Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.     

Phylogenetic diversity of actinomycetes cultured from coastal multipond solar saltern in Tuticorin, India

ABSTRACT: BACKGROUND: Hypersaline solar salterns are extreme environments in many tropical and subtropical regions throughout the world. In India, there are several coastal solar salterns along with the coastal line of the Bay of Bengal and Arabian Sea and inland solar salterns around Sambhar saltlake, from which sodium chloride is obtained for human consumption and industrial needs. Studies on characterization of such coastal and inland solar salterns are scarce and both the bacterial and archaeal diversity of these extreme saline environment remains poorly understood. Moreover, there are no reports on exclusive diversity of actinomycetes inhabiting Indian solar salterns. RESULTS: Soil sediments were collected from both concentrator and crystallizer ponds of solar salterns and subjected to detailed physico-chemical analysis. Actinomycetes were selectively isolated by employing selective processing methods and agar media. A total of 12 representatives were selected from the 69 actinomycete isolates obtained from the saltern soil samples, using Amplified Ribosomal DNA Restriction Analysis. Sequencing and analysis of 16S rDNA from chosen representative isolates displayed the presence of members affiliated to actinobacterial genera: Streptomyces, Micromonospora, Nocardia, Nocardiopsis, Saccharopolyspora and Nonomuraea. The genus Streptomyces was found to be the dominant among the isolates. Furthermore, rare actinomycete genus Nonomuraea was isolated for the first time from Indian solar salterns. CONCLUSIONS: To the best of our knowledge, this study constitutes the first characterization of actinomycete diversity centred on solar salterns located in the eastern coastal region of India. Furthermore, this is the very first report of isolation of Nonomuraea species from solar salterns and also from India. As actinomycetes encompass recurrently foremost sources of biotechnologically important member of the microbial communities, the actinomycetes retrieved from the Indian saltern soil samples laid the platform to search for novel biotechnologically significant bioactive substances.     

Rheology of native dextrans in relation to their primary structure

High molecular weight dextrans were synthesized at five temperatures (3, 10, 20, 25 and 30°C) using an in-vitro enzymatic method. The rheological properties of these dextrans in aqueous solution were assessed through their flow behaviour and their viscoelastic characteristics. The results were interpreted in relation to their primary structure and particularly to their branching.

It was shown that the relatively expanded conformation of the dextrans synthesized at 3, 10 and 20°C gives to these dextrans comparable properties which are not too different from those described in literature for random-coil linear polysaccharides. Dextran synthesized at 30°C exhibited flow properties which are typical of particle suspensions in dilute and semi-dilute solution. In the concentrated domain, this dextran yielded structured systems with properties typical of weak gels. This unexpected behaviour could be related to the highly-ramified structure of this dextran in comparison with the dextrans synthesized between 3 and 20°C. On the other hand, the dextran synthesized at 25°C displayed rheological behaviour which could also be related to an intermediate primary structure between those of dextran synthesized at 20°C and dextran synthesized at 30°C.     

Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.     

Association of lignin and <Emphasis Type="Italic">FLEXIBLE CULM 1 (FC1)</Emphasis> ortholog in imparting culm strength and lodging resistance in kodo millet (<Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L.)

To understand the lodging behavior in kodo millet (Paspalum scrobiculatum L.) at morphological, biochemical, and molecular levels, 22 germplasm accessions selected based on previous trials were characterized for culm strength-related morphological traits such as pulling force, culm weight per unit length, culm diameter, recovery angle after bending and degree of lodging, and biochemical traits such as cellulose, hemicellulose, and lignin content of the culm. Correlation among traits and path analysis with degree of lodging showed that only lignin content per unit length of the culm had a very high negative effect on degree of lodging, followed by culm diameter, which means that higher the lignin content and culm diameter, the lesser will be the degree of lodging. Hence, it was concluded that any improvement for lodging resistance in kodo millet can be achieved through improving the lignin content (mg/cm of the dry culm) and culm diameter. Gene expression studies in kodo millet for FLEXIBLE CULM 1 (FC1), a gene implicated in lignin biosynthetic pathway and lodging resistance in rice, suggests the role of FC1 gene ortholog in lignin accumulation in kodo millet as well. Accordingly, the highest gene expression was recorded in strong culm line “Adari” a land race and the lowest expression in “Aamo 10,” a weak culm line.     

The Landscape of Circular RNA in Cancer

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Genomic outlier profile analysis: mixture models, null hypotheses, and nonparametric estimation

In most analyses of large-scale genomic data sets, differentialexpression analysis is typically assessed by testing for differencesin the mean of the distributions between 2 groups. A recentfinding by Tomlins and others (2005) is of a different typeof pattern of differential expression in which a fraction ofsamples in one group have overexpression relative to samplesin the other group. In this work, we describe a general mixturemodel framework for the assessment of this type of expression,called outlier profile analysis. We start by considering thesingle-gene situation and establishing results on identifiability.We propose 2 nonparametric estimation procedures that have naturallinks to familiar multiple testing procedures. We then developmultivariate extensions of this methodology to handle genome-widemeasurements. The proposed methodologies are compared usingsimulation studies as well as data from a prostate cancer geneexpression study.     

Identification of low molecular weight molecules as new components of the nacre organic matrix

Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da–700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.