SMU.746-SMU.747, a Putative Membrane Permease Complex,Is Involved in Aciduricity,Acidogenesis, and Biofilm Formation in Streptococcus mutans

Dental caries induced by Streptococcus mutans is one of the most prevalent chronic infectious diseases worldwide. The pathogenicity of S. mutans relies on the bacterium''s ability to colonize tooth surfaces and survive a strongly acidic environment. We performed an ISS1 transposon mutagenesis to screen for acid-sensitive mutants of S. mutans and identified an SMU.746-SMU.747 gene cluster that is needed for aciduricity. SMU.746 and SMU.747 appear to be organized in an operon and encode a putative membrane-associated permease. SMU.746- and SMU.747-deficient mutants showed a reduced ability to grow in acidified medium. However, the short-term or long-term acid survival capacity and F₁F₀ ATPase activity remained unaffected in the mutants. Furthermore, deletion of both genes did not change cell membrane permeability and the oxidative and heat stress responses. Growth was severely affected even with slight acidification of the defined medium (pH 6.5). The ability of the mutant strain to acidify the defined medium during growth in the presence of glucose and sucrose was significantly reduced, although the glycolysis rate was only slightly affected. Surprisingly, deletion of the SMU.746-SMU.747 genes triggered increased biofilm formation in low-pH medium. The observed effects were more striking in a chemically defined medium. We speculate that the SMU.746-SMU.747 complex is responsible for amino acid transport, and we discuss its possible role in colonization and survival in the oral environment.     

A Conserved Streptococcal Membrane Protein,LsrS, Exhibits a Receptor-Like Function for Lantibiotics

Streptococcus mutans strain GS-5 produces a two-peptide lantibiotic, Smb, which displays inhibitory activity against a broad spectrum of bacteria, including other streptococci. For inhibition, lantibiotics must recognize specific receptor molecules present on the sensitive bacterial cells. However, so far no such receptor proteins have been identified for any lantibiotics. In this study, using a powerful transposon mutagenesis approach, we have identified in Streptococcus pyogenes a gene that exhibits a receptor-like function for Smb. The protein encoded by that gene, which we named LsrS, is a membrane protein belonging to the CAAX protease family. We also found that nisin, a monopeptide lantibiotic, requires LsrS for its optimum inhibitory activity. However, we found that LsrS is not required for inhibition by haloduracin and galolacticin, both of which are two-peptide lantibiotics closely related to Smb. LsrS appears to be a well-conserved protein that is present in many streptococci, including S. mutans. Inactivation of SMU.662, an LsrS homolog, in S. mutans strains UA159 and V403 rendered the cells refractory to Smb-mediated killing. Furthermore, overexpression of LsrS in S. mutans created cells more susceptible to Smb. Although LsrS and its homolog contain the CAAX protease domain, we demonstrate that inactivation of the putative active sites on the LsrS protein has no effect on its receptor-like function. This is the first report describing a highly conserved membrane protein that displays a receptor-like function for lantibiotics.     

Next generation sequencing in cardiomyopathy: towards personalized genomics and medicine

Next generation sequencing (NGS) is perhaps one of the most exciting advances in the field of life sciences and biomedical research in the last decade. With the availability of massive parallel sequencing, human DNA blueprint can be decoded to explore the hidden information with reduced time and cost. This technology has been used to understand the genetic aspects of various diseases including cardiomyopathies. Mutations for different cardiomyopathies have been identified and cataloging mutations on phenotypic basis are underway and are expected to lead to new discoveries that may translate to novel diagnostic, prognostic and therapeutic targets. With ease in handling NGS, cost effectiveness and fast data output, NGS is now considered as a diagnostic tool for cardiomyopathy by providing targeted gene sequencing. In addition to the number of genetic variants that are identified in cardiomyopathies, there is a need of quicker and easy way to screen multiple genes associated with the disease. In this review, an attempt has been made to explain the NGS technology, methods and applications in cardiomyopathies and their perspective in clinical practice and challenges which are to be addressed.     

Bioinformatics approaches for structural and functional analysis of proteins in secondary metabolism in Withania somnifera

Withania somnifera (Ashwagandha) is an affluent storehouse of large number of pharmacologically active secondary metabolites known as withanolides. These secondary metabolites are produced by withanolide biosynthetic pathway. Very less information is available on structural and functional aspects of enzymes involved in withanolides biosynthetic pathways of Withiana somnifera. We therefore performed a bioinformatics analysis to look at functional and structural properties of these important enzymes. The pathway enzymes taken for this study were 3-Hydroxy-3-methylglutaryl coenzyme A reductase, 1-Deoxy-d-xylulose-5-phosphate synthase, 1-Deoxy-d-xylulose-5-phosphate reductase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, and cycloartenol synthase. The prediction of secondary structure was performed for basic structural information. Three-dimensional structures for these enzymes were predicted. The physico-chemical properties such as pI, AI, GRAVY and instability index were also studied. The current information will provide a platform to know the structural attributes responsible for the function of these protein until experimental structures become available.     

Tetracycline nanoparticles loaded calcium sulfate composite beads for periodontal management

Background

The objective of this study was to fabricate, characterize and evaluate in vitro, an injectable calcium sulfate bone cement beads loaded with an antibiotic nanoformulation, capable of delivering antibiotic locally for the treatment of periodontal disease.

Methods

Tetracycline nanoparticles (Tet NPs) were prepared using an ionic gelation method and characterized using DLS, SEM, and FTIR to determine size, morphology, stability and chemical interaction of the drug with the polymer. Further, calcium sulfate (CaSO₄) control and CaSO₄-Tet NP composite beads were prepared and characterized using SEM, FTIR and XRD. The drug release pattern, material properties and antibacterial activity were evaluated. In addition, protein adsorption, cytocompatibility and alkaline phosphatase activity of the CaSO₄-Tet NP composite beads in comparison to the CaSO₄ control were analyzed.

Results

Tet NPs showed a size range of 130 ± 20 nm and the entrapment efficiency calculated was 89%. The composite beads showed sustained drug release pattern. Further the drug release data was fitted into various kinetic models wherein the Higuchi model showed higher correlation value (R² = 0.9279) as compared to other kinetic models. The composite beads showed antibacterial activity against Staphylococcus aureus and Escherichia coli. The presence of Tet NPs in the composite bead didn't alter its cytocompatibility. In addition, the composite beads enhanced the ALP activity of hPDL cells.

Conclusions

The antibacterial and cytocompatible CaSO₄-Tet NP composite beads could be beneficial in periodontal management to reduce the bacterial load at the infection site.

General significance

Tet NPs would deliver antibiotic locally at the infection site and the calcium sulfate cement, would itself facilitate tissue regeneration.     

Exquisite allele discrimination by toehold hairpin primers

The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a ‘yes/no’ answer.     

2n megagametophyte formed via SDR contributes to tetraploidization in polyembryonic ‘Nadorcott’ tangor crossed by citrus allotetraploids

Key Message

2 n megagametophyte formation plays an important role in polyploidization in polyembryonic citrus and is valuable for plant improvement.

Abstract

Tetraploid plants are frequently observed in the seedlings of diploid polyembryonic citrus genotypes. However, the mechanisms underlying the formation of tetraploids are still indistinct when apomictic citrus genotypes are used as female parent to cross with tetraploids. Herein, 54 tetraploid progenies, which were unexpectedly obtained previously from four 2x × 4x crosses using polyembryonic ‘Nadorcott’ tangor as seed parent, were analyzed by 22 simple sequence repeat (SSR) markers, aiming to reveal their genetic origin and the mechanism underlying 2n megagametophyte formation. The results showed that 13 tetraploids from all these four crosses were doubled diploids as indicated by their identical SSR allelic profile with their female parent; while the remaining 41 tetraploids apparently exhibited paternally derived alleles, which confirmed their zygotic origin. Furthermore, the genotyping of all hybrids indicated that all of them arose from 2n megagametophytes. Based on the genotypes of 2n megagametophytes, the analysis of maternal heterozygosity restitution (HR) for each marker showed that it varied from 0.00 to 87.80 % with a mean value of 40.89 %. In addition, it was observed that 13 markers displayed a lower rate than 50 %. On the basis of the above results, it can be speculated that the second division restitution (SDR) is the mechanism underlying the 2n megagametophyte formation in ‘Nadorcott’ tangor. The elucidation of the mechanism of 2n megagametophyte formation will be of great help to optimize further sexual hybridization for polyploids in citrus.     

Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia–c and IIa–c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes—(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.