Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula. Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.     

Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.     

Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.The gastrointestinal tracts (GIT) of beef cattle are colonized by microorganisms that profoundly impact animal physiology, nutrition, health, and productivity (5). The GIT microbiota potentially impact food safety via pathogen shedding (13) by interacting with organisms such as Salmonella and competing for resources in the GIT. Cattle intestinal microbiota also play an important role in methane emissions, with U.S. beef cattle alone contributing an estimated 3.87 million metric tons of methane into the environment each year, both from rumen and large-intestine fermentations (7). Although the bovine fecal microbiota have been well characterized using culture-based methods, these techniques are necessarily limited to characterizing bacteria that can be grown in the laboratory. Culture-independent methods can reveal community members that are recalcitrant to culture. Only a handful of deep-sequencing studies have been done using culture-independent 16S rRNA-based methods (1, 11, 12, 14), all with dairy cattle, which have a fundamentally different diet and metabolism from beef cattle. Despite the potential contributions of the beef cattle GIT microbiota to animal health, food safety, and global warming, these communities remain poorly characterized. With the advent of pyrosequencing technology, researchers now have the tools to characterize these important communities. Pyrosequencing will allow rapid characterization of large-sample data sets (1). However, the taxonomic information generated by rapid sequencing is approximate by necessity (9), and full-length 16S-rRNA sequencing remains the “gold standard” method. Accordingly, we have characterized fecal bacteria from six feedlot cattle by full-length capillary sequence analysis of 11,171 16S rRNA gene clones (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Bacterial diversity of six feedlot beef cattle. Gray bars represent the percentages of all 16S sequences that were assigned to each taxonomy. Colored dots represent the percentages of 16S sequences from each library that were assigned to each taxonomic group. Asterisks indicate unclassified members of the named taxon. Panel A shows the data for the first 99% of all the sequences. Panel B shows the data for the remaining 1% of sequences. Note differences in scales for panels A and B.Rectal grab fecal samples (n = 6) were collected according to institutional animal care guidelines. All animals were female cross-bred MARCIII beef heifers, 6 to 8 months of age, 214 to 241 kg, housed in the same feedlot pen for 2 months prior to fecal collection, and fed the same typical feedlot beef production growing rations consisting of 61.6% corn silage (41.3% dry matter), 15.2% alfalfa hay, 20.9% corn, and 2.3% liquid supplement.Total fecal DNA was isolated from homogenized samples using MoBio UltraClean fecal kit (Carlsbad, CA). PCR was performed using 27F and 1392R primers (11). Amplification consisted of 25 cycles, with an annealing temperature of 55°C. Amplicons from three reactions per sample were pooled (8), cloned using the Invitrogen TOPO TA cloning kit (Carlsbad, CA), and sequenced bidirectionally with M13 primers using an ABI 3700 sequencer (17). Low-quality and chimeric sequences (6) were excluded from further analysis. Distance matrices were compiled from ClustalW alignments (18) in PHYLIP (4). Pairwise estimates of shared richness were calculated using EstimateS, version 8.2 (R. K. Colwell; http://purl.oclc.org/estimates). DOTUR (16) was used to identify operational taxonomic units (OTUs) and to generate rarefaction curves (Fig. ​(Fig.2),2), richness and evenness estimates, and Shannon''s and Simpson''s diversity indices (Table ​(Table1).1). A 97% similarity cutoff and an 85% similarity cutoff for estimating OTUs were used to approximate species and class-level designations (15). Taxonomies were assigned to one member of each OTU using the RDP “classifier” tool (19), and the RDP taxonomic information was used for Fig. ​Fig.11 and ​and3.3. Common bovine taxa were identified based on inclusion in all three U.S. culture-independent studies (this study and references 1 and 11).Open in a separate windowFIG. 2.Rarefaction curves for six feedlot beef cattle. OTUs were assigned at the 85% DNA sequence similarity level. For comparison purposes, all six curves were truncated after 1,321 sequences.Open in a separate windowFIG. 3.Phylum-level distribution of bacterial sequences from six beef feedlot cattle. Asterisks indicate unclassified members of the named taxon.

TABLE 1.

Richness and diversity indices for 6 beef feedlot cattle

Greater diversity of Shiga toxin-encoding bacteriophage insertion sites among Escherichia coli O157:H7 isolates from cattle than in those from humans

Escherichia coli O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of E. coli O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (P < 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the tir allele sp-1 and the Q933W allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of E. coli O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine E. coli O157:H7 genotypes.     

The selection of serum-independent PC12 cells for a more-reliable manganese cytotoxicity test

A major issue concerning the protocols of heavy metal cytotoxicity tests with PC12 cells was the hypothesis that serum in the culture medium might sequester the metal, thus altering the results obtained. However, serum withdrawal impairs the viability of PC12 cells themselves, thus impeding cytotoxicity testing in the absence of serum. In this study, we repeatedly selected undifferentiated, totally non-adherent PC12 cells in Petri dishes. Surprisingly, we discovered that these cells could survive and proliferate in serum-free medium. Moreover, features such as NGF-responsiveness, resazurin reduction potential, doubling rate, protein content, and basal caspase-3 enzyme activity, were equivalent to those exhibited by standard PC12 cultures. Further experiments aimed at fully characterising these serum-independent PC12 cells are in progress. These cells enabled cytotoxicity experiments to be conducted with manganese, both in serum-supplemented and in serum-deprived medium. The results demonstrated that serum removal decreased the LC50 of manganese from 250microM to 32microM, without affecting the internalisation of the metal. The data exclude an early competitive effect of serum on metal internalisation; rather, they suggest a late protective mechanism mediated by serum against the cytotoxic effect of the already-internalised metal.     

Effect of Proximity to a Cattle Feedlot on Escherichia coli O157:H7 Contamination of Leafy Greens and Evaluation of the Potential for Airborne Transmission

The impact of proximity to a beef cattle feedlot on Escherichia coli O157:H7 contamination of leafy greens was examined. In each of 2 years, leafy greens were planted in nine plots located 60, 120, and 180 m from a cattle feedlot (3 plots at each distance). Leafy greens (270) and feedlot manure samples (100) were collected six different times from June to September in each year. Both E. coli O157:H7 and total E. coli bacteria were recovered from leafy greens at all plot distances. E. coli O157:H7 was recovered from 3.5% of leafy green samples per plot at 60 m, which was higher (P < 0.05) than the 1.8% of positive samples per plot at 180 m, indicating a decrease in contamination as distance from the feedlot was increased. Although E. coli O157:H7 was not recovered from air samples at any distance, total E. coli was recovered from air samples at the feedlot edge and all plot distances, indicating that airborne transport of the pathogen can occur. Results suggest that risk for airborne transport of E. coli O157:H7 from cattle production is increased when cattle pen surfaces are very dry and when this situation is combined with cattle management or cattle behaviors that generate airborne dust. Current leafy green field distance guidelines of 120 m (400 feet) may not be adequate to limit the transmission of E. coli O157:H7 to produce crops planted near concentrated animal feeding operations. Additional research is needed to determine safe set-back distances between cattle feedlots and crop production that will reduce fresh produce contamination.