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Despite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.

Bacterial adherence is an essential virulence factor in pathogenesis and infection. Fusobacterium nucleatum has a central role in oral biofilm architecture by acting as a bridge between early Gram-positive and late Gram-negative colonizers that do not otherwise adhere to each other. In this study, we survey a key adherence interaction of F. nucleatum with Porphyromonas gingivalis, and present evidence that multiple fusobacterial adhesins have a role in the attachment of F. nucleatum ATCC 23726 to P. gingivalis in a highly strain-dependent manner. Interaction between these species displayed varying sensitivities to arginine, galactose and lactose. Arginine was found to hamper coaggregation by at least 62% and up to 89% with several P. gingivalis strains and galactose inhibition ranged from no inhibition up to 58% with the same P. gingivalis strains. Lactose consistently inhibited F. nucleatum interaction with these P. gingivalis strains ranging from 40% to 56% decrease in coaggregation. Among the adhesins involved are the previously described Fap2 and surprisingly, RadD, which was described in an earlier study for its function in attachment of F. nucleatum to Gram-positive species. We also provide evidence for the presence of at least one additional adhesin that is sensitive to arginine but unlike Fap2 and RadD, is not a member of the autotransporter family type of fusobacterial large outer membrane proteins. The strain-specific binding profile of multiple fusobacterial adhesins to P. gingivalis highlights the heterogeneity and complexity of interspecies interactions in the oral cavity.

Background Chickpea ( Cicer arietinum L.) is an economically important cool season grain legume crop that is valued for its nutritive seeds having high protein content. However, several biotic and abiotic stresses and the low genetic variability in the chickpea genome have continuously hindered the chickpea molecular breeding programs. STMS (Sequence Tagged Microsatellite Sites) markers which are preferred for the construction of saturated linkage maps in several crop species, have also emerged as the most efficient and reliable source for detecting allelic diversity in chickpea. However, the number of STMS markers reported in chickpea is still limited and moreover exhibit low rates of both inter and intraspecific polymorphism, thereby limiting the positions of the SSR markers especially on the intraspecific linkage maps of chickpea. Hence, this study was undertaken with the aim of developing additional STMS markers and utilizing them for advancing the genetic linkage map of chickpea which would have applications in QTL identification, MAS and for de novo assembly of high throughput whole genome sequence data. Results A microsatellite enriched library of chickpea (enriched for ( GT/CA) n and (GA/CT) n repeats) was constructed from which 387 putative microsatellite containing clones were identified. From these, 254 STMS primers were designed of which 181 were developed as functional markers. An intraspecific mapping population of chickpea, [ICCV-2 (single podded) × JG-62 (double podded)] and comprising of 126 RILs, was genotyped for mapping. Of the 522 chickpea STMS markers (including the double-podding trait, screened for parental polymorphism, 226 (43.3%) were polymorphic in the parents and were used to genotype the RILs. At a LOD score of 3.5, eight linkage groups defining the position of 138 markers were obtained that spanned 630.9 cM with an average marker density of 4.57 cM. Further, based on the common loci present between the current map and the previously published chickpea intraspecific map, integration of maps was performed which revealed improvement of marker density and saturation of the region in the vicinity of sfl (double-podding) gene thereby bringing about an advancement of the current map. Conclusion An arsenal of 181 new chickpea STMS markers was reported. The developed intraspecific linkage map defined map positions of 138 markers which included 101 new locations.Map integration with a previously published map was carried out which revealed an advanced map with improved density. This study is a major contribution towards providing advanced genomic resources which will facilitate chickpea geneticists and molecular breeders in developing superior genotypes with improved traits.

The oral cavity contains a variety of ecological niches with very different environmental conditions that shape biofilm structure and composition. The space between the periodontal tissue and the tooth surface supports a unique anaerobic microenvironment that is bathed in the nutrient-rich gingival crevicular fluid (GCF). During the development of periodontitis, this environment changes and clinical findings reported a sustained level of calcium ion concentration in the GCF collected from the periodontal pockets of periodontitis patients. Here, we report the effect of calcium ion supplementation on human oral microbial biofilm formation and community composition employing an established SHI medium-based in vitro model system. Saliva-derived human microbial biofilms cultured in calcium-supplemented SHI medium (SHICa) exhibited a significant dose-dependent increase in biomass and metabolic activity. The effect of SHICa medium on the microbial community composition was evaluated by 16S rRNA gene sequencing using saliva-derived microbial biofilms from healthy donors and periodontitis subjects. In this study, intracellular microbial genomic DNA (iDNA) and extracellular DNA (eDNA) were analyzed separately at the genus level. Calcium supplementation of SHI medium had a differential impact on iDNA and eDNA in the biofilms derived from healthy individuals compared to those from periodontitis subjects. In particular, the genus-level composition of the eDNA portion was distinct between the different biofilms. This study demonstrated the effect of calcium in a unique microenvironment on oral microbial complex supporting the dynamic transformation and biofilm formation.

Periodontitis is a highly prevalent disease leading to uncontrolled osteoclastic jawbone resorption and ultimately edentulism; however, the disease onset mechanism has not been fully elucidated. Here we propose a mechanism for initial pathology based on results obtained using a recently developed Osteoadsorptive Fluogenic Sentinel (OFS) probe that emits a fluorescent signal triggered by cathepsin K (Ctsk) activity. In a ligature-induced mouse model of periodontitis, a strong OFS signal is observed before the establishment of chronic inflammation and bone resorption. Single cell RNA sequencing shows gingival fibroblasts to be the primary cellular source of early Ctsk . The in vivo OFS signal is activated when Toll-Like Receptor 9 (TLR9) ligand or oral biofilm extracellular DNA (eDNA) is topically applied to the mouse palatal gingiva. This previously unrecognized interaction between oral microbial eDNA and Ctsk of gingival fibroblasts provides a pathological mechanism for disease initiation and a strategic basis for early diagnosis and treatment of periodontitis. A highly sensitive imaging probe shows that oral microbial extracellular DNA triggers early activity of Ctsk in gingival fibroblasts, initiating periodontitis in mice.

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) n and (CT/GA) n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08-0.91) and 0.74 (0.23-0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.

RadD, a major adhesin of oral fusobacteria, is part of a four-gene operon encoding the small lipoprotein FAD-I and two currently uncharacterized small proteins encoded by the rapA and rapB genes. Previously, we described a role for FAD-I in the induction of human B-defensin 2 (hBD2) upon contact with oral epithelial cells. Here, we investigated potential roles for fad-I, rapA, and rapB in interspecies interaction and biofilm formation. Gene inactivation mutants were generated for each of these genes in the nucleatum and polymorphum subspecies of Fusobacterium nucleatum and characterized for their adherence to partner species, biofilm formation, and operon transcription. Binding to Streptococcus gordonii was increased in all mutant strains with Δfad-I having the most significant effect. This increased adherence was directly proportional to elevated radD transcript levels and resulted in significantly different architecture and height of the biofilms formed by Δfad-I and S. gordonii compared to the wild-type parent. In conclusion, FAD-I is important for fusobacterial interspecies interaction as its lack leads to increased production of the RadD adhesin suggesting a role of FAD-I in its regulation. This regulatory effect does not require the presence of functional RadD.

Denture stomatitis (DS) is a common infection in denture wearers, especially women. This study evaluated the induction of DS using acrylic devices attached to the palate of rats combined with inoculation of Candida spp. Immunocompetent male and female rats received a carbohydrate-rich diet. Impressions were taken from the rats’ palate to individually fabricate acrylic devices. Mono- and multispecies biofilms of C. albicans, C. glabrata, and C. tropicalis were grown on the devices, which were then cemented on posterior teeth and kept in the rats’ palate for four weeks. Microbial samples from the palate and the device were quantified. Oral microbiome of rats inoculated with C. albicans was analyzed by 16S rRNA gene sequencing. Log10(CFU/mL) were analyzed by mixed or two-way MANOVA (α = 0.05). Candida spp. and acrylic device did not induce palatal inflammation macroscopically nor microscopically. Although there was an increase (p < 0.001) of the total microbiota and female rats demonstrated higher (p = 0.007) recovery of Candida spp. from the palate, the gender differences were not biologically relevant. The microbiome results indicate an increase in inflammatory microbiota and reduction in health-associated micro-organisms. Although Candida spp. and acrylic device did not induce DS in immunocompetent rats, the shift in microbiota may precede manifestation of inflammation.

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The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues.