistants (VA) for some groups of older adults in the context of Smart Home Technology (SHT). To research the aspect of older adults' interaction with voice user interfaces (VUI) we organized two workshops and gathered insights concerning possible benefits and barriers to the use of VA combined with SHT by older adults. Apart from evaluating the participants' interaction with the devices during the two workshops we also discuss some improvements to the VA interaction paradigm

Human-Centered Programming: The Design of a Robotic Process Automation Language

RPA (Robotic Process Automation) helps automate repetitive tasks performed by users, often across different software solutions. Regardless of the RPA tool chosen, the key problem in automation is analyzing the steps of these tasks. This is usually done by an analyst with the possible participation of the person responsible for the given activity. However, currently there exists no one-size-fits-all description language, which would allow to record, process, and easily automate steps of specific tasks. Every RPA solution uses a different notation, which is not easily human-readable, editable, and which cannot be applied to a different automation platform. Therefore, in this paper, we propose a new eXtensible Robotic Language (XRL) that can be understood by both programmers and non-programmers to automate repetitive business processes.Comment: 8 page