Export of metabolites by the proteins of the DMT and RhtB families and its possible role in intercellular communication

The earlier published and new experimental data are summarized on the properties of the genes encoding the membrane proteins of the DMT family (RhtA (YbiF), EamA (YdeD), YijE, YddG, YedA, PecM, eukaryotic nucleotide sugar, triose phosphate/phosphate, and hexose phosphate transporters), the RhtB/LysE family (RhtB, RhtC, LeuE, YahN, EamB (YfiK), ArgO (YggA), CmaU), as well as some other families (YicM, YdhC, YdeAB, YdhE (NorE)). These proteins are involved in the export of amino acids, purines, and other metabolites from the cell. The expression of most of the genes encoding these proteins is not induced by the substrates they transport but is controlled by the global regulation systems, such as the Lrp protein, and activated by the signal compounds involved in the intracellular communication. The level of expression, assessed in experiments on translational fusion of the corresponding bacterial genes with the β-galactosidase gene, depends on the growth phase of the bacterial culture, composition of the medium, and some stress factors, such as pH, osmolarity or decreased aeration. The efflux of normal cell metabolites is assumed to be the natural function of these proteins. This function may play a role in density-dependent behavior of cell populations (quorum sensing). It may have been enhanced in the course of evolution via specialization of these proteins in the efflux of compounds derived from metabolic intermediates and adjusted to the role of transmitters.     

Expression of the genes encoding RhtB family proteins depends on global regulator Lrp

In the present work, further study of the genes encoding RhtB family proteins is presented. In our previous work the involvement of two family members, RhtB and RhtC, in efflux of amino acids was demonstrated. Now we investigated regulation of expression of the rhtB, rhtC, yeaS and yahN genes. It is shown that expression of these genes is under control of the global regulator Lrp, depends on the presence of some amino acids in growth medium, and increases during different physiological stresses.     

Expression of Genes Encoding the RhtB Family Proteins Depends on the Global Regulator Lrp

In this work, we continued to study the genes encoding the RhtB family proteins. We studied regulation of four genes of this family: rhtB, rhtC, yeaS, and yahN, two of which (rhtB and rhtC) were previously shown to be involved in amino acid efflux from cells. The results of this study showed that the expression of these genes is regulated by the global regulator Lrp; it depends on the presence of certain amino acids in the growth medium and increases in certain types of physiological stress.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 374–378.Original Russian Text Copyright © 2005 by Kutukova, Zakataeva, Livshits.     

Characterization of the Escherichia coli AaeAB efflux pump: a metabolic relief valve?

Treatment of Escherichia coli with p-hydroxybenzoic acid (pHBA) resulted in upregulation of yhcP, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes yhcQ, encoding a protein of the membrane fusion protein family, and yhcR, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene yhcS, encoding a regulatory protein of the LysR family, in regulating expression of yhcRQP was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of yhcRQP expression. The efflux function encoded by yhcP was proven by the hypersensitivity to pHBA of a yhcP mutant strain. A yhcS mutant strain was also hypersensitive to pHBA. Expression of yhcQ and yhcP was necessary and sufficient for suppression of the pHBA hypersensitivity of the yhcS mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming yhcS, yhcR, yhcQ, and yhcP, to reflect their role in aromatic carboxylic acid efflux, to aaeR, aaeX, aaeA, and aaeB, respectively. The role of pHBA in normal E. coli metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a "metabolic relief valve" to alleviate toxic effects of imbalanced metabolism.     

Influence of threonine exporters on threonine production in Escherichia coli

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.     

Discovery of inhibitors of Pseudomonas aeruginosa virulence through the search for natural-like compounds with a dual role as inducers and substrates of efflux pumps

Multidrug efflux pumps are ancient elements encoded in every genome, from bacteria to humans. In bacteria, in addition to antibiotics, efflux pumps extrude a wide range of substrates, including quorum sensing signals, bacterial metabolites, or plant-produced compounds. This indicates that their original functions may differ from their recently acquired role in the extrusion of antibiotics during human infection. Concerning plant-produced compounds, some of them are substrates and inducers of the same efflux pump, suggesting a coordinated plant/bacteria coevolution. Herein we analyse the ability of 1243 compounds from a Natural Product-Like library to induce the expression of P. aeruginosa mexCD-oprJ or mexAB-oprM efflux pumps' encoding genes. We further characterized natural-like compounds that do not trigger antibiotic resistance in P. aeruginosa and that act as virulence inhibitors, choosing those that were not only inducers but substrates of the same efflux pump. Four compounds impair swarming motility, exotoxin secretion through the Type 3 Secretion System (T3SS) and the ability to kill Caenorhabditis elegans, which might be explained by the downregulation of genes encoding flagellum and T3SS. Our results emphasize the possibility of discovering new anti-virulence drugs by screening natural or natural-like libraries for compounds that behave as both, inducers and substrates of efflux pumps.     

Ecological genomics of marine Roseobacters

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.     

Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems

Multidrug efflux pumps have emerged as relevant elements in the intrinsic and acquired antibiotic resistance of bacterial pathogens. In contrast with other antibiotic resistance genes that have been obtained by virulent bacteria through horizontal gene transfer, genes coding for multidrug efflux pumps are present in the chromosomes of all living organisms. In addition, these genes are highly conserved (all members of the same species contain the same efflux pumps) and their expression is tightly regulated. Together, these characteristics suggest that the main function of these systems is not resisting the antibiotics used in therapy and that they should have other roles relevant to the behavior of bacteria in their natural ecosystems. Among the potential roles, it has been demonstrated that efflux pumps are important for processes of detoxification of intracellular metabolites, bacterial virulence in both animal and plant hosts, cell homeostasis and intercellular signal trafficking.     

Mutations in multidrug efflux homologs, sugar isomerases, and antimicrobial biosynthesis genes differentially elevate activity of the sigma(X) and sigma(W) factors in Bacillus subtilis

The sigma(X) and sigma(W) extracytoplasmic function sigma factors regulate more than 40 genes in Bacillus subtilis. sigma(W) activates genes which function in detoxification and the production of antimicrobial compounds, while sigma(X) activates functions that modify the cell envelope. Transposon mutagenesis was used to identify loci which negatively regulate sigma(W) or sigma(X) as judged by up-regulation from the autoregulatory promoter site P(W) or P(X). Fourteen insertions that activate P(W) were identified. The largest class of insertions are likely to affect transport. These include insertions in genes encoding two multidrug efflux protein homologs (yqgE and yulE), a component of the oligopeptide uptake system (oppA), and two transmembrane proteins with weak similarity to transporters (yhdP and yueF). Expression from P(W) is also elevated as a result of inactivation of at least one member of the sigma(W) regulon (ysdB), an ArsR homolog (yvbA), a predicted rhamnose isomerase (yulE), and a gene (pksR) implicated in synthesis of difficidin, a polyketide antibiotic. In a parallel screen, we identified seven insertions that up-regulate P(X). Remarkably, these insertions were in functionally similar genes, including a multidrug efflux homolog (yitG), a mannose-6-phosphate isomerase gene (yjdE), and loci involved in antibiotic synthesis (srfAB and possibly yogA and yngK). Significantly, most insertions that activate P(W) have little or no effect on P(X), and conversely, insertions that activate P(X) have no effect on P(W). This suggests that these two regulons respond to distinct sets of molecular signals which may include toxic molecules which are exported, cell density signals, and antimicrobial compounds.     

Characterization of SotA and SotB, two Erwinia chrysanthemi proteins which modify isopropyl-beta-D-thiogalactopyranoside and lactose induction of the Escherichia coli lac promoter

The expression, in Escherichia coli, of variants of the Erwinia chrysanthemi secretion genes outB and outS under the Ptac promoter is toxic to the cells. During attempts to clone E. chrysanthemi genes able to suppress this toxicity, I identified two genes, sotA and sotB, whose products are able to reduce the isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of the E. coli lac promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression of sotA and sotB is not regulated by their substrates, but sotA is activated by the cyclic AMP receptor protein (CRP), while sotB is repressed by CRP. Lactose is weakly toxic for E. chrysanthemi. This toxicity is increased in a sotB mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.     

Ecological Genomics of Marine Roseobacters

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.     

A novel ubiquitous family of putative efflux transporters

We describe a novel family of putative efflux transporters (PET) found in bacteria, yeast and plants. None of the members of the PET family has been functionally characterized. The bacterial and yeast proteins display a duplicated internal repeat element consisting of an N-terminal hydrophobic sequence of about 170 residues, exhibiting six putative transmembrane alpha-helical spanners (TMSs), followed by a large (230 residue), C-terminal, hydrophilic, cytoplasmic domain. The plant proteins exhibit only one such unit, but they have a larger C-terminal cytoplasmic domain. Arabidopsis thaliana encodes at least seven paralogues of the PET family. The gram-negative bacterial proteins are sometimes encoded by genes that are found in operons that also contain genes that encode membrane fusion proteins. This fact strongly suggests that PET family proteins are efflux pumps. The sequence, topological and phylogenetic characteristics of these proteins as well as the operonic structures of their encoded genes when relevant are described.     

Nods,Nalps and Naip: intracellular regulators of bacterial-induced inflammation

The innate immune system is the most ancestral and ubiquitous system of defence against microbial infection. The microbial sensing proteins involved in innate immunity recognize conserved and often structural components of microorganisms. One class of these pattern-recognition molecules, the Toll-like receptors (TLRs), are involved in detection of microbes in the extracellular compartment whereas a newly discovered family of proteins, the NBS-LRR proteins (for nucleotide-binding site and leucine-rich repeat), are involved in intracellular recognition of microbes and their products. NBS-LRR proteins are characterized by three structural domains: a C-terminal leucine-rich repeat (LRR) domain able to sense a microbial motif, an intermediary nucleotide binding site (NBS) essential for the oligomerization of the molecule that is necessary for the signal transduction induced by different N-terminal effector motifs, such as a pyrin domain (PYD), a caspase-activating and recruitment domain (CARD) or a baculovirus inhibitor of apoptosis protein repeat (BIR) domain. Two of these family members, Nod1 and Nod2, play a role in the regulation of pro-inflammatory pathways through NF-kappaB induced by bacterial ligands. Recently, it was shown that Nod2 recognizes a specific peptidoglycan motif from bacteria, muramyl dipeptide (MDP). A surprising number of human genetic disorders have been linked to NBS-LRR proteins. For example, mutations in Nod2, which render the molecule insensitive to MDP and unable to induce NF-kappaB activation when stimulated, are associated with susceptibility to a chronic intestinal inflammatory disorder, Crohn's disease. Conversely, mutations in the NBS region of Nod2 induce a constitutive activation of NF-kappaB and are responsible for Blau syndrome, another auto-inflammatory disease. Nalp3, which is an NBS-LRR protein with an N-terminal Pyrin domain, is also implicated in rare auto-inflammatory disorders. In conclusion, NBS-LRR molecules appear as a new family of intracellular receptors of innate immunity able to detect specific bacterial compounds and induce inflammatory response; the dysregulation of these processes due to mutations in the genes encoding these proteins is involved in numerous auto-inflammatory disorders.