Regulation of constant cell elongation and Sfm pili synthesis in Escherichia coli via two active forms of FimZ orphan response regulator.

Escherichia coli (E. coli) has multiple copies of the chaperone-usher (CU) pili operon in five fimbria groups: CU pili, curli, type IV pili, type III secretion pili, and type IV secretion pili. Commensal E. coli K-12 contains 12 CU pili operons. Among these operons, Sfm is expressed by the sfmACDHF operon. Transcriptome analyses, reporter assays, and chromatin immunoprecipitation PCR analyses reported that FimZ directly binds to and activates the sfmA promoter, transcribing sfmACDHF. In addition, FimZ regularly induces constant cell elongation in E. coli, which is required for F-type ATPase function. The bacterial two-hybrid system showed a specific interaction between FimZ and the α subunit of the cytoplasmic F1 domain of F-type ATPase. Studies performed using mutated FimZs have revealed two active forms, I and II. Active form I is required for constant cell elongation involving amino acid residues K106 and D109. Active form II additionally required D56, a putative phosphorylation site, to activate the sfmA promoter. The chromosomal fimZ was hardly expressed in parent strain but functioned in phoB and phoP double-gene knockout strains. These insights may help to understand bacterial invasion restricted host environments by the sfm γ-type pili.

Epistatic Effect of Regulators to the Adaptive Growth of Escherichia coli.

Bacteria survive in the environment with three steps: a sensing environmental conditions, a responding to sensed signals, and an adaptation for proper survival in the environment. An adapting bacterial cell occurs cell division to increase the number of sister cells, termed adaptive growth. Two-component systems (TCSs), representing the main bacterial signal transduction systems, consist of a pair of one sensor kinase (SK) and one response regulator (RR), and RR genes are abundant in most bacterial genomes as part of the core genome. The OmpR gene family, a group of RR genes, is conserved in 95% of known bacterial genomes. The Escherichia coli genome has an estimated 34 RR genes in total, including 14 genes of OmpR family genes. To reveal the contribution of TCSs for fast growth as an adaptive growth strategy of E. coli, we isolated a set of gene knockout strains by using newly developed genome editing technology, the HoSeI (Homologous Sequence Integration) method, based on CRISPR-Cas9. The statistics of single cell observation show a knockout of an arbitrary pair of phoP, phoB, and ompR genes, stably expressed by positive feedback regulation, dramatically inhibit the optimum adaptive growth of E. coli. These insights suggest that the adaptive growth of bacteria is fulfilled by the optimum high intracellular level of regulators acquired during growth under environmental conditions.

Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter.

The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced bioluciferase lux reporter enables easy, fast, and sensitive measurement of the promoter activity without cell lysis because the substrates of bioluminescent reaction are synthesized inside the bacterial cell, thereby allowing low-cost experiments. This protocol describes a high throughput technique to measure the promoter activity in Escherichia coli K-12 using the lux reporter system.

Regulatory roles of pyruvate-sensing two-component system PyrSR (YpdAB) in Escherichia coli K-12.

When the rate of production of metabolites in bacteria exceeds the amounts needed for cell growth, excess metabolites are secreted into the extracellular environment. Upon entry into poor nutrient conditions, overflowed exometabolites are reused to continue cell growth and survival. At present, however, the genetic system for utilization of exometabolites is poorly understood even for the best-characterized model prokaryote Escherichia coli. A two-component system YpdAB of E. coli K-12 was predicted to participate in regulation of this process, and the yhjX gene encoding the MFS-family transporter with an as yet unidentified function was identified as a single regulatory target of YpdB. Using gSELEX screening in vitro, however, we have identified up to eight regulatory targets, including the yhjX gene. The predicted regulatory targets were all confirmed to be under the direct control of YpdB by gel shift assay in vitro and reporter assay in vivo. For induction of YpdAB function, the major exometabolite pyruvate in growing E. coli K-12 was identified as the inducer. We then propose to rename YpdAB as PyrSR (regulator of pyruvate reutilization). One unique feature of PyrSR is its cross-talk with another pyruvate-sensing BtsSR at the TCS stage 1 for fine-tuning of pyruvate reutilization.

Influenza virus replication raises the temperature of cells.

Influenza virus invades the cell by binding sialic acid on the cell membrane through haemagglutinin (HA), and then genome replication and transcription are carried out in the nucleus to produce progeny virus. Multiplication of influenza virus requires metabolites, such as nucleotides and amino acids, as well as cellular machinery to synthesize its genome and proteins, thereby producing viral particles. Influenza virus infection forces the start of several metabolic systems in the cell, which consume or generate large amounts of energy. Thus, the viral multiplication processes involved in both genome replication and transcription are considered to require large numbers of nucleotides. The high-level consumption of nucleotides generates large amounts of energy, some of which is converted into heat, and this heat may increase the temperature of cells. To address this question, we prepared a tool based on rhodamine B fluorescence, which we used to measure the temperatures of influenza virus-infected and uninfected cells. The results indicated that influenza virus multiplication increased the temperature of cells by approximately 4 °C - 5 °C, ATP levels in the cells decreased at 3 h after infection, and mitochondrial membrane potential decreased with multiplication level. Thus, the increase in cellular temperature during influenza virus infection appears to be due to the massive consumption of ATP over a short period.

Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase.

Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase.

Analysis of the Changes in Expression Levels of Sialic Acid on Influenza-Virus-Infected Cells Using Lectin-Tagged Polymeric Nanoparticles.

Viral infections affect millions around the world, sometimes leading to severe consequences or even epidemics. Understanding the molecular dynamics during viral infections would provide crucial information for preventing or stopping the progress of infections. However, the current methods often involve the disruption of the infected cells or expensive and time-consuming procedures. In this study, fluorescent polymeric nanoparticles were fabricated and used as bioimaging nanoprobes that can monitor the progression of influenza viral infection through the changes in the expression levels of sialic acids expressed on the cell membrane. The nanoparticles were composed of a biocompatible monomer to prevent non-specific interactions, a hydrophobic monomer to form the core, a fluorescent monomer, and a protein-binding monomer to conjugate lectin, which binds sialic acids. It was shown that these lectin-tagged nanoparticles that specifically target sialic acids could track the changes in the expression levels of sialic acids caused by influenza viral infections in human lung epithelial cells. There was a sudden drop in the levels of sialic acid at the initial onset of virus infection (t = 0~1 h) and at approximately 4~5 h post-infection. The latter drop correlated with the production of viral proteins that was confirmed using traditional techniques. Thus, the accuracy, the rapidity and the efficacy of the nanoprobes were demonstrated. Such molecular bioimaging tools, which allow easy-handling and in situ monitoring, would be useful to directly observe and decipher the viral infection mechanisms.

Cardiac output and other hemodynamic variables in anesthetized dogs undergoing laparotomy because of abdominal neoplasia.

OBJECTIVE: To measure cardiac output and other hemodynamic variables in anesthetized dogs undergoing laparotomy because of abdominal neoplasia. DESIGN: Prospective case series. ANIMALS: 8 dogs with splenic or hepatic tumors. PROCEDURES: Dogs were anesthetized and underwent abdominal laparotomy. End-tidal isoflurane concentration, heart rate, arterial blood pressures, cardiac output, arterial pH, blood gas partial pressures, PCV, and plasma total protein concentration were measured at set intervals before, during, and after surgery. Cardiac index, stroke index, and systemic vascular resistance index were calculated. RESULTS: End-tidal isoflurane concentration was lowest before and after surgery. Heart rate did not change significantly throughout the anesthetic period. Arterial blood pressures and systemic vascular resistance index were highest shortly after surgery began; cardiac index and stroke volume index did not change significantly during surgery but increased significantly after surgery ended. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that in dogs undergoing laparotomy because of abdominal neoplasia, changes in arterial blood pressures were not necessarily indicative of qualitatively similar changes in cardiac index.

Heterotopic implantation of a porcine bioprosthetic heart valve in a dog with aortic valve endocarditis.

CASE DESCRIPTION: A 5-year-old male German Shepherd Dog was evaluated because of a 5-month history of progressive lethargy, weight loss, and heart failure. CLINICAL FINDINGS: On physical examination, bounding femoral pulses and systolic and diastolic murmurs were detected. Echocardiography revealed severe aortic valve insufficiency (AVI) and a large vegetative lesion on the aortic valve consistent with aortic valve endocarditis. The AVI velocity profile half-time was 130 milliseconds; the calculated peak systolic pressure gradient across the aortic valve was 64 mm Hg. Left ventricular diameter during diastole was 63.6 mm (predicted range, 40.2 to 42 mm) and during systole was 42.9 mm (predicted range, 25.4 to 27 mm). Systolic, diastolic, and mean arterial blood pressures were 120, 43, and 65 mm Hg, respectively. TREATMENT AND OUTCOME: To palliate severe AVI, the descending aorta was occluded (duration, 16.75 minutes) and heterotopic implantation of a porcine bioprosthetic heart valve in that vessel was performed. After surgery, systolic, diastolic, and mean arterial blood pressures were 115, 30, and 61 mm Hg, respectively, in the forelimb and 110, 62, and 77 mm Hg, respectively, in the hind limb. Within 6 months, the AVI velocity profile half-time had increased to 210 milliseconds, indicating diminished severity of AVI. After 24 months, the dog was able to engage in vigorous exercise; no pulmonary edema had developed since surgery. CLINICAL RELEVANCE: Heterotopic bioprosthetic heart valve implantation into the descending aorta during brief aortic occlusion appears feasible in dogs and may provide substantial palliation for dogs with severe AVI.

Evaluation of hemodynamic measurements, including lithium dilution cardiac output, in anesthetized dogs undergoing ovariohysterectomy.

OBJECTIVE: To measure cardiac output in healthy female anesthetized dogs by use of lithium dilution cardiac output and determine whether changes in mean arterial pressure were caused by changes in cardiac output or systemic vascular resistance. DESIGN: Prospective clinical study. ANIMALS: 20 healthy female dogs. PROCEDURE: Dogs were anesthetized for ovariohysterectomy. Ten dogs breathed spontaneously throughout anesthesia, and 10 dogs received intermittent positive-pressure ventilation. Cardiovascular and respiratory measurements, including lithium dilution cardiac output, were performed during anesthesia and surgery. RESULTS: Mean arterial pressure and systemic vascular resistance index were low after induction of anesthesia and just prior to surgery and increased significantly after surgery began. Cardiac index (cardiac output indexed to body surface area) did not change significantly throughout anesthesia and surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Results provide baseline data for cardiac output and cardiac index measurements during clinical anesthesia and surgery in dogs. Changes in mean arterial pressure do not necessarily reflect corresponding changes in cardiac index.