Cell surface CD55 traffics to the nucleus leading to cisplatin resistance and stemness by inducing PRC2 and H3K27 trimethylation on chromatin in ovarian cancer.

BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.

Diffusible Signaling Factor, a Quorum-Sensing Molecule, Interferes with and Is Toxic Towards Bdellovibrio bacteriovorus 109J.

Bdellovibrio bacteriovorus 109J is a predatory bacterium which lives by predating on other Gram-negative bacteria to obtain the nutrients it needs for replication and survival. Here, we evaluated the effects two classes of bacterial signaling molecules (acyl homoserine lactones (AHLs) and diffusible signaling factor (DSF)) have on B. bacteriovorus 109J behavior and viability. While AHLs had a non-significant impact on predation rates, DSF considerably delayed predation and bdelloplast lysis. Subsequent experiments showed that 50 µM DSF also reduced the motility of attack-phase B. bacteriovorus 109J cells by 50% (38.2 ± 14.9 vs. 17 ± 8.9 µm/s). Transcriptomic analyses found that DSF caused genome-wide changes in B. bacteriovorus 109J gene expression patterns during both the attack and intraperiplasmic phases, including the significant downregulation of the flagellum assembly genes and numerous serine protease genes. While the former accounts for the reduced speeds observed, the latter was confirmed experimentally with 50 µM DSF completely blocking protease secretion from attack-phase cells. Additional experiments found that 30% of the total cellular ATP was released into the supernatant when B. bacteriovorus 109J was exposed to 200 µM DSF, implying that this QS molecule negatively impacts membrane integrity.

Programmable Artificial Cells Using Histamine-Responsive Synthetic Riboswitch.

Artificial cells that encapsulate DNA-programmable protein expression machinery are emerging as an attractive platform for studying fundamental cellular properties and applications in synthetic biology. However, interfacing these artificial cells with the complex and dynamic chemical environment remains a major and urgent challenge. We demonstrate that the repertoire of molecules that artificial cells respond to can be expanded by synthetic RNA-based gene switches, or riboswitches. We isolated an RNA aptamer that binds histamine with high affinity and specificity and used it to design robust riboswitches that activate protein expression in the presence of histamine. Finally, the riboswitches were incorporated in artificial cells to achieve controlled release of an encapsulated small molecule and to implement a self-destructive kill-switch. Synthetic riboswitches should serve as modular and versatile interfaces to link artificial cell phenotypes with the complex chemical environment.

Attack-Phase Bdellovibrio bacteriovorus Responses to Extracellular Nutrients Are Analogous to Those Seen During Late Intraperiplasmic Growth.

Bdellovibrio bacteriovorus is a predatory bacterium which lives by invading the periplasm of gram-negative bacteria and consuming them from within. This predator was thought to be dependent upon prey for nutrients since it lacks genes encoding for critical enzymes involved in amino acid biosynthesis. This study, however, found that planktonic attack-phase predators are not just dependent upon prey for nutrients, but rather, they respond to nutrients in the surrounding medium and, subsequently, synthesize and secrete proteases in a nutrient-dependent manner. The major secreted proteases were identified through mass spectrometry analyses. Subsequent RT-qPCR analyses found that the nutrient-induced proteases are similar to those expressed within the prey periplasm during the late intraperiplasmic growth phase. Furthermore, RNA sequencing found that incubating the planktonic attack-phase cells in a nutritious environment for a short period of time (4 h) changes its gene expression pattern to a status that is akin to the late intraperiplasmic phase, with more than 94% of the genes previously identified as being late intraperiplasmic-specific also being induced by nutrient broth in this study. This strong correlation between the gene expression patterns hints that the availability of hydrolyzed prey cell components to the predator is likely the stimulus controlling the expression of late intraperiplasmic B. bacteriovorus genes during predation.

Indole negatively impacts predation by Bdellovibrio bacteriovorus and its release from the bdelloplast.

Bdellovibrio bacteriovorus is a predatory bacterium that attacks a wide range of Gram-negative bacterial pathogens and is proposed to be a potential living antibiotic. In this study, we evaluated the effects of indole, a bacterial signalling molecule commonly produced within the gut, on the predatory ability of B. bacteriovorus HD100. Indole significantly delayed predation on Escherichia coli MG1655 and Salmonella enterica KACC 11595 at physiological concentrations (0.25 to 1 mM) and completely inhibited predation when present at 2 mM. Microscopic analysis revealed that indole blocked the predator from attacking the prey. Furthermore, indole was not toxic to the predator but slowed down its motility. Microarray and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses confirmed that as the gene group showing the greatest downregulation in the presence of indole was flagellar assembly genes. Indole also caused a wide spectrum changes in gene expression including general downregulation of genes involved in ribosome assembly. Furthermore, indole addition to the predatory culture after the entrance of B. bacteriovorus into the prey periplasm slowed down bdelloplast lysis. In conclusion, indole can have significant impacts on the predation efficiency, which should be taken into consideration especially if B. bacteriovorus is to be applied as a probiotic or living antibiotic.

Pretreatment with alum or powdered activated carbon reduces bacterial predation-associated irreversible fouling of membranes.

This study evaluated the co-application of bacterial predation by Bdellovibrio bacteriovorus and either alum coagulation or powdered activated carbon adsorption to reduce fouling caused by Escherichia coli rich feed solutions in dead-end microfiltration tests. The flux increased when the samples were predated upon or treated with 100 ppm alum or PAC, but co-treatment with alum and predation gave the best flux results. The total membrane resistance caused by the predated sample was reduced six-fold when treated with 100 ppm PAC, from 11.8 to 1.98 × 10(11) m(-1), while irreversible fouling (Rp) was 2.7-fold lower. Treatment with 100 ppm alum reduced the total resistance 14.9-fold (11.8 to 0.79 × 10(11) m(-1)) while the Rp decreased 4.25-fold. SEM imaging confirmed this, with less obvious fouling of the membrane after the combined process. This study illustrates that the combination of bacterial predation and the subsequent removal of debris using coagulation or adsorption mitigates membrane biofouling and improves membrane performance.

Assembling the anaerobic gamma-butyrobetaine to TMA metabolic pathway in Escherichia fergusonii and confirming its role in TMA production from dietary L-carnitine in murine models.

IMPORTANCE: The key atherosclerotic TMAO originates from the initial gut microbial conversion of L-carnitine and other dietary compounds into TMA. Developing therapeutic strategies to block gut microbial TMA production needs a detailed understanding of the different production mechanisms and their relative contributions. Recently, we identified a two-step anaerobic pathway for TMA production from L-carnitine through initial conversion by some microbes into the intermediate γBB which is then metabolized by other microbes into TMA. Investigational studies of this pathway, however, are limited by the lack of single microbes harboring the whole pathway. Here, we engineered E. fergusonii strain to harbor the whole two-step pathway and optimized the expression through cloning a specific chaperone from the original host. Inoculating germ-free mice with this recombinant E. fergusonii is enough to raise serum TMAO to pathophysiological levels upon L-carnitine feeding. This engineered microbe will facilitate future studies investigating the contribution of this pathway to cardiovascular disease.

Select symbionts drive high IgA levels in the mouse intestine.

Immunoglobulin A (IgA) is an important factor in maintaining homeostasis at mucosal surfaces, yet luminal IgA levels vary widely. Total IgA levels are thought to be driven by individual immune responses to specific microbes. Here, we found that the prebiotic, pectin oligosaccharide (pec-oligo), induced high IgA levels in the small intestine in a T cell-dependent manner. Surprisingly, this IgA-high phenotype was retained after cessation of pec-oligo treatment, and microbiome transmission either horizontally or vertically was sufficient to retain high IgA levels in the absence of pec-oligo. Interestingly, the bacterial taxa enriched in the overall pec-oligo bacterial community differed from IgA-coated microbes in this same community. Rather, a group of ethanol-resistant microbes, highly enriched for Lachnospiraceae bacterium A2, drove the IgA-high phenotype. These findings support a model of intestinal adaptive immunity in which a limited number of microbes can promote durable changes in IgA directed to many symbionts.

Two distinct gut microbial pathways contribute to meta-organismal production of phenylacetylglutamine with links to cardiovascular disease.

Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardiovascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Using genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decarboxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln formation in vivo.

Microbe-derived uremic solutes enhance thrombosis potential in the host.

p-Cresol sulfate (pCS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of pCS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (n = 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of pCS and IS in an independent validation cohort (n = 3,954). Genetic engineering of human commensals to produce p-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic pCS and IS levels were independently associated with all-cause mortality. Both in vitro and within colonized germ-free mice p-cresol productions were recapitulated by collaboration of two organisms: a Bacteroides strain that converts tyrosine to 4-hydroxyphenylacetate, and a Clostridium strain that decarboxylates 4-hydroxyphenylacetate to p-cresol. We then engineered a single organism, Bacteroides thetaiotaomicron, to produce p-cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains, we show the gut microbial genes for p-cresol (hpdBCA) and indole (tryptophanase) are sufficient to confer a pro-thrombotic phenotype in vivo. Moreover, human fecal metagenomics analyses show that abundances of hpdBCA and tryptophanase are associated with CVD. These studies show that pCS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial p-cresol- and indole-producing pathways represent rational targets for CVD.IMPORTANCEAlterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

Aqueous two-phase system-derived biofilms for bacterial interaction studies.

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a ß-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.

Combined application of bacterial predation and carbon dioxide aerosols to effectively remove biofilms.

This study evaluated predation with Bdellovibrio bacteriovorous and CO(2) aerosol spraying to remove fluorescent Escherichia coli biofilms from silicon chips. Initial tests found that 7.5×10(5) viable E. coli cells were dispersed into the surrounding environment during aerosol treatment. The total number dispersed per test decreased to only 16 for predated biofilms. This is nearly 50,000-fold lower compared to untreated chips and 1000-fold lower compared to chips soaked in HEPES buffer only. Both scanning electron microscopy (SEM) and fluorescent microscopy analyses confirmed that predation alone did not completely eradicate the biofilm population. When used in conjunction with CO(2) aerosols, however, no fluorescent signals remained and the SEM pictures showed a pristine surface devoid of bacteria. Consequently, this study demonstrates these two methods can be used with each other to significantly remove biofilms from surfaces while also significantly reducing the likelihood of human exposure to potential pathogens during their removal.

The future of butyric acid in industry.

In this paper, the different applications of butyric acid and its current and future production status are highlighted, with a particular emphasis on the biofuels industry. As such, this paper discusses different issues regarding butyric acid fermentations and provides suggestions for future improvements and their approaches.

Outer Membrane Porin F in E. coli Is Critical for Effective Predation by Bdellovibrio.

Bdellovibrio and like organisms (BALOs) are a unique bacterial group that live by predating on other bacteria, consuming them from within to grow and replicate before the progeny come out to complete the life cycle. The mechanisms by which these predators recognize their prey and differentiate them from nonprey bacteria, however, are still not clear. Through genetic knockout and complementation studies in different Escherichia coli strains, we found that Bdellovibrio bacteriovorus strain 109J recognizes outer membrane porin F (OmpF) on the E. coli surface and that the activity of the E. coli EnvZ-OmpR regulatory system significantly impacts predation kinetics. OmpF is not the only signal by which BALOs recognize their prey, however, as B. bacteriovorus could eventually predate on the E. coli ΔompF mutant after prolonged incubation. Furthermore, recognizing OmpF as a prey surface structure was dependent on the prey strain, as knocking out OmpF protein homologues in other prey species, including Escherichia fergusonii, Klebsiella pneumoniae, and Salmonella enterica, did not always reduce the predation rate. Consequently, although OmpF was found to be an important surface component used by Bdellovibrio to efficiently recognize and attack E. coli, future work is needed to determine what other prey surface structures are recognized by these predators. IMPORTANCE Bdellovibrio bacteriovorus and like organisms (BALOs) are Gram-negative predatory bacteria that attack other Gram-negative bacteria by penetrating their periplasm and consuming them from within to obtain the nutrients necessary for the predator's growth and replication. How these predators recognize their prey, however, has remained a mystery. Here, we show that the outer membrane porin F (OmpF) in E. coli is recognized by B. bacteriovorus strain 109J and that the loss of this protein leads to severely delayed predation. However, predation of several other prey species was not dependent on the recognition of this protein or its homologues, indicating that there are other structures recognized by the predators on the prey surface that are yet to be discovered.

Disruption of the Gut Microbiota Confers Cisplatin Resistance in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death. Despite initial responses to intervention, up to 80% of patient tumors recur and require additional treatment. Retrospective clinical analysis of patients with ovarian cancer indicates antibiotic use during chemotherapy treatment is associated with poor overall survival. Here, we assessed whether antibiotic (ABX) treatment would impact growth of EOC and sensitivity to cisplatin. Immunocompetent or immunocompromised mice were given untreated control or ABX-containing (metronidazole, ampicillin, vancomycin, and neomycin) water prior to intraperitoneal injection with EOC cells, and cisplatin therapy was administered biweekly until endpoint. Tumor-bearing ABX-treated mice exhibited accelerated tumor growth and resistance to cisplatin therapy compared with control treatment. ABX treatment led to reduced apoptosis, increased DNA damage repair, and enhanced angiogenesis in cisplatin-treated tumors, and tumors from ABX-treated mice contained a higher frequency of cisplatin-augmented cancer stem cells than control mice. Stool analysis indicated nonresistant gut microbial species were disrupted by ABX treatment. Cecal transplants of microbiota derived from control-treated mice was sufficient to ameliorate chemoresistance and prolong survival of ABX-treated mice, indicative of a gut-derived tumor suppressor. Metabolomics analyses identified circulating gut-derived metabolites that were altered by ABX treatment and restored by recolonization, providing candidate metabolites that mediate the cross-talk between the gut microbiome and ovarian cancer. Collectively, these findings indicate that an intact microbiome functions as a tumor suppressor in EOC, and perturbation of the gut microbiota with ABX treatment promotes tumor growth and suppresses cisplatin sensitivity. SIGNIFICANCE: Restoration of the gut microbiome, which is disrupted following antibiotic treatment, may help overcome platinum resistance in patients with epithelial ovarian cancer. See related commentary by Hawkins and Nephew, p. 4511.

The microbial gbu gene cluster links cardiovascular disease risk associated with red meat consumption to microbiota L-carnitine catabolism.

The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine → γBB → TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine → γBB → TMA → TMAO transformation in hosts and contributes to CVD risk.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Development of a histamine aptasensor for food safety monitoring.

Histamine produced by bacteria through decarboxylation of histidine in spoiled foods such as fish is known to cause food poisoning. Therefore, accurate and facile measurement of histamine is of practical importance. Using the recently discovered RNA aptamer that specifically recognizes histamine (A1-949 aptamer), we developed an aptasensor based on the structure-switching mechanism. Specifically, the aptamer A1-949 was fluorescently labeled at the 5' end and hybridized with a short quencher DNA strand that is partially complementary to the aptamer. The quencher strand was modified with a fluorescence quencher at its 3' terminus. Displacement of the quencher strand upon histamine binding results in an increased fluorescence. After optimizing the assay condition, the enantiomeric version of the aptasensor (L-RNA and L-DNA) was synthesized which could detect the achiral analyte with identical sensitivity and improved biochemical stability. The aptasensor performance was validated by measuring fish samples spiked with known concentrations of histamine. Finally, histamine content in spoiled fish samples was measured, and the results were compared with the measurements using a commercial enzymatic assay kit.

Riboswitch Signal Amplification by Controlling Plasmid Copy Number.

Riboswitches are cis-acting RNA devices in mRNAs that control gene expression in response to chemical inputs. As RNA aptamers that recognize diverse classes of molecules can be isolated by in vitro selection, synthetic riboswitches hold promise for various applications in synthetic biology. One of the major drawbacks of riboswitches, however, is their limited dynamic range. A high level of gene expression in the OFF state (leakage) is also a common problem. To address these challenges, we designed and constructed a dual-riboswitch plasmid in which two genes are controlled by theophylline-activated riboswitches. One riboswitch controls the gene of interest, and another riboswitch controls RepL, a phage-derived replication protein that regulates the plasmid copy number. This single-plasmid system afforded an ON/OFF ratio as high as 3900. Furthermore, we used the system to control CRISPR interference (CRISPRi) targeting endogenous genes, and successfully observed expected phenotypic changes in Escherichia coli.

Bdellovibrio bacteriovorus HD100, a predator of Gram-negative bacteria, benefits energetically from Staphylococcus aureus biofilms without predation.

Bdellovibrio bacteriovorus HD100 is a predatory bacterium which lives by invading the periplasm of Gram-negative bacteria and consuming them from within. Although B. bacteriovorus HD100 attacks only Gram-negative bacterial strains, our work here shows attack-phase predatory cells also benefit from interacting with Gram-positive biofilms. Using Staphylococcus aureus biofilms, we show this predator degrades the biofilm matrix, obtains nutrients and uses these to produce and secrete proteolytic enzymes to continue this process. When exposed to S. aureus biofilms, the transcriptome of B. bacteriovorus HD100 was analogous to that seen when present intraperiplasmically, suggesting it is responding similarly as when in a prey. Moreover, two of the induced proteases (Bd2269 and Bd2692) were purified and their activities against S. aureus biofilms verified. In addition, B. bacteriovorus HD100 gained several clear benefits from its interactions with S. aureus biofilms, including increased ATP pools and improved downstream predatory activities when provided prey.