Characterization of Acinetobacter baumannii core oligosaccharide synthesis reveals novel aspects of lipooligosaccharide assembly.

A fundamental feature of Gram-negative bacteria is their outer membrane that protects the cell against environmental stressors. This defense is predominantly due to its asymmetry, with glycerophospholipids located in the inner leaflet and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) confined to the outer leaflet. LPS consists of a lipid A anchor, a core oligosaccharide, and a distal O-antigen while LOS lacks O-antigen. While LPS/LOS is typically essential for growth, this is not the case for Acinetobacter baumannii. Despite this unique property, the synthesis of the core oligosaccharide of A. baumannii LOS is not well-described. Here, we characterized the LOS chemotypes of A. baumannii strains with mutations in a predicted core oligosaccharide locus via tandem mass spectrometry. This allowed for an extensive identification of genes required for core assembly that can be exploited to generate precise structural LOS modifications in many A. baumannii strains. We further investigated two chemotypically identical yet phenotypically distinct mutants, ∆2903 and ∆lpsB, that exposed a possible link between LOS and the peptidoglycan cell wall-two cell envelope components whose coordination has not yet been described in A. baumannii. Selective reconstruction of the core oligosaccharide via expression of 2903 and LpsB revealed that these proteins rely on each other for the unusual tandem transfer of two residues, KdoIII and N-acetylglucosaminuronic acid. The data presented not only allow for better usage of A. baumannii as a tool to study outer membrane integrity but also provide further evidence for a novel mechanism of core oligosaccharide assembly. IMPORTANCE: Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination.

Carotenoid ornaments and the spandrels of physiology: a critique of theory to explain condition dependency.

Even as numerous studies have documented that the red and yellow coloration resulting from the deposition of carotenoids serves as an honest signal of condition, the evolution of condition dependency is contentious. The resource trade-off hypothesis proposes that condition-dependent honest signalling relies on a trade-off of resources between ornamental display and body maintenance. By this model, condition dependency can evolve through selection for a re-allocation of resources to promote ornament expression. By contrast, the index hypothesis proposes that selection focuses mate choice on carotenoid coloration that is inherently condition dependent because production of such coloration is inexorably tied to vital cellular processes. These hypotheses for the origins of condition dependency make strongly contrasting and testable predictions about ornamental traits. To assess these two models, we review the mechanisms of production of carotenoids, patterns of condition dependency involving different classes of carotenoids, and patterns of behavioural responses to carotenoid coloration. We review evidence that traits can be condition dependent without the influence of sexual selection and that novel traits can show condition-dependent expression as soon as they appear in a population, without the possibility of sexual selection. We conclude by highlighting new opportunities for studying condition-dependent signalling made possible by genetic manipulation and expression of ornamental traits in synthetic biological systems.

Chemical manipulation of mitochondrial function affects metabolism of red carotenoids in a marine copepod (Tigriopus californicus).

The shared-pathway hypothesis offers a cellular explanation for the connection between ketocarotenoid pigmentation and individual quality. Under this hypothesis, ketocarotenoid metabolism shares cellular pathways with mitochondrial oxidative phosphorylation such that red carotenoid-based coloration is inextricably linked mitochondrial function. To test this hypothesis, we exposed Tigriopus californicus copepods to a mitochondrially targeted protonophore, 2,4-dinitrophenol (DNP), to induce proton leak in the inner mitochondrial membranes. We then measured whole-animal metabolic rate and ketocarotenoid accumulation. As observed in prior studies of vertebrates, we observed that DNP treatment of copepods significantly increased respiration and that DNP-treated copepods accumulated more ketocarotenoid than control animals. Moreover, we observed a relationship between ketocarotenoid concentration and metabolic rate, and this association was strongest in DNP-treated copepods. These data support the hypothesis that ketocarotenoid and mitochondrial metabolism are biochemically intertwined. Moreover, these results corroborate observations in vertebrates, perhaps suggesting a fundamental connection between ketocarotenoid pigmentation and mitochondrial function that should be explored further.

Acinetobactin-Mediated Inhibition of Commensal Bacteria by Acinetobacter baumannii.

Acinetobacter baumannii is an important hospital-associated pathogen that causes antibiotic resistant infections and reoccurring hospital outbreaks. A. baumannii's ability to asymptomatically colonize patients is a risk factor for infection and exacerbates its spread. However, there is little information describing the mechanisms it employs to colonize patients. A. baumannii often colonizes the upper respiratory tract and skin. Antibiotic use is a risk factor for colonization and infection suggesting that A. baumannii likely competes with commensal bacteria to establish a niche. To begin to investigate this possibility, we cocultured A. baumannii and commensal bacteria of the upper respiratory tract and skin. In conditions that mimic iron starvation experienced in the host, we observed that A. baumannii inhibits Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and Corynebacterium striatum. Then using an ordered transposon library screen we identified the A. baumannii siderophore acinetobactin as the causative agent of the inhibition phenotype. Using mass spectrometry, we show that acinetobactin is released from A. baumannii under our coculture conditions and that purified acinetobactin can inhibit C. striatum and S. hominis. Together our data suggest that acinetobactin may provide a competitive advantage for A. baumannii over some respiratory track and skin commensal bacteria and possibly support its ability to colonize patients. IMPORTANCE The ability of Acinetobacter baumannii to asymptomatically colonize patients is a risk factor for infection and exacerbates its clinical spread. However, there is minimal information describing how A. baumannii asymptomatically colonizes patients. Here we provide evidence that A. baumannii can inhibit the growth of many skin and upper respiratory commensal bacteria through iron competition and identify acinetobactin as the molecule supporting its nutritional advantage. Outcompeting endogenous commensals through iron competition may support the ability of A. baumannii to colonize and spread among patients.

Evidence for hybrid breakdown in production of red carotenoids in the marine invertebrate Tigriopus californicus.

The marine copepod, Tigriopus californicus, produces the red carotenoid pigment astaxanthin from yellow dietary precursors. This 'bioconversion' of yellow carotenoids to red is hypothesized to be linked to individual condition, possibly through shared metabolic pathways with mitochondrial oxidative phosphorylation. Experimental inter-population crosses of lab-reared T. californicus typically produces low-fitness hybrids is due in large part to the disruption of coadapted sets nuclear and mitochondrial genes within the parental populations. These hybrid incompatibilities can increase variability in life history traits and energy production among hybrid lines. Here, we tested if production of astaxanthin was compromised in hybrid copepods and if it was linked to mitochondrial metabolism and offspring development. We observed no clear mitonuclear dysfunction in hybrids fed a limited, carotenoid-deficient diet of nutritional yeast. However, when yellow carotenoids were restored to their diet, hybrid lines produced less astaxanthin than parental lines. We observed that lines fed a yeast diet produced less ATP and had slower offspring development compared to lines fed a more complete diet of algae, suggesting the yeast-only diet may have obscured effects of mitonuclear dysfunction. Astaxanthin production was not significantly associated with development among lines fed a yeast diet but was negatively related to development in early generation hybrids fed an algal diet. In lines fed yeast, astaxanthin was negatively related to ATP synthesis, but in lines fed algae, the relationship was reversed. Although the effects of the yeast diet may have obscured evidence of hybrid dysfunction, these results suggest that astaxanthin bioconversion may still be related to mitochondrial performance and reproductive success.

A Review and Assessment of the Shared-Pathway Hypothesis for the Maintenance of Signal Honesty in Red Ketocarotenoid-Based Coloration.

For decades, scientists have noted connections between individual condition and carotenoid-based coloration in terrestrial and aquatic animals. Organisms that produce more vibrant carotenoid-based coloration tend to have better physiological performance and behavioral displays compared with less colorful members of the same species. Traditional explanations for this association between ornamental coloration and performance invoked the need for color displays to be costly, but evidence for such hypothesized costs is equivocal. An alternative explanation for the condition-dependence of carotenoid-based coloration, the Shared-Pathway Hypothesis (SPH), was developed in response. This hypothesis proposes that red ketocarotenoid-based coloration is tied to core cellular processes involving a shared pathway with mitochondrial energy metabolism, making the concentration of carotenoids an index of mitochondrial function. Since the presentation of this hypothesis, empirical tests of the mechanisms proposed therein have been conducted in several species. In this manuscript, we review the SPH and the growing number of studies that have investigated a connection between carotenoid-based coloration and mitochondrial function. We also discuss future strategies for assessing the SPH to more effectively disentangle evidence that may simultaneously support evidence of carotenoid-resource tradeoffs.

The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport.

The asymmetric outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier to the environment. Perturbations to OM lipid asymmetry sensitize the cell to antibiotics. As such, mechanisms involved in lipid asymmetry are fundamental to our understanding of OM lipid homeostasis. One such mechanism, the Maintenance of lipid asymmetry (Mla) pathway has been proposed to extract mislocalized glycerophospholipids from the outer leaflet of the OM and return them to the inner membrane (IM). Work on this pathway in Acinetobacter baumannii support conflicting models for the directionality of the Mla system being retrograde (OM to IM) or anterograde (IM to OM). Here, we show conclusively that A. baumannii mla mutants exhibit no defects in anterograde transport. Furthermore, we identify an allele of the GTPase obgE that is synthetically sick in the absence of Mla; providing another link between cell envelope homeostasis and stringent response.

Campylobacter jejuni BumSR directs a response to butyrate via sensor phosphatase activity to impact transcription and colonization.

Campylobacter jejuni monitors intestinal metabolites produced by the host and microbiota to initiate intestinal colonization of avian and animal hosts for commensalism and infection of humans for diarrheal disease. We previously discovered that C. jejuni has the capacity to spatially discern different intestinal regions by sensing lactate and the short-chain fatty acids acetate and butyrate and then alter transcription of colonization factors appropriately for in vivo growth. In this study, we identified the C. jejuni butyrate-modulated regulon and discovered that the BumSR two-component signal transduction system (TCS) directs a response to butyrate by identifying mutants in a genetic screen defective for butyrate-modulated transcription. The BumSR TCS, which is important for infection of humans and optimal colonization of avian hosts, senses butyrate likely by indirect means to alter transcription of genes encoding important colonization determinants. Unlike many canonical TCSs, the predicted cytoplasmic sensor kinase BumS lacked in vitro autokinase activity, which would normally lead to phosphorylation of the cognate BumR response regulator. Instead, BumS has likely evolved mutations to naturally function as a phosphatase whose activity is influenced by exogenous butyrate to control the level of endogenous phosphorylation of BumR and its ability to alter transcription of target genes. To our knowledge, the BumSR TCS is the only bacterial signal transduction system identified so far that mediates responses to the microbiota-generated intestinal metabolite butyrate, an important factor for host intestinal health and homeostasis. Our findings suggest that butyrate sensing by this system is vital for C. jejuni colonization of multiple hosts.

Ultraviolet irradiation increases size of the first clutch but decreases longevity in a marine copepod.

An important component of life history theory is understanding how natural variation arises in populations. Both endogenous and exogenous factors contribute to organism survival and reproduction, and therefore, it is important to understand how such factors are both beneficial and detrimental to population dynamics. One ecologically relevant factor that influences the life history of aquatic organisms is ultraviolet (UV) radiation. While the majority of research has focused on the potentially detrimental effects that UV radiation has on aquatic organisms, few studies have evaluated hormetic responses stimulated by radiation under select conditions. The goal of this study was to evaluate the impact of UV-A/B irradiation on life history characteristics in Tigriopus californicus copepods. After exposing copepods to UV-A/B irradiation (control, 1-, and 3-hr UV treatments at 0.5 W/m2), we measured the impact of exposure on fecundity, reproductive effort, and longevity. We found that UV irradiation increased the size of the first clutch among all reproducing females in both the 1- and 3-hr experimental groups and decreased longevity among all females that mated in the 1-hr treatment. UV irradiation had no effect on the number of clutches females produced. These findings indicate a potential benefit of UV irradiation on reproductive performance early in life, although the same exposure came at a cost to longevity.

Plumage redness signals mitochondrial function in the house finch.

Carotenoid coloration is widely recognized as a signal of individual condition in various animals, but despite decades of study, the mechanisms that link carotenoid coloration to condition remain unresolved. Most birds with red feathers convert yellow dietary carotenoids to red carotenoids in an oxidation process requiring the gene encoding the putative cytochrome P450 enzyme CYP2J19. Here, we tested the hypothesis that the process of carotenoid oxidation and feather pigmentation is functionally linked to mitochondrial performance. Consistent with this hypothesis, we observed high levels of red ketolated carotenoids associated with the hepatic mitochondria of moulting wild house finches (Haemorhous mexicanus), and upon fractionation, we found the highest concentration of ketolated carotenoids in the inner mitochondrial membrane. We further found that the redness of growing feathers was positively related to the performance of liver mitochondria. Structural modelling of CYP2J19 supports a direct role of this protein in carotenoid ketolation that may be functionally linked to cellular respiration. These observations suggest that feather coloration serves as a signal of core functionality through inexorable links to cellular respiration in the mitochondria.

Intermembrane transport: Glycerophospholipid homeostasis of the Gram-negative cell envelope.

This perspective addresses recent advances in lipid transport across the Gram-negative inner and outer membranes. While we include a summary of previously existing literature regarding this topic, we focus on the maintenance of lipid asymmetry (Mla) pathway. Discovered in 2009 by the Silhavy group [J. C. Malinverni, T. J. Silhavy, Proc. Natl. Acad. Sci. U.S.A. 106, 8009-8014 (2009)], Mla has become increasingly appreciated for its role in bacterial cell envelope physiology. Through the work of many, we have gained an increasingly mechanistic understanding of the function of Mla via genetic, biochemical, and structural methods. Despite this, there is a degree of controversy surrounding the directionality in which Mla transports lipids. While the initial discovery and subsequent studies have posited that it mediated retrograde lipid transport (removing glycerophospholipids from the outer membrane and returning them to the inner membrane), others have asserted the opposite. This Perspective aims to lay out the evidence in an unbiased, yet critical, manner for Mla-mediated transport in addition to postulation of mechanisms for anterograde lipid transport from the inner to outer membranes.

Top-Down Characterization of Lipooligosaccharides from Antibiotic-Resistant Bacteria.

Modification of structures of lipooligosaccharides (LOS) represents one prevalent mechanism by which Gram-negative bacteria can become resistant to key antibiotics. Owing to the significant complexity of LOS, the structural characterization of these amphipathic lipids has largely focused on elucidation of the lipid A substructures. Analysis of intact LOS enables detection of core oligosaccharide modifications and gives insight into the heterogeneity that results from combinations of lipid A and oligosaccharide substructures. Top-down analysis of intact LOS also provides the opportunity to determine unknown oligosaccharide structures, which is particularly advantageous in the context of glycoconjugate vaccine development. Advances in mass spectrometry technologies, including the development of MSn capabilities and alternative ion activation techniques, have made top-down analysis an indispensable tool for structural characterization of complex biomolecules. Here we combine online chromatographic separations with MS3 utilizing ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD). HCD generally provides information about the presence of labile modifications via neutral loss fragments in addition to the saccharide linkage arrangement, whereas UVPD gives more detailed insight about saccharide branching and the positions of nonstoichiometric modifications. This integrated approach was used to characterize LOS from Acinetobacter baumannii 1205 and 5075. Notably, MS3 analysis of A. baumannii 1205, an antibiotic-resistant strain, confirmed phosphoethanolamine and hexosamine modification of the lipid A substructure and further enabled derivation of a core oligosaccharide structure.

RecET-Mediated Recombineering in Acinetobacter baumannii.

Acinetobacter baumannii rapidly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, enhancing its persistence in hospital settings. Tools to rapidly dissect the A. baumannii genome are needed to understand cellular factors that contribute to its resiliency at a genetic and mechanistic level. While a substantial amount of clinical data has documented the global rise of A. baumannii as an antibiotic-resistant pathogen, genetic tools to dissect its molecular details have been limited. This procedure describes a recombination-mediated genetic engineering (recombineering) system for targeted genome editing of A. baumannii. This system can perform directed mutagenesis on wide-ranging genes and operons and has broad application in various strains of A. baumannii.

Isolation of Lipid Cell Envelope Components from Acinetobacter baumannii.

With the increasing occurrence of antibiotic resistance among Acinetobacter sp., the race is on for researchers to not only isolate resistant isolates but also utilize basic and applied microbiological techniques to study mechanisms of resistance. For many antibiotics, the limit of efficacy against Gram-negative bacteria is dependent on its ability to permeate the outer membrane and access its target. As such, it is critical that researchers be able to isolate and analyze the lipid components of the cell envelope from any number of Acinetobacter sp. that are either resistant or sensitive to antibiotics of interest. The following chapter provides in-depth protocols to confirm the presence or absence of lipooligosaccharide (LOS) in Acinetobacter sp., isolate lipid A, and glycerophospholipids and analyze them using qualitative (mass spectrometry) and semiquantitative (thin-layer chromatography) methods.

Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics.

The outer membrane of Gram-negative bacteria is a critical barrier that prevents entry of noxious compounds. Integral to this functionality is the presence of lipopolysaccharide (LPS) or lipooligosaccharide (LOS), a molecule that is located exclusively in the outer leaflet of the outer membrane. Its lipid anchor, lipid A, is a glycolipid whose hydrophobicity and net negative charge are primarily responsible for the robustness of the membrane. Because of this, lipid A is a hallmark of Gram-negative physiology and is generally essential for survival. Rare exceptions have been described, including Acinetobacter baumannii, which can survive in the absence of lipid A, albeit with significant growth and membrane permeability defects. Here, we show by an evolution experiment that LOS-deficient A. baumannii can rapidly improve fitness over the course of only 120 generations. We identified two factors which negatively contribute to fitness in the absence of LOS, Mla and PldA. These proteins are involved in glycerophospholipid transport (Mla) and lipid degradation (PldA); both are active only on mislocalized, surface-exposed glycerophospholipids. Elimination of these two mechanisms was sufficient to cause a drastic fitness improvement in LOS-deficient A. baumannii The LOS-deficient double mutant grows as robustly as LOS-positive wild-type bacteria while remaining resistant to the last-resort polymyxin antibiotics. These data provide strong biological evidence for the directionality of Mla-mediated glycerophospholipid transport in Gram-negative bacteria and furthers our knowledge of asymmetry-maintenance mechanisms in the context of the outer membrane barrier.

Design of synthetic bacterial communities for predictable plant phenotypes.

Specific members of complex microbiota can influence host phenotypes, depending on both the abiotic environment and the presence of other microorganisms. Therefore, it is challenging to define bacterial combinations that have predictable host phenotypic outputs. We demonstrate that plant-bacterium binary-association assays inform the design of small synthetic communities with predictable phenotypes in the host. Specifically, we constructed synthetic communities that modified phosphate accumulation in the shoot and induced phosphate starvation-responsive genes in a predictable fashion. We found that bacterial colonization of the plant is not a predictor of the plant phenotypes we analyzed. Finally, we demonstrated that characterizing a subset of all possible bacterial synthetic communities is sufficient to predict the outcome of untested bacterial consortia. Our results demonstrate that it is possible to infer causal relationships between microbiota membership and host phenotypes and to use these inferences to rationally design novel communities.

Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens.

UNLABELLED: Interspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain (phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria. IMPORTANCE: Biofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that inhibited biofilm gene expression in Bacillus subtilis. We identified Pseudomonas protegens as one such bacterium and found that the biofilm-inhibiting compound it produces was the antibiotic 2,4-diacetylphloroglucinol (DAPG). We showed that even at subinhibitory concentrations, DAPG inhibits biofilm formation and sporulation in B. subtilis. These findings have potential implications for understanding the interactions between these two microbes in the natural world and support the idea that many compounds considered antibiotics can impact bacterial development at subinhibitory concentrations.