Portable, On-Demand Biomolecular Manufacturing.

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.

Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long-read sequencing.

Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, but genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. Here, we introduce nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin. We took advantage of compound heterozygous COL7A1 mutations in a patient with recessive dystrophic epidermolysis bullosa who showed revertant skin spots. Cas9-mediated enrichment of genomic DNA (gDNA) covering the two mutation sites (>8 kb) in COL7A1 and subsequent MinION sequencing successfully detected intragenic crossover in the epidermis of the clinically revertant skin. This method enables the discernment of haplotypes of up to a few tens of kilobases of gDNA. Moreover, it is devoid of polymerase chain reaction amplification, which can technically induce recombination. We, therefore, propose that nCATS is a powerful tool for understanding complicated gene modifications, including RM.

Cas9-guided haplotyping of three truncation variants in autosomal recessive disease.

An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5' and 3' variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5' and 3' variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.

Identification, expression, and purification of DNA cytosine 5-methyltransferases with short recognition sequences.

BACKGROUND: DNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences. RESULTS: We identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising. CONCLUSIONS: In the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously.

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines.

The role of lymphocytes as a cornerstone of the inflammatory response in the invasive pathogenesis of Chlamydia trachomatis (Ct) LGV (L1-3) infection is unclear. Therefore, we assessed whether the adaptation of CtL2 to immortal lymphoid Jurkat cells under hypoxic conditions occurred through proinflammatory cytokine profile modification. The quantities of inclusion-forming units with chlamydial 16S rDNA confirmed that CtL2 grew well under hypoxic rather than normoxic conditions in the cells. Confocal microscopic imaging and transmission electron microscopy revealed the presence of bacterial progeny in the inclusions and showed that the inclusions were larger under hypoxic rather than normoxic conditions; this was supported by the results of 3D image construction. Furthermore, PCR-based analysis of proinflammatory cytokines revealed that the gene expression levels under hypoxic conditions were significantly higher than those under normoxic conditions. In particular, the expression of two genes (CXCL8 and CXCR3) was significantly diminished under normoxic conditions. Taken together, the results indicated that hypoxia promoted CtL2 growth in Jurkat cells while maintaining the levels of proinflammatory cytokines. Thus, Ct LGV infection in lymphocytes under hypoxic conditions might be crucial to a complete understanding of the invasive pathogenesis.

Distribution of amoebal endosymbiotic environmental chlamydia Neochlamydia S13 via amoebal cytokinesis.

We previously isolated a symbiotic environmental amoeba, harboring an environmental chlamydia, Neochlamydia S13. Interestingly, this bacterium failed to survive outside of host cells and was immediately digested inside other amoebae, indicating bacterial distribution via cytokinesis. This may provide a model for understanding organelle development and chlamydial pathogenesis and evolution; therefore, we assessed our hypothesis of Neochlamydia S13 distribution via cytokinesis by comparative analysis with other environmental Chlamydiae (Protochlamydia R18 and Parachlamydia Bn9 ). Dual staining with 4',6-diamidino-2-phenylindole and phalloidin revealed that the progeny of Neochlamydia S13 and Protochlamydia R18 existed in both daughter cells with a contractile ring on the verge of separation. However, in contrast to other environmental Chlamydiae, little Neochlamydia S13 16S ribosomal DNA was amplified from the culture supernatant. Interestingly, Neochlamydia S13 failed to infect aposymbiotic amoebae, indicating an intimate interaction with the host cells. Furthermore, its infectious rates in cultures expanded from a single amoeba were always maintained at 100%, indicating distribution via cytokinesis. We concluded that unlike other environmental Chlamydiae, Neochlamydia S13 has a unique ability to divide its progeny only via host amoebal cytokinesis. This may be a suitable model to elucidate the mechanism of cell organelle distribution and of chlamydial pathogenesis and evolution.

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality.

Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROS-dependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.

Novel Sequence Type in Bacillus cereus Strains Associated with Nosocomial Infections and Bacteremia, Japan.

Bacillus cereus is associated with foodborne illnesses characterized by vomiting and diarrhea. Although some B. cereus strains that cause severe extraintestinal infections and nosocomial infections are recognized as serious public health threats in healthcare settings, the genetic backgrounds of B. cereus strains causing such infections remain unknown. By conducting pulsed-field gel electrophoresis and multilocus sequence typing, we found that a novel sequence type (ST), newly registered as ST1420, was the dominant ST isolated from the cases of nosocomial infections that occurred in 3 locations in Japan in 2006, 2013, and 2016. Phylogenetic analysis showed that ST1420 strains belonged to the Cereus III lineage, which is much closer to the Anthracis lineage than to other Cereus lineages. Our results suggest that ST1420 is a prevalent ST in B. cereus strains that have caused recent nosocomial infections in Japan.

Restriction glycosylases: involvement of endonuclease activities in the restriction process.

All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction­modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

Population Evolution of Helicobacter pylori through Diversification in DNA Methylation and Interstrain Sequence Homogenization.

Decoding of closely related genomes is now revealing the process of population evolution. In bacteria, population divergence appears associated with a unique set of sequence-specific epigenetic DNA methylation systems, often within restriction-modification (RM) systems. They might define a unique gene expression pattern and limit genetic flux between lineages in population divergence. We addressed the contribution of methylation systems to population diversification in panmictic bacterial species, Helicobacter pylori, which shows an interconnected population structure through frequent mutual recombination. We analyzed complete genome sequences of 28 strains collected in Fukui, Japan. Their nucleotide sequences are closely related although fine-scale analyses revealed two subgroups likely reflecting human subpopulations. Their sequences are tightly connected by homologous recombination. Our extensive analysis of RM systems revealed an extreme variability in DNA methyltransferases, especially in their target recognition domains. Their diversity was, however, not immediately related to the genome sequence diversity, except for very closely related strains. An interesting exception is a hybrid strain, which likely has conserved the methylation gene repertoire from one parent but diversified in sequence by massive acquisition of fragmentary DNA sequences from the other parent. Our results demonstrate how a bacterial population can be extremely divergent in epigenetics and yet homogenized in sequence.

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

Methylome diversification through changes in DNA methyltransferase sequence specificity.

Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement--the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.

Chromosome painting in silico in a bacterial species reveals fine population structure.

Identifying population structure forms an important basis for genetic and evolutionary studies. Most current methods to identify population structure have limitations in analyzing haplotypes and recombination across the genome. Recently, a method of chromosome painting in silico has been developed to overcome these shortcomings and has been applied to multiple human genome sequences. This method detects the genome-wide transfer of DNA sequence chunks through homologous recombination. Here, we apply it to the frequently recombining bacterial species Helicobacter pylori that has infected Homo sapiens since their birth in Africa and shows wide phylogeographic divergence. Multiple complete genome sequences were analyzed including sequences from Okinawa, Japan, that we recently sequenced. The newer method revealed a finer population structure than revealed by a previous method that examines only MLST housekeeping genes or a phylogenetic network analysis of the core genome. Novel subgroups were found in Europe, Amerind, and East Asia groups. Examination of genetic flux showed some singleton strains to be hybrids of subgroups and revealed evident signs of population admixture in Africa, Europe, and parts of Asia. We expect this approach to further our understanding of intraspecific bacterial evolution by revealing population structure at a finer scale.

Movement of DNA sequence recognition domains between non-orthologous proteins.

Comparisons of proteins show that they evolve through the movement of domains. However, in many cases, the underlying mechanisms remain unclear. Here, we observed the movements of DNA recognition domains between non-orthologous proteins within a prokaryote genome. Restriction-modification (RM) systems, consisting of a sequence-specific DNA methyltransferase and a restriction enzyme, contribute to maintenance/evolution of genomes/epigenomes. RM systems limit horizontal gene transfer but are themselves mobile. We compared Type III RM systems in Helicobacter pylori genomes and found that target recognition domain (TRD) sequences are mobile, moving between different orthologous groups that occupy unique chromosomal locations. Sequence comparisons suggested that a likely underlying mechanism is movement through homologous recombination of similar DNA sequences that encode amino acid sequence motifs that are conserved among Type III DNA methyltransferases. Consistent with this movement, incongruence was observed between the phylogenetic trees of TRD regions and other regions in proteins. Horizontal acquisition of diverse TRD sequences was suggested by detection of homologs in other Helicobacter species and distantly related bacterial species. One of these RM systems in H. pylori was inactivated by insertion of another RM system that likely transferred from an oral bacterium. TRD movement represents a novel route for diversification of DNA-interacting proteins.

Birth and death of genes linked to chromosomal inversion.

The birth and death of genes is central to adaptive evolution, yet the underlying genome dynamics remain elusive. The availability of closely related complete genome sequences helps to follow changes in gene contents and clarify their relationship to overall genome organization. Helicobacter pylori, bacteria in our stomach, are known for their extreme genome plasticity through mutation and recombination and will make a good target for such an analysis. In comparing their complete genome sequences, we found that gain and loss of genes (loci) for outer membrane proteins, which mediate host interaction, occurred at breakpoints of chromosomal inversions. Sequence comparison there revealed a unique mechanism of DNA duplication: DNA duplication associated with inversion. In this process, a DNA segment at one chromosomal locus is copied and inserted, in an inverted orientation, into a distant locus on the same chromosome, while the entire region between these two loci is also inverted. Recognition of this and three more inversion modes, which occur through reciprocal recombination between long or short sequence similarity or adjacent to a mobile element, allowed reconstruction of synteny evolution through inversion events in this species. These results will guide the interpretation of extensive DNA sequencing results for understanding long- and short-term genome evolution in various organisms and in cancer cells.

Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.

Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.

Large variations in bacterial ribosomal RNA genes.

Ribosomal RNA (rRNA) genes, essential to all forms of life, have been viewed as highly conserved and evolutionarily stable, partly because very little is known about their natural variations. Here, we explored large-scale variations of rRNA genes through bioinformatic analyses of available complete bacterial genomic sequences with an emphasis on formation mechanisms and biological significance. Interestingly, we found bacterial genomes in which no 16S rRNA genes harbor the conserved core of the anti-Shine-Dalgarno sequence (5'-CCTCC-3'). This loss was accompanied by elimination of Shine-Dalgarno-like sequences upstream of their protein-coding genes. Those genomes belong to 1 or 2 of the following categories: primary symbionts, hemotropic Mycoplasma, and Flavobacteria. We also found many rearranged rRNA genes and reconstructed their history. Conjecturing the underlying mechanisms, such as inversion, partial duplication, transposon insertion, deletion, and substitution, we were able to infer their biological significance, such as co-orientation of rRNA transcription and chromosomal replication, lateral transfer of rRNA gene segments, and spread of rRNA genes with an apparent structural defect through gene conversion. These results open the way to understanding dynamic evolutionary changes of rRNA genes and the translational machinery.

Detection of DNA Modification Using Nanopore Sequencers.

DNA modification is a crucial factor of epigenetic modification and has vital functions for gene regulation and phenotype control. A profound understanding of DNA modification requires precise mapping of the modified bases on genomic DNA. In addition to methods such as bisulfite sequencing and single-molecule real-time (SMRT) sequencing of PacBio sequencers, nanopore sequencers can be also utilized for the detection of DNA modification. Here, I will briefly review the three methods for the detection of DNA modification with nanopore sequencers and introduce a protocol using MinION and Megalodon.

Genome comparison and context analysis reveals putative mobile forms of restriction-modification systems and related rearrangements.

The mobility of restriction-modification (RM) gene complexes and their association with genome rearrangements is a subject of active investigation. Here we conducted systematic genome comparisons and genome context analysis on fully sequenced prokaryotic genomes to detect RM-linked genome rearrangements. RM genes were frequently found to be linked to mobility-related genes such as integrase and transposase homologs. They were flanked by direct and inverted repeats at a significantly high frequency. Insertion by long target duplication was observed for I, II, III and IV restriction types. We found several RM genes flanked by long inverted repeats, some of which had apparently inserted into a genome with a short target duplication. In some cases, only a portion of an apparently complete RM system was flanked by inverted repeats. We also found a unit composed of RM genes and an integrase homolog that integrated into a tRNA gene. An allelic substitution of a Type III system with a linked Type I and IV system pair, and allelic diversity in the putative target recognition domain of Type IIG systems were observed. This study revealed the possible mobility of all types of RM systems, and the diversity in their mobility-related organization.