Effect of temperature, pH and plasmids on in vitro biofilm formation in Escherichia coli

Acid resistance (AR) in Escherichia coli is an important trait that protects this microorganism from the deleterious effect of low-pH environments. Reports on biofilm formation in E. coli K12 showed that the genes participating in AR were differentially expressed. Herein, we investigated the relationship between AR genes, in particular those coding for specific transcriptional regulators, and their biofilm-forming ability at the phenotypic level. The latter was measured in 96-well plates by staining the bacteria attached to the well, following 24-hour growth under static conditions, with crystal violet. The growth conditions were as follows: Luria Bertani (LB) medium at neutral and acidic pH, at 37°C or 25°C. We observed that the three major transcriptional regulators of the AR genes (gadX, gadE, gadW) only marginally affected biofilm formation in E. coli. However, a striking and novel finding was the different ability of all the tested E. coli strains to form a biofilm depending on the temperature and pH of the medium: LB, pH 7.4, strongly supported biofilm formation at 25°C, with biofilm being hardly detectable at 37°C. On the contrary, LB, pH 5.5, best supported biofilm formation at 37°C. Moreover, we observed that when E. coli carried a plasmid, the presence of the plasmid itself affected the ability to develop a biofilm, typically by increasing its formation. This phenomenon varies from plasmid to plasmid, depends on growth conditions, and, to the best of our knowledge, remains largely uninvestigated

Gian Carlo Ferretti - Giulia Iannuzzi, Storie di uomini e libri

Review of the book Storie di uomini e libri Anyone who is familiar with the history of Italian publishing will have come across the name of Gian Carlo Ferretti. He is one of the leading critics and scholars in the field and has engaged in extensive research since the sixties, writing an impressive number of volumes and contributions – among them, the seminal text Storia dell’editoria letteraria in Italia, 1945-2003, published by Einaudi in 2004. Storie di uomini e libri, co-authored with Giulia Iannuzzi, relates to the line of research opened and developed by Ferretti throughout his life on the relation between literature, market, and cultural production. The premise of this volume, and of Ferretti’s entire work, is that literature, culture, and publishing industry are inextricably linked..

The yhiM gene codes for an inner membrane protein involved in GABA export in Escherichia coli

In order to survive the exposure to acid pH, Escherichia coli activates molecular circuits leading from acid tolerance to extreme acid resistance (AR). The activation of the different circuits involves several global and specific regulators affecting the expression of membrane, periplasmic and cytosolic proteins acting at different levels to dampen the harmful consequences of the uncontrolled entry of protons intracellularly. Many genes coding for the structural components of the AR circuits (protecting from pH ≤ 2.5) and their specific transcriptional regulators cluster in a genomic region named AFI (acid fitness island) and respond in the same way to global regulators (such as RpoS and H-NS) as well as to anaerobiosis, alkaline, cold and respiratory stresses, in addition to the acid stress. Notably some genes coding for structural components of AR, though similarly regulated, are non-AFI localised. Amongst these the gadBC operon, coding for the major structural components of the glutamate-based AR system, and the ybaS gene, coding for a glutaminase required for the glutamine-based AR system. The yhiM gene, a non-AFI gene, appears to belong to this group. We mapped the transcription start of the 1.1 kb monocistronic yhiM transcript: it is an adenine residue located 22 nt upstream a GTG start codon. By real-time PCR we show that GadE and GadX equally affect the expression of yhiM under oxidative growth conditions. While YhiM is partially involved in the RpoS-dependent AR, we failed to detect a significant involvement in the glutamate- or glutamine-dependent AR at pH ≤ 2.5. However, when grown in EG at pH 5.0, the yhiM mutant displays impaired GABA export, whereas when YhiM is overexpressed, an increases of GABA export in EG medium in the pH range 2.5–5.5 is observed. Our data suggest that YhiM is a GABA transporter with a physiological role more relevant at mildly acidic pH, but not a key component of AR at pH < 2.5

Telemetrische Messung des intrakraniellen Drucks in der pädiatrischen Neurochirurgie

Introduction: Telemetric intracranial pressure measurement (tICPM) enables the measurement of intracranial pressure (ICP) in different conditions, such as body posture and activity. Neurovent-P-Tel (P-Tel, Raumedic, Helmbrechts, Germany) and sensor reservoir (SR, Miethke, Potsdam, Germany) are among the most used systems, and their employment influences the management of patients with cerebrospinal fluid (CSF) circulation disturbances. The SR has added several advantages in the follow-up of patients. The aim of this work is to assess the in vitro and in vivo performance of tICPM devices and to describe new achievements in maneuver measurements in pediatric neurosurgery. Materials and methods: Six SRs were tested and compared to standard invasive ICP measurement methods in an experimental setting. In vivo data from 21 patients in a mixed P-Tel and SR population (8 with a P-Tel and 13 with an SR) was retrospectively evaluated. Additionally, a specific measurement protocol, combining different body positions, ventilation patterns and jugular compression, was developed and applied in 17 SR-implanted patients. Results: In vitro testing showed a significant similarity between SRs and invasive pressure measuring systems. The in vivo dataset contained measurements from a total of 21 patients (median age 16.5 years, range 10–39.5 years). A maneuver protocol was applied in one population with a ventriculoperitoneal shunt system, and in another with a stand-alone SR for diagnostic purposes only. There were 13 shunted patients (median age 15.8 years, range 4.0–35.2 years), and 6 were non-shunted (median age 11.9 years, range 3.6–17.7 years). A total of 480 measurements were performed. tICPM-guided shunt adjustments led to clinical improvements in 7 P-Tel (75%) and 6 SR (76.9%) patients, with shunt survival rates of 44.4% at 77.9 months and 83.3% at 42 months, respectively. Three secondary shunt implantations were indicated in the stand-alone population. Comparative analysis in intracranial pressure and amplitude among stand-alone and shunted SR patients showed differences in the changes of these two parameters: in the shunted group, intracranial pressure showed more significant variations, while the non-shunted population showed a more drastic change in amplitude. Conclusions: SRs were confirmed to have a good reliability in vitro. tICPM is well-established and helpful in clinical practice – especially in complex shunt-dependent patients with chronic symptomatology. The introduction of an analysis tool improved the throughput of the data analysis, but data processing remains cumbersome. Future improvements will focus on the elaboration of relevant parameters and performing tests.Fragestellung: Die telemetrische intrakranielle Druckmessung (tICPM) ermöglicht die Messung des intrakraniellen Drucks (ICP) unter verschiedenen Bedingungen (Körperhaltung, Aktivität). Neurovent-P-Tel (P-Tel, Raumedic, Helmbrechts, Deutschland) und Sensor-Reservoir (SR, Miethke, Potsdam, Deutschland) gehören zu den am häufigsten verwendeten Systemen, und ihr Einsatz hat Einfluss auf das Management von Patienten mit Liquorzirkulationsstörungen. Das SR hat mehrere Vorteile in der Patientenbehandlung gebracht. Ziel dieser Arbeit ist es, die In-vitro- und In-vivo-Leistung von tICPM-Geräten zu bewerten und neue Perspektiven bei Manövermessungen in der pädiatrischen Neurochirurgie zu beschreiben. Materialien und Methoden: Sechs SR wurden experimentell getestet und mit invasiven Standardmethoden zur ICP-Messung verglichen. In-vivo-Daten von 21 Patienten aus einer gemischten P-Tel- und SR-Population (8 P-Tel und 13 SR) wurden retrospektiv ausgewertet. Darüber hinaus wurde ein spezifisches Messprotokoll entwickelt, das verschiedene Körperpositionen, Beatmungsmuster und Jugularkompression kombiniert und bei 17 SR-implantierten Patienten angewendet wurde. Ergebnisse: In-vitro-Tests zeigten eine signifikante Ähnlichkeit zwischen SRs und invasiven Druckmesssystemen. Der In-vivo-Datensatz enthielt Messungen von 21 Patienten (mittleres Alter 16.5 Jahre, Range 10-39.5 Jahre). Ein Manöverprotokoll wurde bei einer Population mit einem Shuntsystem und bei einer mit „stand-alone“ SR zu rein diagnostischen Zwecken angewendet. An 13 Shunt-Patienten (medianes Alter 15.8 Jahre, Range 4.0-35.2 Jahre) und 6 Nicht-Shunt-Patienten (medianes Alter 11.9 Jahre, Range 3.6-17.7 Jahre) wurden 480 Messungen durchgeführt. Die tICPM-gesteuerten Shuntanpassungen führten bei 7 P-Tel- (75 %) und 6 SR-Patienten (76.9 %) zu klinischen Verbesserungen, mit Shunt-Überlebensraten von 44.4 % nach 77.9 Monaten bzw. 83.3 % nach 42 Monaten. Drei sekundäre Shunt-Implantationen waren in der Stand-alone-Population indiziert. Eine Vergleichsanalyse des intrakraniellen Drucks und der Amplitude zwischen „stand-alone“- und Shunt-SR-Patienten zeigte Unterschiede in den Veränderungen dieser beiden Parameter: In der Shunt-Gruppe wies der intrakranielle Druck deutlichere Veränderungen auf, während die nicht geshuntete Population eine drastischere Veränderung der Amplitude zeigte. Schlussfolgerungen: Es wurde bestätigt, dass die SRs in vitro eine gute Zuverlässigkeit aufweisen. tICPM ist in der klinischen Praxis gut etabliert und hilfreich - insbesondere bei komplexen shuntabhängigen Patienten mit chronischer Symptomatik. Die Einführung eines Analysetools verbesserte den Durchsatz der Datenanalyse, aber die Datenverarbeitung ist umständlich. Künftige Verbesserungen werden sich auf die Ausarbeitung der relevanten Parameter und die Durchführung von Tests konzentrieren

Sull’etimologia del termine tarbusc “fez”

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Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH < 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

The glutaminase-dependent acid resistance system. Qualitative and quantitative assays and analysis of its distribution in enteric bacteria

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilised bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress

Different electrostatic forces drive the binding kinetics of SARS-CoV, SARS-CoV-2 and MERS-CoV Envelope proteins with the PDZ2 domain of ZO1

The Envelope protein (E) is a structural protein encoded by the genome of SARS-CoV, SARS-CoV-2 and MERS-CoV Coronaviruses. It is poorly present in the virus but highly expressed in the host cell, with prominent role in virus assembly and virulence. The E protein possesses a PDZ-binding motif (PBM) at its C terminus that allows it to interact with host PDZ domain containing proteins. ZO1 is a key protein in assembling the cytoplasmic plaque of epithelial and endothelial Tight Junctions (TJs) as well as in determining cell diferentiation, proliferation and polarity. The PDZ2 domain of ZO1 is known to interact with the Coronaviruses Envelope proteins, however the molecular details of such interaction have not been established. In this paper we directly measured, through Fluorescence Resonance Energy Transfer and Stopped-Flow methodology, the binding kinetics of the PDZ2 domain of ZO1 with peptides mimicking the C-terminal portion of the Envelope protein from SARS-CoV, SARSCoV-2 and MERS-CoV in diferent ionic strength conditions. Interestingly, the peptide mimicking the E protein from MERS-CoV display much higher microscopic association rate constant with PDZ2 compared to SARS-CoV and SARS-CoV-2 suggesting a stronger contribution of electrostatic forces in the early events of binding. A comparison of thermodynamic and kinetic data obtained at increasing ionic strengths put in evidence diferent contribution of electrostatics in the recognition and complex formation events for the three peptides. Our data are discussed under the light of available structural data of PDZ2 domain of ZO1 and of previous works about these protein systems

E.T.A. Hoffmann e W.A. Mozart: confronto tra la composizione letteraria di Don Juan e la composizione musicale di Don Giovanni

L'oggetto del presente elaborato è un confronto tra l'opera Don Giovanni di Wolfgang Amadeus Mozart e il racconto Don Juan di Ernst Theodor Amadeus Hoffmann. A una sezione dedicata alla vita dello scrittore e alle sue opere segue un'analisi della figura del Don Giovanni nella storia, in Hoffmann e in Mozart. Un parallelo tra i due capolavori è invece il tema dell'ultimo capitolo, con un focus specifico sui personaggi, i temi, e l'interpretazione