Michiko E. Taga

How rhizobial symbionts invade plants: the Sinorhizobium – Medicago model

Nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex signal exchange mechanisms that allow a specific bacterial species to induce its host plant to form invasion structures through which the bacteria can enter the plant root. Once the bacteria have been endocytosed within a host-membrane-bound compartment by root cells, the bacteria differentiate into a new form that can convert atmospheric nitrogen into ammonia. Bacterial differentiation and nitrogen fixation are dependent on the microaerobic environment and other support factors provided by the plant. In return, the plant receives nitrogen from the bacteria, which allows it to grow in the absence of an external nitrogen source. Here, we review recent discoveries about the mutual recognition process that allows the model rhizobial symbiont Sinorhizobium meliloti to invade and differentiate inside its host plant alfalfa (Medicago sativa) and the model host plant barrel medic (Medicago truncatula).

The LuxS-dependent autoinducer AI-2 controls the expression of an ABC transporter that functions in AI-2 uptake in Salmonella typhimurium

In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI‐2, originally discovered in the quorum‐sensing bacterium Vibrio harveyi, is made by many species of Gram‐negative and Gram‐positive bacteria. In every case, production of AI‐2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI‐2 in most of these luxS‐containing species of bacteria are not known. Here, we describe the identification and characterization of AI‐2‐regulated genes in Salmonella typhimurium. We find that LuxS and AI‐2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)‐type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium. A gene encoding a DNA‐binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI‐2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI‐2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI‐2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI‐2 into the cells. It is intriguing that an operon regulated by AI‐2 encodes functions resembling the ribose transporter, given recent findings that AI‐2 is derived from the ribosyl moiety of S‐ribosylhomocysteine.

Lsr‐mediated transport and processing of AI‐2 in Salmonella typhimurium

The LuxS‐dependent autoinducer AI‐2 is proposed to function in interspecies cell–cell communication in bacteria. In Salmonella typhimurium, AI‐2 is produced and released during exponential growth and is subsequently imported into the bacteria via the Lsr (luxS regulated) ATP binding cassette (ABC) transporter. AI‐2 induces transcription of the lsrACDBFGE operon, the first four genes of which encode the Lsr transport apparatus. In this report, we identify and characterize LsrK, a new protein that is required for the regulation of the lsr operon and the AI‐2 uptake process. LsrK is a kinase that phosphorylates AI‐2 upon entry into the cell. Our data indicate that phosphorylation of AI‐2 results in its sequestration in the cytoplasm. We suggest that phospho‐AI‐2 is the inducer responsible for inactivation of LsrR, the repressor of the lsr operon. We also show that two previously uncharacterized members of the lsr operon, LsrF and LsrG, are necessary for the further processing of phospho‐AI‐2. Transport and processing of AI‐2 could be required for removing the quorum‐sensing signal, conveying the signal to an internal detector and/or scavenging boron.

BluB cannibalizes flavin to form the lower ligand of vitamin B12.

Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms. The biosynthesis of the lower ligand of vitamin B12, 5,6-dimethylbenzimidazole (DMB), is poorly understood. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and d-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease.

Human Gut Microbes Use Multiple Transporters to Distinguish Vitamin B12 Analogs and Compete in the Gut

Genomic and metagenomic sequencing efforts, including human microbiome projects, reveal that microbes often encode multiple systems that appear to accomplish the same task. Whether these predictions reflect actual functional redundancies is unclear. We report that the prominent human gut symbiont Bacteroides thetaiotaomicron employs three functional, homologous vitamin B₁₂ transporters that in at least two cases confer a competitive advantage in the presence of distinct B₁₂ analogs (corrinoids). In the mammalian gut, microbial fitness can be determined by the presence or absence of a single transporter. The total number of distinct corrinoid transporter families in the human gut microbiome likely exceeds those observed in B. thetaiotaomicron by an order of magnitude. These results demonstrate that human gut microbes use elaborate mechanisms to capture and differentiate corrinoids in vivo and that apparent redundancies observed in these genomes can instead reflect hidden specificities that determine whether a microbe will colonize its host.

Nutrient cross-feeding in the microbial world.

The stability and function of a microbial community depends on nutritional interactions among community members such as the cross-feeding of essential small molecules synthesized by a subset of the population. In this review, we describe examples of microbe–microbe and microbe–host cofactor cross-feeding, a type of interaction that influences the forms of metabolism carried out within a community. Cofactor cross-feeding can contribute to both the health and nutrition of a host organism, the virulence and persistence of pathogens, and the composition and function of environmental communities. By examining the impact of shared cofactors on microbes from pure culture to natural communities, we stand to gain a better understanding of the interactions that link microbes together, which may ultimately be a key to developing strategies for manipulating microbial communities with human health, agricultural, and environmental implications.

Vitamin B12 as a Modulator of Gut Microbial Ecology

The microbial mechanisms and key metabolites that shape the composition of the human gut microbiota are largely unknown, impeding efforts to manipulate dysbiotic microbial communities toward stability and health. Vitamins, which by definition are not synthesized in sufficient quantities by the host and can mediate fundamental biological processes in microbes, represent an attractive target for reshaping microbial communities. Here, we discuss how vitamin B12 (cobalamin) impacts diverse host-microbe symbioses. Although cobalamin is synthesized by some human gut microbes, it is a precious resource in the gut and is likely not provisioned to the host in significant quantities. However, this vitamin may make an unrecognized contribution in shaping the structure and function of human gut microbial communities.

Versatility in Corrinoid Salvaging and Remodeling Pathways Supports Corrinoid-Dependent Metabolism in Dehalococcoides mccartyi

ABSTRACT Corrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism in Dehalococcoides mccartyi strain 195. Dehalococcoides bacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolated D. mccartyi strains require exogenous corrinoids such as vitamin B12 for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterized D. mccartyi strains has been shown to be capable of synthesizing corrinoids de novo. In this study, we investigate the ability of D. mccartyi strain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification by D. mccartyi provides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.

Sinorhizobium meliloti, a bacterium lacking the autoinducer‐2 (AI‐2) synthase, responds to AI‐2 supplied by other bacteria

Many bacterial species respond to the quorum‐sensing signal autoinducer‐2 (AI‐2) by regulating different niche‐specific genes. Here, we show that Sinorhizobium meliloti, a plant symbiont lacking the gene for the AI‐2 synthase, while not capable of producing AI‐2 can nonetheless respond to AI‐2 produced by other species. We demonstrate that S. meliloti has a periplasmic binding protein that binds AI‐2. The crystal structure of this protein (here named SmlsrB) with its ligand reveals that it binds (2R,4S)‐2‐methyl‐2,3,3,4‐tetrahydroxytetrahydrofuran (R‐THMF), the identical AI‐2 isomer recognized by LsrB of Salmonella typhimurium. The gene encoding SmlsrB is in an operon with orthologues of the lsr genes required for AI‐2 internalization in enteric bacteria. Accordingly, S. meliloti internalizes exogenous AI‐2, and mutants in this operon are defective in AI‐2 internalization. S. meliloti does not gain a metabolic benefit from internalizing AI‐2, suggesting that AI‐2 functions as a signal in S. meliloti. Furthermore, S. meliloti can completely eliminate the AI‐2 secreted by Erwinia carotovora, a plant pathogen shown to use AI‐2 to regulate virulence. Our findings suggest that S. meliloti is capable of ‘eavesdropping’ on the AI‐2 signalling of other species and interfering with AI‐2‐regulated behaviours such as virulence.

Methods for Analysis of Bacterial Autoinducer‐2 Production

The quorum-sensing signal molecule autoinducer-2 (AI-2) is produced by over 50 diverse bacterial species and controls many different processes, including antibiotic production, biofilm formation, and virulence. AI-2 production often varies according to growth phase, media conditions, and the presence of specific factors. This unit describes a biological assay for AI-2 activity produced by a bacterial strain of interest. The assay employs an AI-2 reporter strain, Vibrio harveyi BB170, which produces light in response to AI-2. In the first stage of the assay, culture fluids of the bacterial strain of interest are collected over a time course of growth and filtered to remove cells. In the next stage, these culture fluids are mixed with BB170, and the light produced in response to AI-2 in the culture fluids is measured using a luminometer. BB170 is exquisitely sensitive to AI-2, and therefore, even low amounts of AI-2 can be detected using this bioassay.