Arabidopsis HECT and RING-type E3 Ligases Promote MAPKKK18 Degradation to Regulate Abscisic Acid Signaling.

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling pathways that transduce extracellular signals into diverse cellular responses. Arabidopsis MAPKKK18 is a component of the MAPKKK17/18-MKK3-MPK1/2/7/14 cascades, which play critical roles in abscisic acid (ABA) signaling, drought tolerance and senescence. A very important aspect of MAP kinase signaling is both its activation and its termination, which must be tightly controlled to achieve appropriate biological responses. Recently, the ubiquitin-proteasome system (UPS) has received increasing attention as a key mechanism for maintaining the homeostasis of MAPK cascade components and other ABA signaling effectors. Previous studies have shown that the stability of MAPKKK18 is regulated by the UPS via the ABA core pathway. Here, using multiple proteomic approaches, we found that MAPKKK17/18 turnover is tightly controlled by three E3 ligases, UPL1, UPL4 and KEG. We also identified lysines 154 and 237 as critical for MAPKKK18 stability. Taken together, this study sheds new light on the mechanism that controls MAPKKK17/18 activity and function.

Genomic and Metabolic Characterization of Plant Growth-Promoting Rhizobacteria Isolated from Nodules of Clovers Grown in Non-Farmed Soil.

The rhizosphere microbiota, which includes plant growth-promoting rhizobacteria (PGPR), is essential for nutrient acquisition, protection against pathogens, and abiotic stress tolerance in plants. However, agricultural practices affect the composition and functions of microbiota, reducing their beneficial effects on plant growth and health. Among PGPR, rhizobia form mutually beneficial symbiosis with legumes. In this study, we characterized 16 clover nodule isolates from non-farmed soil to explore their plant growth-promoting (PGP) potential, hypothesizing that these bacteria may possess unique, unaltered PGP traits, compared to those affected by common agricultural practices. Biolog profiling revealed their versatile metabolic capabilities, enabling them to utilize a wide range of carbon and energy sources. All isolates were effective phosphate solubilizers, and individual strains exhibited 1-aminocyclopropane-1-carboxylate deaminase and metal ion chelation activities. Metabolically active strains showed improved performance in symbiotic interactions with plants. Comparative genomics revealed that the genomes of five nodule isolates contained a significantly enriched fraction of unique genes associated with quorum sensing and aromatic compound degradation. As the potential of PGPR in agriculture grows, we emphasize the importance of the molecular and metabolic characterization of PGP traits as a fundamental step towards their subsequent application in the field as an alternative to chemical fertilizers and supplements.

The first genomic characterization of a stable, hemin-dependent small colony variant strain of Staphylococcus epidermidis isolated from a prosthetic-joint infection.

Phenotype switching from a wild type (WT) to a slow-growing subpopulation, referred to as small colony variants (SCVs), supports an infectious lifestyle of Staphylococcus epidermidis, the leading cause of medical device-related infections. Specific mechanisms underlying formation of SCVs and involved in the shaping of their pathogenic potential are of particular interest for stable strains as they have been only rarely cultured from clinical specimens. As the SCV phenotype stability implies the existence of genetic changes, the whole genome sequence of a stable, hemin-dependent S. epidermidis SCV strain (named 49SCV) involved in a late prosthetic joint infection was analyzed. The strain was isolated in a monoculture without a corresponding WT clone, therefore, its genome was compared against five reference S. epidermidis strains (ATCC12228, ATCC14990, NBRC113846, O47, and RP62A), both at the level of the genome structure and coding sequences. According to the Multilocus Sequence Typing analysis, the 49SCV strain represented the sequence type 2 (ST2) regarded as the most prominent infection-causing lineage with a worldwide dissemination. Genomic features unique to 49SCV included the absence of the Staphylococcal Cassette Chromosome (SCC), ~12 kb deletion with the loss of genes involved in the arginine deiminase pathway, and frameshift-generating mutations within the poly(A) and poly(T) homopolymeric tracts. Indels were identified in loci associated with adherence, metabolism, stress response, virulence, and cell wall synthesis. Of note, deletion in the poly(A) of the hemA gene has been considered a possible trigger factor for the phenotype transition and hemin auxotrophy in the strain. To our knowledge, the study represents the first genomic characterization of a clinical, stable and hemin-dependent S. epidermidis SCV strain. We propose that previously unreported indels in the homopolymeric tracts can constitute a background of the SCV phenotype due to a resulting truncation of the corresponding proteins and their possible biological dysfunction. Streamline of genetic content evidenced by the loss of the SCC and a large genomic deletion can represent a possible strategy associated both with the SCV phenotype and its adaptation to chronicity.

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction.

BRM Complex in Arabidopsis Adopts ncBAF-like Composition and Requires BRD Subunits for Assembly and Stability.

ATP-dependent SWI/SNF chromatin remodelling complexes are conserved multi-subunit assemblies that control genome activity. Functions of SWI/SNF complexes in plant development and growth have been well established, but the architecture of particular assemblies is unclear. In this study, we elucidate the organization of Arabidopsis SWI/SNF complexes formed around a BRM catalytic subunit, and define the requirement of bromodomain-containing proteins BRD1/2/13 for the formation and stability of the entire complex. Using affinity purification followed by mass spectrometry, we identify a set of BRM-associated subunits and demonstrate that the BRM complexes strongly resemble mammalian non-canonical BAF complexes. Furthermore, we identify BDH1 and 2 proteins as components of the BRM complex and, using mutant analyses, show that BDH1/2 are important for vegetative and generative development, as well as hormonal responses. We further show that BRD1/2/13 represent unique subunits of the BRM complexes, and their depletion severely affects the integrity of the complex, resulting in the formation of residual assemblies. Finally, analyses of BRM complexes after proteasome inhibition revealed the existence of a module consisting of the ATPase, ARP, and BDH proteins, assembled with other subunits in a BRD-dependent manner. Together, our results suggest modular organization of plant SWI/SNF complexes and provide a biochemical explanation for mutant phenotypes.

A New Face of the Old Gene: Deletion of the PssA, Encoding Monotopic Inner Membrane Phosphoglycosyl Transferase in Rhizobium leguminosarum, Leads to Diverse Phenotypes That Could Be Attributable to Downstream Effects of the Lack of Exopolysaccharide.

The biosynthesis of subunits of rhizobial exopolysaccharides is dependent on glycosyltransferases, which are usually encoded by large gene clusters. PssA is a member of a large family of phosphoglycosyl transferases catalyzing the transfer of a phosphosugar moiety to polyprenol phosphate; thus, it can be considered as priming glycosyltransferase commencing synthesis of the EPS repeating units in Rhizobium leguminosarum. The comprehensive analysis of PssA protein features performed in this work confirmed its specificity for UDP-glucose and provided evidence that PssA is a monotopic inner membrane protein with a reentrant membrane helix rather than a transmembrane segment. The bacterial two-hybrid system screening revealed interactions of PssA with some GTs involved in the EPS octasaccharide synthesis. The distribution of differentially expressed genes in the transcriptome of the ΔpssA mutant into various functional categories indicated complexity of cell response to the deletion, which can mostly be attributed to the lack of exopolysaccharide and downstream effects caused by such deficiency. The block in the EPS biosynthesis at the pssA step, potentially leading to an increased pool of UDP-glucose, is likely to be filtered through to other pathways, and thus the absence of EPS may indirectly affect the expression of proteins involved in these pathways.

Genome-Wide Association Study Identifies Two Novel Loci Associated with Female Stress and Urgency Urinary Incontinence.

PURPOSE: Genome-wide association studies have not identified replicable genetic risk loci for stress or urgency urinary incontinence. MATERIALS AND METHODS: We carried out a discovery stage, case control, genome-wide association study in 3 independent discovery cohorts of European women (8,979) for stress incontinence, urgency incontinence, and any incontinence phenotypes. We conducted replication in 6 additional studies of European ancestry (4,069). We collected bladder biopsies from women with incontinence (50) to further investigate bladder expression of implicated genes and pathways and used symptom questionnaires for phenotyping. We conducted meta-analyses using inverse variance fixed effects models and whole transcriptome analyses using Affymetrix® arrays with replication with TaqMan® polymerase chain reaction. RESULTS: In the discovery stage, we identified 16 single nucleotide polymorphisms genotyped or imputed at 5 loci that reached genome-wide significance (p <5×10-8). In replication, rs138724718 on chromosome 2 near the macrophage receptor with collagenous structure (MARCO) gene (replication p=0.003) was associated with stress incontinence. In addition, rs34998271 on chromosome 6 near the endothelin 1 (EDN1) gene (replication p=0.0008) was associated with urgency incontinence. In combined meta-analyses of discovery and replication cohorts, associations with genome-wide significance for these 2 single nucleotide polymorphisms were confirmed. Transcriptomics analyses showed differential expression of 7 of 19 genes in the endothelin pathway between stress and urgency incontinence (p <0.0001). CONCLUSIONS: We uncovered 2 new risk loci near the genes endothelin 1 (EDN1), associated with urgency incontinence, and macrophage receptor with collagenous structure (MARCO), associated with stress incontinence. These loci are biologically plausible given their roles in smooth muscle contraction and innate host defense, respectively.

Structure of the disaccharide repeating unit of O-specific polysaccharide isolated from Aeromonas veronii strain Bs8 pathogenic to common carp (Cyprinus carpio).

The O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Aeromonas veronii strain Bs8, which is pathogenic to common carp (Cyprinus carpio), after mild acid hydrolysis followed by gel-permeation chromatography. The high-molecular-mass OPS fraction was investigated using chemical methods, mass spectrometry, and 1H and 13C NMR spectroscopy techniques, including 2D homonuclear 1H,1H TOCSY, DQF COSY, NOESY, and heteronuclear 1H-detected 1H,13C HSQC, and HMBC experiments. The analysis revealed that the O-specific polysaccharide contains sugars with the galacto configuration of the ring and is composed of a disaccharide repeating unit with the following structure.

Identification of Novel Inhibitors of a Plant Group A Protein Phosphatase Type 2C Using a Combined In Silico and Biochemical Approach.

Type 2C protein phosphatases (PP2Cs) of group A play a significant role in the regulation of various processes in plants including growth, development, ion transport, and stress acclimation. In this study, we selected potential PP2C group A inhibitors using a structure-based virtual screening method followed by biochemical and in vitro validation. Over twenty million chemical compounds from the ZINC database were used for docking studies. The precision of the calculations was increased by an induced-fit docking protocol and the molecular mechanics/generalized Born surface area (MM/GBSA) method, which yielded approximate values for the binding energy of the protein-ligand complex. After clustering and ranking their activity, the top-ranking compounds were tested against PP2C group A members in vitro and their in vivo activity was also explored. Phosphatase activity assays identified two compounds with significant inhibitory activity against ABI1 protein ranging from around 57 to 91% at a concentration of 100 µM. Importantly, this in vitro activity correlated well with in vivo inhibition of seed germination, as expected for PP2C inhibitors. The results should promote the design of novel inhibitors with improved potency against ABI1-like and other PP2Cs that might be used in agriculture for the protection of crops against stress.

PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium Leguminosarum bv. Trifolii.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a ΔpssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

Protein Phosphatases Type 2C Group A Interact with and Regulate the Stability of ACC Synthase 7 in Arabidopsis.

Ethylene is an important plant hormone that controls growth, development, aging and stress responses. The rate-limiting enzymes in ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylate synthases (ACSs), are strictly regulated at many levels, including posttranslational control of protein half-life. Reversible phosphorylation/dephosphorylation events play a pivotal role as signals for ubiquitin-dependent degradation. We showed previously that ABI1, a group A protein phosphatase type 2C (PP2C) and a key negative regulator of abscisic acid signaling regulates type I ACS stability. Here we provide evidence that ABI1 also contributes to the regulation of ethylene biosynthesis via ACS7, a type III ACS without known regulatory domains. Using various approaches, we show that ACS7 interacts with ABI1, ABI2 and HAB1. We use molecular modeling to predict the amino acid residues involved in ABI1/ACS7 complex formation and confirm these predictions by mcBiFC-FRET-FLIM analysis. Using a cell-free degradation assay, we show that proteasomal degradation of ACS7 is delayed in protein extracts prepared from PP2C type A knockout plants, compared to a wild-type extract. This study therefore shows that ACS7 undergoes complex regulation governed by ABI1, ABI2 and HAB1. Furthermore, this suggests that ACS7, together with PP2Cs, plays an essential role in maintaining appropriate levels of ethylene in Arabidopsis.

Mgl2 Is a Hypothetical Methyltransferase Involved in Exopolysaccharide Production, Biofilm Formation, and Motility in Rhizobium leguminosarum bv. trifolii.

In this study, functional characterization of the mgl2 gene located near the Pss-I exopolysaccharide biosynthesis region in Rhizobium leguminosarum bv. trifolii TA1 is described. The hypothetical protein encoded by the mgl2 gene was found to be similar to methyltransferases (MTases). Protein homology and template-based modeling facilitated prediction of the Mgl2 structure, which greatly resembled class I MTases with a S-adenosyl-L-methionine-binding cleft. The Mgl2 protein was engaged in exopolysaccharide, but not lipopolysaccharide, synthesis. The mgl2 deletion mutant produced exopolysaccharide comprised of only low molecular weight fractions, while overexpression of mgl2 caused overproduction of exopolysaccharide with a normal low to high molecular weight ratio. The deletion of the mgl2 gene resulted in disturbances in biofilm formation and a slight increase in motility in minimal medium. Red clover (Trifolium pratense) inoculated with the mgl2 mutant formed effective nodules, and the appearance of the plants indicated active nitrogen fixation. The mgl2 gene was preceded by an active and strong promoter. Mgl2 was defined as an integral membrane protein and formed homodimers in vivo; however, it did not interact with Pss proteins encoded within the Pss-I region. The results are discussed in the context of the possible involvement of the newly described potential MTase in various metabolic traits, such as the exopolysaccharide synthesis and motility that are important for rhizobial saprophytic and symbiotic relationships.

A Mutation in the Mesorhizobium loti oatB Gene Alters the Physicochemical Properties of the Bacterial Cell Wall and Reduces Survival inside Acanthamoeba castellanii.

In our previous report, we had shown that the free-living amoeba Acanthamoeba castellanii influenced the abundance, competiveness, and virulence of Mesorhizobium loti NZP2213, the microsymbiont of agriculturally important plants of the genus Lotus. The molecular basis of this phenomenon; however, had not been explored. In the present study, we demonstrated that oatB, the O-acetyltransferase encoding gene located in the lipopolysaccharide (LPS) synthesis cluster of M. loti, was responsible for maintaining the protective capacity of the bacterial cell envelope, necessary for the bacteria to fight environmental stress and survive inside amoeba cells. Using co-culture assays combined with fluorescence and electron microscopy, we showed that an oatB mutant, unlike the parental strain, was efficiently destroyed after rapid internalization by amoebae. Sensitivity and permeability studies of the oatB mutant, together with topography and nanomechanical investigations with the use of atomic force microscopy (AFM), indicated that the incomplete substitution of lipid A-core moieties with O-polysaccharide (O-PS) residues rendered the mutant more sensitive to hydrophobic compounds. Likewise, the truncated LPS moieties, rather than the lack of O-acetyl groups, made the oatB mutant susceptible to the bactericidal mechanisms (nitrosative stress and the action of lytic enzymes) of A. castellanii.

Mitogen-Activated Protein Kinase Cascades in Plant Hormone Signaling.

Mitogen-activated protein kinase (MAPK) modules play key roles in the transduction of environmental and developmental signals through phosphorylation of downstream signaling targets, including other kinases, enzymes, cytoskeletal proteins or transcription factors, in all eukaryotic cells. A typical MAPK cascade consists of at least three sequentially acting serine/threonine kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and finally, the MAP kinase (MAPK) itself, with each phosphorylating, and hence activating, the next kinase in the cascade. Recent advances in our understanding of hormone signaling pathways have led to the discovery of new regulatory systems. In particular, this research has revealed the emerging role of crosstalk between the protein components of various signaling pathways and the involvement of this crosstalk in multiple cellular processes. Here we provide an overview of current models and mechanisms of hormone signaling with a special emphasis on the role of MAPKs in cell signaling networks. One-sentence summary: In this review we highlight the mechanisms of crosstalk between MAPK cascades and plant hormone signaling pathways and summarize recent findings on MAPK regulation and function in various cellular processes.

Synthesis of Rhizobial Exopolysaccharides and Their Importance for Symbiosis with Legume Plants.

Rhizobia dwell and multiply in the soil and represent a unique group of bacteria able to enter into a symbiotic interaction with plants from the Fabaceae family and fix atmospheric nitrogen inside de novo created plant organs, called nodules. One of the key determinants of the successful interaction between these bacteria and plants are exopolysaccharides, which represent species-specific homo- and heteropolymers of different carbohydrate units frequently decorated by non-carbohydrate substituents. Exopolysaccharides are typically built from repeat units assembled by the Wzx/Wzy-dependent pathway, where individual subunits are synthesized in conjunction with the lipid anchor undecaprenylphosphate (und-PP), due to the activity of glycosyltransferases. Complete oligosaccharide repeat units are transferred to the periplasmic space by the activity of the Wzx flippase, and, while still being anchored in the membrane, they are joined by the polymerase Wzy. Here we have focused on the genetic control over the process of exopolysaccharides (EPS) biosynthesis in rhizobia, with emphasis put on the recent advancements in understanding the mode of action of the key proteins operating in the pathway. A role played by exopolysaccharide in Rhizobium-legume symbiosis, including recent data confirming the signaling function of EPS, is also discussed.

A Role for Barley Calcium-Dependent Protein Kinase CPK2a in the Response to Drought.

Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L.), one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index (NBI), an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

RepB proteins of the multipartite Rhizobium leguminosarum bv. trifolii genome discriminate between centromere-like parS sequences for plasmid segregational stability.

The plasmids of the Rhizobiaceae family members and other Alphaproteobacteria are usually large, low copy-number and contain all elements necessary for active segregation and replication located in one operon comprising repABC genes. The genome of Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) consists of a chromosome and four plasmids (pRleTA1a-d) with repABC operons. In this work, centromere-binding RepB proteins of four RtTA1 plasmids were studied. Stability assays of the truncated derivatives of repABC cassettes demonstrated that RepA, RepB proteins and parS-like elements constituted plasmid partitioning systems, while RepC were sufficient for their replication. Individual RepB proteins bound specifically to centromere-like parS elements of the parental plasmids, which was crucial step toward the proper segregation of plasmids into daughter cells. RtTA1 RepB proteins formed dimers and oligomers in the solution. The C-terminal part of RepB was responsible for dimerization, while the domain engaged in parS binding was located in the middle of the protein. It was concluded that the specific interaction between individual RepB proteins and their target sequences together with the substantial diversity of the Rep proteins and parS originating from different plasmids strongly contributed to the coexistence of several plasmids equipped with similar repABC cassettes in the multipartite bacterial genome.

Optic disc pit with maculopathy ­ a case report. / [Dolek rozwojowy tarczy nerwu wzrokowego z makulopatia ­ opis przypadku].

The article presents a case of an 18-year old man with bilateral optic disc pit associated with serous macular detachment in the left eye. Optic disc pit is a rare congenital abnormality of the optic nerve head, which affects 1:11 000 people, with no gender predilection. Optic disc pits are usually incidental findings on fundus examination. In ophthalmic examination, optic disc pit presents as an oval, gray, white or yellowish depression in the optic disc, commonly involving temporal quadrants, but may be situated in any sector. Approximately 25­75% of eyes with optic disc pits have a poor visual prognosis, as a result of serous macular detachment and macular holes. There are no guidelines on the management of patients with optic disc pit maculopathy. Numerous techniques have been described, including laser photocoagulation, intravitreal gas injection and pars plana vitrectomy with many different modifications.

Plasmid-Encoded RepA Proteins Specifically Autorepress Individual repABC Operons in the Multipartite Rhizobium leguminosarum bv. trifolii Genome.

Rhizobia commonly have very complex genomes with a chromosome and several large plasmids that possess genes belonging to the repABC family. RepA and RepB are members of the ParA and ParB families of partitioning proteins, respectively, whereas RepC is crucial for plasmid replication. In the repABC replicons, partitioning and replication functions are transcriptionally linked resulting in complex regulation of rep gene expression. The genome of R. leguminosarum bv. trifolii TA1 (RtTA1) consists of a chromosome and four plasmids (pRleTA1a-d), equipped with functional repABC genes. In this work, the regulation of transcription of the individual repABC cassettes of the four RtTA1 plasmids was studied. The involvement of the RepA and RepB as well as parS-like centromere sites in this process was depicted, demonstrating some dissimilarity in expression of respective rep regions. RtTA1 repABC genes of individual plasmids formed operons, which were negatively regulated by RepA and RepB. Individual RepA were able to bind to DNA without added nucleotides, but in the presence of ADP, bound specifically to their own operator sequences containing imperfect palindromes, and caused operon autorepression, whereas the addition of ATP stimulated non-specific binding of RepA to DNA. The RepA proteins were able to dimerize/oligomerize: in general dimers formed independently of ATP or ADP, although ATP diminished the concentration of oligomers that were produced. By the comprehensive approach focusing on a set of plasmids instead of individual replicons, the work highlighted subtle differences between the organization and regulation of particular rep operons as well as the structures and specificity of RepA proteins, which contribute to the fine-tuned coexistence of several replicons with similar repABC cassettes in the complex bacterial genome.

PssP2 is a polysaccharide co-polymerase involved in exopolysaccharide chain-length determination in Rhizobium leguminosarum.

Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.