A Natural Plasmid Uniquely Encodes Two Biosynthetic Pathways Creating a Potent Anti-MRSA Antibiotic

Background Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin. Methodology/Principal Findings High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via “mutasynthesis” that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates. Conclusions/Significance Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA

New Vectors for Urea-Inducible Recombinant Protein Production

We have developed a novel urea-inducible recombinant protein production system by exploiting the Proteus mirabilis urease ureR-ureD promoter region and the ureR AraC-family transcriptional regulator. Experiments using the expression of β-galactosidase and green fluorescent protein (GFP) showed that promoter activity is tightly regulated and that varying the concentration of urea can give up to 100-fold induction. Production of proteins of biopharmaceutical interest has been demonstrated, including human growth hormone (hGH), a single chain antibody fragment (scFv) against interleukin-1β and a potential Neisserial vaccine candidate (BamAENm). Expression levels can be fine-tuned by temperature and different urea concentrations, and can be induced with readily available garden fertilisers and even urine. As urea is an inexpensive, stable inducer, a urea-induced expression system has the potential to considerably reduce the costs of large-scale recombinant protein production

Inexpensive protein overexpression driven by the NarL transcription activator protein

Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. However, these can be expensive and, sometimes, chemically unstable. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate-responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high-level controlled RPP. We show that target proteins, such as green fluorescent protein, human growth hormone, and single-chain variable region antibody fragments can be expressed to high levels using our promoter systems. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products

On the vegetation of Mosor

Im vorliegenden Beitrag wird ein Überblick über die Vegetation des Mosor-Gebirges, die sämtlich zu der mediterranen Region gehört, gegeben. Dies hängt von den klimatischen Verhältnissen bzw. von der geographischen Lage des Mosor-Gebirges, das gänzlich im Hintergrund des zentralen Teiles des mittleren immergrünen Gebietes Kroatiens verläuft, ab. Gewisse Pflanzengesellschaften und einige Pflanzenarten befinden sich hier auf der Nordwest- bzw. Südgrenze ihres Verbreitungsgebietes.Mosor se s obzirom na svoj fitogeografski položaj odlikuje nekim specifičnostima u biljnom pokrovu. Iako ima visinu od 1340 m/nm, vegetacija na Mosoru pripada u cijelosti mediteranskoj regiji. Šumska zajednica Carpinetum orientalis adriaticum zauzima ondje položaje od 400 do 900 m/nm, a zajednica Seslerio-Ostryetum od 900 m/nm naviše. Na obroncima Mosora zajednica Andropogoni-Diplachnetum serotinae dosiže, koliko je dosad poznato, najjužniju granicu svoje raširenosti. S druge strane, zajednica Erico-Cistetum cretici i Brachypodio-Trifolietum stellati imaju, prema dosadašnjim istraživanjima, na području Mosora i široj okolici Splita svoju sjevernu granicu raširenosti. Isto tako, po podacima iz literature, zajednica Campanulo-Moltkietum petraeae ima na Mosoru (uz Kozjak i Dinaru) svoju sjeverozapadnu granicu. Inače biljni pokrov Mosora, iako jako utjecajan, odlikuje se gotovo svim najznačajnijim tipovima vegetacije mediteranske regije.The papeir gives a short survey of the vegetational cover of Mosor, starting from climatozonal vegetation to the various stages of its degradation. In respect to its phytogeographic position, the mountain of Mosor has certain specific features in its vegetational cover. Although the mountain is 1340 m high, the vegetation of Mosor belongs entirely to the Mediterranean region. The forest community Carpinetum orientalis adriaticum is situated here at places between 400 to 900 m above sea, and the community Seslerio-Ostryetum from 900 m upwards. On the slopes of Mosor, the community Andropogoni-Diplachnetum reaches, as far as it is known today, the southernmost border of its distribution. On the other hand, the communities Erico-Cistetum cretici and Brachypodio-Trijolietum stellati reach, according to current investigations, their northern border in the area of Mosor and the wider surroundings of Split. Also, according to the literature, the community Campanulo-Moltkietum petraeae has its north-western border at Mosor (together with Kozjak and Dinara mts). Otherwise the vegetational cover of Mosor, although of great influence, is characterized by all the most significant types of the vegetation of the Mediterranean region

A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules