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

Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR

Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about two-fold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure

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

Intelligence, reason of state and the art of governing risk and opportunity in early modern Europe

Drawing upon primary and secondary historical material, this paper explores the role of intelligence in early modern government. It focuses upon developments in seventeenth- and early-eighteenth-century England, a site-specific genealogical moment in the broader history of state power/knowledges. Addressing a tendency in Foucauldian work to neglect pre-eighteenth-century governance, the analysis reveals a set of interrelated processes which gave rise to an innovative technique for anticipating hazard and opportunity for the state. At the intersection of raison d’État, the evolving art of government, widespread routines of secrecy and a post-Westphalia field of European competition and exchange, intelligence was imagined as a fundamental solution to the concurrent problems of ensuring peace and stability while improving state forces. In the administrative offices of the English Secretary of State, an assemblage of complex and interrelated procedures sought to produce and manipulate information in ways which exposed both possible risks to the state and potential opportunities for expansion and gain. As this suggests, the art of intelligence played an important if largely unacknowledged role in the formation and growth of the early modern state. Ensuring strategic advantage over rivals, intelligence also limited the ability of England's neighbours to dominate trade, control the seas and master the colonies, functioning as a constitutive feature of European balance and equilibrium. As the analysis concludes, understanding intelligence as a form of governmental technique – a way of doing something – reveals an entirely novel way of thinking about and investigating its myriad (historical and contemporary) formations

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

High quality genome annotation and expression visualisation of a mupirocin-producing bacterium

Pseudomonas strain NCIMB10586, in the P. fluorescens subgroup, produces the polyketide antibiotic mupirocin, and has potential as a host for industrial production of a range of valuable products. To underpin further studies on its genetics and physiology, we have used a combination of standard and atypical approaches to achieve a quality of the genome sequence and annotation, above current standards for automated pathways. Assembly of Illumina reads to a PacBio genome sequence created a retrospectively hybrid assembly, identifying and fixing 415 sequencing errors which would otherwise affect almost 5% of annotated coding regions. Our annotation pipeline combined automation based on related well-annotated genomes and stringent, partially manual, tests for functional features. The strain was close to P. synxantha and P. libaniensis and was found to be highly similar to a strain being developed as a weed-pest control agent in Canada. Since mupirocin is a secondary metabolite whose production is switched on late in exponential phase, we carried out RNAseq analysis over an 18 h growth period and have developed a method to normalise RNAseq samples as a group, rather than pair-wise. To review such data we have developed an easily interpreted way to present the expression profiles across a region, or the whole genome at a glance. At the 2-hour granularity of our time-course, the mupirocin cluster increases in expression as an essentially uniform bloc, although the mupirocin resistance gene stands out as being expressed at all the time points