Tn5 Transposon Research Articles

Transcriptional Profiling and Transposon Mutagenesis Study of the Endophyte Pantoea eucalypti FBS135 Adapting to Nitrogen Starvation.

The research on plant endophytes has been drawing a lot of attention in recent years. Pantoea belongs to a group of endophytes with plant growth-promoting activity and has been widely used in agricultural fields. In our earlier studies, Pantoea eucalypti FBS135 was isolated from healthy-growing Pinus massoniana and was able to promote pine growth. P. eucalypti FBS135 can grow under extremely low nitrogen conditions. To understand the mechanism of the low-nitrogen tolerance of this bacterium, the transcriptome of FBS135 in the absence of nitrogen was examined in this study. We found that FBS135 actively regulates its gene expression in response to nitrogen deficiency. Nearly half of the number (4475) of genes in FBS135 were differentially expressed under this condition, mostly downregulated, while it significantly upregulated many transportation-associated genes and some nitrogen metabolism-related genes. In the downregulated genes, the ribosome pathway-related ones were significantly enriched. Meanwhile, we constructed a Tn5 transposon library of FBS135, from which four genes involved in low-nitrogen tolerance were screened out, including the gene for the host-specific protein J, RNA polymerase σ factor RpoS, phosphoribosamine-glycine ligase, and serine acetyltransferase. Functional analysis of the genes revealed their potential roles in the adaptation to nitrogen limitation. The results obtained in this work shed light on the mechanism of endophytes represented by P. eucalypti FBS135, at the overall transcriptional level, to an environmentally limited nitrogen supply and provided a basis for further investigation on this topic.

PtrA regulates prodigiosin synthesis and biological functions in Serratia marcescens FZSF02.

Serratia marcescens is a gram-negative bacterium that is able to produce many secondary metabolites, such as the prominent red pigment prodigiosin (PG). In this work, a ptrA-disrupted mutant strain with reduced PG production was selected from Tn5 transposon mutants. RT-qPCR results indicated that ptrA promoted elevated transcription of the pig gene cluster in S. marcescens FZSF02. Furthermore, we found that ptrA also controls several other important biological functions of S. marcescens, including swimming and swarming motilities, biofilm formation, hemolytic activity, and stress tolerance. In conclusion, this study demonstrates that ptrA is a PG synthesis-promoting factor in S. marcescens and provides a brief understanding of the regulatory mechanism of ptrA in S. marcescens cell motility and hemolytic activity.

Modularity and diversity of target selectors in Tn7 transposons

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, whichwe show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.

Tn5 Transposon-based Mutagenesis for Engineering Phage-resistant Strains ofEscherichia coliBL21 (DE3).

Escherichia coli is a preferred strain for recombinant protein production, however, it is often plagued by phage infection during experimental studies and industrial fermentation. While the existing methods of obtaining phage-resistant strains by natural mutation are not efficient enough and time-consuming. Herein, a high-throughput method by combining Tn5 transposon mutation and phage screening was used to produce Escherichia coli BL21 (DE3) phage-resistant strains. Mutant strains PR281-7, PR338-8, PR339-3, PR340-8, and PR347-9 were obtained, and they could effectively resist phage infection. Meanwhile, they had good growth ability, did not contain pseudolysogenic strains, and were controllable. The resultant phage-resistant strains maintained the capabilities of producing recombinant proteins since no difference in mCherry red fluorescent protein expression was found in phage-resistant strains. Comparative genomics showed that PR281-7, PR338-8, PR339-3, and PR340-8 mutated in ecpE, nohD, nrdR, and livM genes, respectively. In this work, a strategy was successfully developed to obtain phage-resistant strains with excellent protein expression characteristics by Tn5 transposon mutation. This study provides a new reference to solve the phage contamination problem.

HexR Transcription Factor Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Coordinating Type Three Secretion System Genes.

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. We previously conducted a screening for reduced virulence using Tn5 transposon mutants and identified one of the transcriptional factors, HexR, as a potential Pcal virulence factor. However, the role of HexR in plant pathogenic Pseudomonas virulence has not been investigated well. Here, we demonstrated that the Pcal hexR mutant showed reduced disease symptoms and bacterial populations on cabbage, indicating that HexR contributes to Pcal virulence. We used RNA-seq analysis to characterize the genes regulated by HexR. We found that several type three secretion system (T3SS)-related genes had lower expression of the Pcal hexR mutant. Five genes were related to T3SS machinery, two genes were related to type three helper proteins, and three genes encoded type three effectors (T3Es). We also confirmed that T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, were also down-regulated in the Pcal hexR mutant both in culture and in vivo by using RT-qPCR. T3SS functions to suppress plant defense in host plants and induce hypersensitive response (HR) cell death in non-host plants. Therefore, we investigated the expression profiles of cabbage defense-related genes, including PR1 and PR5, and found that the expressions of these genes were greater in the Pcal hexR mutant. We also demonstrated that the hexR mutant did not induce HR cell death in non-host plants, indicating that HexR contributes in causing HR in nonhost plants. Together, these results indicate that the mutation in hexR leads to a reduction in the T3SS-related gene expression and thus an impairment in plant defense suppression, reducing Pcal virulence.

Tn5 tagments and transposes oligos to single-stranded DNA for strand-specific RNA sequencing.

Tn5 transposon tagments double-stranded DNA and RNA/DNA hybrids to generate nucleic acids that are ready to be amplified for high-throughput sequencing. The nucleic acid substrates for the Tn5 transposon must be explored to increase the applications of Tn5. Here, we found that the Tn5 transposon can transpose oligos into the 5' end of single-stranded DNA longer than 140 nucleotides. Based on this property of Tn5, we developed a tagmentation-based and ligation-enabled single-stranded DNA sequencing method called TABLE-seq. Through a series of reaction temperature, time, and enzyme concentration tests, we applied TABLE-seq to strand-specific RNA sequencing, starting with as little as 30 pg of total RNA. Moreover, compared with traditional dUTP-based strand-specific RNA sequencing, this method detects more genes, has a higher strand specificity, and shows more evenly distributed reads across genes. Together, our results provide insights into the properties of Tn5 transposons and expand the applications of Tn5 in cutting-edge sequencing techniques.

High-Throughput Mutagenesis Reveals a Role for Antimicrobial Resistance- and Virulence-Associated Mobile Genetic Elements in Staphylococcus aureus Host Adaptation.

Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex 398 (CC398) is the dominant livestock-associated (LA) MRSA lineage in European livestock and an increasing cause of difficult-to-treat human disease. LA-CC398 MRSA evolved from a diverse human-associated methicillin-sensitive population, and this transition from humans to livestock was associated with three mobile genetic elements (MGEs). In this study, we apply transposon-directed insertion site sequencing (TraDIS), a high-throughput transposon mutagenesis approach, to investigate genetic signatures that contribute to LA-CC398 causing disease in humans. We identified 26 genes associated with LA-CC398 survival in human blood and 47 genes in porcine blood. We carried out phylogenetic reconstruction on 1,180 CC398 isolates to investigate the genetic context of all identified genes. We found that all genes associated with survival in human blood were part of the CC398 core genome, while 2/47 genes essential for survival in porcine blood were located on MGEs. Gene SAPIG0966 was located on the previously identified Tn916 transposon carrying a tetracycline resistance gene, which has been shown to be stably inherited within LA-CC398. Gene SAPIG1525 was carried on a phage element, which in part, matched phiSa2wa_st1, a previously identified bacteriophage carrying the Panton-Valentine leucocidin (PVL) virulence factor. Gene deletion mutants constructed in two LA-CC398 strains confirmed that the SAPIG0966 carrying Tn916 and SAPIG1525 were important for CC398 survival in porcine blood. Our study shows that MGEs that carry antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for host adaptation. IMPORTANCE CC398 is the dominant type of methicillin-resistant Staphylococcus aureus (MRSA) in European livestock and a growing cause of human infections. Previous studies have suggested MRSA CC398 evolved from human-associated methicillin-sensitive Staphylococcus aureus and is capable of rapidly readapting to human hosts while maintaining antibiotic resistance. Using high-throughput transposon mutagenesis, our study identified 26 and 47 genes important for MRSA CC398 survival in human and porcine blood, respectively. Two of the genes important for MRSA CC398 survival in porcine blood were located on mobile genetic elements (MGEs) carrying resistance or virulence genes. Our study shows that these MGEs carrying antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for blood infection and host adaptation.

Evaluation of qPCR to Detect Shifts in Population Composition of the Rhizobial Symbiont Mesorhizobium japonicum during Serial in Planta Transfers.

Microbial symbionts range from mutualistic to commensal to antagonistic. While these roles are distinct in their outcome, they are also fluid in a changing environment. Here, we used the Lotus japonicus-Mesorhizobium japonicum symbiosis to investigate short-term and long-term shifts in population abundance using an effective, fast, and low-cost tracking methodology for M. japonicum. We use quantitative polymerase chain reaction (qPCR) to track previously generated signature-tagged M. japonicum mutants targeting the Tn5 transposon insertion and the flanking gene. We used a highly beneficial wild type and moderately beneficial and non-beneficial mutants of M. japonicum sp. nov. to demonstrate the specificity of these primers to estimate the relative abundance of each genotype within individual nodules and after serial transfers to new hosts. For the moderate and non-beneficial genotypes, qPCR allowed us to differentiate genotypes that are phenotypically indistinguishable and investigate host control with suboptimal symbionts. We consistently found the wild type increasing in the proportion of the population, but our data suggest a potential reproductive trade-off between the moderate and non-beneficial genotypes. The multi-generation framework we used, coupled with qPCR, can easily be scaled up to track dozens of M. japonicum mutants simultaneously. Moreover, these mutants can be used to explore M. japonicum genotype abundance in the presence of a complex soil community.

In vitro-obtained meropenem-vaborbactam resistance mechanisms among clinical Klebsiella pneumoniae carbapenemase-producing K. pneumoniae isolates

A novel ß-lactam-β-lactamase inhibitor (BLBI), meropenem (MEM), combined with the boronate-based inhibitor vaborbactam (VAB), has recently been introduced for the treatment of infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. The purpose of this study was to select for MEM-VAB resistance using a collection of eight KPC-producing K. pneumoniae clinical isolates, including three that produce KPC variants conferring ceftazidime-avibactam (CAZ-AVI) resistance, and subsequently decipher the corresponding resistance mechanisms. Mutants were selected in a stepwise process on agar plates containing different MEM-VAB concentrations. Susceptibility testing was performed by broth microdilution, and complementation assays were performed with wildtype ompK36. Whole genome sequencing was performed on mutants, and KPC copy number was assessed by quantitative polymerase chain reaction . Mutants were obtained from 6/8 tested isolates and reduced susceptibility to all tested β-lactams, and BLBIs, including CAZ-AVI, imipenem-relebactam, and aztreonam-AVI, were observed. No mutations were identified in the blaKPC. However, mutations in ompK36 were observed in four mutant lineages, and complementation with a wild-type ompK36 resulted in a reduction of minimal inhibitory concentrations to both MEM-VAB and other ß-lactams/BLBIs. blaKPC gene copy numbers were significantly increased in four mutant lineages. Whole genome sequencing identified genomic rearrangements in two lineages comprising mutations in the plasmid replicon encoding gene and duplication of the Tn4401 transposon bearing the blaKPC gene into a ColE-like, high copy number plasmid. In contrast to what is observed with KPC-producing mutants exhibiting resistance to CAZ-AVI, mainly corresponding to mutated KPC enzymes, here the MEM-VAB-resistant mutants showed permeability defects combined with increased KPC production, resulting from genomic rearrangement.

Contamination of Proteus mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products from food markets in China.

Proteus mirabilis is an opportunistic pathogen frequently associated with nosocomial infection and food poisoning cases. Contamination of P. mirabilis in retail meat products may be important transmission routes for human infection with P. mirabilis. In this study a total of 89 P. mirabilis strains were isolated from 347 samples in 14 food markets in China and subjected to whole-genome sequencing. Phylogenetic analysis showed that all 89 strains were divided into 81 different clones (SNPs >5), indicating high genetic diversity of P. mirabilis in food markets. Antimicrobial susceptibility testing showed that 81 (91.01%) strains displayed multidrug resistance profiles. Seventy-three different resistance genes (or variants) were found, including various clinically important antimicrobial resistance genes aac(6')-Ib-cr (77.53%), bla CTX-M (39.33%), fosA3 (30.34%), as well as multiresistance gene cfr (4.50%), tigecycline resistance gene cluster tmexCD3-toprJ1 (4.50%) and carbapenemase gene bla NDM-1 (1.12%). Diverse genetic elements including Tn7 transposon, plasmid, SXT/R391 integrative conjugative element were associated with the horizontal transfer of cfr. tmexCD3-toprJ1 and bla NDM-1 were located on ICEPmiChnJZ26 and Salmonella genomic island 1, respectively. Our study emphasized high contamination of P. mirabilis harbouring various clinically important antimicrobial resistance genes in retail meat and aquatic products, which might be an important issue in terms of food safety and human health.

His-Ala-Phe-Lys peptide from Burkholderia arboris possesses antifungal activity.

Burkholderia arboris, which belongs to the Burkholderia cepacia complex, has been shown to possess antifungal activity against several plant fungal pathogens; however, the antifungal compounds are yet to be identified. Here, we identified the antifungal compounds produced by B. arboris using genetic and metabolomic approaches. We generated a Tn5 transposon mutation library of 3,000 B. arboris mutants and isolated three mutants with reduced antifungal activity against the plant fungal pathogen Fusarium oxysporum. Among the mutants, the M464 mutant exhibited the weakest antifungal activity. In the M464 genome, the transposon was inserted into the cobA gene, encoding uroporphyrin-III methyltransferase. Deletion of the cobA gene also resulted in reduced antifungal activity, indicating that the cobA gene contributed to the antifungal activity of B. arboris. Furthermore, a comparison of the differential metabolites between wild type B. arboris and the ∆cobA mutant showed a significantly decreased level of tetrapeptide His-Ala-Phe-Lys (Hafk) in the ∆cobA mutant. Therefore, a Hafk peptide with D-amino acid residues was synthesized and its antifungal activity was evaluated. Notably, the Hafk peptide displayed significant antifungal activity against F. oxysporum and Botrytis cinerea, two plant pathogens that cause destructive fungal diseases. Overall, a novel antifungal compound (Hafk) that can be used for the biocontrol of fungal diseases in plants was identified in B. arboris.

Elucidation of Essential Genes and Mutant Fitness during Adaptation toward Nitrogen Fixation Conditions in the Endophyte Azoarcus olearius BH72 Revealed by Tn-Seq.

Azoarcus olearius BH72 is a diazotrophic model endophyte that contributes fixed nitrogen to its host plant, Kallar grass, and expresses nitrogenase genes endophytically. Despite extensive studies on biological nitrogen fixation (BNF) of diazotrophic endophytes, little is known about global genetic players involved in survival under respective physiological conditions. Here, we report a global genomic screen for putatively essential genes of A. olearius employing Tn5 transposon mutagenesis with a modified transposon combined with high-throughput sequencing (Tn-Seq). A large Tn5 master library of ~6 × 105 insertion mutants of strain BH72 was obtained. Next-generation sequencing identified 183,437 unique insertion sites into the 4,376,040-bp genome, displaying one insertion every 24 bp on average. Applying stringent criteria, we describe 616 genes as putatively essential for growth on rich medium. COG (Clusters of Orthologous Groups) assignment of the 564 identified protein-coding genes revealed enrichment of genes related to core cellular functions and cell viability. To mimic gradual adaptations toward BNF conditions, the Tn5 mutant library was grown aerobically in synthetic medium or microaerobically on either combined or atmospheric nitrogen. Enrichment and depletion analysis of Tn5 mutants not only demonstrated the role of BNF- and metabolism-related proteins but also revealed that, strikingly, many genes relevant for plant-microbe interactions decrease bacterial competitiveness in pure culture, such type IV pilus- and bacterial envelope-associated genes. IMPORTANCE A constantly growing world population and the daunting challenge of climate change demand new strategies in agricultural crop production. Intensive usage of chemical fertilizers, overloading the world's fields with organic input, threaten terrestrial and marine ecosystems as well as human health. Long overlooked, the beneficial interaction of endophytic bacteria and grasses has attracted ever-growing interest in research in the last decade. Capable of biological nitrogen fixation, diazotrophic endophytes not only provide a valuable source of combined nitrogen but also are known for diverse plant growth-promoting effects, thereby contributing to plant productivity. Elucidation of an essential gene set for a prominent model endophyte such as A. olearius BH72 provides us with powerful insights into its basic lifestyle. Knowledge about genes detrimental or advantageous under defined physiological conditions may point out a way of manipulating key steps in the bacterium's lifestyle and plant interaction toward a more sustainable agriculture.

Horizontal transfer of intracellular and extracellular ARGs in sludge compost under sulfamethoxazole stress

Aerobic composting has been considered a safe way to recycle sludge, providing a green recycling system that conserves resources. Sludge from sewage treatment plants has been identified as a significant reservoir for ARGs. However, it remains unclear whether the residual antibiotics in sludge affect the horizontal transfer mechanism of intracellular and extracellular antibiotic resistance genes (iARGs, eARGs) in compost. Here, intracellular, extracellular and total genomic DNA was extracted during sulfamethoxazole (SMX) selective pressure sludge composting. After composting, most of the sul in the sludge was removed, the removal rate of iARGs and eARGs was around 80 % and 90 %. The presence of antibiotic stress in sludge would promote the horizontal transfer of ARGs. Metagenomic sequencing confirmed that the sul gene cluster was carried by multiple mobile genetic elements, such as the Tn3 transposon and intl1, which promoted the horizontal transfer of iARGs. SMX selective pressure increased the expression of genes responsible for SOS response (29.52 %), type IV secretion systems (28.09 %) and type IV pili (40.45 %). Combined with the KEGG annotation analysis, SMX facilitated intake to eARGs by hosts, such as Pseudomonas, via type IV pili. Overall, this is the first study to verify that the horizontal transfer of iARGs and eARGs is promoted by the presence of antibiotic residues in sludge during composting.

Siderophore Synthesis Ability of the Nitrogen-Fixing Bacterium (NFB) GXGL-4A is Regulated at the Transcriptional Level by a Transcriptional Factor (trX) and an Aminomethyltransferase-Encoding Gene (amt).

Kosakonia radicincitans GXGL-4A, a gram-negative nitrogen-fixing (NF) bacterial strain is coated with a thick capsulatus on the surface of cell wall, which becomes a physical barrier for exogenous DNA to enter the cell, so the operation of genetic transformation is difficult. In this study, an optimized Tn5 transposon mutagenesis system was established by using a high osmotic HO-1 medium combined with the electroporation transformation. Eventually, a mutant library containing a total of 1633 Tn5 insertional mutants were established. Of these mutants, the mutants M81 and M107 were found to have an enhanced capability to synthesize siderophore through the CAS agar plate assay and the spectrophotometric determination. The bacterial cells of two mutants were applied in cucumber growth-promoting experiment. Cucumber seedlings treated with M81 and M107 cells had a significant increase in biomass including seedling height, seedling fresh weight, root fresh weight, and root length. The whole genome sequencing of the mutants M81 and M107 showed that the integration sites of Tn5 transposon element were located in MmyB-like helix-turn-helix transcription regulator (locus tag: A3780_19720, trX) and aminomethyltransferase-encoding genes (locus tag: A3780_01680, amt) in the genome of GXGL-4A, respectively. The ability of siderophore synthesis of the target mutants was improved by Tn5 insertion mutagenesis, and the mutants obtained showed a good plant growth-promoting effect when applied to the cucumber seedlings. The results suggest that the identified functional genes regulates the biosynthesis of siderophore in azotobacter GXGL-4A, and the specific mechanism needs to be further investigated.

Structural basis for topological regulation of Tn3 resolvase.

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδtransposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Spectinomycin resistance in Lysobacter enzymogenes is due to its rRNA target but also relies on cell-wall recycling and purine biosynthesis

Resistance to spectinomycin emerged after widely used for treatment of gonorrhea. Previous studies revealed that Lysobacter enzymogenes strain C3 (LeC3) exhibited elevated level of intrinsic resistance to spectinomycin. In this study, we screened a Tn5 transposon mutant library of LeC3 to elucidate the underlying molecular mechanisms of spectinomycin resistance. Insertion sites in 15 out of 19 mutants recovered with decreased spectinomycin resistance were located on two ribosomal RNA operons at different loci, indicating the pivotal role of ribosomal RNAs in conferring spectinomycin resistance in L. enzymogenes. The other mutants harbored mutations in the tuf, rpoD, mltB, and purB genes. Among them, the tuf and rpoD genes, respectively, encode a translation elongation factor Tu and an RNA polymerase primary sigma factor. They both contribute to protein biosynthesis, where ribosomal RNAs play essential roles. The mltB gene, whose product is involved in cell-wall recycling, was not only associated with resistance against spectinomycin, but also conferred resistance to osmotic stress and ampicillin. In addition, mutation of the purB gene, for which its product is involved in the biosynthesis of inosine and adenosine monophosphates, led to decreased spectinomycin resistance. Addition of exogenous adenine at lower concentration in medium restored the growth deficiency in the purB mutant and increased bacterial resistance to spectinomycin. These results suggest that while cell-wall recycling and purine biosynthesis might contribute to spectinomycin resistance, target rRNAs play critical role in spectinomycin resistance in L. enzymogenes.

Phage Resistance Reduced the Pathogenicity of Xanthomonas oryzae pv. oryzae on Rice.

Plants grow together with microbes that have both negative and positive impacts on the host, while prokaryotes are in turn also hosts for viruses, co-evolving together in a complex interrelationship. Most research focuses on the interaction of either bacterial pathogens interacting with the plant host, or the impact on viruses on their pathogenic bacterial hosts. Few studies have investigated the co-evolution of bacterial pathogens with their host plants as well as with their bacterial viruses. In this work, we aimed to identify the genes that were associated with both phage sensitivity and host pathogenicity of the bacterium Xanthomonas oryzae pv. oryzae (Xoo), which is the most important bacterial rice pathogen. Using the Tn5 transposon mutation technology, we created a library of Xoo strain C2 comprising 4524 mutants, which were subsequently tested for phage infectability. The phage infection tests showed that less than 1% of the mutants (n = 36) were resistant to phage infection, which was attributed to the Tn5 insertion in 19 genes. Interestingly, three out of 19 genes that conveyed resistance to the phage resulted in reduced pathogenicity to rice seedlings compared to the wild type. We identified three genes involved in both phage infection and bacterial virulence, which were studied by knockout mutants and complementation experiments. All of the three knockout mutants were resistant to infection by phage X2, while the complemented strains restored the susceptibility to the bacterial virus. Surprisingly, the genes are also essential for pathogenicity, which we confirmed by single knockout mutants corresponding to the Tn5 mutants. All three genes are involved in lipopolysaccharide synthesis, thus changing the cell envelope surface molecule composition. Our work shows a possible balance in terms of the connection between bacterial virulence and phage resistance, supporting the deployment of phages for the biocontrol of plant pathogens.

Nigella sativa oil extract: A natural novel specific conjugal transfer inhibitor of vancomycin resistance from vanA/B-resistant Enterococcus faecium to Staphylococcus aureus.

The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) has been identified as one of the most challenging problems in healthcare settings worldwide. Specific conjugation inhibitors' development is critical in the fight against the spread of emerging VRSA. The impact of Nigella sativa oil on VR genes conjugal transfer from Enterococcus faecium (VREtfm) to vancomycin-sensitive S. aureus (VSSA) was investigated in this study. Enterococciwere isolated from retail broilers, fish, cows' milk, and human urine. VR E. faecalis and VREtfm VanA phenotypes were prevalent in retail broiler samples. The VREtfm isolates were dominant, exhibiting high levels of resistance to gentamycin and ciprofloxacin antibiotics, as well as the existence of both vanA and vanB genes and virulence traits (ESP+ , asa1+ ) as determined by PCR. Transconjugant VREtfm strains containing vanA/vabB and 20 kb plasmids (transfer frequency around 103 ) and carrying the Tn1546 transposon were identified. Tn1546 transposon transfer with its VR markers to VSSA was effectively inhibited in treated VREtfm donor strains with a sub-minimum inhibitory concentration of N. sativa oil. This work offers new insights for overcoming VR conjugal transfer utilizing natural N. sativa oil, as well as a suggestion for a novel specialized conjugation inhibitor that could effectively facilitate the difficulty of eliminating VR bacteria from healthcare settings.

Stable antibiotic resistance and rapid human adaptation in livestock-associated MRSA.

Mobile genetic elements (MGEs) are agents of horizontal gene transfer in bacteria, but can also be vertically inherited by daughter cells. Establishing the dynamics that led to contemporary patterns of MGEs in bacterial genomes is central to predicting the emergence and evolution of novel and resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex (CC) 398 is the dominant MRSA in European livestock and a growing cause of human infections. Previous studies have identified three categories of MGEs whose presence or absence distinguishes livestock-associated CC398 from a closely related and less antibiotic-resistant human-associated population. Here, we fully characterise the evolutionary dynamics of these MGEs using a collection of 1180 CC398 genomes, sampled from livestock and humans, over 27 years. We find that the emergence of livestock-associated CC398 coincided with the acquisition of a Tn916 transposon carrying a tetracycline resistance gene, which has been stably inherited for 57 years. This was followed by the acquisition of a type V SCCmec that carries methicillin, tetracycline, and heavy metal resistance genes, which has been maintained for 35 years, with occasional truncations and replacements with type IV SCCmec. In contrast, a class of prophages that carry a human immune evasion gene cluster and that are largely absent from livestock-associated CC398 have been repeatedly gained and lost in both human- and livestock-associated CC398. These contrasting dynamics mean that when livestock-associated MRSA is transmitted to humans, adaptation to the human host outpaces loss of antibiotic resistance. In addition, the stable inheritance of resistance-associated MGEs suggests that the impact of ongoing reductions in antibiotic and zinc oxide use in European farms on livestock-associated MRSA will be slow to be realised.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, andfliR), 4 curli fimbriae genes (csgD, csgA, csgF, andcsgG) and 3 type 1 fimbriae genes (fimA, fimD, andfimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, andcsgG) and other functional genes (fimC, glxK, yehE, 07045, and11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.