The mycobacterial mutasome: composition and recruitment in live cells

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome', minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins, remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Cas9-Assisted Biological Containment of a Genetically Engineered Human Commensal Bacterium and Genetic Elements

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Biosensing bacteria can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination for at least 10 days. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

Molecular Characteristics and Antimicrobial Resistance of Salmonella enterica Serovar Schwarzengrund from Chicken Meat in Japan

Our previous study revealed that Salmonella enterica serovar Schwarzengrund-contaminated areas of broiler chickens have expanded from West Japan to East Japan. The present study investigated the antimicrobial resistance and molecular characteristics of 124 S. Schwarzengrund isolates obtained from chicken meat produced in East and West Japan from 2008 to 2019. Comparing the isolates obtained in 2008 and 2015–2019, an increase in the proportion of those resistant to kanamycin [51.4–89.7% (p < 0.001)] was observed. In contrast, the proportion of isolates resistant to both streptomycin and tetracycline and those that harbored a 1.0-kb class 1 integron, aadA1, and tetA, significantly decreased from 100% in 2008 to 47.1% in 2015–2019 (p < 0.001). A 1.0-kb class 1 integron containing aadA1, harbored by 78 isolates, was different from that reported in globally distributed S. Schwarzengrund strains (1.9 kb, containing the dfrA12-aadA2 gene cassette). Twenty-five isolates from different product districts and years of isolation were typed as sequence type (ST) 241 with multilocus sequence typing. Our results suggest that S. Schwarzengrund, which contaminates chicken meat in Japan, shares a common ancestor regardless of the product district from 2008 to recent years. Moreover, S. Schwarzengrund ST241 may have spread from western to eastern Japan.

Carriage of antibiotic resistant bacteria in endangered and declining Australian pinniped pups

The rapid emergence of antimicrobial resistance (AMR) is a major concern for wildlife and ecosystem health globally. Genetic determinants of AMR have become indicators of anthropogenic pollution due to their greater association with humans and rarer presence in environments less affected by humans. The objective of this study was to determine the distribution and frequency of the class 1 integron, a genetic determinant of AMR, in both the faecal microbiome and in Escherichia coli isolated from neonates of three pinniped species. Australian sea lion ( Neophoca cinerea ), Australian fur seal ( Arctocephalus pusillus doriferus ) and long-nosed fur seal ( Arctocephalus forsteri ) pups from eight breeding colonies along the Southern Australian coast were sampled between 2016-2019. DNA from faecal samples ( n =309) and from E. coli ( n =795) isolated from 884 faecal samples were analysed for class 1 integrons using PCRs targeting the conserved integrase gene ( intI ) and the gene cassette array. Class 1 integrons were detected in A. p. doriferus and N. cinerea pups sampled at seven of the eight breeding colonies investigated in 4.85% of faecal samples ( n =15) and 4.52% of E. coli isolates ( n =36). Integrons were not detected in any A. forsteri samples. DNA sequencing of the class 1 integron gene cassette array identified diverse genes conferring resistance to four antibiotic classes. The relationship between class 1 integron carriage and the concentration of five trace elements and heavy metals was also investigated, finding no significant association. The results of this study add to the growing evidence of the extent to which antimicrobial resistant bacteria are polluting the marine environment. As AMR determinants are frequently associated with bacterial pathogens, their occurrence suggests that these pinniped species are vulnerable to potential health risks. The implications for individual and population health as a consequence of AMR carriage is a critical component of ongoing health investigations.

Genetic Background of Antimicrobial Resistance in Multiantimicrobial-Resistant Escherichia coli Isolates from Feces of Healthy Broiler Chickens in Tunisia

Multiantimicrobial-resistant Escherichia coli isolates are a global human health problem causing increasing morbidity and mortality. Genes encoding antimicrobial resistance are mainly harbored on mobile genetic elements (MGEs) such as transposons and plasmids as well as integrons, which enhance their rapid spread. The aim of this study was to characterize 83 multiantimicrobial-resistant E. coli isolates recovered from healthy broiler chickens. Among 78 tetracycline-resistant isolates, the tetA, tetB, and tetC genes were detected in 59 (75.6%), 14 (17.9%), and one (1.2%) isolates, respectively. The sul1, sul2, and sul3 genes were detected 31 (46.2%), 16 (23.8%), and 6 (8.9%) isolates, respectively, among 67 sulfonamide-resistant isolates. The PCR-based replicon typing method showed plasmids in 29 isolates, IncFIB (19), IncI1-Iγ (17), IncF (14), IncK (14), IncFIC (10), IncP (8), IncY (3), IncHI2 (1), and IncX (1). The class 1 and 2 integrons were detected in 57 and 2 isolates, respectively; one isolate harbored both integrons. Seven and one gene cassette arrays were identified in class 1 and class 2 integrons, respectively. Our findings show that multiantimicrobial-resistant E. coli isolates from chickens serve as reservoirs of highly diverse and abundant tet and sul genes and plasmid replicons. Such isolates and MGEs pose a potential health threat to the public and animal farming.

A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production.

The Psilocybe genus is well known for the synthesis of valuable psychoactive compounds such as Psilocybin, Psilocin, Baeocystin and Aeruginascin. The ubiquity of Psilocybin synthesis in Psilocybe has been attributed to a horizontal gene transfer mechanism of a ~20Kb gene cassette. A recently published highly contiguous reference genome derived from long read single molecule sequencing has underscored interesting variation in this Psilocybin synthesis gene cassette. This reference genome has also enabled the shotgun sequencing of spores from many Psilocybe strains to better catalog the genomic diversity in the Psilocybin synthesis pathway. Here we present the de novo assembly of genomes of 81 Psilocybe genomes compared to the P.envy reference genome. Surprisingly, the genomes of Psilocybe galindoi, Psilocybe tampanensis and Psilocybe azurescens lack sequence coverage over the previously described Psilocybin synthesis pathway but do demonstrate amino acid sequence homology to an alternative pathway and may illuminate previously proposed convergent evolution of Psilocybin synthesis.

Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities

Integrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the use of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

Global distribution of anaerobic dichloromethane degradation potential

Anthropogenic activities and natural processes release dichloromethane (DCM), a toxic chemical with substantial ozone-depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial species revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising eight to ten genes with predicted 79.6 – 99.7% amino acid identity. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system, and shotgun proteomics applied to two DCM-catabolizing cultures revealed high expression of proteins encoded on the mec gene cluster during anaerobic growth with DCM. In a DCM-contaminated groundwater plume, the abundance of mec genes strongly correlated with DCM concentrations (R2 = 0.71 – 0.85) indicating their value as process-specific bioremediation biomarkers. mec gene clusters were identified in metagenomes representing peat bogs, the deep subsurface, and marine ecosystems including oxygen minimum zones (OMZs), suggesting DCM turnover in diverse habitats. The broad distribution of anaerobic DCM catabolic potential suggests a relevant control function for emissions to the atmosphere, and a role for DCM as a microbial energy source in critical zone environments. The findings imply that the global DCM flux might be far greater than emission measurements suggest.ImportanceDichloromethane (DCM) is an increasing threat to stratospheric ozone with both anthropogenic and natural emission sources. Anaerobic bacterial metabolism of DCM has not yet been taken into consideration as a factor in the global DCM cycle. The discovery of the mec gene cassette associated with anaerobic bacterial DCM metabolism and its widespread distribution in environmental systems highlight a strong attenuation potential for DCM. Knowledge of the mec cassette offers new opportunities to delineate DCM sources, enables more robust estimates of DCM fluxes, supports refined DCM emission modeling and simulation of the stratospheric ozone layer, reveals a novel, ubiquitous C1 carbon metabolic system, and provides prognostic and diagnostic tools supporting bioremediation of groundwater aquifers impacted by DCM.

Metabolic Engineering of Pediococcus acidilactici BD16 for Heterologous Expression of Synthetic alaD Gene Cassette and L-Alanine Production in the Recombinant Strain Using Fed-Batch Fermentation

Metabolic engineering substantially aims at the development of more efficient, robust and industrially competitive microbial strains for the potential applications in food, fermentation and pharmaceutical industries. An efficient lab scale bioprocess was developed for high level fermentative production of L-alanine using metabolically engineered Pediococcus acidilactici BD16 (alaD+). Computational biology tools assisted the designing of a synthetic alaD gene cassette, which was further cloned in shuttle vector pLES003 and expressed using an auto-inducible P289 promoter. Further, L-alanine production in the recombinant P. acidilactici BD16 (alaD+) strain was carried out using fed-batch fermentation under oxygen depression conditions, which significantly enhanced L-alanine levels. The recombinant strain expressing the synthetic alaD gene produced 229.12 g/L of L-alanine after 42 h of fed-batch fermentation, which is the second highest microbial L-alanine titer reported so far. After extraction and crystallization, 95% crystal L-alanine (217.54 g/L) was recovered from the culture broth with an enantiomeric purity of 97%. The developed bioprocess using recombinant P. acidilactici BD16 (alaD+) is suggested as the best alternative to chemical-based commercial synthesis of L-alanine for potential industrial applications.