Identification of the agr Peptide of Listeria monocytogenes

Artículo científicoListeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco- 2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulatio

Identifying the Growth Modulon of Corynebacterium glutamicum

The growth rate (μ) of industrially relevant microbes, such as Corynebacterium glutamicum, is a fundamental property that indicates its production capacity. Therefore, understanding the mechanism underlying the growth rate is imperative for improving productivity and performance through metabolic engineering. Despite recent progress in the understanding of global regulatory interactions, knowledge of mechanisms directing cell growth remains fragmented and incomplete. The current study investigated RNA-Seq data of three growth rate transitions, induced by different pre-culture conditions, in order to identify transcriptomic changes corresponding to increasing growth rates. These transitions took place in minimal medium and ranged from 0.02 to 0.4 h-1 μ. This study enabled the identification of 447 genes as components of the growth modulon. Enrichment of genes within the growth modulon revealed 10 regulons exhibiting a significant effect over growth rate transition. In summary, central metabolism was observed to be regulated by a combination of metabolic and transcriptional activities orchestrating control over glycolysis, pentose phosphate pathway, and the tricarboxylic acid cycle. Additionally, major responses to changes in the growth rate were linked to iron uptake and carbon metabolism. In particular, genes encoding glycolytic enzymes and the glucose uptake system showed a positive correlation with the growth rate

Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate

Graf M, Haas T, Mueller F, et al. Continuous Adaptive Evolution of a Fast-Growing Corynebacterium glutamicum Strain Independent of Protocatechuate. Frontiers in Microbiology . 2019;10: 1648.Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h(-1) in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (RCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h(-1) and 1.03g(Glc) g(CDW)(-1) h(-1) in minimal medium without above mentioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes

Vibrio natriegens as Host for Expression of Multisubunit Membrane Protein Complexes

Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Bio-based production of organic acids with Corynebacterium glutamicum

The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, l-and d-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers. Funding Information Work in the laboratories of the authors was supported by the Fachagentur Nachwachsende Rohstoffe (FNR) of the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV; FNR Grants 220-095-08A and 220-095-08D; Bio-ProChemBB project, ERA-IB programme), by the Deutsche Bundesstiftung Umwelt (DBU Grant AZ13040/05) and the Evonik Degussa AG

Application of metabolic engineering for the biotechnological production of L-valine

The branched chain amino acid l-valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, l-valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient l-valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in l-valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial l-valine production, genetic strain engineering and bioprocess development for l-valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and bioprocess engineering in the near future