Reloj

miércoles, 27 de marzo de 2013

Asymmetric growth and division in Mycobacterium spp.: compensatory mechanisms for non-medial septa

Mycobacterium spp., rod-shaped cells belonging to the phylum Actinomycetes, lack the Min- and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid-cell. We show that the position for establishment of the FtsZ-ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non-medially, producing two unequal but viable daughters. Septal sites and cellular
growth disclosed by staining with the membrane specific dye FM4-64 and fluorescent antibiotic vancomycin (FL-Vanco), respectively, showed that many division sites were off-centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time-lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post-septal DNA transport and unequal polar growth may compensate for the non-medial division site placement in Mycobacterium spp.

Acceso a la publicación

sábado, 23 de marzo de 2013

A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

B. subtilis biofilm formation is tightly regulated by elaborate signaling pathways. In contrast to domesticated lab strains of B. subtilis, which form smooth, essentially featureless colonies, undomesticated strains such as NCIB3610 form architecturally complex biofilms.  NCIB3610 also encodes an 80-kb plasmid absent from laboratory strains, and mutations in a plasmid-encoded homolog of a Rap protein, RapP, caused a hyper-rugose biofilm phenotype.

Here we explored the role of rapP-phrP in biofilm formation. We found that RapP is a phosphatase that dephosphorylates the intermediate response regulator Spo0F. RapP appears to employ a catalytic glutamate to dephosphorylate the Spo0F aspartylphosphate, and the implications of the RapP catalytic glutamate are discussed. In addition to regulating B. subtilis biofilm formation, we found that RapP regulates sporulation and genetic competence as a result of its ability to dephosphorylate Spo0F. Interestingly, while rap-phr gene cassettes routinely form regulatory pairs, i.e., the mature phr gene product inhibits the activity of the rap gene product, the phrP gene product did not inhibit RapP activity in our assays. RapP activity was, however, inhibited by PhrH in vivo but not in vitro.

Additional genetic analysis suggests that RapP is directly inhibited by peptide binding. We speculate that PhrH could be subject to post-translational modification in vivo and directly inhibit RapP activity, or, more likely, PhrH upregulates the expression of a peptide that in turn directly binds to RapP and inhibits its Spo0F phosphatase activity.

Acceso al trabajo

Improvement of Natamycin Production by Engineering of Phosphopantetheinyl Transferases in Streptomyces chattanoogensis L10

Phosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and non ribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and non ribosomal peptides.

The industrial natamycin (NTM) producer, Streptomyces chattanoogensis L10, contains two PPTases (SchPPT and SchACPS), and five PKSs. Biochemical characterization of these two PPTases shows: SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs; SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs); the specificity of SchPPT is possibly controlled by its C-terminus.

Inactivation of SchPPT in S. chattanoogensis L10 abolished production of NTM but not the spore pigment, while overexpression of SchPPT not only increased the NTM production by about 40% but also accelerated productions of both NTM and the spore pigment.

Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved the polyketide production by engineering the cognate PPTase in bacteria.

Acceso al trabajo

miércoles, 20 de marzo de 2013

Microbial metabolic exchange in 3D

Mono- and multispecies microbial populations alter the chemistry of their surrounding environments during colony development thereby influencing multicellular behavior and interspecies interactions of neighboring microbes. Here we present a methodology that enables the creation of three-dimensional (3D) models of a microbial chemotype that can be correlated to the colony phenotype through multimodal imaging analysis. 

These models are generated by performing matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS) on serial cross-sections of microbial colonies grown on 8mm deep agar, registering data sets of each serial section in MATLAB to create a model, and then superimposing the model with a photograph of the colonies themselves. 

As proof-of-principle, 3D models were used to visualize metabolic exchange during microbial interactions between Bacillus subtilis and Streptomyces coelicolor, as well as, Candida albicans and Pseudomonas aeruginosa. The resulting models were able to capture the depth profile of secreted metabolites within the agar medium and revealed properties of certain mass signals that were previously not observable using two-dimensional MALDI-TOF IMS.



Enlace al trabajo

Observing the invisible through imaging mass spectrometry

Many microbes can be cultured as single-species communities. The microbial communities, or colonies, curate their environment via metabolic exchange factors such as released natural products. To date, there are very few tools available that can monitor, in a systematic and informative fashion, the metabolic release patterns by microbes grown in a pure or mixed culture. There are significant challenges in the ability to monitor the metabolic secretome from growing microbial colonies. For example, the chemistries of such molecules can be extremely diverse, ranging from polyketides (e.g. erythromycin), non-ribosomal peptides (e.g. penicillin), isoprenoids (e.g. artemisinin), fatty acids (e.g. octanoic acid), microcins (e.g. Nisin), to peptides (e.g. microcin C7), poly-nucleotides and proteins.  Because of this chemical diversity, most of these molecules are extracted prior to analysis and studied one at a time and apart from the native spatial context of a microbial colony. Thus, limited information is obtained about the metabolic output of colonies in a synergetic or multiplexed fashion.

Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) is a powerful tool for simultaneously investigating the spatial distribution of multiple different biological molecules. The technique offers a molecular view of the peptides, proteins, polymers and lipids produced by a microbial colony without the need of exogenous labels or radioactive trace material



Target compounds can be measured and visualized simultaneously and in a high throughput manner within a single experiment. IMS extends beyond techniques such as MALDI profiling or MALDI intact cell analysis. Although invaluable, these techniques give a broad view of the metabolites produced in reference to a growing colony, where discretely secreted low global concentration but high local concentration metabolites could be missed.


jueves, 7 de marzo de 2013

Nuevas funciones para los sistemas de restricción-modificación

Kommireddy Vasu and Valakunja Nagaraja

SUMMARY 

Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

Copia del trabajo en la Moodle.

Bacterias que degradan el iboprufeno

 El ibuprofeno es un antiinflamatorio no esteroideo (AINE), utilizado frecuentemente como antipirético y para el alivio sintomático del dolor de cabeza (cefalea), dolor dental (odontalgia), dolor muscular o mialgia, molestias de la menstruación (dismenorrea), dolor neurológico de carácter leve y dolor postquirúrgico. También se usa para tratar cuadros inflamatorios, como los que se presentan en artritis, artritis reumatoide (AR) y artritis gotosa. En el último nº de Microbiology (SGM) se ha publicado un trabajo en el que se describe un microorganismo que degrada el iboprufeno.

Abstract

Sphingomonas Ibu-2 has the unusual ability to cleave the acid side chain from the pharmaceutical ibuprofen and related arylacetic acid derivatives to yield corresponding catechols under aerobic conditions via a previously uncharacterized mechanism. Screening a chromosomal library of Ibu-2 DNA in Escherichia coli EPI300 allowed us to identify one fosmid clone (pFOS3G7) that conferred the ability to metabolize ibuprofen to isobutylcatechol.

Iboprufeno




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viernes, 1 de marzo de 2013

Base de datos de enzimas de restricción

Para saber casi todo sobre los enzimas de restricción deben visitar la siguiente página web