Region C of the Escherichia coli heat shock sigma factor RpoH (σ32) contains a turnover element for proteolysis by the FtsH protease

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor σ32 (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma facto

Low copy expression vectors for use in Yersinia sp. and related organisms

In Yersinia, the most commonly used expression vectors for genetic studies such as gene complementation do not effectively allow for both induction and repression of gene expression. Additionally, there is no expression system available that can be induced in bacteria growing in vitro as well as in vivo, e.g. in eukaryotic cell lines or in living animal models. Here, we present a series of novel inducible low copy expression vectors that are well suited for use in the Yersinia species. Their tet operator/promoter/repressor system makes them distinct from other vectors, and gene transcription in bacteria can easily be induced by addition of anhydrotetracyline (ATc) either to the growth medium, to tissue culture medium during bacterial infections of cell lines or by injection into animals infected with bacteria. Researchers can choose between two different antibiotic resistances (kanamycin or spectinomycin), between two copy numbers (5 or 12-22) as well as between two different versions for expression from either the native RBS and ATG or RBS and ATG encoded in the plasmid. The whole vector series contains the same multi-cloning site from pBluescript II KS+ that allows for easy subcloning. Moreover, these vectors are built in a modular fashion that makes it simple to adapt them for other purposes. Finally, in addition to their use in Yersinia they are suitable for use in many other Enterobacteriaceae

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System

ABSTRACT The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS , respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS , but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes encoding a type III secretion system (T3SS) whose function is not yet evident. The significance of this work lies in finding that two HKs regulate this T3SS, highlighting its importance

A novel soft cardiac assist device based on a dielectric elastomer augmented aorta: an in vivo study

Although heart transplant is the preferred solution for patients suffering from heart failures, cardiac assist devices remain key substitute therapies. Among them, aortic augmentation using dielectric elastomer actuators (DEAs) might be an alternative technological application for the future. The electrically driven actuator does not require bulky pneumatic elements (such as conventional intra-aortic balloon pumps) and conforms tightly to the aorta thanks to the manufacturing method presented here. In this study, the proposed DEA-based device replaces a section of the aorta and acts as a counterpulsation device. The feasibility and validation of in vivo implantation of the device into the descending aorta in a porcine model, and the level of support provided to the heart are investigated. Additionally, the influence of the activation profile and delay compared to the start of systole is studied. We demonstrate that an activation of the DEA just before the start of systole (30 ms at 100 bpm) and deactivation just after the start of diastole (0-30 ms) leads to an optimal assistance of the heart with a maximum energy provided by the DEA. The end-diastolic and left ventricular pressures were lowered by up to 5% and 1%, respectively, compared to baseline. The early diastolic pressure was augmented in average by up to 2%

CcpA-Independent Glucose Regulation of Lactate Dehydrogenase 1 in Staphylococcus aureus

Lactate Dehydrogenase 1 (Ldh1) is a key enzyme involved in Staphylococcus aureus NO·-resistance. Full ldh1-induction requires the presence of glucose, and mutants lacking the Carbon-Catabolite Protein (CcpA) exhibit decreased ldh1 transcription and diminished Ldh1 activity. The redox-regulator Rex represses ldh1 directly by binding to Rex-sites within the ldh1 promoter (Pldh1). In the absence of Rex, neither glucose nor CcpA affect ldh1 expression implying that glucose/CcpA-mediated activation requires Rex activity. Rex-mediated repression of ldh1 depends on cellular redox status and is maximal when NADH levels are low. However, compared to WT cells, the ΔccpA mutant exhibited impaired redox balance with relatively high NADH levels, yet ldh1 was still poorly expressed. Furthermore, CcpA did not drastically alter Rex transcript levels, nor did glucose or CcpA affect the expression of other Rex-regulated genes indicating that the glucose/CcpA effect is specific for Pldh1. A putative catabolite response element (CRE) is located ∼30 bp upstream of the promoter-distal Rex-binding site in Pldh1. However, CcpA had no affinity for Pldh1 in vitro and a genomic mutation of CRE upstream of Pldh1 in S. aureus had no affect on Ldh1 expression in vivo. In contrast to WT, ΔccpA S. aureus preferentially consumes non-glycolytic carbon sources. However when grown in defined medium with glucose as the primary carbon source, ΔccpA mutants express high levels of Ldh1 compared to growth in media devoid of glucose. Thus, the actual consumption of glucose stimulates Ldh1 expression rather than direct CcpA interaction at Pldh1

Identification of a Turnover Element in Region 2.1 of Escherichia coli σ(32) by a Bacterial One-Hybrid Approach

Induction of the heat shock response in Escherichia coli requires the alternative sigma factor σ(32) (RpoH). The cellular concentration of σ(32) is controlled by proteolysis involving FtsH, other proteases, and the DnaKJ chaperone system. To identify individual σ(32) residues critical for degradation, we used a recently developed bacterial one-hybrid system and screened for stabilized versions of σ(32). The five single point mutations that rendered the sigma factor more stable mapped to positions L47, A50, and I54 in region 2.1. Strains expressing the stabilized σ(32) variants exhibited elevated transcriptional activity, as determined by a groE-lacZ fusion. Structure calculations predicted that the three mutated residues line up on the same face of an α-helix in region 2.1, suggesting that they are positioned to interact with proteins of the degradation machinery

Region C of the Escherichia coli heat-shock sigma factor RpoH (σ32) contains a turnover element for proteolysis by the FtsH protease

ISSN:0378-1097ISSN:1574-6968ISSN:0168-6496ISSN:0920-8534ISSN:0168-644

Identification of YsrT and Evidence that YsrRST Constitute a Unique Phosphorelay System in Yersinia enterocolitica▿

Two-component systems (TCS) and phosphorelay systems are mechanisms used by bacteria and fungi to quickly adapt to environmental changes to produce proteins necessary for survival in new environments. Bacterial pathogens use TCS and phosphorelay systems to regulate genes necessary to establish infection within their hosts, including type III secretion systems (T3SS). The Yersinia enterocolitica ysa T3SS is activated in response to NaCl by YsrS and YsrR, a putative hybrid sensor kinase and a response regulator, respectively. Hybrid TCS consist of a sensor kinase that typically has three well-conserved sites of phosphorylation: autophosphorylation site H1, D1 within a receiver domain, and H2 in the histidine phosphotransferase (HPt) domain. From H2, the phosphoryl group is transferred to D2 on the response regulator. A curious feature of YsrS is that it lacks the terminal HPt domain. We report here the identification of the HPt-containing protein (YsrT) that provides this activity for the Ysr system. YsrT is an 82-residue protein predicted to be cytosolic and α-helical in nature and is encoded by a gene adjacent to ysrS. To demonstrate predicted functions of YsrRST as a phosphorelay system, we introduced alanine substitutions at H1, D1, H2, and D2 and tested the mutant proteins for the ability to activate a ysaE-lacZ reporter. As expected, substitutions at H1, H2, and D2 resulted in a loss of activation of ysaE expression. This indicates an interruption of normal protein function, most likely from loss of phosphorylation. A similar result was expected for D1; however, an intriguing “constitutive on” phenotype was observed. In addition, the unusual feature of a separate HPt domain led us to compare the sequences surrounding the ysrS-ysrT junction in several Yersinia strains. In every strain examined, ysrT is a separate gene, leading to speculation that there is a functional advantage to YsrT being an independent protein

Novel para-aortic cardiac assistance using a pre-stretched dielectric elastomer actuator.

OBJECTIVES We propose an evolution of a dielectric elastomer actuator based cardiac assist device that acts as a counterpulsation system. We introduce a new pre-stretched actuator and implant the device in a graft bypass between the ascending and descending aorta to redirect all blood through the device (ascending aorta clamped). The objective was to evaluate the influence of these changes on the assistance provided to the heart. METHODS The novel para-aortic device and the new implantation technique were tested in-vivo in 5 pigs. We monitored the pressure and flow in the aorta as well as the pressure-volume characteristics of the left-ventricle. Different activation timings were tested to identify the optimal device actuation. RESULTS The proposed device helps reducing the end-diastolic pressure in the aorta by up to 13±4.0% as well as the peak systolic pressure by up to 16±3.6%. The early diastolic pressure was also increased up to 10±3.5%. With different activation we also showed that the device could increase or decrease the stroke volume. CONCLUSIONS The new setup and the novel para-aortic device presented here helped improve cardiac assistance compared to previous studies. Moreover, we revealed a new way to assist the heart by actuating the device at different starting time to modify the left ventricular stroke volume and stroke work

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System

The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes encoding a type III secretion system (T3SS) whose function is not yet evident. The significance of this work lies in finding that two HKs regulate this T3SS, highlighting its importance