Internal translational initiation in the mRNA from the Neurospora crassa albino-3 gene.

The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA

The Neurospora crassa carotenoid biosynthetic gene (albino 3) reveals highly conserved regions among prenyltransferases.

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al-3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may be involved in the formation of the catalytic site of the prenyltransferases

Variation in expression of HMW1 and HMW2 adhesins in invasive nontypeable Haemophilus influenzae isolates

<p>Abstract</p> <p>Background</p> <p>Among surface antigens of nontypeable <it>Haemophilus influenzae </it>(NTHi), the HMW1 and HMW2 proteins are the major adhesins promoting colonization of the upper respiratory tract. Since they are potential vaccine candidates, knowledge concerning variation in HMW proteins expression among clinical isolates is of great interest. In this study, expression of <it>hmw1A </it>and <it>hmw2A </it>genes was evaluated by quantitative real-time reverse transcription-PCR in 3 NTHi invasive isolates (strains 56, 72, 91) and in the prototype strain 12. Number of 7-bp repeats within the <it>hmwA </it>promoters and presence of HMW proteins by Western blotting were also determined.</p> <p>Results</p> <p>Results showed that gene transcription varied not only among different isolates but also between the <it>hmw1A </it>and <it>hmw2A </it>genes from the same isolate. Compared to that found in prototype strain 12, up-regulation of the <it>hmw1A </it>gene expression was found in strain 56, down-regulation of both <it>hmw1A </it>and <it>hmw2A </it>genes transcripts was observed in strain 72 whereas the two <it>hmwA </it>genes appeared differentially expressed in strain 91 with the <it>hmw1A </it>transcript enhanced but the <it>hmw2A </it>transcript reduced.</p> <p>Conclusion</p> <p>Increasing numbers of 7-bp repeats within the <it>hmwA </it>promoters generally correlated with decreased amounts of mRNA transcript, however additional control mechanisms contributing to modulation of <it>hmw1A </it>gene seem to be present.</p

A novel IncQ plasmid type harbouring a class 3 integron from Escherichia coli

Objectives To determine the genetic structures at the origin of the mobilization of the extended-spectrum β-lactamase (ESBL) blaGES-1 gene in an Escherichia coli clinical isolate. Methods ESBL-encoding genes and class 1 or class 3 integron-specific motifs were screened. Conjugation experiments were performed to determine whether the plasmid-carrying blaGES-1 gene was self-transferable. Plasmid sequencing was achieved by a primer-walking approach. Results The blaGES-1 gene was located in a class 3 integron. That unusual genetic structure was itself located on an ∼9 kb plasmid, pQ7, which was not self-transferable. Sequence analysis revealed that plasmid pQ7 belonged to a novel subtype of the IncQ group. Conclusions This study identified for the first time the blaGES-1 gene in E. coli and in Switzerland. It describes a novel IncQ-type plasmid subgroup that possesses original features, in particular iteron sequences that constitute a hot spot for integration of foreign DN

A novel plasmid carrying blaCTX-M-15 identified in commensal Escherichia coli from healthy pregnant women in Ibadan, Nigeria

AbstractThe aim of this study was to investigate the molecular characteristics of commensal Escherichia coli producing extended-spectrum β-lactamases and showing fluoroquinolone resistance circulating in a healthy population in Ibadan, Nigeria. In total, 101 faecal samples from healthy pregnant women on the day of admission to hospital were collected and plated on eosin–methylene blue agar supplemented with cefotaxime. Genotyping demonstrated the presence of the blaCTX-M-15 gene in all of the cefotaxime-resistant isolates (n=32), and there was circulation of prevalent clones. The aac(6′)-Ib-cr, qnrS1, qepA1 and qnrB1 genes were identified in several strains. A novel plasmid supporting the spread of the blaCTX-M-15, blaTEM-1 and qnrS1 genes was identified in these isolates by complete DNA sequencing

Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems

Genome-based retrospective analysis of a Providencia stuartii outbreak in Rome, Italy. Broad spectrum IncC plasmids spread the NDM carbapenemase within the hospital

Providencia stuartii is a member of the Morganellaceae family, notorious for its intrinsic resistance to several antibiotics, including last-resort drugs such as colistin and tigecycline. Between February and March 2022, a four-patient outbreak sustained by P. stuartii occurred in a hospital in Rome. Phenotypic analyses defined these strains as eXtensively Drug-Resistant (XDR). Wholegenome sequencing was performed on the representative P. stuartii strains and resulted in fully closed genomes and plasmids. The genomes were highly related phylogenetically and encoded various virulence factors, including fimbrial clusters. The XDR phenotype was primarily driven by the presence of the (NDM)-N-bla- 1 metallo- beta-lactamase alongside the rmtC 16S rRNA methyltransferase, conferring resistance to most beta-lactams and every aminoglycoside, respectively. These genes were found on an IncC plasmid that was highly related to an NDM-IncC plasmid retrieved from a ST15 Klebsiella pneumoniae strain circulating in the same hospital two years earlier. Given its ability to acquire resistance plasmids and its intrinsic resistance mechanisms, P. stuartii is a formidable pathogen. The emergence of XDR P. stuartii strains poses a significant public health threat. It is essential to monitor the spread of these strains and develop new strategies for their control and treatment

Ceftazidime-avibactam resistance in Klebsiella pneumoniae sequence type 37: a decade of persistence and concealed evolution

The first reports of carbapenem-resistant Enterobacterales in our hospital date back to 2006. In that period, few ertapenem-resistant but meropenem-susceptible Klebsiella pneumoniae isolates belonging to sequence type (ST) 37 were retrieved from clinical samples. These strains produced the CTX-M-15 extended spectrum β-lactamase, OmpK35 was depleted due to a nonsense mutation, and a novel OmpK36 variant was identified. Yet, starting from 2010, Klebsiella pneumoniae carbapenemase (KPC)-producing ST512 isolates started prevailing and ST37 vanished from sight. Since 2018 the clinical use of the combination of ceftazidime-avibactam (CZA) has been introduced in clinical practice for the treatment of bacteria producing serine-β-lactamases, but KPC-producing, CZA-resistant K. pneumoniae are emerging. In 2021, four CZA-resistant ST37 isolates producing KPC variants were isolated from the same number of patients. blaKPC gene cloning in Escherichia coli was used to define the role of those KPC variants on CZA resistance, and whole genome sequencing was performed on these isolates and on three ST37 historical isolates from 2011. CZA resistance was due to mutations in the blaKPC genes carried on related pKpQIL-type plasmids, and three variants of the KPC enzyme have been identified in the four ST37 strains. The four ST37 isolates were closely related to each other and to the historical isolates, suggesting that ST37 survived without notice in our hospital for 10 years, waiting to re-emerge as a CZA-resistant K. pneumoniae clone. The ancestor of these contemporary isolates derives from ST37 wild-type porin strains, with no other mutations in chromosomal genes involved in conferring antibiotic resistance (parC, gyrA, ramR, mgrB, pmrB)