tRNATyr has an unusually short half-life in Trypanosoma brucei.

Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes. Differential subcellular localization of the cytoplasmic splicing machinery and a nuclear enzyme responsible for queuosine modification at the anticodon "wobble" position appear to be important quality control mechanisms for tRNATyr, the only intron-containing tRNA in T. brucei Since tRNA-guanine transglycosylase (TGT), the enzyme responsible for Q formation, cannot act on an intron-containing tRNA, retrograde nuclear transport is an essential step in maturation. Unlike maturation/processing pathways, the general mechanisms of tRNA stabilization and degradation in T. brucei are poorly understood. Using a combination of cellular and molecular approaches, we show that tRNATyr has an unusually short half-life. tRNATyr, and in addition tRNAAsp, also show the presence of slow-migrating bands during electrophoresis; we term these conformers: alt-tRNATyr and alt-tRNAAsp, respectively. Although we do not know the chemical or structural nature of these conformers, alt-tRNATyr has a short half-life resembling that of tRNATyr; the same is not true for alt-tRNAAsp We also show that RRP44, which is usually an exosome subunit in other organisms, is involved in tRNA degradation of the only intron-containing tRNA in T. brucei and is partly responsible for its unusually short half-life.

Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei.

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for 'normal' growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.

The Power of Asymmetry: Architecture and Assembly of the Gram-Negative Outer Membrane Lipid Bilayer.

Determining the chemical composition of biological materials is paramount to the study of natural phenomena. Here, we describe the composition of model gram-negative outer membranes, focusing on the predominant assembly, an asymmetrical bilayer of lipid molecules. We also give an overview of lipid biosynthetic pathways and molecular mechanisms that organize this material into the outer membrane bilayer. An emphasis is placed on the potential of these pathways as targets for antibiotic development. We discuss deviations in composition, through bacterial cell surface remodeling, and alternative modalities to the asymmetric lipid bilayer. Outer membrane lipid alterations of current microbiological interest, such as lipid structures found in commensal bacteria, are emphasized. Additionally, outer membrane components could potentially be engineered to develop vaccine platforms. Observations related to composition and assembly of gram-negative outer membranes will continue to generate novel discoveries, broaden biotechnologies, and reveal profound mysteries to compel future research.

Loss of a Cardiolipin Synthase in Helicobacter pylori G27 Blocks Flagellum Assembly.

Helicobacter pylori uses a cluster of polar, sheathed flagella for motility, which it requires for colonization of the gastric epithelium in humans. As part of a study to identify factors that contribute to localization of the flagella to the cell pole, we disrupted a gene encoding a cardiolipin synthase (clsC) in H. pylori strains G27 and B128. Flagellum biosynthesis was abolished in the H. pylori G27 clsC mutant but not in the B128 clsC mutant. Transcriptome sequencing analysis showed that flagellar genes encoding proteins needed early in flagellum assembly were expressed at wild-type levels in the G27 clsC mutant. Examination of the G27 clsC mutant by cryo-electron tomography indicated the mutant assembled nascent flagella that contained the MS ring, C ring, flagellar protein export apparatus, and proximal rod. Motile variants of the G27 clsC mutant were isolated after allelic exchange mutagenesis using genomic DNA from the B128 clsC mutant as the donor. Genome resequencing of seven motile G27 clsC recipients revealed that each isolate contained the flgI (encodes the P-ring protein) allele from B128. Replacing the flgI allele in the G27 clsC mutant with the B128 flgI allele rescued flagellum biosynthesis. We postulate that H. pylori G27 FlgI fails to form the P ring when cardiolipin levels in the cell envelope are low, which blocks flagellum assembly at this point. In contrast, H. pylori B128 FlgI can form the P ring when cardiolipin levels are low and allows for the biosynthesis of mature flagella.IMPORTANCEH. pylori colonizes the epithelial layer of the human stomach, where it can cause a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. To colonize the stomach, H. pylori must penetrate the viscous mucous layer lining the stomach, which it accomplishes using its flagella. The significance of our research is identifying factors that affect the biosynthesis and assembly of the H. pylori flagellum, which will contribute to our understanding of motility in H. pylori, as well as other bacterial pathogens that use their flagella for host colonization.

AlmG, responsible for polymyxin resistance in pandemic Vibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferases.

Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. However, some bacteria have developed resistance mechanisms to survive these compounds. Current pandemic O1 Vibrio cholerae biotype El Tor is resistant to polymyxins, whereas a previous pandemic strain of the biotype Classical is polymyxin-sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to antimicrobial peptides through aminoacylation of lipopolysaccharide (LPS), expected to decrease the negatively charged surface of the V. cholerae outer membrane. This Gram-negative system bears striking resemblance to a related Gram-positive cell-wall remodeling strategy that also promotes CAMP resistance. Mutants defective in AlmEF-dependent LPS modification exhibit reduced fitness in vivo Here, we present investigation of AlmG, the hitherto uncharacterized member of the AlmEFG pathway. Evidence for AlmG glycyl to lipid substrate transferase activity is demonstrated in vivo by heterologous expression of V. cholerae pathway enzymes in a specially engineered Escherichia coli strain. Development of a minimal keto-deoxyoctulosonate (Kdo)-lipid A domain in E. coli was necessary to facilitate chemical structure analysis and to produce a mimetic Kdo-lipid A domain AlmG substrate to that synthesized by V. cholerae. Our biochemical studies support a uniquely nuanced pathway of Gram-negative CAMPs resistance and provide a more detailed description of an enzyme of the pharmacologically relevant lysophosphospholipid acyltransferase (LPLAT) superfamily.

Novel coordination of lipopolysaccharide modifications in Vibrio cholerae promotes CAMP resistance.

In the environment and during infection, the human intestinal pathogen Vibrio cholerae must overcome noxious compounds that damage the bacterial outer membrane. The El Tor and classical biotypes of O1 V. cholerae show striking differences in their resistance to membrane disrupting cationic antimicrobial peptides (CAMPs), such as polymyxins. The classical biotype is susceptible to CAMPs, but current pandemic El Tor biotype isolates gain CAMP resistance by altering the net charge of their cell surface through glycine modification of lipid A. Here we report a second lipid A modification mechanism that only functions in the V. cholerae El Tor biotype. We identify a functional EptA ortholog responsible for the transfer of the amino-residue phosphoethanolamine (pEtN) to the lipid A of V. cholerae El Tor that is not functional in the classical biotype. We previously reported that mildly acidic growth conditions (pH 5.8) downregulate expression of genes encoding the glycine modification machinery. In this report, growth at pH 5.8 increases expression of eptA with concomitant pEtN modification suggesting coordinated regulation of these LPS modification systems. Similarly, efficient pEtN lipid A substitution is seen in the absence of lipid A glycinylation. We further demonstrate EptA orthologs from non-cholerae Vibrio species are functional.

What drives interaction strengths in complex food webs? A test with feeding rates of a generalist stream predator.

Describing the mechanisms that drive variation in species interaction strengths is central to understanding, predicting, and managing community dynamics. Multiple factors have been linked to trophic interaction strength variation, including species densities, species traits, and abiotic factors. Yet most empirical tests of the relative roles of multiple mechanisms that drive variation have been limited to simplified experiments that may diverge from the dynamics of natural food webs. Here, we used a field-based observational approach to quantify the roles of prey density, predator density, predator-prey body-mass ratios, prey identity, and abiotic factors in driving variation in feeding rates of reticulate sculpin (Cottus perplexus). We combined data on over 6,000 predator-prey observations with prey identification time functions to estimate 289 prey-specific feeding rates at nine stream sites in Oregon. Feeding rates on 57 prey types showed an approximately log-normal distribution, with few strong and many weak interactions. Model selection indicated that prey density, followed by prey identity, were the two most important predictors of prey-specific sculpin feeding rates. Feeding rates showed a positive relationship with prey taxon densities that was inconsistent with predator saturation predicted by current functional response models. Feeding rates also exhibited four orders-of-magnitude in variation across prey taxonomic orders, with the lowest feeding rates observed on prey with significant anti-predator defenses. Body-mass ratios were the third most important predictor variable, showing a hump-shaped relationship with the highest feeding rates at intermediate ratios. Sculpin density was negatively correlated with feeding rates, consistent with the presence of intraspecific predator interference. Our results highlight how multiple co-occurring drivers shape trophic interactions in nature and underscore ways in which simplified experiments or reliance on scaling laws alone may lead to biased inferences about the structure and dynamics of species-rich food webs.

UVliPiD: A UVPD-Based Hierarchical Approach for De Novo Characterization of Lipid A Structures.

The lipid A domain of the endotoxic lipopolysaccharide layer of Gram-negative bacteria is comprised of a diglucosamine backbone to which a variable number of variable length fatty acyl chains are anchored. Traditional characterization of these tails and their linkages by nuclear magnetic resonance (NMR) or mass spectrometry is time-consuming and necessitates databases of pre-existing structures for structural assignment. Here, we introduce an automated de novo approach for characterization of lipid A structures that is completely database-independent. A hierarchical decision-tree MS(n) method is used in conjunction with a hybrid activation technique, UVPDCID, to acquire characteristic fragmentation patterns of lipid A variants from a number of Gram-negative bacteria. Structural assignments are derived from integration of key features from three to five spectra and automated interpretation is achieved in minutes without the need for pre-existing information or candidate structures. The utility of this strategy is demonstrated for a mixture of lipid A structures from an enzymatically modified E. coli lipid A variant. A total of 27 lipid A structures were discovered, many of which were isomeric, showcasing the need for a rapid de novo approach to lipid A characterization.

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence.

Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model.

Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10.

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.

Are large macroalgal blooms necessarily bad? Nutrient impacts on seagrass in upwelling-influenced estuaries.

Knowledge of nutrient pathways and their resulting ecological interactions can alleviate numerous environmental problems associated with nutrient increases in both natural and managed systems. Although not unique, coastal systems are particularly prone to complex ecological interactions resulting from nutrient inputs from both the land and sea. Nutrient inputs to coastal systems often spur ulvoid macroalgal blooms, with negative consequences for seagrasses, primarily through shading, as well as through changes in local biogeochemistry. We conducted complementary field and mesocosm experiments in an upwelling-influenced estuary, where marine-derived nutrients dominate, to understand the direct and indirect effects of nutrients on the macroalgal-eelgrass (Zostera marina L.) interaction. In the field experiment, we found weak evidence that nutrients and/or macroalgal treatments had a negative effect on eelgrass. However, in the mesocosm experiment, we found that a combination of nutrient and macroalgal treatments led to strongly negative eelgrass responses, primarily via indirect effects associated with macroalgal additions. Together, increased total light attenuation and decreased sediment oxygen levels were associated with larger effects on eelgrass than shading alone, which was evaluated using mimic algae treatments that did not alter sediment redox potential. Nutrient addition in the mesocosms directly affected seagrass density; biomass, and morphology, but not as strongly as macroalgae. We hypothesize that the contrary results from these parallel experiments are a consequence of differences in the hydrodynamics between field and mesocosm settings. We suggest that the high rates of water movement and tidal submersion of our intertidal field experiments alleviated the light reduction and negative biogeochemical changes in the sediment associated with macroalgal canopies, as well as the nutrient effects observed in the mesocosm experiments. Furthermore, adaptation of ulvoids and eelgrass to high, but variable, background nutrient concentrations in upwelling-influenced estuaries may partly explain the venue-specific results reported here. In order to manage critical seagrass habitats, nutrient criteria and macroalgal indicators must consider variability in marine-based nutrient delivery and local physical conditions among estuaries.

LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis enhances immunogenicity against pneumonic plague.

Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC P(BAD) crp) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δasd-206 ΔmsbB868::P(msbB) msbB(EC) ΔP(crp21)::TT araC P(BAD) crp] for use with a balanced-lethal Asd-positive (Asd(+)) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopE(Nt138)-LcrV) was synthesized in χ10057 harboring an Asd(+) plasmid (pYA5199, yopE(Nt138)-lcrV) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ~240 median lethal doses (LD50) (2.4 × 10(4) CFU) of Y. pestis KIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.

193 nm ultraviolet photodissociation mass spectrometry for the structural elucidation of lipid A compounds in complex mixtures.

Here we implement ultraviolet photodissociation (UVPD) in an online liquid chromatographic tandem mass spectrometry (MS/MS) strategy to support analysis of complex mixtures of lipid A combinatorially modified during development of vaccine adjuvants. UVPD mass spectrometry at 193 nm was utilized to characterize the structures and fragment ion types of lipid A from Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa using an Orbitrap mass spectrometer. The fragment ions generated by UVPD were compared to those from collision induced dissociation (CID) and higher energy collision dissociation (HCD) with respect to the precursor charge state. UVPD afforded the widest array of fragment ion types including acyl chain C-O, C-N, and C-C bond cleavages and glycosidic C-O and cross ring cleavages, thus providing the most comprehensive structural analysis of the lipid A. UVPD exhibited virtually no dependence on precursor ion charge state and was best at determining lipid A structure including acyl chain length and composition, giving it an advantage over collision based methods. UVPD was incorporated into an LC-MS/MS methodology for the analysis of a number of structural variants in a complex mixture of combinatorially engineered Escherichia coli lipid A.

Community ecology of invasions: direct and indirect effects of multiple invasive species on aquatic communities.

With many ecosystems now supporting multiple nonnative species from different trophic levels, it can be challenging to disentangle the net effects of invaders within a community context. Here, we combined wetland surveys with a mesocosm experiment to examine the individual and combined effects of nonnative fish predators and nonnative bullfrogs on aquatic communities. Among 139 wetlands, nonnative fish (bass, sunfish, and mosquitofish) negatively influenced the probability of occupancy of Pacific treefrogs (Pseudacris regilla), but neither invader correlated strongly with occupancy by California newts (Taricha torosa), western toads (Anaxyrus boreas), or California red-legged frogs (Rana draytonii). In mesocosms, mosquitofish dramatically reduced the abundance of zooplankton and palatable amphibian larvae (P. regilla and T. torosa), leading to increases in nutrient concentrations and phytoplankton (through loss of zooplankton), and rapid growth of unpalatable toad larvae (through competitive release). Bullfrog larvae reduced the growth of native anurans but had no effect on survival. Despite strong effects on natives, invaders did not negatively influence one another, and their combined effects were additive. Our results highlight how the net effects of multiple nonnative species depend on the trophic level of each invader, the form and magnitude of invader interactions, and the traits of native community members.

Species diversity reduces parasite infection through cross-generational effects on host abundance.

With growing interest in the effects of biodiversity on disease, there is a critical need for studies that empirically identify the mechanisms underlying the diversity-disease relationship. Here, we combined wetland surveys of host community structure with mechanistic experiments involving a multi-host parasite to evaluate competing explanations for the dilution effect. Sampling of 320 wetlands in California indicated that snail host communities were strongly nested, with competent hosts for the trematode Ribeiroia ondatrae predominating in low-richness assemblages and unsuitable hosts increasingly present in more diverse communities. Moreover, competent host density was negatively associated with increases in snail species richness. These patterns in host community assembly support a key prerequisite underlying the dilution effect. Results of multigenerational mesocosm experiments designed to mimic field-observed community assemblages allowed us to evaluate the relative importance of host density and diversity in influencing parasite infection success. Increases in snail species richness (from one to four species) had sharply negative effects on the density of infected hosts (-90% reduction). However, this effect was indirect; competition associated with non-host species led to a 95% reduction in host density (susceptible host regulation), owing primarily to a reduction in host reproduction. Among susceptible hosts, there were no differences in infection prevalence as a function of community structure, indicating a lack of support for a direct effect of diversity on infection (encounter reduction). In monospecific conditions, higher initial host densities increased infection among adult hosts; however, compensatory reproduction in the low-density treatments equalized the final number of infected hosts by the next generation, underscoring the relevance of multigenerational studies in understanding the dilution effect. These findings highlight the role of interspecific competition in mediating the relationship between species richness and parasite infection and emphasize the importance of field-informed experimental research in understanding mechanisms underlying the diversity-disease relationship.

Multi-Substrate Specificity and the Evolutionary Basis for Interdependence in tRNA Editing and Methylation Enzymes.

Among tRNA modification enzymes there is a correlation between specificity for multiple tRNA substrates and heteromultimerization. In general, enzymes that modify a conserved residue in different tRNA sequences adopt a heterodimeric structure. Presumably, such changes in the oligomeric state of enzymes, to gain multi-substrate recognition, are driven by the need to accommodate and catalyze a particular reaction in different substrates while maintaining high specificity. This review focuses on two classes of enzymes where the case for multimerization as a way to diversify molecular recognition can be made. We will highlight several new themes with tRNA methyltransferases and will also discuss recent findings with tRNA editing deaminases. These topics will be discussed in the context of several mechanisms by which heterodimerization may have been achieved during evolution and how these mechanisms might impact modifications in different systems.

Food-web interaction strength distributions are conserved by greater variation between than within predator-prey pairs.

Species interactions in food webs are usually recognized as dynamic, varying across species, space, and time because of biotic and abiotic drivers. Yet food webs also show emergent properties that appear consistent, such as a skewed frequency distribution of interaction strengths (many weak, few strong). Reconciling these two properties requires an understanding of the variation in pairwise interaction strengths and its underlying mechanisms. We estimated stream sculpin feeding rates in three seasons at nine sites in Oregon to examine variation in trophic interaction strengths both across and within predator-prey pairs. Predator and prey densities, prey body mass, and abiotic factors were considered as putative drivers of within-pair variation over space and time. We hypothesized that consistently skewed interaction strength distributions could result if individual interaction strengths show relatively little variation, or alternatively, if interaction strengths vary but shift in ways that conserve their overall frequency distribution. Feeding rate distributions remained consistently and positively skewed across all sites and seasons. The mean coefficient of variation in feeding rates within each of 25 focal species pairs across surveys was less than half the mean coefficient of variation seen across species pairs within a survey. The rank order of feeding rates also remained conserved across streams, seasons and individual surveys. On average, feeding rates on each prey taxon nonetheless varied by a hundredfold, with some feeding rates showing more variation in space and others in time. In general, feeding rates increased with prey density and decreased with high stream flows and low water temperatures, although for nearly half of all species pairs, factors other than prey density explained the most variation. Our findings show that although individual interaction strengths exhibit considerable variation in space and time, they can nonetheless remain relatively consistent, and thus predictable, compared to the even larger variation that occurs across species pairs. These results highlight how the ecological scale of inference can strongly shape conclusions about interaction strength consistency and help reconcile how the skewed nature of interaction strength distributions can persist in highly dynamic food webs.

A systematic review of context bias in invasion biology.

The language that scientists use to frame biological invasions may reveal inherent bias-including how data are interpreted. A frequent critique of invasion biology is the use of value-laden language that may indicate context bias. Here we use a systematic study of language and interpretation in papers drawn from invasion biology to evaluate whether there is a link between the framing of papers and the interpretation of results. We also examine any trends in context bias in biological invasion research. We examined 651 peer-reviewed invasive species competition studies and implemented a rigorous systematic review to examine bias in the presentation and interpretation of native and invasive competition in invasion biology. We predicted that bias in the presentation of invasive species is increasing, as suggested by several authors, and that bias against invasive species would result in misinterpreting their competitive dominance in correlational observational studies compared to causative experimental studies. We indeed found evidence of bias in the presentation and interpretation of invasive species research; authors often introduced research with invasive species in a negative context and study results were interpreted against invasive species more in correlational studies. However, we also found a distinct decrease in those biases since the mid-2000s. Given that there have been several waves of criticism from scientists both inside and outside invasion biology, our evidence suggests that the subdiscipline has somewhat self-corrected apparent biases.

Antimicrobial peptide resistance of Vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases.

The current pandemic El Tor biotype of O1 Vibrio cholerae is resistant to polymyxins, whereas the previous pandemic strain of the classical biotype is polymyxin sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to polymyxins through the glycylation of lipopolysaccharide. Here, we present the mechanistic determination of initial steps in the AlmEFG pathway. We verify that AlmF is an aminoacyl carrier protein and identify AlmE as the enzyme required to activate AlmF as a functional carrier protein. A combination of structural information and activity assays was used to identify a pair of active site residues that are important for mediating AlmE glycine specificity. Overall, the structure of AlmE in complex with its glycyl-adenylate intermediate reveals that AlmE is related to Gram-positive d-alanine/d-alanyl carrier protein ligase, while the trio of proteins in the AlmEFG system forms a chemical pathway that resembles the division of labor in nonribosomal peptide synthetases.