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Structure and expression of the ato operon coding for F(1)F(o)-ATP synthase from the antibiotic-producing actinomycete Nonomuraea sp- ATCC 39727
No full textNonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the
glycopeptide antibiotic A40926. In this study, the nucleotide sequence of the atp
operon coding for F1F0-ATP synthase of Nonomuraea sp. ATCC 39727 was determined.
It consisted of ten open reading frames arranged in the order atpI (encoding the
i protein), orfX, atpB (a subunit), atpE (c subunit), atpF (b subunit), atpH
(delta subunit), atpA (alpha subunit), atpG (gamma subunit), atpD (beta subunit)
and atpC (epsilon subunit). The orfX coded for a putative small hydrophobic 71
amino acid peptide of unknown function related to several bacterial permeases.
Its presence appeared to be a distinctive feature of the atp operon of
phylogenetically distant actinobacteria. Transcription of the atp operon was
evaluated. The results of northern blot and RT-PCR experiments demonstrated that
the atp genes were co-transcribed into a single polycistronic mRNA. Real-time
RT-PCR data provided evidence showing that transcription of the atp operon was
biphasic during Nonomuraea growth. The amount of the atpD transcript decreased at
the end of the exponential growth phase, and then moderately increased during the
early stationary phase when, in contrast, the levels of ctaC, encoding the
cytochrome c oxidase subunit II, progressively decreased. Western blot analysis
confirmed that ATP synthase was also present in the membrane during the
stationary phase. These results together with previous data demonstrate that
oligomycin-sensitive ATP-driven proton pumping activity remained constant in the
stationary phase; in contrast, the activity and cytochrome content of the
respiratory enzymes became negligible
Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a 'rare' actinomycete.
No full textWith the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)-encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild-type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic cluster-specific dpgA mRNA were stabilized during the idiophase in the wild-type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse-labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Due distinte RNA polimerasi controllano il metabolismo secondario di un attinomicete antibiotico-produttore
No full textActinomadura ATCC 39727 è attinomicete produttore dell’antibiotico glicopeptidico A40927. Questo è il precursore dell’antibiotico glicopeptidico semisintetico dalbavancina.
La produzione degli antibiotici negli attinomiceti è controllata da molteplici e coordinati meccanismi di regolazione, la cui precisa natura è ancora oggi poco nota.
Sta divenendo sempre più consistente un ruolo della risposta stringente e del ppGpp. La risposta stringente è un meccanismo di adattamento allo stress ambientale, in particolare alla carenza di nutrienti. In queste condizioni viene sintetizzato il ppGpp che si lega alla RNA pol e mediante una regolazione trascrizionale, induce un cambiamento dell’espressione genica che risulta in una regolazione della crescita, del differenziamento e del metabolismo secondario. Negli attinomiceti l’attivazione della risposta stringente e i livelli del ppGpp correlano positivamente con i livelli di alcuni antibiotici.
Sulla base di queste premesse siamo andati ad indagare il ruolo della risposta stringente e del ppGpp nella produzione del A40926 in Actinomadura ATCC 39727.
Il nostro studio ha richiesto l’isolamento di mutanti della risposta stringente. Abbiamo isolato due tipi di mutanti:
Mutanti con compromessa sintesi di ppGpp. Questi si possono ottenere selezionando con il tiostreptone. Tra i tiostreptone resistenti è possibile isolare alcuni ceppi con una difettiva sintesi della proteina ribosomale L11, implicata nella regolazione della sintesi del ppGpp da parte della sintasi ribosomiale.
Il secondo tipo di mutanti è caratterizzato da mutanti rifampicina-resistenti con fenotipo stringente. Tra i mutanti resistenti alla rifampicina si possono selezionare dei mutanti che presentano una risposta stringente attivata. Tra gli attinomiceti questi presentano una attivazione nei patway coinvolti nella sintesi degli antibiotici e in altri metaboliti secondari e in alcuni processi del differenziamento.
Abbiamo isolato alcuni mutanti di entrambi i tipi e li abbiamo caratterizzati per la produzione del A4092
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Starvation-induced posttranscriptional control of rat liver mitochondrial citrate carrier expression
No full textStarvation has been associated with a reduced citrate carrier (CTP) activity in rat liver mitochondria. In the present study the molecular mechanism responsible for this reduction was investigated. Northern blot analysis performed with hepatic total RNA showed a decrease of about 40% in the CTP mRNA abundance in starved rats, when compared to fed animals. Nuclear run-on assay did not reveal any appreciable difference in the rate of CTP mRNA synthesis between the two groups of animals, while the apparent half-life of CTP mRNA in hepatocytes from fed and starved rats was 11 and 6h, respectively. Therefore, these results suggest that in starved rats the regulation of hepatic CTP expression occurs at posttranscriptional level. Moreover, the reduced CTP activity in starved animals gradually increased by refeeding. The carrier activity reached fed rat values 6-9h following refeeding. Interestingly, the accumulation of CTP mRNA raised in parallel with the transport activity. © 2002 Elsevier Science (USA). All rights reserved
Natural merodiploidy involving duplicated rpoB alleles affects secondary metabolism in a producer actinomycete.
No full textActinomadura
sp. ATCC 39727 produces the glycopeptide
antibiotic A40926, structurally similar to teicoplanin.
Production of A40926 is governed by the
stringent response at the transcriptional level. In fact,
addition of an amino acid pool prevented the transcription of
dbv cluster genes involved in the A40926 biosynthesis and the antibiotic production in chemically defined media, and a thiostrepton-resistant relaxed mutant was severely impaired in its ability to produce the antibiotic. The derivative strain
rif19, highly resistant to rifampicin (minimal inhibitory
concentration, MIC>200mg ml-1), was isolated from the wild type strain that exhibited low resistance torifampicin (MIC<25mg ml-1). In this strain A40926 production started earlier than in the wild type, and reached higher final levels. Moreover, the antibiotic
production was not subjected to the stringent control. Molecular analysis led to the identification of two distinct rpoBalleles,rpoBS
and rpoB R, in both the wildtype and therif19. rpoBR harboured the H426N missense which is responsible for rifampicin-resistance
in bacteria, in addition to other nucleotide substitutions affecting the primary structure of theRNA polymerase b-chain. Transcriptanalysis revealed thatrpoB R was expressed at a very low level in the wild type strain during the pseudo-exponential
growth phase, and that the amount of rpoB RmRNA increased during the transition to the stationary phase. In contrast, expression of rpoB R was constitutive in the rif19. The results of mRNA half-life analysis did not support the hypothesis that post-transcriptional events are responsible for the different
rpoB expression patterns in the two strains, suggesting a
role of transcriptional mechanisms
Differential effects of coconut oil- and fish oil-enriched diets on tricarboxylate carrier in rat liver mitochondria.
No full textThe mitochondrial tricarboxylate carrier (TCC) plays an important role in lipogenesis being TCC-responsible for the efflux from the mitochondria to the cytosol of acetyl-CoA, the primer for fatty acid synthesis. In this study, we investigated the effects of two high-fat diets with different fatty acid composition on the hepatic TCC activity. Rats were fed for 3 weeks on a basal diet supplemented with 15% of either coconut oil (CO), abundant in medium-chain saturated fatty acids, or fish oil (FO), rich in n-3 polyunsaturated fatty acids. Mitochondrial fatty acid composition was differently influenced by the dietary treatments, while no appreciable change in phospholipid composition and cholesterol level was observed. Compared with CO, the TCC activity was markedly decreased in liver mitochondria from FO-fed rats; kinetic analysis of the carrier revealed a decrease of the Vmax, with no change of the Km. No difference in the Arrhenius plot between the two groups was observed. Interestingly, the carrier protein level and the corresponding mRNA abundance decreased following FO treatment.
These data indicate that FO administration markedly decreased the TCC activity as compared with CO. This effect is most likely due to a reduced gene expression of the carrier protein
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