The E. coli dicarboxylic acid transporters DauA act as a signal transducer by interacting with the DctA uptake system.
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ABSTRACT:
SUBMITTER: Karinou E
PROVIDER: S-EPMC5703999 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The dctA gene, coding for the dicarboxylate transport protein, has an inducible promoter dependent on activation by the two-component sensor-regulator pair DctB and DctD. LacZ fusion analysis indicates that there is a single promoter for dctB and dctD. The dctA promoter is also induced by nitrogen limitation, an effect that requires DctB-DctD and NtrC. DctB alone is able to detect dicarboxylates in the absence of DctA and initiate transcription via DctD. However, DctA modifies signal detection by DctB such that in the absence of DctA, the ligand specificity of DctB is broader. dctAp also responds to heterologous induction by osmotic stress in the absence of DctA. This effect requires both DctB and DctD. A transposon insertion in the dctA-dctB intergenic region (dctA101) which locks transcription of dctA at a constitutive level independent of DctB-DctD results in improper signalling by DctB-DctD. Strain RU150, which carries this insertion, is defective in nitrogen fixation (Fix-) and grows very poorly on ammonia as a nitrogen source whenever the DctB-DctD signalling circuit is activated by the presence of a dicarboxylate ligand. Mutation of dctB or dctD in strain RU150 reinstates normal growth on dicarboxylates. This suggests that DctD-P improperly regulates a heterologous nitrogen-sensing operon. Increased expression of DctA, either via a plasmid or by chromosomal duplication, restores control of DctB-DctD and allows strain RU150 to grow on ammonia in the presence of a dicarboxylate. Thus, while DctB is a sensor for dicarboxylates in its own right, it is regulated by DctA. The absence of DctA allows DctB and DctD to become promiscuous with regard to signal detection and cross talk with other operons. This indicates that DctA contributes significantly to the signalling specificity of DctB-DctD and attenuates cross talk with other operons.
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Project description:The cytoplasmic PASC domain of the fumarate responsive sensor kinase DcuS of Escherichia coli links the transmembrane to the kinase domain. PASC is also required for interaction with the transporter DctA serving as a cosensor of DcuS. Earlier studies suggested that PASC functions as a hinge and transmits the signal to the kinase. Reorganizing the PASC dimer interaction and, independently, removal of DctA, converts DcuS to the constitutive ON state (active without fumarate stimulation). ON mutants were categorized with respect to these two biophysical interactions and the functional state of DcuS: type I-ON mutations grossly reorganize the homodimer, and decrease interaction with DctA. Type IIA-ON mutations create the ON state without grossly reorganizing the homodimer, whereas interaction with DctA is decreased. The type IIB-ON mutations were neither in PASC /PASC , nor in DctA/DcuS interaction affected, similar to fumarate activated wild-typic DcuS. OFF mutations never affected dimer stability. The ON mutations provide novel mechanistic insight: PASC dimerization is essential to silence the kinase. Reorganizing the homodimer and its interaction with DctA activate the kinase. The study suggests a novel ON homo-dimer conformation (type IIB) and an OFF conformation for PASC . Type IIB-ON corresponds to the fumarate induced wild-type conformation, representing an interesting target for structural biology.
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Project description:RNA-seq libraries were constructed for three samples, including (I) ΔURA3 strain grown in SD medium containing 2% w/v glucose(pH=5.0);(II)ΔURA3 strain grown in SD medium containing 2%w/v glucose and 2% w/v succinic acid(pH=5.0);(III)ΔURA3 strain grown in SD medium containing 2% w/v succinic acid(pH=5.0). For preparation of RNA samples, ΔURA3 cells grown overnight were inoculated into 50 ml liquid Synthetic Dextrose (SD) medium with the initial inoculation amount of OD600= 0.1, and cultured for 24 hours to the logarithmic growth phase (OD600= 2-3). The cells were collected by centrifugation at 6000g for 5 minutes. After washing twice with phosphate buffer saline (PBS), they were inoculated into new 50 mL SD medium containing carbon sources of different combinations of glucose and succinic acid .The pH of the culture sample is adjusted to 5.0 with NaOH. After flask culturing at 30°C and 250 rpm for an additional three hours, the yeast cells were collected by centrifugation for total RNA isolation and Illumina RNA-seq library construction. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The 150-nt paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). Each sample contains two biological replicates.
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Project description:Phytochromes are plant photoreceptors that regulate plant growth and development with respect to the light environment. Following the initial light-perception event, the phytochromes initiate a signal-transduction process that eventually results in alterations in cellular behavior, including gene expression. Here we describe the molecular cloning and functional characterization of Arabidopsis FHY1. FHY1 encodes a product (FHY1) that specifically transduces signals downstream of the far-red (FR) light-responsive phytochrome A (PHYA) photoreceptor. We show that FHY1 is a novel light-regulated protein that accumulates in dark (D)-grown but not in FR-grown hypocotyl cells. In addition, FHY1 transcript levels are regulated by light, and by the product of FHY3, another gene implicated in FR signaling. These observations indicate that FHY1 function is both FR-signal transducing and FR-signal regulated, suggesting a negative feedback regulation of FHY1 function. Seedlings homozygous for loss-of-function fhy1 alleles are partially blind to FR, whereas seedlings overexpressing FHY1 exhibit increased responses to FR, but not to white (WL) or red (R) light. The increased FR-responses conferred by overexpression of FHY1 are abolished in a PHYA-deficient mutant background, showing that FHY1 requires a signal from PHYA for function, and cannot modulate growth independently of PHYA.
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Project description:In Escherichia coli, the catabolism of C4-dicarboxylates is regulated by the DcuS-DcuR two-component system. The functional state of the sensor kinase DcuS is controlled by C4-dicarboxylates (like fumarate) and complexation with the C4-dicarboxylate transporters DctA and DcuB, respectively. Free DcuS (DcuSF) is known to be constantly active even in the absence of fumarate, whereas the DcuB-DcuS and DctA-DcuS complexes require fumarate for activation. To elucidate the impact of the transporters on the functional state of DcuS and the concentrations of DcuSF and DcuB-DcuS (or DctA-DcuS), the absolute levels of DcuS, DcuB, and DctA were determined in aerobically or anaerobically grown cells by mass spectrometry. DcuS was present at a constant very low level (10 to 20 molecules DcuS/cell), whereas the levels of DcuB and DctA were higher (minimum, 200 molecules/cell) and further increased with fumarate (12.7- and 2.7-fold, respectively). Relating DcuS and DcuB contents with the functional state of DcuS allowed an estimation of the proportions of DcuS in the free (DcuSF) and the complexed (DcuB-DcuS) states. Unexpectedly, DcuSF levels were always low (<2% of total DcuS), ruling out earlier models that show DcuSF as the major species under noninducing conditions. In the absence of fumarate, when DcuSF is responsible for basal dcuB expression, up to 8% of the maximal DcuB levels are formed. These suffice for DcuB-DcuS complex formation and basal transport activity. In the presence of fumarate (>100 ?M), the DcuB-DcuS complex drives the majority of dcuB expression and is thus responsible for induction.IMPORTANCE Two-component systems (TCS) are major devices for sensing by bacteria and adaptation to environmental cues. Membrane-bound sensor kinases of TCS often use accessory proteins of unknown function. The DcuS-DcuR TCS responds to C4-dicarboxylates and requires formation of the complex of DcuS with C4-dicarboxylate transporters DctA or DcuB. Free DcuS (DcuSF) is constitutively active in autophosphorylation and was supposed to have a major role under specific conditions. Here, absolute concentrations of DcuS, DcuB, and DctA were determined under activating and nonactivating conditions by mass spectrometry. The relationship of their absolute contents to the functional state of DcuS revealed their contribution to the control of DcuS-DcuR in vivo, which was not accessible by other approaches, leading to a revision of previous models.
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| S-EPMC3256229 | biostudies-literature
Project description:The isolation of aerobic citrate-utilizing Escherichia coli (Cit(+)) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of E. coli Cit(+) mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type E. coli strain B and with K-12 and three K-12 derivatives: an E. coli ?rpoS::kan mutant (impaired for stationary-phase survival), an E. coli ?citT::kan mutant (deleted for the anaerobic citrate/succinate antiporter), and an E. coli ?dctA::kan mutant (deleted for the aerobic succinate transporter). E. coli underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent E. coli Cit(+) mutants were isolated from all E. coli derivatives except the E. coli ?citT::kan mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry. E. coli Cit(+) mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four E. coli Cit(+) mutants revealed the required sequence of mutational events leading to a refined Cit(+) mutant. These events showed amplified citT and dctA loci followed by DNA rearrangements consistent with promoter capture events for citT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE.IMPORTANCE E. coli cannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and R. E. Lenski, Nature 489:513-518, 2012, http://dx.doi.org/10.1038/nature11514 ) found a single aerobic, citrate-utilizing E. coli strain after 33,000 generations (15 years). This was interpreted as a speciation event. Here we show why it probably was not a speciation event. Using similar media, 46 independent citrate-utilizing mutants were isolated in as few as 12 to 100 generations. Genomic DNA sequencing revealed an amplification of the citT and dctA loci and DNA rearrangements to capture a promoter to express CitT, aerobically. These are members of the same class of mutations identified by the LTEE. We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved.
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