Complete Genome Sequences of Mycobacterium chimaera Strains 850 and 852, Isolated from Heater-Cooler Unit Water

Pinzauti, David; Giorgi, Stefano De; Fox, Valeria; Lazzeri, Elisa; Messina, Gabriele; Santoro, Francesco; Iannelli, Francesco; Ricci, Susanna; Pozzi, Gianni

doi:10.1128/mra.01021-21

Cited by 3 publications

(2 citation statements)

References 14 publications

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“…Nanopore sequencing was carried out essentially as already described [ 47 ]. Briefly, a DNA size selection of genomic DNA was performed with 0.5 volumes of AMPure XP beads (Beckman Coulter, Milano, Italy).…”

Section: Methodsmentioning

confidence: 99%

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages

Bivona,

Bonomo,

Colombini

et al. 2024

Antibiotics

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Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

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Section: Methodsmentioning

confidence: 99%

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages

Bivona,

Bonomo,

Colombini

et al. 2024

Antibiotics

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“…Nanopore sequencing was carried out essentially as already described [27,28]. Briefly, sequencing reactions were carried out in 1.5 ml LoBind tubes (Sarstedt) using wide bore (∅1.2 mm) tips for DNA manipulation to reduce physical shearing.…”

Section: Nanopore Sequencingmentioning

confidence: 99%

The mobilome of Lactobacillus crispatus M247 includes two novel genetic elements: Tn7088 coding for a putative bacteriocin and the siphovirus prophage ΦM247

Colombini,

Santoro,

Tirziu

et al. 2023

Microbial Genomics

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Lactobacillus crispatus is a member of the vaginal and gastrointestinal human microbiota. Here we determined the complete genome sequence of the probiotic strain M247 combining Nanopore and Illumina technologies. The M247 genome is organized in one circular chromosome of 2 336 109 bp, with a GC content of 37.04 % and 2303 ORFs, of which 1962 could be annotated. Analysis of the M247 mobilome, which accounts for 14 % of the whole genome, revealed the presence of: (i) Tn7088, a novel 14 105 bp long integrative and mobilizable element (IME) containing 16 ORFs; (ii) ΦM247, a novel 42 510 bp long siphovirus prophage containing 52 ORFs; (iii) three clustered regularly interspaced short palindromic repeats (CRISPRs); and (iv) 226 insertion sequences (ISs) belonging to 14 different families. Tn7088 has a modular organization including a mobilization module encoding FtsK homologous proteins and a relaxase, an integration/excision module coding for an integrase and an excisionase, and an adaptation module coding for a class I bacteriocin and homologous to the listeriolysin S (lls) locus of Listeria monocytogenes . Genome-wide homology search analysis showed the presence of Tn7088-like elements in 12 out of 23 L . crispatus complete public genomes. Mobilization and integration/excision modules are essentially conserved, while the adaptation module is variable since it is the target site for the integration of different ISs. Prophage ΦM247 contains genes for phage structural proteins, DNA replication and packaging, lysogenic and lytic cycles. ΦM247-like prophages are present in seven L . crispatus complete genomes, with sequence variability mainly due to the integration of ISs. PCR and sequencing showed that the Tn7088 IME excises from the M247 chromosome producing a circular form at a concentration of 4.32×10−5 copies per chromosome, and reconstitution of the Tn7088 chromosomal target site occurred at 6.65×10−4 copies per chromosome. The ΦM247 prophage produces an excised form and a reconstituted target site at a level of 3.90×10−5 and 2.48×10−5 copies per chromosome, respectively. This study identified two novel genetic elements in L. crispatus . Tn7088 represents the first example of an IME carrying a biosynthetic gene cluster for a class I bacteriocin in L. crispatus .

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A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci

Tirziu,

Colombini,

Stincarelli

et al. 2024

World J Microbiol Biotechnol

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The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

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Complete Genome Sequences of Mycobacterium chimaera Strains 850 and 852, Isolated from Heater-Cooler Unit Water

Cited by 3 publications

References 14 publications

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages

The mobilome of Lactobacillus crispatus M247 includes two novel genetic elements: Tn7088 coding for a putative bacteriocin and the siphovirus prophage ΦM247

A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci

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