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jueves, 26 de mayo de 2016

Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation

Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and wellbeing1, 2. Intestinal colonization begins at birth and climaxes with the acquisition of two dominant groups of strict anaerobic bacteria belonging to the Firmicutes and Bacteroidetes phyla2. Culture-independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases1. However, owing to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown3

Here we describe a novel workflow based on targeted phenotypic culturing linked to large-scale whole-genome sequencing, phylogenetic analysis and computational modelling that demonstrates that a substantial proportion of the intestinal bacteria are culturable. 

Applying this approach to healthy individuals, we isolated 137 bacterial species from characterized and candidate novel families, genera and species that were archived as pure cultures. Whole-genome and metagenomic sequencing, combined with computational and phenotypic analysis, suggests that at least 50–60% of the bacterial genera from the intestinal microbiota of a healthy individual produce resilient spores, specialized for host-to-host transmission. Our approach unlocks the human intestinal microbiota for phenotypic analysis and reveals how a marked proportion of oxygen-sensitive intestinal bacteria can be transmitted between individuals, affecting microbiota heritability.


 a, Relative abundance of bacteria in faecal samples (x axis) compared with relative abundance of bacteria growing on YCFA agar plates (y axis) as determined by metagenomic sequencing. Bacteria grown on YCFA agar are representative of the complete faecal samples as indicated by Spearman ρ = 0.75 (n = 6). b, Principal component analysis plot of 16S rRNA gene sequences detected from six donor faecal samples (n = 6), representing bacteria in complete faecal samples (green), faecal bacterial colonies recovered from YCFA agar plates without ethanol pre-treatment (black) or with ethanol pre-treatment to select for ethanol-resistant spore-forming bacteria (red). Culturing without ethanol selection is representative of the complete faecal sample, ethanol treatment shifts the profile, enriching for ethanol-resistant spore-forming bacteria and allowing their subsequent isolation. c, Phylogenetic tree of bacteria cultured from the six donors constructed from full-length 16S rRNA gene sequences. Novel candidate species (red), genera (blue) and families (green) are shown by dot colours. Major phyla and family names are indicated. Proteobacteria were not cultured, but are included for context.

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miércoles, 25 de mayo de 2016

Making a decision: to stay or move


Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole
 
Making a right decision to stay or move is critical for survival in a changing environment. Here we identify a novel mechanism for glucose-dependent on-off switching of flagellar synthesis in Vibrio vulnificus: When enzyme IIAGlc of the bacterial PEP:carbohydrate phosphotransferase system is dephosphorylated in the presence glucose, it delocalizes a protein required for flagellar biosynthesis from the flagellated pole. This leads to a loss of motility and enables bacteria to stay in a favorable habitat.

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viernes, 20 de mayo de 2016

Antibiotics From Scratch (desde la línea de salida)

Researchers have devised an approach for synthesizing new macrolide antibiotics from simple chemical building blocks. Using this method,Andrew Myers of Harvard University and colleagues synthesized more than 300 new antibiotic candidates, several of which were effective against some of the most stubbornly drug-resistant bacterial strains, according to the study published today (May 18) in Nature.

Here they present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. 

“It’s a tour de force in synthetic chemistry,” Kim Lewis of Northeastern University in Boston, who was not involved in the study, told The Scientist. “This is the first time there is a relatively easy path to synthesize macrolide erythromycin-type antibiotics from scratch.”
For most of the field’s history, natural products have been the starting point for new antibiotics. Most of them have been made by chemically modifying natural products in a process known as semisynthesis. Every existing antibiotic in a class called macrolides—including the commonly prescribed drug azithromycin—has been made by modifying erythromycin, which was first discovered in a soil sample in 1949. But some bacteria are developing resistance to these drugs at a startling rate, and semisynthesis is limited by the difficulty of modifying such complex molecules.
In the present study, Myers and his colleagues have developed a method for synthesizing macrolides from eight simple chemical building blocks to produce a diverse collection of structures that would be practically impossible to make using semisynthetic methods. In the same way you might build any complex device, from a cell phone to an airplane, “you divide it into building blocks,” and assemble those blocks, Myers told The Scientist. This approach “allows for, theoretically, tens of thousands of compounds” or more, he added.
Macrolides consist of a macrolactone ring with one or two sugars. “Decorating” these rings with different chemical groups by combining the building blocks in various combinations, Myers’s team created more than 300 synthetic macrolides, including the US Food and Drug Administration-approved drug telithromycin and the candidate solithromycin, which is currently being tested in Phase 3 clinical trials.
Next, the researchers evaluated the newly synthesized compounds with a panel of different bacteria, including two strains of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) isolated from clinical samples. “Some of these are really scary bugs,” said Myers.
Most of the compounds were effective against garden-variety pneumonia bacteria, and a few of them were more potent than approved antibiotics against MRSA, VRE, and other highly resistant strains, the researchers found. One of the synthetic compounds, called FSM-100573, was especially effective against gram-negative bacteria, which are traditionally less susceptible to macrolides. (The technology for synthesizing these macrolides has been licensed to the Watertown, Massachusetts-based Macrolide Pharmaceuticals, which Myers cofounded.)
These compounds have yet to be tested preclinically. And, as with all antibiotics, bacteria will ultimately develop resistance to them. However, this approach allows scientists to develop an exponentially larger number of new drug candidates. “At the end of the day, it’s kind of a numbers game,” Myers said.
“The work represents a paradigm shift in the discovery of novel antibiotics from the macrolide class,”Rodrigo Andrade of Temple University in Philadelphia, Pennsylvania, who was not involved in the work, told The Scientist. Synthesis of complex molecules can be a lengthy and expensive process, but Myers has developed a route that is more efficient, Andrade added. “This opens the door to myriad macrolide drug candidates that can help offset the incessant and inevitable onset of antibiotic resistance.”
“I’m impressed by the scale of the work and elegance of approach,” said Gerry Wright of McMaster University in Canada, who was also not involved in the research. “At the end of the day, it’s impossible to know whether or not they’re going to generate anything in clinic,” he said. Even so, “this is a significant step forward to regain the upper hand” against drug-resistant bugs,” Wright added.
Myers and his colleagues have previously synthesized novel tetracycline antibiotics, one of which is now a Phase 3 clinical trial candidate. His team is also developing methods to synthesize other classes of antibiotics, he said.

miércoles, 4 de mayo de 2016

Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ

Abstract:
Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called “nucleoid occlusion” (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator–CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA–DNA–FtsZ CTD regulatory interaction and provide insight into FtsZ–regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA–DNA–FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA–DNA–FtsZ interaction with implications for SlmA’s NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.

Importancia:
The bacterial protein FtsZ polymerizes into protofilaments to create the cytokinetic ring responsible for directing cell division. Cellular levels of FtsZ are above the concentration required for Z-ring formation. Hence, FtsZ-binding proteins have evolved that control its spatiotemporal formation. The SlmA protein is one such factor that, when bound to specific chromosomal DNA, inhibits FtsZ polymerization to prevent Z rings from forming through the bacterial chromosome. This inhibition depends on complex formation between SlmA-DNA and the FtsZ C-terminal domain (CTD). Here we describe SlmA–DNA–FtsZ CTD structures. These structures and complementary biochemistry unveil the molecular basis for the unique requirement that SlmA be DNA-bound to interact with FtsZ, a mechanism that appears to be conserved among SlmA-containing bacteria.