11 research outputs found
Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms
Pseudomonas aeruginosa produces several phenazines including the recently described 5-methyl-phenazine-1-carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans . Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro , suggesting that 5MPCA modification by protein amine groups occurs in vivo . The red-pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA-induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed-species biofilms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79382/1/j.1365-2958.2010.07414.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/79382/2/MMI_7414_sm_Figures_Table.pd
Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression
Until recently, engineering strategies
for altering gene expression
have focused on transcription control using strong inducible promoters
or one of several methods to knock down wasteful genes. Recently,
synthetic riboregulators have been developed for translational regulation
of gene expression. Here, we report a new modular synthetic riboregulator
class that has the potential to finely tune protein expression and
independently control the concentration of each enzyme in an engineered
metabolic pathway. This development is important because the most
straightforward approach to altering the flux through a particular
metabolic step is to increase or decrease the concentration of the
enzyme. Our design includes a <i>cis</i>-repressor at the
5âČ end of the mRNA that forms a stem-loop helix, occluding
the ribosomal binding sequence and blocking translation. A <i>trans</i>-expressed activating-RNA frees the ribosomal-binding
sequence, which turns on translation. The overall architecture of
the riboregulators is designed using WatsonâCrick base-pairing
stability. We describe here a <i>cis</i>-repressor that
can completely shut off translation of antibiotic-resistance reporters
and a <i>trans</i>-activator that restores translation.
We have established that it is possible to use these riboregulators
to achieve translational control of gene expression over a wide dynamic
range. We have also found that a targeting sequence can be modified
to develop riboregulators that can, in principle, independently regulate
translation of many genes. In a selection experiment, we demonstrated
that by subtly altering the sequence of the <i>trans</i>-activator it is possible to alter the ratio of the repressed and
activated states and to achieve intermediate translational control
Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems
Engineered microbes
can be used for producing value-added chemicals
from renewable feedstocks, relieving the dependency on nonrenewable
resources such as petroleum. These microbes often are composed of
synthetic metabolic pathways; however, one major problem in establishing
a synthetic pathway is the challenge of precisely controlling competing
metabolic routes, some of which could be crucial for fitness and survival.
While traditional gene deletion and/or coarse overexpression approaches
do not provide precise regulation, cis-repressors
(CRs) are RNA-based regulatory elements that can control the production
levels of a particular protein in a tunable manner. Here, we describe
a protocol for a generally applicable fluorescence-activated cell
sorting technique used to isolate eight subpopulations of CRs from
a semidegenerate library in Escherichia coli, followed by deep sequencing that permitted the identification of
15 individual CRs with a broad range of protein production profiles.
Using these new CRs, we demonstrated a change in production levels
of a fluorescent reporter by over two orders of magnitude and further
showed that these CRs are easily ported from E. coli to Pseudomonas putida. We next used
four CRs to tune the production of the enzyme PpsA, involved in pyruvate
to phosphoenolpyruvate (PEP) conversion, to alter the pool of PEP
that feeds into the shikimate pathway. In an engineered P. putida strain, where carbon flux in the shikimate
pathway is diverted to the synthesis of the commodity chemical cis,cis-muconate, we found that tuning
PpsA translation levels increased the overall titer of muconate. Therefore,
CRs provide an approach to precisely tune protein levels in metabolic
pathways and will be an important tool for other metabolic engineering
efforts