Multi-species host range of staphylococcal phages isolated from wastewater

The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment.

Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3′-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.

Engineering a circular riboregulator inEscherichia coli

Circular RNAs have recently been shown to be important gene expression regulators in mammalian cells. However, their role in prokaryotes remains elusive. Here, we engineered a synthetic riboregulator that self-splice to produce a circular molecule, exploiting group I permuted intron-exon (PIE) sequences. We demonstrated that the resulting circular riboregulator can activate gene expression, showing increased dynamic range compared to the linear form. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker. Thanks to the increased regulatory activity by higher stability, isolation due to self-splicing, and modularity of PIE, we envisage engineered circular riboregulators in further synthetic biology applications.

Role of VltAB, an ABC Transporter Complex, in Viologen Tolerance inStreptococcus mutans

ABSTRACTStreptococcus mutans, a Gram-positive organism, is the primary causative agent in the formation of dental caries in humans. To persist in the oral cavity,S. mutansmust be able to tolerate rapid environmental fluctuations and exposure to various toxic chemicals. However, the mechanisms underlying the ability of this cariogenic pathogen to survive and proliferate under harsh environmental conditions remain largely unknown. Here, we wanted to understand the mechanisms by whichS. mutanswithstands exposure to methyl viologen (MV), a quaternary ammonium compound (QAC) that generates superoxide radicals in the cell. To elucidate the essential genes for MV tolerance, screening of ∼3,500 mutants generated by ISS1mutagenesis, revealed 15 MV-sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906 genes, respectively. These two genes are appeared to be organized in an operon and encode a putative ABC transporter complex; we designated the genes asvltAandvltB, forviologentransporter. To verify our results,vltAwas deleted by using an antibiotic resistance marker; the mutant was just as sensitive to MV as the ISS1insertion mutants. Furthermore,vltAandvltBmutants were also sensitive to other viologen compounds such as benzyl and ethyl viologens. Complementation assays were also carried out to confirm the role of VltA and VltB in viologen tolerance. Sensitivity to various drugs, including a wide range of QACs, was evaluated. It appears that a functional VltA is also required for full resistance toward acriflavin, ethidium bromide, and safranin; all are well-known QACs. These results indicate that VltA/B constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that homologs of VltA/B are widely present in streptococci, enterococci, and other important Gram-positive pathogens.

Targeted Chromosomal Knockouts in Mycoplasma pneumoniae

ABSTRACT Most gene knockouts in mycoplasmas are achieved through labor-intensive transposon mutagenesis. Here, we describe a method for making targeted deletions in Mycoplasma pneumoniae by use of homologous recombination. In this method, M. pneumoniae is transformed with a plasmid carrying an antibiotic resistance marker flanked by 1-kb regions surrounding the target gene. Following selection for the antibiotic resistance, colonies are screened for double crossovers which indicate complete deletion of the target open reading frame.