Genetically stable kill-switch using "demon and angel" expression construct of essential genes.

Genetic instability of synthetic genetic devices is a key obstacle for practical use. This problem is particularly critical in kill-switches for conditional host killing. Here, we propose a genetically stable kill-switch based on a "demon and angel" expression construct of a toxic essential gene. The kill-switch conditionally overexpresses the toxic essential gene. Additionally, the identical essential gene is deleted in the genome. The essential gene is expressed at a low level to maintain host survival in the OFF state and kills the host by the overexpression in the ON state. The single expression construct is responsible for both killing the hosts and maintaining viability, reducing the emergence of loss-of-function mutants. We constructed the kill-switch using the toxic essential gene encoding tyrosyl-tRNA synthetase, tyrS, in Escherichia coli. The bacteria harboring the kill-switch were conditionally suicidal over 300 generations. Toxic overexpression of essential genes has also been found in other organisms, suggesting that the "demon and angel" kill switch is scalable to various organisms.

Genetic mutation in Escherichia coli genome during adaptation to the murine intestine is optimized for the host diet.

Mammalian gut microbes colonize the intestinal tract of their host and adapt to establish a microbial ecosystem. The host diet changes the nutrient profile of the intestine and has a high impact on microbiota composition. Genetic mutations in Escherichia coli, a prevalent species in the human gut, allow for adaptation to the mammalian intestine, as reported in previous studies. However, the extent of colonization fitness in the intestine elevated by genetic mutation and the effects of diet change on these mutations in E. coli are still poorly known. Here, we show that notable mutations in sugar metabolism-related genes (gatC, araC, and malI) were detected in the E. coli K-12 genome just 2 weeks after colonization in the germ-free mouse intestine. In addition to elevated fitness by deletion of gatC, as previously reported, deletion of araC and malI also elevated E. coli fitness in the murine intestine in a host diet-dependent manner. In vitro cultures of medium containing nutrients abundant in the intestine (e.g., galactose, N-acetylglucosamine, and asparagine) also showed increased E. coli fitness after deletion of the genes-of-interest associated with their metabolism. Furthermore, the host diet was found to influence the developmental trajectory of gene mutations in E. coli. Taken together, we suggest that genetic mutations in E. coli are selected in response to the intestinal environment, which facilitates efficient utilization of nutrients abundant in the intestine under laboratory conditions. Our study offers some insight into the possible adaptation mechanisms of gut microbes.IMPORTANCEThe gut microbiota is closely associated with human health and is greatly impacted by the host diet. Bacteria such as Escherichia coli live in the gut all throughout the life of a human host and adapt to the intestinal environment. Adaptive mutations in E. coli are reported to enhance fitness in the mammalian intestine, but to what extent is still poorly known. It is also unknown whether the host diet affects what genes are mutated and to what extent fitness is affected. This study suggests that genetic mutations in the E. coli K-12 strain are selected in response to the intestinal environment and facilitate efficient utilization of abundant nutrients in the germ-free mouse intestine. Our study provides a better understanding of these intestinal adaptation mechanisms of gut microbes.

Functional analysis of Rickettsia monacensis strain humboldt folA dihydrofolate reductase gene via complementation assay.

Nutritive symbiosis between bacteria and ticks is observed across a range of ecological contexts; however, little characterization on the molecular components responsible for this symbiosis has been done. Previous studies in our lab demonstrated that Rickettsia monacensis str. Humboldt (strain Humboldt) can synthesize folate de novo via the folate biosynthesis pathway involving folA, folC, folE, folKP, and ptpS genes. In this study, expression of the strain Humboldt folA gene within a folA mutant Escherichia coli construct was used to functionally characterize the strain Humboldt folA folate gene in vivo. The strain Humboldt folA folate gene was subcloned into a TransBac vector and transformed into a folA mutant E. coli construct. The mutant containing strain Humboldt folA subclone and a pFE604 clone of the knocked-out folA gene was cured of pFE604. Curing of the folA mutant E. coli construct was successful using acridine orange and 43.5 °C incubation temperature. The plasmid curing assay showed curing efficiency of the folA mutant at 100%. Functional complementation was assessed by growth phenotype on minimal media with and without IPTG between strain Humboldt folA and E. coli folA. Large and homogenous wild-type colony growth was observed for both strain Humboldt and E. coli folA on minimal media with 0.1 mM IPTG, wild-type growth for strain Humboldt folA and pin-point growth for E. coli folA on 0.01 mM IPTG, and pin-point growth without IPTG for both strain Humboldt and E. coli folA. This study provides evidence substantiating the in vivo functionality of strain Humboldt folA in producing functional gene products for folate biosynthesis.

Past, Present, and Future of Genome Modification in Escherichia coli.

Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design.

Bacterial persisters are a stochastically formed subpopulation of low-energy cells.

Persisters represent a small subpopulation of non- or slow-growing bacterial cells that are tolerant to killing by antibiotics. Despite their prominent role in the recalcitrance of chronic infections to antibiotic therapy, the mechanism of their formation has remained elusive. We show that sorted cells of Escherichia coli with low levels of energy-generating enzymes are better able to survive antibiotic killing. Using microfluidics time-lapse microscopy and a fluorescent reporter for in vivo ATP measurements, we find that a subpopulation of cells with a low level of ATP survives killing by ampicillin. We propose that these low ATP cells are formed stochastically as a result of fluctuations in the abundance of energy-generating components. These findings point to a general "low energy" mechanism of persister formation.

Cell Lysis Directed by SulA in Response to DNA Damage in Escherichia coli.

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.

Evolved bacterial resistance against fluoropyrimidines can lower chemotherapy impact in the Caenorhabditis elegans host.

Metabolism of host-targeted drugs by the microbiome can substantially impact host treatment success. However, since many host-targeted drugs inadvertently hamper microbiome growth, repeated drug administration can lead to microbiome evolutionary adaptation. We tested if evolved bacterial resistance against host-targeted drugs alters their drug metabolism and impacts host treatment success. We used a model system of Caenorhabditis elegans, its bacterial diet, and two fluoropyrimidine chemotherapies. Genetic screens revealed that most of loss-of-function resistance mutations in Escherichia coli also reduced drug toxicity in the host. We found that resistance rapidly emerged in E. coli under natural selection and converged to a handful of resistance mechanisms. Surprisingly, we discovered that nutrient availability during bacterial evolution dictated the dietary effect on the host - only bacteria evolving in nutrient-poor media reduced host drug toxicity. Our work suggests that bacteria can rapidly adapt to host-targeted drugs and by doing so may also impact the host.

Optimization of RK2-based gene introduction system for Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis plays important roles in both industrial applications and basic research. However, transformation of competent B. subtilis cells is more difficult to achieve compared with that of Escherichia coli. It has been reported that the conjugative broad host range plasmid RK2 can be transferred to various organisms, including B. subtilis. Nevertheless, the protocol for conjugation from E. coli to B. subtilis has not been properly established. Thus, we optimized interspecies conjugation from E. coli to B. subtilis using the RK2 system. We constructed mobilizable shuttle and integrative vectors pEB1 and pEB2, respectively. pEB1 was used to evaluate the effect of mating media, time, temperature, and genetic background of the recipient and donor strains. We found that conjugation was not significantly affected by the conjugation time or genetic background of the recipient and donor strains. Conjugation on agar was more efficient than that in a liquid medium. A low temperature (16°C and lower) drastically decreased conjugation efficiency. When using the optimized protocol for homologous recombination after conjugation, we could not obtain double crossover mutants, as only single crossover mutants were observed in the initial selection. We then established a two-step homologous recombination method whereby positive colonies were cultivated further, which finally allowed efficient yield of double crossover recombinants. The optimized conjugation method described here allowed facility and efficient gene introduction into B. subtilis from E. coli.

A cAMP/CRP-controlled mechanism for the incorporation of extracellular ADP-glucose in Escherichia coli involving NupC and NupG nucleoside transporters.

ADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identified during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specific RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions.

Extensive impact of non-antibiotic drugs on human gut bacteria.

A few commonly used non-antibiotic drugs have recently been associated with changes in gut microbiome composition, but the extent of this phenomenon is unknown. Here, we screened more than 1,000 marketed drugs against 40 representative gut bacterial strains, and found that 24% of the drugs with human targets, including members of all therapeutic classes, inhibited the growth of at least one strain in vitro. Particular classes, such as the chemically diverse antipsychotics, were overrepresented in this group. The effects of human-targeted drugs on gut bacteria are reflected on their antibiotic-like side effects in humans and are concordant with existing human cohort studies. Susceptibility to antibiotics and human-targeted drugs correlates across bacterial species, suggesting common resistance mechanisms, which we verified for some drugs. The potential risk of non-antibiotics promoting antibiotic resistance warrants further exploration. Our results provide a resource for future research on drug-microbiome interactions, opening new paths for side effect control and drug repurposing, and broadening our view of antibiotic resistance.

Update of thermotolerant genes essential for survival at a critical high temperature in Escherichia coli.

Previous screening of a single-gene knockout library consisting of 3,908 disrupted-mutant strains allowed us to identify 51 thermotolerant genes that are essential for survival at a critical high temperature (CHT) in Escherichia coli [Murata M, Fujimoto H, Nishimura K, Charoensuk K, Nagamitsu H, Raina S, Kosaka T, Oshima T, Ogasawara N, Yamada M (2011) PLoS ONE 6: e20063]. In this study, we identified another 21 thermotolerant genes. E. coli thus has 72 thermotolerant genes in total. The genes are classified into 8 groups: genes for energy metabolism, outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control, cell division and transporters. This classification and physiological analysis indicate the existence of fundamental strategies for survival at a CHT, which seems to exclude most of the heat shock responses.

Chemical Genomics, Structure Elucidation, and in Vivo Studies of the Marine-Derived Anticlostridial Ecteinamycin.

A polyether antibiotic, ecteinamycin (1), was isolated from a marine Actinomadura sp., cultivated from the ascidian Ecteinascidia turbinata. 13C enrichment, high resolution NMR spectroscopy, and molecular modeling enabled elucidation of the structure of 1, which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ecteinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficile NAP1/B1/027 (MIC = 59 ng/µL), as well as other toxigenic and nontoxigenic C. difficile isolates both in vitro and in vivo. Additionally, chemical genomics studies using Escherichia coli barcoded deletion mutants led to the identification of sensitive mutants such as trkA and kdpD involved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. coli chemical genomics. On the basis of these data, we propose ecteinamycin as an ionophore antibiotic that causes C. difficile detoxification and cell death via potassium transport dysregulation.

Accumulation of heme biosynthetic intermediates contributes to the antibacterial action of the metalloid tellurite.

The metalloid tellurite is highly toxic to microorganisms. Several mechanisms of action have been proposed, including thiol depletion and generation of hydrogen peroxide and superoxide, but none of them can fully explain its toxicity. Here we use a combination of directed evolution and chemical and biochemical approaches to demonstrate that tellurite inhibits heme biosynthesis, leading to the accumulation of intermediates of this pathway and hydroxyl radical. Unexpectedly, the development of tellurite resistance is accompanied by increased susceptibility to hydrogen peroxide. Furthermore, we show that the heme precursor 5-aminolevulinic acid, which is used as an antimicrobial agent in photodynamic therapy, potentiates tellurite toxicity. Our results define a mechanism of tellurite toxicity and warrant further research on the potential use of the combination of tellurite and 5-aminolevulinic acid in antimicrobial therapy.

Combinatorial selection for replicable RNA by Qß replicase while maintaining encoded gene function.

Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qß RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qß replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of ß-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qß replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qß replicase while maintaining the encoded gene function.

In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes.

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach.

Comprehensive identification of translation start sites by tetracycline-inhibited ribosome profiling.

Tetracycline-inhibited ribosome profiling (TetRP) provides a powerful new experimental tool for comprehensive genome-wide identification of translation initiation sites in bacteria. We validated TetRP by confirming the translation start sites of protein-coding genes in accordance with the 2006 version of Escherichia coli K-12 annotation record (GenBank U000962) and found ∼150 new start sites within 60 nucleotides of the annotated site. This analysis revealed 72 per cent of the genes whose initiation site annotations were changed from the 2006 GenBank record to the newer 2014 annotation record (GenBank U000963), indicating a high sensitivity. Also, results from reporter fusion and proteomics of N-terminally enriched peptides showed high specificity of the TetRP results. In addition, we discovered over 300 translation start sites within non-coding, intergenic regions of the genome, using a threshold that retains ∼2,000 known coding genes. While some appear to correspond to pseudogenes, others may encode small peptides or have previously unforeseen roles. In summary, we showed that ribosome profiling upon translation inhibition by tetracycline offers a simple, reliable and comprehensive experimental tool for precise annotation of translation start sites of expressed genes in bacteria.

A robust platform for chemical genomics in bacterial systems.

While genetic perturbation has been the conventional route to probing bacterial systems, small molecules are showing great promise as probes for cellular complexity. Indeed, systematic investigations of chemical-genetic interactions can provide new insights into cell networks and are often starting points for understanding the mechanism of action of novel chemical probes. We have developed a robust and sensitive platform for chemical-genomic investigations in bacteria. The approach monitors colony volume kinetically using transmissive scanning measurements, enabling acquisition of growth rates and conventional endpoint measurements. We found that chemical-genomic profiles were highly sensitive to concentration, necessitating careful selection of compound concentrations. Roughly 20,000,000 data points were collected for 15 different antibiotics. While 1052 chemical-genetic interactions were identified using the conventional endpoint biomass approach, adding interactions in growth rate resulted in 1564 interactions, a 50-200% increase depending on the drug, with many genes uncharacterized or poorly annotated. The chemical-genetic interaction maps generated from these data reveal common genes likely involved in multidrug resistance. Additionally, the maps identified deletion backgrounds exhibiting class-specific potentiation, revealing conceivable targets for combination approaches to drug discovery. This open platform is highly amenable to kinetic screening of any arrayable strain collection, be it prokaryotic or eukaryotic.

Toward Network Biology in E. coli Cell.

E. coli has been a critically important model research organism for more than 50 years, particularly in molecular biology. In 1997, the E. coli draft genome sequence was published. Post-genomic techniques and resources were then developed that allowed E. coli to become a model organism for systems biology. Progress made since publication of the E. coli genome sequence will be summarized.

Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein.