McpT, a broad range carboxylate chemoreceptor in Sinorhizobium meliloti

Chemoreceptors enable the legume symbiont Sinorhizobium meliloti to detect and respond to specific chemicals released from their host plant alfalfa, which allows the establishment of a nitrogen-fixing symbiosis. The periplasmic region (PR) of transmembrane chemoreceptors act as the sensory input module for chemotaxis systems via binding of specific ligands, either directly or indirectly. S. meliloti has six transmembrane and two cytosolic chemoreceptors. However, only the function of three of the transmembrane receptors have been characterized so far, with McpU, McpV, and McpX serving as general amino acid, short-chain carboxylate, and quaternary ammonium compound sensors, respectively. In the present study, we analyzed the S. meliloti chemoreceptor McpT. High-throughput differential scanning fluorimetry assays, using Biolog Phenotype Microarray TM plates, identified fifteen potential ligands for McpT PR , the majority classified as mono-, di-, and tri-carboxylates. S. meliloti exhibited positive chemotaxis toward seven selected carboxylates, namely, α-ketobutyrate, citrate, glyoxylate, malate, malonate, oxalate, and succinate. These carboxylates were detected in seed exudates of the alfalfa host. Deletion of mcpT resulted in a significant decrease of chemotaxis to all carboxylates except for citrate. Isothermal titration calorimetry revealed that McpT PR bound preferentially to the monocarboxylate glyoxylate, and with lower affinity to the dicarboxylates malate, malonate and oxalate. However, no direct binding was detected for the remaining three carboxylates that elicited an McpT-dependent chemotaxis response. Taken together, these results demonstrate that McpT is a broad range carboxylate chemoreceptor that mediates chemotactic response via direct ligand binding and an indirect mechanism that yet needs to be identified. IMPORTANCE Nitrate pollution is one of the most widespread and challenging environmental problems, mainly caused by the agricultural over-application of nitrogen fertilizers. Biological nitrogen fixation by the endosymbiont Sinorhizobium meliloti enhances the growth of its host Medicago sativa (alfalfa), which also efficiently supplies the soil with nitrogen. Establishment of the S. meliloti - alfalfa symbiosis relies on the early exchange and recognition of chemical signals. The present study contributes to the disclosure of this complex molecular dialogue by investigating the underlying mechanisms of carboxylate sensing in S. meliloti . Understanding individual steps that govern S. meliloti -alfalfa molecular cross-talk helps in the development of efficient, commercial bacterial inoculants that promote the growth of this most cultivated forage legume in the world and improves soil fertility.

Biolog Phenotype Microarray Is a Tool for the Identification of Multidrug Resistance Efflux Pump Inducers

ABSTRACTMultidrug resistance efflux pumps frequently present low levels of basal expression. However, antibiotic-resistant mutants that overexpress these resistance determinants are selected during infection. In addition, increased expression of efflux pumps can be induced by environmental signals/cues, which can lead to situations of transient antibiotic resistance. In this study, we have applied a novel high-throughput methodology in order to identify inducers able to trigger the expression of theStenotrophomonas maltophiliaSmeVWX and SmeYZ efflux pumps. To that end, bioreporters in which the expression of the yellow fluorescent protein (YFP) is linked to the activity of eithersmeVWXorsmeYZpromoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. YFP production was also measured by flow cytometry, and the levels of expression ofsmeVandsmeYin the presence of a set of selected compounds were also determined by real-time reverse transcription-PCR (RT-PCR). The expression ofsmeVWXwas induced by iodoacetate, clioquinol, and selenite, while boric acid, erythromycin, chloramphenicol, and lincomycin triggeredsmeYZexpression. The susceptibility to antibiotics that are known substrates of the efflux pumps decreased in the presence of the inducers. However, the analyzed multidrug efflux systems did not contribute toS. maltophiliaresistance to the studied inducers. To sum up, the use of fluorescent bioreporters in combination with Biolog plates is a valuable tool for identifying inducers of the expression of bacterial multidrug resistance efflux pumps, and likely of other bacterial systems whose expression is regulated in response to signals/cues.

Metabolic phenotype of clinical and environmentalMycobacterium aviumsubsp.hominissuisisolates

BackgroundMycobacterium aviumsubsp.hominissuis(MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited.MethodsIn this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTMtechnology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes.ResultsThe ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307).DiscussionOur study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections.

RamA Confers Multidrug Resistance in Salmonella enterica via Increased Expression of acrB, Which Is Inhibited by Chlorpromazine

ABSTRACT Salmonella enterica serovar Typhimurium SL1344, in which efflux pump genes (acrB, acrD, acrF, tolC) or regulatory genes thereof (marA, soxS, ramA) were inactivated, was grown in the presence of 240 antimicrobial and nonantimicrobial agents in the Biolog Phenotype MicroArray. Mutants lacking tolC, acrB, and ramA grew significantly worse than other mutants in the presence of 48 agents (some of which have not previously been identified as substrates of AcrAB-TolC) and particularly poorly in the presence of phenothiazines, which are human antipsychotics. MIC testing revealed that the phenothiazine chlorpromazine had antimicrobial activity and synergized with common antibiotics against different Salmonella serovars and SL1344. Chlorpromazine increased the intracellular accumulation of ethidium bromide, which was ablated in mutants lacking acrB, suggesting an interaction with AcrB. High-level but not low-level overexpression of ramA increased the expression of acrB; conferred resistance to chloramphenicol, tetracycline, nalidixic acid, and triclosan and organic solvent tolerance; and increased the amount of ethidium bromide accumulated. Chlorpromazine induced the modest overproduction of ramA but repressed acrB. These data suggest that phenothiazines are not efflux pump inhibitors but influence gene expression, including that of acrB, which confers the synergy with antimicrobials observed.

A screening system for carbon sources enhancing β-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride)

To identify carbon sources that trigger β-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on α-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis Δnag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Δnag1 strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available.