Regulation underlying hierarchical and simultaneous utilization of carbon substrates by flux sensors in Escherichia coli.

Many microorganisms exhibit nutrient preferences, exemplified by the 'hierarchical' consumption of certain carbon substrates. Here, we systematically investigate under which physiological conditions hierarchical substrate utilization occurs and its mechanisms of implementation. We show utilization hierarchy of Escherichia coli to be ordered by the carbon-uptake flux rather than the identity of the substrates. A detailed study of glycerol uptake finds that it is fully suppressed if the uptake flux of another glycolytic substrate exceeds a threshold, which is set to the influx obtained when grown on glycerol alone. Below this threshold, limited glycerol uptake is 'supplemented' such that the total carbon uptake is maintained at the threshold. This behaviour results from total-flux feedback mediated by cAMP-Crp signalling but also requires inhibition by the regulator fructose 1,6-bisphosphate, which senses the upper-glycolytic flux and ensures that glycerol uptake defers to other glycolytic substrates but not to gluconeogenic ones. A quantitative model reproduces all of the observed utilization patterns, including those of key mutants. The proposed mechanism relies on the differential regulation of uptake enzymes and requires a specific operon organization. This organization is found to be conserved across related species for several uptake systems, suggesting the deployment of similar mechanisms for hierarchical substrate utilization by a spectrum of microorganisms.

Next-generation sequencing-based analysis of reverse transcriptase fidelity.

In this study, we devised a simple and rapid method to analyze fidelity of reverse transcriptase (RT) using next-generation sequencing (NGS). The method comprises a cDNA synthesis reaction from standard RNA with a primer containing a tag of 14 randomized bases and the RT to be tested, PCR using high-fidelity DNA polymerase, and NGS. By comparing the sequence of each read with the reference sequence, mutations were identified. The mutation can be identified to be due to an error introduced by either cDNA synthesis, PCR, or NGS based on whether the sequence reads with the same tag contain the same mutation or not. The error rates in cDNA synthesis with Moloney murine leukemia virus (MMLV) RT thermostable variant MM4 or the recently developed 16-tuple variant of family B DNA polymerase with RT activity, RTX, from Thermococcus kodakarensis, were 0.75-1.0 × 10-4 errors/base, while that in the reaction with the wild-type human immunodeficiency virus type 1 (HIV-1) RT was 2.6 × 10-4 errors/base. Overall, our method could precisely evaluate the fidelity of various RTs with different reaction conditions in a high-throughput manner without the use of expensive optics and troublesome adaptor ligation.

Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.

Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4polL329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.

Expression of moloney murine leukemia virus reverse transcriptase in a cell-free protein expression system.

OBJECTIVE: To characterize Moloney murine leukemia virus (MMLV) reverse transcriptases (RTs) expressed in a cell-free system and in Escherichia coli. RESULTS: We previously expressed MMLV RT using an E. coli expression system and generated a highly thermostable quadruple variant MM4 (E286R/E302K/L435R/D524A) by site-directed mutagenesis. In this study, we expressed the wild-type MMLV RT (WT) and MM4 using a cell-free protein expression system from insect cells. WT exhibited DNA polymerase and RNase H activities, while MM4, in which the catalytic residue for RNase H activity, Asp524 is changed into Ala, exhibited only DNA polymerase activity. MM4, when held at 60 °C for 10 min, retained DNA polymerase activity, while WT, held at 54 °C for 10 min, lost this activity. In the cDNA synthesis reaction (0.5 µl) in which WT or MM4 were exposed to various temperatures and amounts of target RNA in a microarray chip, MM4 exhibited higher thermostability than WT. CONCLUSION: MMLV RT expressed in the cell-free system is indistinguishable from that expressed in E. coli.

Overflow metabolism in Escherichia coli results from efficient proteome allocation.

Overflow metabolism refers to the seemingly wasteful strategy in which cells use fermentation instead of the more efficient respiration to generate energy, despite the availability of oxygen. Known as the Warburg effect in the context of cancer growth, this phenomenon occurs ubiquitously for fast-growing cells, including bacteria, fungi and mammalian cells, but its origin has remained unclear despite decades of research. Here we study metabolic overflow in Escherichia coli, and show that it is a global physiological response used to cope with changing proteomic demands of energy biogenesis and biomass synthesis under different growth conditions. A simple model of proteomic resource allocation can quantitatively account for all of the observed behaviours, and accurately predict responses to new perturbations. The key hypothesis of the model, that the proteome cost of energy biogenesis by respiration exceeds that by fermentation, is quantitatively confirmed by direct measurement of protein abundances via quantitative mass spectrometry.

A growth-rate composition formula for the growth of E.coli on co-utilized carbon substrates.

When bacteria are cultured in medium with multiple carbon substrates, they frequently consume these substrates simultaneously. Building on recent advances in the understanding of metabolic coordination exhibited by Escherichia coli cells through cAMP-Crp signaling, we show that this signaling system responds to the total carbon-uptake flux when substrates are co-utilized and derive a mathematical formula that accurately predicts the resulting growth rate, based only on the growth rates on individual substrates.

Coordination of bacterial proteome with metabolism by cyclic AMP signalling.

The cyclic AMP (cAMP)-dependent catabolite repression effect in Escherichia coli is among the most intensely studied regulatory processes in biology. However, the physiological function(s) of cAMP signalling and its molecular triggers remain elusive. Here we use a quantitative physiological approach to show that cAMP signalling tightly coordinates the expression of catabolic proteins with biosynthetic and ribosomal proteins, in accordance with the cellular metabolic needs during exponential growth. The expression of carbon catabolic genes increased linearly with decreasing growth rates upon limitation of carbon influx, but decreased linearly with decreasing growth rate upon limitation of nitrogen or sulphur influx. In contrast, the expression of biosynthetic genes showed the opposite linear growth-rate dependence as the catabolic genes. A coarse-grained mathematical model provides a quantitative framework for understanding and predicting gene expression responses to catabolic and anabolic limitations. A scheme of integral feedback control featuring the inhibition of cAMP signalling by metabolic precursors is proposed and validated. These results reveal a key physiological role of cAMP-dependent catabolite repression: to ensure that proteomic resources are spent on distinct metabolic sectors as needed in different nutrient environments. Our findings underscore the power of quantitative physiology in unravelling the underlying functions of complex molecular signalling networks.

Predictors of gastric myoelectrical activity in type 2 diabetes mellitus.

BACKGROUND: Previous studies have clearly demonstrated the delayed gastric emptying of solid meals in diabetics, whereas their gastric myoelectrical activity, which primarily determines gastric motility, has not yet been fully confirmed. GOALS: This study aimed to clarify the characteristics and potential predictors of gastric myoelectrical activity in type 2 diabetics. STUDY: Twenty-eight diabetics and 18 healthy controls participated. Duodenal biopsy sample was used for reverse transcription-polymerase chain reaction to evaluate cholecystokinin and motilin mRNA contents. Electrogastrography was performed before and after the test meal, and was assessed in terms of dominant frequency; dominant frequency instability coefficient; and the percentage of bradygastria, normogastria, and tachygastria. RESULTS: Over the entire recording period, dominant frequency was significantly lower, and dominant frequency instability coefficient and the percentage of bradygastria were significantly higher in diabetics than in controls. In diabetics, the multiple regression analysis demonstrated that dominant frequency instability coefficient and the percentage of tachygastria in the fasting period were dependent on fasting plasma glucose level and HbA1c, respectively. Moreover, dominant frequency over the entire period and the postprandial percentage of bradygastria were significantly associated with body mass index; the fasting percentage of bradygastria and postprandial dominant frequency instability coefficient were associated with fasting serum leptin level; the postprandial percentage of bradygastria was also associated with cholecystokinin mRNA content. CONCLUSIONS: Gastric myoelectrical activity in type 2 diabetics is impaired on dominant frequency, dominant frequency instability coefficient, and the percentage of bradygastria and predicted by body mass index, fasting serum leptin level, and cholecystokinin mRNA content besides the glycemic status.

[Group B streptococcal vertebral osteomyelitis following superficial suppurative thrombophlebitis].

An 80-year-old woman with type II diabetes mellitus was admitted to hospital with high-grade fever and leg pain for the previous three days. Physical examination revealed marked distention of the peripheral veins in both lower legs and she complained of pain. Spontaneous superficial suppurative thrombophlebitis was diagnosed and transfusion of cefazolin every 8 hours was started immediately after blood cultures. After 48 hours, the distention of the peripheral veins was improved; however, she suffered from a severe back pain thereafter. Two sets of blood culture yielded Group B streptococcus. Therefore the antibiotic was changed to ampicillin every 6 hours. To investigate the cause of back pain, MRI of the lumbar vertebral body was taken. Saggital gadolinium T1-weighted MRI demonstrated a high signal intensity lesion from Th7 to Th11, suggesting vertebral osteomyelitis following Group B streptococcal bacteremia from superficial suppurative thrombophlebitis. One week later, the clinical symptoms mostly disappeared. After six weeks of treatment, she was discharged. Suppurative thrombophlebitis is an inflammation of the vein wall by microorganisms and sometimes causes secondary metastatic abscess. Aging and diabetes are also risk factors for group B streptococcal invasive infection. This case suggests vertebral osteomyelitis should be taken into consideration during the course of group B streptococcal bacteremia in an elderly patient complaining back pain.

A newborn case of congenital laryngeal cyst complicated with pneumothorax and pneumomediastinum.

Benign congenital laryngeal cysts are rare entities. They often cause chronic hoarseness and severe stridor. Case reports of congenital laryngeal cyst complicated with pneumothorax and pneumomediastinum are very rare. A 3,112 g full-term male newborn developed stridor which got worse during crying for 12 h after birth. Chest retractions were present with inspiration. Chest X-rays showed the presence of right pneumothorax and pneumomediastinum. Transnasal flexible laryngoscopic examination revealed a large cystic mass, which occupied almost the entire supraglottic airway. The operation was performed with the techniques of laryngomicrosurgery under general anesthesia. The cystic wall was punctured and serous liquid contents were aspirated. Excision of the entire cystic lesion was performed. The next day, extubation was performed without any troubles. The stridor had disappeared and the pneumothorax and pneumomediastinum were improved without further medical intervention. The histopathological examination revealed that the cystic wall consisted of normal squamous epithelial cells. It is reasonable to think that the high airway pressure due to congenital laryngeal cyst was responsible for pneumothorax and pneumomediastinum.

Distribution of vanilloid receptors in the rat laryngeal innervation.

OBJECTIVE: Capsaicin is known to selectively activate nociceptic sensory neurons through vanilloid receptors. In this study we investigated the distribution of vanilloid receptor subtype 1 (VR1) and vanilloid receptor-like protein 1 (VRL-1) in the rat larynx. MATERIAL AND METHODS: The distributions of VR1 and VRL-1 were determined immunohistochemically. The colocalization of vanilloid receptors with common choline acetyltransferase (cChAT), vasoactive intestinal polypeptide (VIP), substance P (SP) and neuronal nitric oxide synthase (nNOS) was also studied using an immunohistochemical double-labeling technique. RESULTS: VRL-1-positive fibers were detected in the laryngeal epithelium and lamina propria. VR1-positive nerve fibers were seen in the lamina propria but not in the mucosal epithelium. VR1- and VRL-1-positive cells were distributed in the intralaryngeal ganglia and colocalization of capsaicin receptors with VIP, nNOS and cChAT was seen. CONCLUSION: These findings suggest that these capsaicin receptors participate in the parasympathetic innervation as well as in nociception of the rat larynx.