Host-Specific Evolutionary and Transmission Dynamics Shape the Functional Diversification of Staphylococcus epidermidis in Human Skin.

Metagenomic inferences of bacterial strain diversity and infectious disease transmission studies largely assume a dominant, within-individual haplotype. We hypothesize that within-individual bacterial population diversity is critical for homeostasis of a healthy microbiome and infection risk. We characterized the evolutionary trajectory and functional distribution of Staphylococcus epidermidis-a keystone skin microbe and opportunistic pathogen. Analyzing 1,482 S. epidermidis genomes from 5 healthy individuals, we found that skin S. epidermidis isolates coalesce into multiple founder lineages rather than a single colonizer. Transmission events, natural selection, and pervasive horizontal gene transfer result in population admixture within skin sites and dissemination of antibiotic resistance genes within-individual. We provide experimental evidence for how admixture can modulate virulence and metabolism. Leveraging data on the contextual microbiome, we assess how interspecies interactions can shape genetic diversity and mobile gene elements. Our study provides insights into how within-individual evolution of human skin microbes shapes their functional diversification.

A host-microbiota interactome reveals extensive transkingdom connectivity.

The myriad microorganisms that live in close association with humans have diverse effects on physiology, yet the molecular bases for these impacts remain mostly unknown1-3. Classical pathogens often invade host tissues and modulate immune responses through interactions with human extracellular and secreted proteins (the 'exoproteome'). Commensal microorganisms may also facilitate niche colonization and shape host biology by engaging host exoproteins; however, direct exoproteome-microbiota interactions remain largely unexplored. Here we developed and validated a novel technology, BASEHIT, that enables proteome-scale assessment of human exoproteome-microbiome interactions. Using BASEHIT, we interrogated more than 1.7 million potential interactions between 519 human-associated bacterial strains from diverse phylogenies and tissues of origin and 3,324 human exoproteins. The resulting interactome revealed an extensive network of transkingdom connectivity consisting of thousands of previously undescribed host-microorganism interactions involving 383 strains and 651 host proteins. Specific binding patterns within this network implied underlying biological logic; for example, conspecific strains exhibited shared exoprotein-binding patterns, and individual tissue isolates uniquely bound tissue-specific exoproteins. Furthermore, we observed dozens of unique and often strain-specific interactions with potential roles in niche colonization, tissue remodelling and immunomodulation, and found that strains with differing host interaction profiles had divergent interactions with host cells in vitro and effects on the host immune system in vivo. Overall, these studies expose a previously unexplored landscape of molecular-level host-microbiota interactions that may underlie causal effects of indigenous microorganisms on human health and disease.

Enterococcus faecalis 6-phosphogluconolactonase is required for both commensal and pathogenic interactions with Manduca sexta.

Enterococcus faecalis is a commensal and pathogen of humans and insects. In Manduca sexta, E. faecalis is an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigate E. faecalis factors required for commensalism, we identified E. faecalis genes that are upregulated in the gut of M. sexta using recombinase-based in vivo expression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designated pglA. A pglA deletion mutant was impaired in both pathogenesis and gut persistence in M. sexta and produced enhanced biofilms compared with the wild type in an in vitro polystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants for E. faecalis commensal and pathogenic interactions with M. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to prevent E. faecalis infections.

Extracellular Vesicles Derived from Plasmodium-infected Hosts as Stimuli of "Trained" Innate Immunity.

Although the burden of malaria has been successfully controlled globally, this disease remains a major public health issue. To date, neither existing drugs nor vaccines against malaria are sufficient in eliminating malaria worldwide. To achieve the eradication of malaria by 2040, effective interventions targeting all Plasmodium species are urgently needed. As the cornerstone of vaccine design, immune memory serves a significant role in the host's defense against Plasmodium infections. It has long been considered that innate immunity is non-specific and lacks immunologic memory. However, emerging evidence has suggested that innate immunity can be trained following exposure of the body to infectious agents, such as Plasmodium or its products, which, in turn, promotes the onset of a type of memory in innate immune cells. The above "trained" innate immune cells, whose phenotype is modified in response to epigenetic modifications, metabolic recombination, or cytokine secretion, exhibit differential pathophysiology after the exposure of the body to a pathogen. In addition, Plasmodium-infected red blood cells and other host cells can secrete exosomes that contain conserved parasite-specific information, such as proteins, RNA, non-coding RNA molecules, and nucleic acids. These molecules can act as stimuli for promoting the establishment of "trained" innate immunity against malaria, thereby altering the onset and progression of the parasitic disease. A deeper understanding of the role of exosomes in the development of "trained" innate immunity during Plasmodium infection could provide novel therapeutic and prevention strategies against malaria infections.

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression.

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

Engineering a "detect and destroy" skin probiotic to combat methicillin-resistant Staphylococcus aureus.

The prevalence and virulence of pathogens such as methicillin-resistant Staphylococcus (S.) aureus (MRSA), which can cause recurrent skin infections, are of significant clinical concern. Prolonged antibiotic exposure to treat or decolonize S. aureus contributes to development of antibiotic resistance, as well as depletion of the microbiome, and its numerous beneficial functions. We hypothesized an engineered skin probiotic with the ability to selectively deliver antimicrobials only in the presence of the target organism could provide local bioremediation of pathogen colonization. We constructed a biosensing S. epidermidis capable of detecting the presence of S. aureus quorum sensing autoinducer peptide and producing lysostaphin in response. Here, we demonstrate in vitro activity of this biosensor and present and discuss challenges to deployment of this and other engineered topical skin probiotics.

Large-Scale CRISPRi and Transcriptomics of Staphylococcus epidermidis Identify Genetic Factors Implicated in Lifestyle Versatility.

Staphylococcus epidermidis is a ubiquitous human commensal skin bacterium that is also one of the most prevalent nosocomial pathogens. The genetic factors underlying this remarkable lifestyle plasticity are incompletely understood, mainly due to the difficulties of genetic manipulation, precluding high-throughput functional profiling of this species. To probe the versatility of S. epidermidis to survive across a diversity of environmental conditions, we developed a large-scale CRISPR interference (CRISPRi) screen complemented by transcriptional profiling (RNA sequencing) across 24 diverse conditions and piloted a droplet-based CRISPRi approach to enhance throughput and sensitivity. We identified putative essential genes, importantly revealing amino acid metabolism as crucial to survival across diverse environments, and demonstrated the importance of trace metal uptake for survival under multiple stress conditions. We identified pathways significantly enriched and repressed across our range of stress and nutrient-limited conditions, demonstrating the considerable plasticity of S. epidermidis in responding to environmental stressors. Additionally, we postulate a mechanism by which nitrogen metabolism is linked to lifestyle versatility in response to hyperosmotic challenges, such as those encountered on human skin. Finally, we examined the survival of S. epidermidis under acid stress and hypothesize a role for cell wall modification as a vital component of the survival response under acidic conditions. Taken together, this study integrates large-scale CRISPRi and transcriptomics data across multiple environments to provide insights into a keystone member of the human skin microbiome. Our results additionally provide a valuable benchmarking analysis for CRISPRi screens and are a rich resource for other staphylococcal researchers. IMPORTANCE Staphylococcus epidermidis is a bacteria that broadly inhabits healthy human skin, yet it is also a common cause of skin infections and bloodstream infections associated with implanted medical devices. Because human skin has many different types of S. epidermidis, each containing different genes, our goal is to determine how these different genes allow S. epidermidis to switch from healthy growth in the skin to being an infectious pathogen. Understanding this switch is critical to developing new strategies to prevent and treat S. epidermidis infections.

Integrating genomics and metabolomics for scalable non-ribosomal peptide discovery.

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.

A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes.

The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful-focusing on CRISPRi as a potential application-as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species.IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

A Chemical Counterpunch: Chromobacterium violaceum ATCC 31532 Produces Violacein in Response to Translation-Inhibiting Antibiotics.

Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an antibiotic-induced response (air) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules.IMPORTANCE Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration.

Seroprevalence of severe fever with thrombocytopenia syndrome virus antibodies among inhabitants of Dachen Island, eastern China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) has been emerging and has caused many human cases in China, Japan and Korea. Some studies speculated that SFTSV was transmitted with bird migration among these countries. Notably, SFTS cases have been identified in a Chinese island named Dachen which is situated southwest of Japan and Korea. In this study, we conducted a serum survey of SFTSV antibodies among inhabitants of the island. A total of 439 serum specimens were collected in June 2018. All serum samples were tested for total antibodies and IgM antibody with double-antigen sandwich ELISA method. The rates of seropositivity for SFTSV total antibodies and IgM antibody were 3.0% (95% CI 1.4-4.6) and 0.5% (2/439), respectively. The median age of all participants was 61 years and all seropositive samples were all from inhabitants aged >50 years. The differences of seroprevalence between different gender groups and different age groups were not significant. However, seroprevalence varied significantly among different villages (P = 0.033). Our results showed that some inhabitants of Dachen Island had been infected with SFTSV, and some ticks and host animals of the island carry SFTSV. Comprehensive measures should be conducted to prevent the occurrence of SFTS cases in the island.

Evaluation of INSeq To Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization.

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in Paeruginosa PGPR2 for root colonization.

Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families.

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).

Resident microbiota of the gypsy moth midgut harbors antibiotic resistance determinants.

Little is known about the significance of insects as environmental reservoirs of antibiotic-resistant bacteria. We characterized the antibiotic resistome of the microbial community in gypsy moth larval midguts by applying functional metagenomics to cultured isolates. The minimum inhibitory concentrations of 12 antibiotics were determined for 44 cultured isolates, and antibiotic resistance genes were selected from metagenomic libraries derived from DNA extracted from a pool of the isolates. Six unique clones were identified. Two were highly resistant to penicillin-type beta-lactams, two were moderately resistant to erythromycin, and two were moderately resistant to a range of antibiotics, including erythromycin, carbenicillin, and chloramphenicol. Sequence analysis predicted that the active genes encoded efflux pumps, a transcriptional activator of efflux pump protein expression, and an extended-spectrum class A beta-lactamase. Insect guts are a reservoir of antibiotic resistance genes with the potential for dissemination.

Signal mimics derived from a metagenomic analysis of the gypsy moth gut microbiota.

Bacterial signaling is an important part of community life, but little is known about the signal transduction pathways of the as-yet-uncultured members of microbial communities. To address this gap, we aimed to identify genes directing the synthesis of signals in uncultured bacteria associated with the midguts of gypsy moth larvae. We constructed a metagenomic library consisting of DNA extracted directly from the midgut microbiota and analyzed it using an intracellular screen designated METREX, which detects inducers of quorum sensing. In this screen, the metagenomic DNA and a biosensor reside in the same cell. The biosensor consists of a quorum-sensing promoter, which requires an acylhomoserine lactone or other small molecule ligand for activation, driving the expression of the reporter gene gfp. We identified an active metagenomic clone encoding a monooxygenase homologue that mediates a pathway of indole oxidation that leads to the production of a quorum-sensing inducing compound. The signal from this clone induces the activities of LuxR from Vibrio fischeri and CviR from Chromobacterium violaceum. This study is the first to identify a new structural class of quorum-sensing inducer from uncultured bacteria.

Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor.

The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.

Complex physiological and molecular processes underlying root gravitropism.

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway.

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

Arabidopsis thaliana: A Model for the Study of Root and Shoot Gravitropism.

UNLABELLED: For most plants, shoots grow upward and roots grow downward. These growth patterns illustrate the ability for plant organs to guide their growth at a specified angle from the gravity vector (gravitropism). They allow shoots to grow upward toward light, where they can photosynthesize, and roots to grow downward into the soil, where they can anchor the plant as well as take up water and mineral ions.Gravitropism involves several steps organized in a specific response pathway. These include the perception of a gravistimulus (reorientation within the gravity field), the transduction of this mechanical stimulus into a physiological signal, the transmission of this signal from the site of sensing to the site of response, and a curvature-response which allows the organ tip to resume growth at a predefined set angle from the gravity vector.The primary sites for gravity sensing are located in the cap for roots, and in the endodermis for shoots. The curvature response occurs in the elongation zones for each organ. Upon gravistimulation, a gradient of auxin appears to be generated across the stimulated organ, and be transmitted to the site of response where it promotes a differential growth response. Therefore, while the gravity-induced auxin gradient has to be transmitted from the cap to the elongation zones in roots, there is no need for a longitudinal transport in shoots, as sites for gravity sensing and response overlap in this organ.A combination of molecular genetics, physiology, biochemistry and cell biology, coupled with the utilization of Arabidopsis thaliana as a model system, have recently allowed the identification of a number of molecules involved in the regulation of each phase of gravitropism in shoots and roots of higher plants. In this review, we attempt to summarize the results of these experiments, and we conclude by comparing the molecular and physiological mechanisms that underlie gravitropism in these organs. ABBREVIATIONS: GSPA: gravitational set point angle; IAA: indole-3-acetic acid; NAA: 1-naphthalene acetic acid; NPA: 1-N-naphthylphthalamic acid; 2,4-D: 2,4-dichlorphenoxy acetic acid; TIBA: 2,3,5-triiodobenzoic acid.