Multiplex genome editing by natural transformation Dalia, Ankur B.; McDonough, EmilyKate; Camilli, Andrew
Proceedings of the National Academy of Sciences - PNAS,
06/2014, Letnik:
111, Številka:
24
Journal Article
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Editing bacterial genomes is an essential tool in research and synthetic biology applications. Here, we describe multiplex genome editing by natural transformation (MuGENT), a method for accelerated ...evolution based on the cotransformation of unlinked genetic markers in naturally competent microorganisms. We found that natural cotransformation allows scarless genome editing at unprecedented frequencies of ∼50%. Using DNA substrates with randomized nucleotides, we found no evidence for bias during natural cotransformation, indicating that this method can be used for directed evolution studies. Furthermore, we found that natural cotransformation is an effective method for multiplex genome editing. Because MuGENT does not require selection at edited loci in cis , output mutant pools are highly complex, and strains may have any number and combination of the multiplexed genome edits. We demonstrate the utility of this technique in metabolic and phenotypic engineering by optimizing natural transformation in Vibrio cholerae . This was accomplished by combinatorially editing the genome via gene deletions and promoter replacements and by tuning translation initiation of five genes involved in the process of natural competence and transformation. MuGENT allowed for the generation of a complex mutant pool in 1 wk and resulted in the selection of a genetically edited strain with a 30-fold improvement in natural transformation. We also demonstrate the efficacy of this technique in Streptococcus pneumoniae and highlight the potential for MuGENT to be used in multiplex genetic interaction analysis. Thus, MuGENT is a broadly applicable platform for accelerated evolution and genetic interaction studies in diverse naturally competent species.
This study investigated neuroanatomic, genetic, cognitive, sociodemographic and emotional underpinnings of the Negative Urgency subscale of the Urgency, Premeditation, Perseverance, Sensation-Seeking ...and Positive Urgency Impulsive Behavior Scale in a healthy developmental sample. The goal of the investigation is to contribute to the harmonisation of behavioural, brain and neurogenetic aspects of behavioural self-control. Three domains – (1) Demographic, developmental, psychiatric and cognitive ability; (2) Regional brain volumes (neurobiological); and (3) Genetic variability (single nucleotide polymorphisms) – were examined, and models with relevant predictor variables were selected. Least absolute shrinkage and selection operator and best subset regressions were used to identify sparse models predicting negative urgency scores, which revealed that variables related to emotional regulation and right cingulate volume, as well as single nucleotide polymorphisms in CADM2 and SLC6A4, were associated with negative urgency. Our results contribute to the construct and criterion validity of negative urgency and support the hypothesis that negative urgency is a result of a complex array of influences across domains whose integration furthers developmental psychopathology research.
Summary
Phosphate is essential for life, being used in many core processes such as signal transduction and synthesis of nucleic acids. The waterborne agent of cholera, Vibrio cholerae, encounters ...phosphate limitation in both the aquatic environment and human intestinal tract. This bacterium can utilize extracellular DNA (eDNA) as a phosphate source, a phenotype dependent on secreted endo‐ and exonucleases. However, no transporter of nucleotides has been identified in V. cholerae, suggesting that in order for the organism to utilize the DNA as a phosphate source, it must first separate the phosphate and nucleoside groups before transporting phosphate into the cell. In this study, we investigated the factors required for assimilation of phosphate from eDNA. We identified PhoX, and the previously unknown proteins UshA and CpdB as the major phosphatases that allow phosphate acquisition from eDNA and nucleotides. We demonstrated separable but partially overlapping roles for the three phosphatases and showed that the activity of PhoX and CpdB is induced by phosphate limitation. Thus, this study provides mechanistic insight into how V. cholerae can acquire phosphate from extracellular DNA, which is likely to be an important phosphate source in the environment and during infection.
V. cholerae was previously shown to use extracellular DNA as a source of phosphate. We have identified three phosphatases/nucleotidases that are essential for this phenotype: PhoX, UshA, and CpdB. We also show that UshA is a 5′nucleotidase and that CpdB is a low phosphate activated 3′nucleotidase.
Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar ...environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo-specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non-inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn-seq to identify potential mechanisms of xds regulation. Our selection identified the two-component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate-limited environment.
Summary
Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar ...environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo‐specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non‐inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn‐seq to identify potential mechanisms of xds regulation. Our selection identified the two‐component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate‐limited environment.
Summary
Phosphate is essential for life, being used in many core processes such as signal transduction and synthesis of nucleic acids. The waterborne agent of cholera,
V
ibrio cholerae
, encounters ...phosphate limitation in both the aquatic environment and human intestinal tract. This bacterium can utilize extracellular
DNA
(e
DNA
) as a phosphate source, a phenotype dependent on secreted endo‐ and exonucleases. However, no transporter of nucleotides has been identified in
V
. cholerae
, suggesting that in order for the organism to utilize the
DNA
as a phosphate source, it must first separate the phosphate and nucleoside groups before transporting phosphate into the cell. In this study, we investigated the factors required for assimilation of phosphate from e
DNA
. We identified
PhoX
, and the previously unknown proteins
UshA
and
CpdB
as the major phosphatases that allow phosphate acquisition from e
DNA
and nucleotides. We demonstrated separable but partially overlapping roles for the three phosphatases and showed that the activity of
PhoX
and
CpdB
is induced by phosphate limitation. Thus, this study provides mechanistic insight into how
V
. cholerae
can acquire phosphate from extracellular
DNA
, which is likely to be an important phosphate source in the environment and during infection.
Prions are self-propagating protein aggregates formed by specific proteins that can adopt alternative folds. Prions were discovered as the cause of the fatal transmissible spongiform encephalopathies ...in mammals, but prions can also constitute nontoxic protein-based elements of inheritance in fungi and other species. Prion propagation has recently been shown to occur in bacteria for more than a hundred cell divisions, yet a fraction of cells in these lineages lost the prion through an unknown mechanism. Here, we investigate prion propagation in single bacterial cells as they divide using microfluidics and fluorescence microscopy. We show that the propagation occurs in two distinct modes. In a fraction of the population, cells had multiple small visible aggregates and lost the prion through random partitioning of aggregates to one of the two daughter cells at division. In the other subpopulation, cells had a stable large aggregate localized to the pole; upon division the mother cell retained this polar aggregate and a daughter cell was generated that contained small aggregates. Extending our findings to prion domains from two orthologous proteins, we observe similar propagation and loss properties. Our findings also provide support for the suggestion that bacterial prions can form more than one self-propagating state. We implement a stochastic version of the molecular model of prion propagation from yeast and mammals that recapitulates all the observed single-cell properties. This model highlights challenges for prion propagation that are unique to prokaryotes and illustrates the conservation of fundamental characteristics of prion propagation.
Summary
V
ibrio cholerae
, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen,
V
. cholerae
moves between two dissimilar ...environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene,
xds
, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the
in vivo
‐specific induction of
xds
. A transcriptional fusion of
xds
to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the
xds
fusion under non‐inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn‐seq to identify potential mechanisms of
xds
regulation. Our selection identified the two‐component system
PhoB
/
R
as the dominant activator of
xds
expression.
In vitro
validation confirmed that
PhoB
, a protein which is only active during phosphate limitation, was responsible for
xds
activation. Using
xds
expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate‐limited environment.
The Dimensional Change Card Sort (DCCS) is a measure of cognitive flexibility for children, which requires rule-use and shifting. Demographic, cognitive, regional cortical thickness, and genetic ...variables, including those related to language and executive function, were used to build predictive models of DCCS scores in 556 healthy pediatric participants. Gender, age, frontal, and temporal lobe regions of interest, and measures of sustained attention, inhibition, and word reading were selected as the best predictors of DCCS performance. Results indicated that DCCS performance is related to a broad range of cognitive functions and anatomic regions associated with various levels of cognitive function.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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