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Author(s):  
Muhammad Kamruzzaman ◽  
Amy J. Mathers ◽  
Jonathan R. Iredell

Conjugative plasmids are the principal mediator in the emergence and spread of antibiotic resistance genes in Enterobacterales. Plasmid entry-exclusion (EEX) systems can restrict their transfer into the recipient bacteria carrying closely related plasmids. In this study, we have identified and characterized a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, responsible for widespread dissemination of the bla KPC carbapenemase gene among Enterobacterales in the United States. The identified eex gene in the recipient strain of different Enterobacterales species inhibits the conjugation transfer of pKPC_UVA01 plasmids at a range of 200-400 fold, and this inhibition was found to be a dose-dependent function of the EEX protein in recipient cells. The C-terminus truncated version of eex or eex with an early termination codon at the C-terminus region alleviates inhibition of conjugative transfer. Unlike the strict specificity of plasmid exclusion by the known EEX protein, the newly identified EEX in the recipient strain can inhibit the transfer of IncP and IncN plasmids. The eex gene from the plasmid pKPC_UVA01 is not required for conjugative transfer but is essential in the donor bacteria for entry exclusion of this plasmid. This is a novel function of a single protein that is essential in both donor and recipient bacteria for entry exclusion of a plasmid. This eex gene is found to be distributed in multi-drug resistance plasmids similar to pKPC_UVA01 in different Enterobacterales species and may contribute to the stability of this plasmid type by controlling its transfer.


Author(s):  
Mariana Handelman ◽  
Zohar Meir ◽  
Jennifer Scott ◽  
Yona Shadkchan ◽  
Wei Liu ◽  
...  

Aspergillus fumigatus is the most common cause of invasive fungal mold infections in immunocompromised individuals. Current antifungal treatment relies heavily on the triazole antifungals which inhibit fungal Erg11/Cyp51 activity and subsequent ergosterol biosynthesis. However, resistance, due primarily to cyp51 mutation, is rapidly increasing. A. fumigatus contains two Cyp51 isoenzymes, Cyp51A and Cyp51B. Overexpression and mutation of Cyp51A is a major cause of triazole resistance in A. fumigatus . The role of Cyp51B in generating resistance is unclear. Here we show that overexpression or mutation of cyp51B results in triazole resistance. We demonstrate that introduction of a G457S Cyp51B mutation identified in a resistant clinical isolate, results in voriconazole resistance in the naïve recipient strain. Our results indicate that mutations in cyp51B resulting in clinical resistance do exist and should be monitored.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Polonca Stefanic ◽  
Katarina Belcijan ◽  
Barbara Kraigher ◽  
Rok Kostanjšek ◽  
Joseph Nesme ◽  
...  

AbstractBacillus subtilis is a soil bacterium that is competent for natural transformation. Genetically distinct B. subtilis swarms form a boundary upon encounter, resulting in killing of one of the strains. This process is mediated by a fast-evolving kin discrimination (KD) system consisting of cellular attack and defence mechanisms. Here, we show that these swarm antagonisms promote transformation-mediated horizontal gene transfer between strains of low relatedness. Gene transfer between interacting non-kin strains is largely unidirectional, from killed cells of the donor strain to surviving cells of the recipient strain. It is associated with activation of a stress response mediated by sigma factor SigW in the donor cells, and induction of competence in the recipient strain. More closely related strains, which in theory would experience more efficient recombination due to increased sequence homology, do not upregulate transformation upon encounter. This result indicates that social interactions can override mechanistic barriers to horizontal gene transfer. We hypothesize that KD-mediated competence in response to the encounter of distinct neighbouring strains could maximize the probability of efficient incorporation of novel alleles and genes that have proved to function in a genomically and ecologically similar context.


2021 ◽  
Vol 37 (6) ◽  
pp. 25-33
Author(s):  
V.Yu. Matys ◽  
V.A. Nemashkalov ◽  
A.M. Rozhkova ◽  
I.A. Shashkov ◽  
A.D. Satrutdinov ◽  
...  

A new recombinant Aspergillus niger tannase (tannin acyl hydrolase) produced by the Penicillium verruculosum fungus has been studied. A strain with a high level of extracellular tannase (TAN2) secretion (80% of the total extracellular protein) was obtained by cloning the tan2 gene (PDB Acc. No: MT828303) into the recipient strain. The tannase enzyme preparation degraded tannins in black tea extracts. TAN2 was isolated in homogeneous form using chromatographic methods; the enzyme had a high activity against gallotannin (53 U/mg) and less activity against propyl gallate (4.7 U/mg). Homogeneous TAN2 showed temperature and pH optima of 45 °C and 3.5, respectively. At a temperature of 50 °C, TAN2 retained above 80% activity for 3 h; at 60 °C, it retained about 75% of its activity for 90 min; at 70 °C, the enzyme was completely inactivated within 10 min. Tannase was characterized by a high tolerance to NaCl, the activity against gallotannin exceeded 50% of the initial value in solutions with a salt concentration of up to 5 M. The tannase activity was stimulated by Ca2+, Mg2+, Zn2+, Mn2+, Cu2+, Cd2+, Pb2+ by 3--64% and inhibited by 4-65% in the presence of Co2+, Fe3+ and Fe2+ ions. tannase, tannins, Aspergillus niger, Penicillium verruculosum This work was supported by the Ministry of Science and Higher Education of the Russian Federation.


2020 ◽  
Vol 85 (4) ◽  
pp. 1005-1015
Author(s):  
Kentaro Ochi ◽  
Maho Tokuda ◽  
Kosuke Yanagiya ◽  
Chiho Suzuki-Minakuchi ◽  
Hideaki Nojiri ◽  
...  

ABSTRACT The frequency of transconjugants were compared for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1 under aerobic and anaerobic conditions. Filter mating assays were performed with one donor strain and one recipient strain using different donors of Pseudomonas and recipient strains, including Pseudomonas, Pantoea, and Buttiauxella. Under anaerobic condition, frequencies of transconjugants for both plasmids were 101-103-fold lower than those under aerobic condition regardless of whether aerobically or anaerobically grown donors and recipients were used. To compare the transconjugant ranges under aerobic and anaerobic conditions, conjugation was performed between the donor of pBP136 and recipient bacteria extracted from environmental samples. Several transconjugants were uniquely obtained from each aerobic or anaerobic condition. Our findings indicate that a plasmid can differently spread among bacteria depending on the oxygen concentrations of the environment.


2020 ◽  
Author(s):  
Nadia Morson ◽  
Olivia Molenda ◽  
Katherine J. Picott ◽  
Ruth E. Richardson ◽  
Elizabeth A. Edwards

ABSTRACTVinyl chloride (VC) is a human carcinogen that accumulates in soil and groundwater due to incomplete dechlorination of chlorinated ethenes. Some strains of obligate organohalide respiring Dehalococcoides mccartyi can synthesize the VC reductase that catalyzes the dechlorination of VC to ethene. The gene encoding the VC reductase, vcrA, is found on a mobile genetic element called the vcrA-Genomic Island (GI) that may participate in horizontal gene transfer. We designed an experiment to try to induce horizontal gene transfer of the vcrA-GI by mixing two enrichment cultures: one containing the donor D. mccartyi strain with the vcrA-GI that could not fix nitrogen and the second containing the recipient strain devoid of the vcrA-GI that could fix nitrogen. Therefore, mixing the two cultures in medium without ammonium while providing VC as the sole electron acceptor was hypothesized to select for a mutant strain of D. mccartyi that could both fix nitrogen and respire VC. However, after over 4 years of incubation, no evidence for horizontal gene transfer of the vcrA-GI was found. Rather, we observed VC-dechlorinating activity attributed to the TCE reductase, TceA, in the recipient strain. We also observed that D. mccartyi can grow by scavenging low concentrations of fixed nitrogen sources. During this experiment we identified two additional D. mccartyi strains in the KB-1 TCE-enriched culture that could fix nitrogen. The presence of multiple strains of D. mccartyi with distinct phenotypes may enhance bioaugmentation success, but here it may have undermined attempts to force horizontal gene transfer of the vcrA-GI.IMPORTANCEDehalococcoides mccartyi are a powerful bioremediation tool for the degradation of chlorinated solvent contamination in soil and groundwater. Only a few D. mccartyi strains have the ability to dechlorinate toxic chlorinated compounds like vinyl chloride. Interestingly, the genetic ability to dechlorinate vinyl chloride is theorized to be shared among D. mccartyi strains. In this study we attempted to promote the genetic transfer of vinyl chloride degrading ability from one D. mccartyi strain to another. Although we did not observe this exchange, our findings suggest there may be restrictions of genetic transfer between specific clades or sub-groups of D. mccartyi strains. Developing our understanding of genetic transfer among D. mccartyi strains could allow for enhanced degradation of chlorinated solvent contamination in situ.


2020 ◽  
Vol 8 (10) ◽  
pp. 1538
Author(s):  
Abraham Fikru Mechesso ◽  
Dong Chan Moon ◽  
Hee Young Kang ◽  
Hyun-Ju Song ◽  
Su-Jeong Kim ◽  
...  

We examined the prevalence and molecular characteristics of mcr-3 carrying colistin-resistant Escherichia coli among cattle, pig, and chicken isolates in South Korea. Among a total of 185 colistin-resistant E. coli isolates determined in this study (47 from cattle, 90 from pigs, and 48 from chicken), PCR amplification detected mcr-3 genes in 17 isolates predominantly from diseased pigs. The mcr-3 genes were characterized as mcr-3.1 in 15 isolates and mcr-3.5 in 2 isolates. The mcr-3 gene was transferred to the E. coli J53 recipient strain from more than 50% of the mcr-3-carrying isolates. The mcr-3.1 and mcr-3.5 genes were identified predominantly in IncHI2 and IncP plasmids, respectively. Multi-locus sequence typing analysis revealed eight previously reported sequence types (ST), including ST1, ST10, and ST42. We identified isolates with similar pulsed-field gel electrophoresis patterns from diseased pigs in three farms. Besides, the isolates carried various virulence factors and demonstrated resistance to multiple antimicrobials, including β-lactams and quinolones. Further, the mcr-3.5 encodes three amino acid substitutions compared with mcr-3.1. To the best of our knowledge, this is the first report of pathogenic E. coli carrying mcr-3.5 in South Korea, which implies that mcr-3 variants may have already been widely spread in the pig industry.


2020 ◽  
Author(s):  
JVE Chan-Hyams ◽  
David Ackerley

© 2020 The Authors Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.


2020 ◽  
Author(s):  
JVE Chan-Hyams ◽  
David Ackerley

© 2020 The Authors Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two Escherichia coli strains grown in co-culture. The first of these was an activator strain that overexpressed the E. coli nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.


2020 ◽  
Author(s):  
Dmitri Mikhailovich Muzaev ◽  
Andrey Mikhailovitch Rumyantsev ◽  
Ousama Raek Al Shanaa ◽  
Elena Viktorovna Sambuk

Background. A selective system based on the M1 virus of the yeast Saccharomyces cerevisiae was proposed. Methods. To create a recipient strain, a DNA fragment encoding the killer toxin of the M1 virus under the control of the regulated promoter of the GAL1 gene was inserted into the genome of S. cerevisiae strains Y-1236 and Y-2177. Results. Integration of such expression cassette leads to the conditional lethality - resulting strains die on a medium with galactose when killer toxin synthesis occurs. A linear DNA fragment containing the gene of interest flanked by sequences homologous to the promoter of the GAL1 gene and the termination region of the CYC1 gene is used to transform the obtained strains. During transformation due to homologous recombination, the sequence encoding the killer toxin is cleaved and the transformants grow on a medium with galactose. Conclusion. The proposed selective system combines the main advantages of other systems: the use of simple media, without the need to add expensive antibiotics, and a simplified technique for constructing expression cassettes and selecting transformants.


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