Type I BREX system defends against antibiotic-resistant plasmids in Escherichia coli.

The Bacteriophage Exclusion (BREX) system is a novel antiphage defense system identified in Bacillus cereus in 2015. The purpose of this study was to investigate the presence of the BREX system defenses against antibiotic-resistant plasmids such as blaKPC and blaNDM invasion in Escherichia coli. The BREX system was present in 5.4% (23/424) of E. coli clinical isolates and 6.5% (84/1283) of E. coli strains with completely sequenced genomes in the GenBank database. All 23 BREX-positive E. coli clinical isolates were susceptible to carbapenems, while all five isolates carrying blaKPC and 11 carrying blaNDM were BREX-negative. For E. coli strains in the GenBank database, 37 of 38 strains carrying blaKPC and 109 of 111 strains carrying blaNDM were BREX negative. The recognition site sequence of methyltransferase PglX in a clinical E. coli 3756 was 5'-CANCATC-3' using PacBio single-molecular real-time sequencing. The transformation efficiency of plasmid psgRNA-ColAori-target with the PglX recognition site was reduced by 100% compared with the plasmid without the recognition site in E. coli DH5α-pHSG398-BREX. The BREX showed lower defense efficacy against plasmid psgRNA-15Aori-target which had the same plasmid backbone but different surrounding sequences of recognition sites with psgRNA-ColAori-target. The conjugation frequency of the KPC-2 plasmid and NDM-5 plasmid in E. coli 3756-ΔBREX was higher than that in E. coli 3756 clinical isolate (1.0 × 10-6 vs 1.3 × 10-7 and 5.5 × 10-7 vs 1.7 × 10-8, respectively). This study demonstrated that the type I BREX system defends against antibiotic-resistant plasmids in E. coli.

Mutations in the C-terminal region of the bacteriophage exclusion protein PglX can selectively inactivate restriction in Salmonella.

Salmonella enterica serovar Typhimurium strain LT2 is protected by two DNA restriction-modification systems (HsdRMS and Mod-Res) and a Type I bacteriophage exclusion (BREX) system (BrxA-L). The LB5000 strain was constructed to inactivate restriction but not methylation in all three systems and has been available for decades (L. R. Bullas and J. I. Ryu, J Bacteriol 156:471-474, 1983, https://doi.org/10.1128/jb.156.1.471-474.1983). However, this strain had been heavily mutagenized and contains hundreds of other mutations, including a few in DNA repair genes. Here, we describe the development of a strain that is only mutated for DNA restriction by the three systems and remains competent for DNA modification. We transferred mutations specific to DNA restriction from LB5000 to a wild-type LT2 background. The hsdR and res mutations affected only restriction in the wild-type background, but the brxC and pglZ mutations for the poorly understood BREX system also reduced modification. Amino acids in an unannotated conserved region of PglX in the BREX system were then randomized. Mutations were identified that specifically affected restriction at 37°C but were found to be temperature sensitive for restriction and methylation when tested at 30°C and 42°C. These mutations in PglX are consistent with a domain that communicates DNA methylation information to other BREX effector proteins. Finally, mutations generated in the specificity domain of PglX may have changed the DNA binding site recognized by the BREX system. IMPORTANCE The restriction system mutants constructed in this study will be useful for cloning DNA and transferring plasmids from other bacterial species into Salmonella. We verified which mutations in strain LB5000 resulted in loss of restriction for each restriction-modification system and the BREX system by moving these mutations to a wild-type Salmonella background. The methylase PglX was then mutagenized, which adds to our knowledge of the BREX system that is found in many bacteria but is not well understood. These PglX mutations affected restriction and methylation at different temperatures, which suggests that the C-terminal region of PglX may coordinate interactions between the methylase and other BREX system proteins.

Diverse Durham collection phages demonstrate complex BREX defense responses.

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.

Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase.

Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows T3 to evade the BREX defense. Although SAM depletion weakly affects BREX methylation, it completely inhibits the defensive function of BREX, suggesting that SAM could be a co-factor for BREX-mediated exclusion of phage DNA, similar to its anti-defense role in type I R-M. The anti-BREX activity of T3 SAMase is mediated not just by enzymatic degradation of SAM but also by direct inhibition of MetK, the host SAM synthase. We present a 2.8 Å cryoelectron microscopy (cryo-EM) structure of the eight-subunit T3 SAMase-MetK complex. Structure-guided mutagenesis reveals that this interaction stabilizes T3 SAMase in vivo, further stimulating its anti-BREX activity. This work provides insights in the versatility of bacteriophage counterdefense mechanisms and highlights the role of SAM as a co-factor of diverse bacterial immunity systems.

CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae.

Genomic integration is the preferred method for gene expression in microbial industrial production. However, traditional homologous recombination based multiplexed integration methods often suffer from low integration efficiency and complex experimental procedures. Here, we report a CRISPR/Cas9 based multiplexed integration (CMI) system in Saccharomyces cerevisiae, which can achieve quadruple integration at an individual locus without pre-engineering the host. A fused protein, Cas9-Brex27, was used as a bait to attract Rad51 recombinase to the proximity of the double-strand breaks introduced by the CRISPR/Cas9 system. The efficiency of quadruple integration was increased to 53.9% with 40 bp homology arms (HAs) and 78% with 100 bp HAs. CMI was applied to integrate a heterologous mogrol biosynthetic pathway consisting of four genes in a one-step transformation and offered an efficient solution for multiplexed integration. This method expands the synthetic biology toolbox of S. cerevisiae.

Analysis of pCl107 a large plasmid carried by an ST25 Acinetobacter baumannii strain reveals a complex evolutionary history and links to multiple antibiotic resistance and metabolic pathways.

Acinetobacter baumannii has successfully spread during the last decades as one of the main critically important pathogens. However, many aspects including plasmids, are still under-investigated. Here, we report the complete sequence of an Acinetobacter baumannii strain, belonging to the ST25IP (Institut Pasteur) sequence type recovered in 2012 in Lebanon, using a combination of Illumina MiSeq and Oxford Nanopore sequencing and a hybrid assembly approach. This strain (Cl107) carries a 198 kb plasmid called pCl107 that encodes the MPFI conjugative transfer system. The plasmid carries the aacA1, aacC2, sul2, strAB, and tetA(B) antibiotic resistance genes. pCl107 region encompassing the sul2, strAB, tetA(B) is closely related to AbGRI1 chromosomal resistance islands, which are widespread in A. baumannii strains belonging to Global Clone 2. The resistance region found in pCl107 is one of the missing links in the evolutionary history of the AbGRI1 islands. pCl107 also contains a BREX Type 1 region and represents one of the two main evolution patterns observed in BREX clusters found in plasmids related to pCl107. pCl107 also harbours a ptx phosphonate metabolism module, which plays an ancestral structure compared to other large plasmids in ST25 strains. While the uric acid metabolic module found in pCl107 is incomplete, we identified possible ancestors from plasmids and chromosomes of Acinetobacter spp. Our analyses indicate a complex evolutionary history of plasmids related to pCl107 with many links to multiple antibiotic resistance and metabolic pathways.

Acinetobacter defence mechanisms against biological aggressors and their use as alternative therapeutic applications.

Several Acinetobacter strains are important nosocomial pathogens, with Acinetobacter baumannii being the species of greatest worldwide concern due to its multi-drug resistance and the recent appearance of hyper-virulent strains in the clinical setting. Colonisation of this environment is associated with a multitude of bacterial factors, and the molecular features that promote environmental persistence in abiotic surfaces, including intrinsic desiccation resistance, biofilm formation and motility, have been previously addressed. On the contrary, mechanisms enabling Acinetobacter spp. survival when faced against other biological competitors are starting to be characterised. Among them, secretion systems (SS) of different types, such as the T5bSS (Contact-dependent inhibition systems) and the T6SS, confer adaptive advantages against bacterial aggressors. Regarding mechanisms of defence against bacteriophages, such as toxin-antitoxin, restriction-modification, Crispr-Cas and CBASS, among others, have been identified but remain poorly characterised. In view of this, we aimed to summarise the present knowledge on defence mechanisms that enable niche establishment in members of the Acinetobacter genus. Different proposals are also described for the use of some components of these systems as molecular tools to treat Acinetobacter infections.

Microbial Arsenal of Antiviral Defenses - Part I.

Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). Constant threat of phage infection is a major force that shapes evolution of the microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering have been deeply intertwined with the study of phages and host defenses. Nowadays, owing to the rise of phage therapy, broad application of CRISPR-Cas technologies, and development of bioinformatics approaches that facilitate discovery of new systems, phage biology experiences a revival. This review describes variety of strategies employed by microbes to counter phage infection, with a focus on novel systems discovered in recent years. First chapter covers defense associated with cell surface, role of small molecules, and innate immunity systems relying on DNA modification.

Hybrid genome de novo assembly with methylome analysis of the anaerobic thermophilic subsurface bacterium Thermanaerosceptrum fracticalcis strain DRI-13T.

BACKGROUND: There is a dearth of sequenced and closed microbial genomes from environments that exceed > 500 m below level terrestrial surface. Coupled with even fewer cultured isolates, study and understanding of how life endures in the extreme oligotrophic subsurface environments is greatly hindered. Using a de novo hybrid assembly of Illumina and Oxford Nanopore sequences we produced a circular genome with corresponding methylome profile of the recently characterized thermophilic, anaerobic, and fumarate-respiring subsurface bacterium, Thermanaerosceptrum fracticalcis, strain DRI-13T to understand how this microorganism survives the deep subsurface. RESULTS: The hybrid assembly produced a single circular genome of 3.8 Mb in length with an overall GC content of 45%. Out of the total 4022 annotated genes, 3884 are protein coding, 87 are RNA encoding genes, and the remaining 51 genes were associated with regulatory features of the genome including riboswitches and T-box leader sequences. Approximately 24% of the protein coding genes were hypothetical. Analysis of strain DRI-13T genome revealed: 1) energy conservation by bifurcation hydrogenase when growing on fumarate, 2) four novel bacterial prophages, 3) methylation profile including 76.4% N6-methyladenine and 3.81% 5-methylcytosine corresponding to novel DNA methyltransferase motifs. As well a cluster of 45 genes of unknown protein families that have enriched DNA mCpG proximal to the transcription start sites, and 4) discovery of a putative core of bacteriophage exclusion (BREX) genes surrounded by hypothetical proteins, with predicted functions as helicases, nucleases, and exonucleases. CONCLUSIONS: The de novo hybrid assembly of strain DRI-13T genome has provided a more contiguous and accurate view of the subsurface bacterium T. fracticalcis, strain DRI-13T. This genome analysis reveals a physiological focus supporting syntrophy, non-homologous double stranded DNA repair, mobility/adherence/chemotaxis, unique methylome profile/recognized motifs, and a BREX defense system. The key to microbial subsurface survival may not rest on genetic diversity, but rather through specific syntrophy niches and novel methylation strategies.

A Novel Bacteriophage Exclusion (BREX) System Encoded by the pglX Gene in Lactobacillus casei Zhang.

The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N6-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (ΔpglX) was constructed. Interestingly, m6A methylation of the 5'-ACRCAG-3' motif was eliminated in the ΔpglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ΔpglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5'-ACRCAG-3'). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ΔpglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system.IMPORTANCELactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria.

Viral Communities of Shark Bay Modern Stromatolites.

Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients.

Recycling of ferromanganese gas cleaning plant (GCP) sludge by novel agglomeration.

Ferroalloy industries rely on minerals which are not available in pure form. The total value chain is very cost intensive and market driven. From mineral and environment conservation point of view every possible aspect must be explored for the economic utilisation of waste and low-grade ores. Unlike other metallurgical processes, Ferro alloys production generates a wide variety of waste rich in manganese/chrome which has a potential for recycling back. Fine size, high moisture content and presence of alkalis categorise this material into hazardous waste and economically non-viable. The challenge is to convert such anthropogenic material into a suitable feedstock for the furnace. In this article, the results of smelting trials carried out in electric arc furnace (EAF) using novel extruded briquettes (BREX) produced from the wastes of ferroalloy plant is discussed. The briquette produced by this technique exhibit high physical and metallurgical property and can replace the natural ore as a charge to some extent. Extruded briquettes (BREX) can be efficiently used as one of the essential charge component (up to 30% of the ore part of the charge) for the Silicomanganese smelting thus improving technical and economical parameters of the furnace and decreasing the self-cost of the Silicomanganese production.

BacteRiophage EXclusion (BREX): A novel anti-phage mechanism in the arsenal of bacterial defense system.

The arm race between bacteria and phages leads to the development of many phage resistance mechanisms. Bacteria are perpetually changing their strategy to avert phage infection and killing. Recently, a study published that shed light on a novel bacterial defense system called bacteriophage exclusion (BREX). BREX system is a six-gene cassette in Bacillus cereus which provides complete phage resistance to a broad range of phages, including lytic and temperate ones. It is a novel defense strategy which involves DNA methylation of the host cell and block phage DNA replication.