The gut microbiota is implicated in numerous aspects of health and disease, but dissecting these connections is challenging because genetic tools for gut anaerobes are limited. Inducible promoters ...are particularly valuable tools because these platforms allow real-time analysis of the contribution of microbiome gene products to community assembly, host physiology, and disease. We developed a panel of tunable expression platforms for the prominent genus Bacteroides in which gene expression is controlled by a synthetic inducer. In the absence of inducer, promoter activity is fully repressed; addition of inducer rapidly increases gene expression by four to five orders of magnitude. Because the inducer is absent in mice and their diets, Bacteroides gene expression inside the gut can be modulated by providing the inducer in drinking water. We use this system to measure the dynamic relationship between commensal sialidase activity and liberation of mucosal sialic acid, a receptor and nutrient for pathogens.
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•We introduce tools for modulating microbiome gene expression inside the gut•Engineered promoters couple gene expression in Bacteroides to a synthetic inducer•These promoters provide over 105-fold dynamic range and can be tightly repressed•We measure dynamics of commensal liberation of host sialic acid, a pathogen nutrient
New tools enable modulating gene expression of anaerobe members of the microbiome inside the gut through introduction of a synthetic inducer in drinking water.
Tetracycline antibiotics (TCs) including tetracycline (TTC), chlorotetracycline (CTC) and oxytetracycline (OTC) adsorb strongly to aluminum oxide (Al
2O
3), and the surface interaction promotes ...structural transformation of TCs. The latter phenomenon was not widely recognized previously. Typically, rapid adsorption of TCs to Al
2O
3 occurs in the first 3
h (TC
=
40
μM, Al
2O
3
=
1.78
g
L
−1, pH
=
5, and
T
=
22
°C), followed by continuous first-order decay of the parent compound (
k
obs
=
15
±
1.0, 18
±
1.0 and 6.2
±
0.9
×
10
−3
h
−1 for TTC, CTC and OTC, respectively) and product formation. The transformation reaction rate of TCs strongly correlates with adsorption to Al
2O
3 surfaces. Both adsorption and transformation occur at the highest rate at around neutral pH conditions. Product evaluation indicates that Al
2O
3 promotes dehydration of TTC to yield anhydrotetracycline (AHTTC), epimerization of TTC, and formation of Al-TTC complexes. Al
2O
3 promotes predominantly the transformation of CTC to iso-CTC. The surface-bound Al(+III) acts as a Lewis acid site to promote the above transformation of TCs. Formation of AHTTC is of special concern because of its higher cytotoxicity. Results of this study indicate that aluminum oxide will likely affect the fate of TC antibiotics in the aquatic environment via both adsorption and transformation.
is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in
genetics have opened the door to conducting targeted and random mutagenesis ...experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for
using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the
genome.
The low transformation efficiency of
necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the
promoter system with the addition of a riboswitch).
transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition.
Here we present the preliminary sequencing results of transposon mutant pools of both
biovars, using two plasmid-free representatives: urogenital strain
SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis.
This significant advance provides an efficient method for assaying
gene function and will enable the identification of the essential gene set of
. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.
In this study we report the design, construction and validation of a novel transposon aimed to systematically screen for protein localization and expression patterns in prokaryotes using fluorescence ...microscopy. Upon random insertion in an open reading frame in the proper frame and orientation, the transposon creates an N-terminal fluorescent protein fusion to the msfGFP reporter. Moreover, in order to examine the localization of fusion proteins whose native expression might be too low or absent, the transposon was fitted with a PLtetO-1 promoter that makes the expression of the generated fluorescent protein fusions controllable by anhydrotetracycline. Importantly, upon flipping out the PLtetO-1 promoter and neighboring antibiotic resistance marker, an in-frame “sandwich” msfGFP fusion is created in which the N- and C-terminal portions of the targeted protein are again controlled by its native promoter.
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•Creation of a transposon to screen for bacterial protein localization and expression patterns.•Transposon enables conditional expression of msfGFP fusion proteins by PLtetO-1 promoter.•Flipping out PLtetO-1 creates an in-frame sandwich fusion with native promoter control.•Random transposon conjugation and its functionality was validated in S. Typhimurium.
An important toxin‐antitoxin (TA) system hok/sok, encoded by R1 plasmid of Escherichia coli, is involved in the post segregation killing of cells that have lost the plasmid. The lethal properties of ...hok protein have been utilized for the environmental containment of microbes and the development of potential vaccine candidates. This study aimed to demonstrate the potent anti‐microbial property of a 19 amino acid (AA) long N‐terminal fragment of hok peptide. This was accomplished by designing a conditional suicide system based on hok gene expression cloned in an anhydrotetracycline (aTc) inducible vector – pASK75. Heat shock and electroporation were utilized for the transformation of Escherichia coli and Vibrio cholerae cells, respectively. The minimal induction concentration (MIdC) of aTc, determined by analyzing the expression of green fluorescent protein cloned separately into pASK75 vector, was 30 ng/mL. As hok gene was synthesized de novo (using recombinant polymerase chain reaction) in our study, various random sized hok fragments were generated (as a result of the error‐prone nature of Taq polymerase). The smallest hok fragment able to bring about effective antimicrobial killing was a 19 AA long N‐terminal fragment of hok having the wild type sequence, except for the carboxy terminus AA residue. The MIdC of aTc in our experiments was 6‐fold lower than previously reported, making our bacterial clones suitable for use in mammalian systems as potential vaccine candidates. Based on our experiments, we hypothesize the 19 AA long N‐terminal fragment of hok peptide to be the smallest possible hok fragment sufficient to bring about effective antimicrobial killing.
remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and ...transmission. However, important differences exist between the sporulation pathways of
and the model Gram-positive organism
. Amongst the challenges in studying sporulation in
is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed
regulatory elements upstream of the master regulator of sporulation,
, generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the
regulon both
and
, as demonstrated using a mouse model of
infection (CDI). Insights into differences between the sporulation pathways in
and
gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The
strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in
Antibiotic resistance is a growing global health threat, demanding urgent responses. Tetracyclines, a widely used antibiotic class, are increasingly succumbing to antibiotic resistance; generating ...novel analogues is therefore a top priority for public health. Fungal tetracyclines provide structural and enzymatic diversity for novel tetracycline analogue production in tractable heterologous hosts, like yeasts, to combat antibiotic-resistant pathogens. Here, we successfully engineered Saccharomyces cerevisiae (baker’s yeast) and Saccharomyces boulardii (probiotic yeast) to produce the nonantibiotic fungal anhydrotetracycline, TAN-1612, in synthetic defined medianecessary for clean purificationsthrough heterologously expressing TAN-1612 genes mined from the fungus, Aspergillus niger ATCC 1015. This was accomplished via (i) a promoter library-based combinatorial pathway optimization of the biosynthetic TAN-1612 genes coexpressed with a putative TAN-1612 efflux pump, reducing TAN-1612 toxicity in yeasts while simultaneously increasing supernatant titers and (ii) the development of a medium-throughput UV–visible spectrophotometric assay that facilitates TAN-1612 combinatorial library screening. Through this multipronged approach, we optimized TAN-1612 production, yielding an over 450-fold increase compared to previously reported S. cerevisiae yields. TAN-1612 is an important tetracycline analogue precursor, and we thus present the first step toward generating novel tetracycline analogue therapeutics to combat current and emerging antibiotic resistance. We also report the first heterologous production of a fungal polyketide, like TAN-1612, in the probiotic S. boulardii. This highlights that engineered S. boulardii can biosynthesize complex natural products like tetracyclines, setting the stage to equip probiotic yeasts with synthetic therapeutic functionalities to generate living therapeutics or biocontrol agents for clinical and agricultural applications.
represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in ...pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in
remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of
, encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of
in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any
gene during host infection.
The development of drug delivery vehicles is in significant demand in the context of precision medicine. With the development of synthetic biology, the use of genetically engineered bacteria as drug ...delivery vectors has attracted more and more attention. Herein, we reviewed the research advances in bioengineered bacteria as drug carriers, with emphasis on the synthetic biology strategies for modifying these bacteria, including the targeted realization method of engineered bacteria, the designing scheme of genetic circuits, and the release pathways of therapeutic compounds. Based on this, the essential components, design principles, and health concerns of engineering bacteria as drug carriers and the development prospects in this field have been discussed.
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The synthesis and biological evaluation of semisynthetic anhydrotetracycline analogues as small molecule inhibitors of tetracycline-inactivating enzymes are reported. Inhibitor potency was found to ...vary as a function of enzyme (major) and substrate-inhibitor pair (minor), and anhydrotetracycline analogue stability to enzymatic and nonenzymatic degradation in solution contributes to their ability to rescue tetracycline activity in whole cell Escherichia coli expressing tetracycline destructase enzymes. Taken collectively, these results provide the framework for the rational design of next-generation inhibitor libraries en route to a viable and proactive adjuvant approach to combat the enzymatic degradation of tetracycline antibiotics.
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