Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant 10 homologue of FleQ, which begins to commandeer control of the fleQ regulon at the cost 11 of disrupting nitrogen uptake and assimilation.Step 2 is a switch-of-function mutation 12 that redirects NtrC away from nitrogen uptake and towards its novel function as a 13 flagellar regulator. Our results demonstrate that natural selection can rapidly rewire 14 regulatory networks in very few, repeatable mutational steps. 25Here we monitor the recovery of microbial populations from a catastrophic gene 26 deletion: bacteria engineered to lack a particular function are exposed to environments 27 that impose strong selection to re-evolve it, sometimes by recruitment of new genes to 28 regulatory networks (6, 7, 8, 9). 29In the plant-associated soil bacterium P. fluorescens, the master regulator of The starting P. fluorescens strain is AR2; this strain lacks flagella, due to deletion of 33 fleQ, and is unable to move by spreading motility due to mutation of viscosin synthase 34 (viscB), resulting in a distinctive, point-like colony morphology on spreading motility 35 medium (SMM) (12) ( Figure 1A). We grew replicate populations of AR2 on SMM; when 36 local nutrients became depleted, starvation imposed strong selection to re-evolve 37 motility. To demonstrate that this finding was not strain-specific, these experiments were 38 replicated in a different strain of P. fluorescens, Pf0-2x. This strain is a ∆fleQ variant of 39 Pf0-1, already viscosin-deficient, and is thus unable to move by spreading or swimming 40 motility. 41After 96 hours incubation of AR2 and Pf0-2x at room temperature on SMM, two 42 breakout mutations were visible conferring first slow (AR2S and Pf0-2xS) and then fast 43 (AR2F and Pf0-2xF) spreading over the agar surface (Fig. 1A). The AR2F strain 44 produces flagella, but we could not detect flagella in EM samples for AR2S (Fig. 1B). Table S1). The expression of 55 genes required for flagellum biosynthesis and chemotaxis was abolished in AR2 56 compared to wild-type SBW25 (Fig. 2A). The ntrB mutation in AR2S partially restores 57 the expression of flagellar genes, and over-activates the expression of genes involved 58 in nitrogen regulation, uptake and metabolism. The subsequent ntrC mutation in AR2F 59 reduces the expression of nitrogen uptake and metabolism genes, while further up-60 regulating flagellar and chemotaxis gene expression to wild-type levels (Fig. 2B). While 61 AR2S and AR2F showed higher growth rates than the ancestor in LB medium (the 62 medium on which the mutants arose; Tukey-Kramer HSD test, growth in LB compared 63 to AR2: AR2S, P < 0.001; AR2F, P < 0.001) (Fig. 1C), both mutants grew poorly in 64 minimal medium with ammonium as the sole nitrogen source (Tukey-Kramer HSD test, 65 growth in M9 + ammonium compared to AR2: AR2S, P < 0.001; AR2F, P = 0.001). This 66 is likely to be the result of ammonium toxicity due to the strong up-regulation of genes 67 5 involved in ammonium uptake and assimilation, ind...

In legume-Rhizobium symbioses, specialised soil bacteria fix atmospheric nitrogen in return for carbon. However, ineffective strains can arise, making discrimination essential. Discrimination can occur via partner choice, where legumes prevent ineffective strains from entering, or via sanctioning, where plants provide fewer resources. Several studies have inferred that legumes exercise partner choice, but the rhizobia compared were not otherwise isogenic. To test when and how plants discriminate ineffective strains we developed sets of fixing and non-fixing strains that differed only in the expression of nifH – essential for nitrogen fixation – and could be visualised using marker genes. We show that the plant is unable to select against the non-fixing strain at the point of entry, but that non-fixing nodules are sanctioned. We also used the technique to characterise mixed nodules (containing both a fixing and a non-fixing strain), whose frequency could be predicted using a simple diffusion model. We discuss that sanctioning is likely to evolve in preference to partner choice in any symbiosis where partner quality cannot be adequately assessed until goods or services are actively exchanged.

Transcriptomics has the potential to discover new RNA virus genomes by sequencing total intracellular RNA pools. In this study, we have searched publicly available transcriptomes for sequences similar to viruses of the Nidovirales order. We report two potential nidovirus genomes, a highly divergent 35.9 kb likely complete genome from the California sea hare Aplysia californica, which we assign to a nidovirus named Aplysia abyssovirus 1 (AAbV), and a coronavirus-like 22.3 kb partial genome from the ornamented pygmy frog Microhyla fissipes, which we assign to a nidovirus named Microhyla alphaletovirus 1 (MLeV). AAbV was shown to encode a functional main proteinase, and a translational readthrough signal. Phylogenetic analysis suggested that AAbV represents a new family, proposed here as Abyssoviridae. MLeV represents a sister group to the other known coronaviruses. The importance of MLeV and AAbV for understanding nidovirus evolution, and the origin of terrestrial nidoviruses are discussed.

This paper describes the modification of nonwoven fabric such that it responds by releasing an encapsulated antimicrobial from within an attached vesicle in response to two species of pathogenic bacteria (Staphylococcus aureus MSSA 476 and Pseudomonas aeruginosa PAO1), but does not respond to nonpathogenic Escherichia coli DH5alpha. This concept is based on the generalization that a majority of pathogenic bacteria secrete virulence factors such as toxins and lipases that actively damage cell membranes, typically observed as tissue damage around infected wounds, while nonpathogenic bacteria do not (or not at high concentration). The eventual aim of this work is to produce responsive dressings which release antimicrobials and change color only on infected wounds. This paper details preliminary approaches to achieving this goal, including vesicle-bacteria studies in aqueous suspension, and fluorescence imaging of fluorescein containing vesicles lysed by S. aureus and P. aeruginosa, but not by E. coli.

Discovery of new natural products by heterologous expression reaches its limits, especially when specific building blocks are missing in the heterologous host or the production medium. Here, we describe the insect-specific production of the new GameXPeptides E-H (5-8) from Photorhabdus luminescens TTO1, which can be produced heterologously from expression of the GameXPeptide synthetase GxpS only upon supplementation of the production media with the missing building blocks, and thus must be regarded as the true natural products under natural conditions.

There has been a recent surge in the use of silver as an antimicrobial agent in a wide range of domestic and clinical products, intended to prevent or treat bacterial infections and reduce bacterial colonization of surfaces. It has been reported that the antibacterial and cytotoxic properties of silver are affected by the assay conditions, particularly the type of growth media used in vitro. The toxicity of Ag+ to bacterial cells is comparable to that of human cells. We demonstrate that biologically relevant compounds such as glutathione, cysteine and human blood components significantly reduce the toxicity of silver ions to clinically relevant pathogenic bacteria and primary human dermal fibroblasts (skin cells). Bacteria are able to grow normally in the presence of silver nitrate at >20-fold the minimum inhibitory concentration (MIC) if Ag+ and thiols are added in a 1∶1 ratio because the reaction of Ag+ with extracellular thiols prevents silver ions from interacting with cells. Extracellular thiols and human serum also significantly reduce the antimicrobial activity of silver wound dressings Aquacel-Ag (Convatec) and Acticoat (Smith & Nephew) to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli in vitro. These results have important implications for the deployment of silver as an antimicrobial agent in environments exposed to biological tissue or secretions. Significant amounts of money and effort have been directed at the development of silver-coated medical devices (e.g. dressings, catheters, implants). We believe our findings are essential for the effective design and testing of antimicrobial silver coatings.

SummaryMutation of ptsP encoding EI Ntr of the PTS Ntr system in Rhizobium leguminosarum strain Rlv3841 caused a pleiotropic phenotype as observed with many bacteria. The mutant formed dry colonies and grew poorly on organic nitrogen or dicarboxylates. Most strikingly the ptsP mutant had low activity of a broad range of ATP-dependent ABC transporters. This lack of activation, which occurred post-translationally, may explain many of the pleiotropic effects. In contrast protoncoupled transport systems were not inhibited in a ptsP mutant. Regulation by PtsP also involves two copies of ptsN that code for EIIA Ntr , resulting in a phosphorylation cascade. As in Escherichia coli, the Rlv3841 PTS Ntr system also regulates K + homeostasis by transcriptional activation of the high-affinity ATP-dependent K + transporter KdpABC. This involves direct interaction of a two-component sensor regulator pair KdpDE with unphosphorylated EIIA Ntr . Critically, ptsP mutants, which cannot phosphorylate PtsN1 or PtsN2, had a fully activated KdpABC transporter. This is the opposite pattern from that observed with ABC transporters which apparently require phosphorylation of PtsN. These results suggest that ATP-dependent transport might be regulated via PTS Ntr responding to the cellular energy charge. ABC transport may be inactivated at low energy charge, conserving ATP for essential processes including K + homeostasis.

SummaryMutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Gln : Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post-translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute-binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem-loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N 2 was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N2 on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.