Topological and transcriptional analysis of <Emphasis Type="Italic">pssL</Emphasis> gene product: a putative Wzx-like exopolysaccharide translocase in <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii</Emphasis> TA1

An identified pssL gene is yet another one, besides the pssT, pssN and pssP genes, encoding for a protein engaged in polysaccharide polymerization and export in Rhizobium leguminosarum bv. trifolii strain TA1 (RtTA1). Amino acid sequence similarity and hypothetical protein secondary structure placed the PssL protein within Wzx (RfbX) translocases with putative flippase function that belong to the polysaccharide specific transport (PST) family. The predicted secondary structure of the PssL membrane protein was examined with a series of PssL-PhoA and PssL-LacZ translational fusions. The results support the hypothesis of PssL being a member of PST protein family comprising transporters with 12 membrane spanning segments and amino and carboxyl termini located in the cytoplasm. Results of semi-quantitative RT-PCR showed that the initial abundance of mRNA encoding PssL protein was relatively lower when compared to the quantity of the previously identified PssT membrane protein. PssL might be a good candidate for Wzx-like protein that together with PssT (Wzy protein) could be responsible for Wzx/Wzy-like-dependent EPS polymerization and translocation in RtTA1.     

<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii rosR</Emphasis> is required for interaction with clover,biofilm formation and adaptation to the environment

Background  

Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.     

Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume <Emphasis Type="Italic">Vavilovia formosa</Emphasis> Form an Isolated Group within <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.     

Genetic Diversity of a Natural Population of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis> Nodulating Plants of <Emphasis Type="Italic">Vicia faba</Emphasis> in the Vesuvian Area

A total of 98 rhizobial strains, isolated during the winter of the years 2003 (35 isolates), 2004 (33 isolates), and 2005 (30 isolates) were analyzed to determine the genetic diversity of the natural population nodulating Vicia faba plants and to identify dominant genotypes. All isolates were identified as Rhizobium leguminosarum bv. viciae by biovar-specific polymerase chain reaction amplification of the nodC gene. Intraspecific DNA polymorphism was evaluated through the restriction endonucleases analysis combined with pulsed-field gel electrophoresis. Four genotypes characterized 53% of the isolates, showing a high occurrence; moreover, they were recovered over the 3 years, thus showing a lasting persistence in the soil, which could mean a high degree of saprophytic competitiveness. The richness, diversity, and dominance indexes of genotypes were calculated to monitor the evolution of the rhizobial population during the 3 years. The genetic diversity of the analyzed strains decreased along the 3 years. In fact, the biodiversity index H′ decreased from 2.6 in the first and second year to 1.9 in the third year; probably, as a result of bean monocropping, specific genotypes of Rh. leguminosarum bv. viciae were naturally selected.     

Comparison of the adaptive potential for <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viceae</Emphasis> nodule bacterial populations isolated in natural ecosystems and agrocenoses

Polymorphism analysis was performed in Rhizobium leguminosarum bv. viceae populations isolated from geographically distant regions of Ukraine and Middle Asia. Examination of cultural, biochemical, and symbiotic traits revealed interpopulation differences, which were attributed to the difference in conditions between natural ecosystems and agrocenoses. Vetch has high species diversity and is not cultivated in Middle Asia, and the corresponding rhizobial population displayed higher genetic diversity and higher polymorphism of adaptive traits ensuring saprophytic development in soil and the rhizosphere, including melanin synthesis (35%) and active exopolysaccharide production (90%). Strains of the Ukrainian population had a lower exopolysaccharide production (10%), did not produce melanin, had higher herbicide resistance, and utilized glucose and succinate (main components of plant root exudation) as carbon sources. Strains capable of efficient symbiosis with Vicia villosa Roth. had a higher frequency in the Middle Asian than in the Ukrainian population, especially among strains isolated from soil (80 and 35%, respectively). In addition, strains of the Middle Asian population better competed for nodulation. It was assumed that the formation of rhizobial populations in vetch cultivation regions (Ukraine) is aimed at adaptation to ectosymbiotic (rhizospheric) interactions with plants and anthropogenic stress factors, while strains of the vetch original center (Middle Asia) are mostly adapted to the endosymbiotic interaction and to natural edaphic stress factors.     

Complexity of phenotypes and symbiotic behaviour of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> exopolysaccharide mutants

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 -lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of -lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l^–1). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of -lactamase was observed only in strain EHA101.Abbreviations CFU Colony-forming unit - MBC Minimum bactericidal concentration - MIC Minimum inhibitory concentration - PBP Penicillin-binding protein     

Effect of the synthetic polysaccharide MOD-19 on the formation and function of symbiosis between <Emphasis Type="Italic">Pisum sativum</Emphasis> L. and <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

The physiological action of the MOD-19 polysaccharide (PS), synthesized similarly to bacterial glucans, on the nodule bacteria Rhizobium leguminosarum bv. viciae and pea seeds was studied. It was found that MOD-19 stimulated nodule bacterium growth and bacterial biomass accumulation. It also altered metabolism in rhizobia grown in solid and liquid media containing this polymer. Treatment of pea seeds with MOD-19 before sowing increased the intensity of root formation, plant tissue peroxidase activity, and general symbiosis efficiency owing to secondary nodule formation on lateral roots and prolongation of their intense nitrogen fixation.     

A study of the competitive properties of the aluminum-tolerant strain <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii</Emphasis> 9-4A by the antibiotic resistance method

The virulence, competitive ability, and symbiotic efficiency of 2 Rhizobium leguminosarum bv. trifolii strains—the wild aluminum tolerant strain 9-4A and the commercial strain 348a—were compared when introducting their variants marked with antibiotic resistance into the rhizosphere of red clover (Trifolium pratense L.) plants. High virulence and competitive ability of the strain tolerant to aluminum was demonstrated by a concurrent inoculation of the seeds with these two strains. The resistance acquisition by the commercial strain was accompanied by a decrease in its symbiotic efficiency. Presumably, the resistant variant of aluminum-tolerant isolate retains its symbiotic properties due to its adaptation to acidity factors at the level of membrane function.     

Two of the three <Emphasis Type="Italic">groEL</Emphasis> homologues in <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> are dispensable for normal growth

Although many bacteria contain only a single groE operon encoding the essential chaperones GroES and GroEL, examples of bacteria containing more than one groE operon are common. The root-nodulating bacterium Rhizobium leguminosarum contains at least three operons encoding homologues to Escherichia coli GroEL, referred to as Cpn60.1, Cpn60.2 and Cpn60.3, respectively. We report here a detailed analysis of the requirement for and relative levels of these three proteins. Cpn60.1 is present at higher levels than Cpn60.2, and Cpn60.3 protein could not be detected under any conditions although the cpn60.3 gene is transcribed under anaerobic conditions. Insertion mutations could not be constructed in cpn60.1 unless a complementing copy was present, showing that this gene is essential for growth under the conditions used here. Both cpn60.2 and cpn60.3 could be inactivated with no loss of viability, and a double cpn60.2 cpn60.3 mutant was also constructed which was fully viable. Thus only Cpn60.1 is required for growth of this organism.Dedicated to the memory of Professor V. Javier Benedí, 1957–2002     

The <Emphasis Type="Italic">pssA</Emphasis> gene encodes UDP-glucose: Polyprenyl phosphate-glucosyl phosphotransferase initiating biosynthesis of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> exopolysaccharide

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum bv. viciae VF39 secrete an acidic heteropolysaccharide, the biosynthesis of which involves the stage of polyprenyl diphosphate octasaccharide formation with its carbohydrate fragment corresponding to the repeating polymer unit. The amino acid analysis of the product of the pssA gene, we have earlier identified, showed its homology to bacterial polyisoprenyl phosphate hexose 1-phosphate transferases catalyzing the formation of phosphodiester bonds between polyprenyl phosphates and hexose 1-phosphates, whose donors are nucleotide sugars. The immunoblotting demonstrated that Rhizobium cells synthesize a protein with a molecular mass of 25 kDa, which implies the translation of the open reading frame occurring from the second initiating codon followed by the protein processing. It was shown that PssA is an integral membrane-bound protein involved in glucose 1-phosphate transfer from UDP-glucose to polyprenyl phosphate to form polyprenyl diphosphate glucose. These results suggest that the pssA gene encodes UDP-glucose:polyprenyl phosphate-glucosyl phosphotransferase.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.     

Cadmium biosorption by <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic"> circulans</Emphasis> strain EB1

Summary A heavy metal resistant bacterium, Bacillus circulans strain EB1 showed a high cadmium biosorption capacity coupled with a high tolerance to this metal when grown in its presence. Bacillus circulans EB1 cells grown in the presence of 28.1 mg cadmium/l were capable of removing cadmium with a specific biosorption capacity of 5.8 mg Cd/g dry wt biomass in the first 8 h. When the cells were pre-conditioned with low concentrations of cadmium in pre-grown medium, the uptake was increased to 6.7 mg Cd/g dry wt biomass. The maximum uptake of␣cadmium was during mid-logarithmic phase of growth. The resting cells (both wet and dry) of EB1 were also able to biosorb cadmium. Specific biosorption capacities of wet and dry biomass were 9.8 and 26.5 mg Cd/g dry wt biomass, respectively. Maximum cadmium removals by both wet and dry cells were at pH 7.0. The results showed that the cadmium removal capacity of resting cells was markedly higher than that of growing cells. Since both growing and resting cells had a high biosorption capacity for cadmium, EB1 cells could serve as an excellent biosorbent for removal of cadmium from natural environments.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.