12 research outputs found
Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius
Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli-Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2-0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30-50h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1mgL−1 culture. In addition we also determined the promoter strength of several constitutive promoter
Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius
Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli–Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2–0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30–50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L−1 culture. In addition we also determined the promoter strength of several constitutive promoters
Macromolecular Fingerprinting of Sulfolobus Species in Biofilm: A Transcriptomic and Proteomic Approach Combined with Spectroscopic Analysis
Microorganisms in nature often live in surfaceassociated
sessile communities, encased in a self-produced
matrix, referred to as biofilms. Biofilms have been well studied in
bacteria but in a limited way for archaea. We have recently characterized
biofilm formation in three closely related hyperthermophilic
crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and
S. tokodaii. These strains form different communities ranging
from simple carpet structures in S. solfataricus to high density
tower-like structures in S. acidocaldarius under static condition.
Here, we combine spectroscopic, proteomic, and transcriptomic
analyses to describe physiological and regulatory features
associated with biofilms. Spectroscopic analysis reveals that in
comparison to planktonic life-style, biofilm life-style has distinctive
influence on the physiology of each Sulfolobus spp.
Proteomic and transcriptomic data show that biofilm-forming
life-style is strain specific (eg ca. 15% of the S. acidocaldarius
genes were differently expressed, S. solfataricus and S. tokodaii
had ∼3.4 and ∼1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions,
including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most
striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key
regulatory factor in biofilm development
Podocyte Purinergic P2X4 channels are mechanotransducers that mediate cytoskeletal disorganization
Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X(4)) blocker 5-BDBD. Moreover, mechanical P2X(4) channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X(4) channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X(4) channels as mechanotransducers in podocytes
Podocyte Purinergic P2X 4
Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X(4)) blocker 5-BDBD. Moreover, mechanical P2X(4) channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X(4) channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X(4) channels as mechanotransducers in podocytes
Macromolecular Fingerprinting of <i>Sulfolobus</i> Species in Biofilm: A Transcriptomic and Proteomic Approach Combined with Spectroscopic Analysis
Microorganisms in nature often live in surface-associated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: <i>Sulfolobus acidocaldarius</i>, <i>S. solfataricus</i>, and <i>S. tokodaii</i>. These strains form different communities ranging from simple carpet structures in <i>S. solfataricus</i> to high density tower-like structures in <i>S. acidocaldarius</i> under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each <i>Sulfolobus</i> spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the <i>S. acidocaldarius</i> genes were differently expressed, S. <i>solfataricus</i> and <i>S. tokodaii</i> had ∼3.4 and ∼1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development