Publications

simultaneous catabolite repression between glucose and toluene metabolism in pseudomonas putida is channeled through different signaling pathways.	pseudomonas putida kt2440(pww0) can use toluene via the tol plasmid-encoded catabolic pathways and can use glucose via a series of three peripheral chromosome-encoded routes that convert glucose into 6-phosphogluconate (6pg), namely, the glucokinase pathway, in which glucose is transformed to 6pg through the action of glucokinase and glucose-6-phosphate dehydrogenase. alternatively, glucose can be oxidized to gluconate, which can be phosphorylated by gluconokinase to 6pg or oxidized to 2-ketoglu ...	2007	17616587
tol plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo.	molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pww0 was conjugated from pseudomonas to rhizobium. the xyle gene was detected both in rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. tol plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-cont ...	2007	17000041
new insights on toluene biodegradation by pseudomonas putida f1: influence of pollutant concentration and excreted metabolites.	the influence of toluene concentration on the specific growth rate, cellular yield, specific co(2), and metabolite production by pseudomonas putida f1 (ppf1) was investigated. both cellular yield and specific co(2) production remained constant at 1.0 +/- 0.1 g biomass dry weight (dw) g(-1) toluene and 1.91 +/- 0.31 g co(2) g(-1) biomass, respectively, under the tested range of concentrations (2-250 mg toluene l(-1)). the specific growth rate increased up to 70 mg toluene l(-1). further increases ...	2007	17136537
growth of the genetically engineered strain cupriavidus necator rw112 with chlorobenzoates and technical chlorobiphenyls.	cupriavidus necator (formerly ralstonia eutropha) strain h850 is known to grow on biphenyl, and to co-oxidize congeners of polychlorinated biphenyls (pcbs). using a tn5-based minitransposon shuttle system and the tol plasmid, the rational construction of hybrids of h850 was achieved by subsequent introduction of three distinct elements carrying 11 catabolic loci from three other biodegrading bacteria into the parent strain, finally yielding c. necator rw112. the new genetic elements introduced i ...	2007	17185547
complete sequence determination combined with analysis of transposition/site-specific recombination events to explain genetic organization of incp-7 tol plasmid pww53 and related mobile genetic elements.	recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (tol) plasmids and their residing transposons. to elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pww53, a tol plasmid from pseudomonas putida mt53. pww53 was found to belong to the incp-7 incompatibility group that play important roles in the catabolism of se ...	2007	17408691
toluene biodegradation by pseudomonas putida f1: targeting culture stability in long-term operation.	the stability of pseudomonas putida f1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h(-1)). each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the tol plasmid. at all tested conditions ...	2008	17487552
xyls-pm promoter interactions through two helix-turn-helix motifs: identifying xyls residues important for dna binding and activation.	the xyls protein is the positive transcription regulator of the tol plasmid meta-cleavage pathway operon pm. xyls belongs to the arac family of transcriptional regulators and exhibits an n-terminal domain involved in effector recognition, and a c-terminal domain, made up of seven alpha-helices conforming two helix-turn-helix dna-binding domains. alpha-helix 3 and alpha-helix 6 are the recognition helices. in consonance with xyls structural organization, pm exhibits a bipartite dna-binding motif ...	2008	18005985
bioaugmentation of aerobic microbial granules with pseudomonas putida carrying tol plasmid.	this paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using pseudomonas putida kt2442 cells bearing the tol (pwwo) plasmid. the methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. expression of reporter proteins (gfp and dsred) allowed in situ monitoring of donor cell attachment and plas ...	2008	18076969
[study on method of acclimation and screening of a highly effective toluene-degrading bacterium].	to establish a highly effective toluene-degrading bacterium acclimation and screening method.	2008	18589590
transcriptional wiring of the tol plasmid regulatory network to its host involves the submission of the sigma54-promoter pu to the response regulator ppra.	implantation of the regulatory circuit of the degradation pathway of tol plasmid pww0 in the native transcriptional network of the host pseudomonas putida involves interplay between plasmid- and chromosome-encoded factors. we have employed a reverse genetics approach to investigate such a molecular wiring by identifying host proteins that form stable complexes with pu, the sigma(54)-dependent promoter of the upper tol operon of pww0. this approach revealed that the pu upstream activating sequenc ...	2008	19138193
conditions required for the stimulation of bioluminescence activity of the genetically engineered bacteria, p. putida mt-2 kg1206, preserved by deep-freezing.	herein the conditions required for the stimulation of bioluminescence activity in a genetically engineered strain of pseudomonas putida mt-2 kg1206, containing the intact tol plasmid and a constructed plasmid with the p(m)-lux gene, are reported upon. both sodium lactate (sl) and potassium nitrate (kno(3)) were able to stimulate the bioluminescence activity, but a greater increase was observed with nitrogen amendment. this selected stimulant was then tested on reconstituted cells that had been p ...	2009	19176233
competition triggers plasmid-mediated enhancement of substrate utilisation in pseudomonas putida.	competition between species plays a central role in the activity and structure of communities. stable co-existence of diverse organisms in communities is thought to be fostered by individual tradeoffs and optimization of competitive strategies along resource gradients. outside the laboratory, microbes exist as multispecies consortia, continuously interacting with one another and the environment. survival and proliferation of a particular species is governed by its competitive fitness. therefore, ...	2009	19557171
bioaugmentation of microbial communities in laboratory and pilot scale sequencing batch biofilm reactors using the tol plasmid.	the aim of this study was to investigate the effectiveness of bioaugmentation and transfer of plasmid pwwo (tol plasmid) to mixed microbial populations in pilot and laboratory scale sequencing batch biofilm reactors (sbbrs) treating synthetic wastewater containing benzyl alcohol (ba) as a model xenobiotic. the plasmid donor was a pseudomonas putida strain chromosomally tagged with the gene for the red fluorescent protein carrying a green fluorescent protein labeled tol plasmid, which confers deg ...	2009	19010662
a new extant respirometric assay to estimate intrinsic growth parameters applied to study plasmid metabolic burden.	start-up phenomena in microbial biokinetic assays are not captured by the most commonly used growth-related equations. in this study we propose a new respirometric experimental design to estimate intrinsic growth parameters that allow us to avoid these limitations without data omission, separate mathematical treatment, or wake-up pulses prior to the analysis. identifiability and sensitivity analysis were performed to confirm the robustness of the new approach for obtaining unique and accurate es ...	2010	19718700
effect of carbon source addition on toluene biodegradation by an escherichia coli dh5alpha transconjugant harboring the tol plasmid.	horizontal gene transfer (hgt) of plasmids is a naturally occurring phenomenon which could be manipulated for bioremediation applications. specifically, hgt may prove useful to enhance bioremediation through genetic bioaugmentation. however, because the transfer of a plasmid between donor and recipient cells does not always result in useful functional phenotypes, the conditions under which hgt events result in enhanced degradative capabilities must first be elucidated. the objective of this stud ...	2010	20506384
the regulatory logic of m-xylene biodegradation by pseudomonas putida mt-2 exposed by dynamic modelling of the principal node ps/pr of the tol plasmid.	the structure of the extant transcriptional control network of the tol plasmid pww0 born by pseudomonas putida mt-2 for biodegradation of m-xylene is far more complex than one would consider necessary from a mere engineering point of view. in order to penetrate the underlying logic of such a network, which controls a major environmental cleanup bioprocess, we have developed a dynamic model of the key regulatory node formed by the ps/pr promoters of pww0, where the clustering of control elements ...	2010	20553551
complete nucleotide sequence of tol plasmid pdk1 provides evidence for evolutionary history of incp-7 catabolic plasmids.	to understand the mechanisms for structural diversification of pseudomonas-derived toluene-catabolic (tol) plasmids, the complete sequence of a self-transmissible plasmid pdk1 with a size of 128,921 bp from pseudomonas putida hs1 was determined. comparative analysis revealed that (i) pdk1 consisted of a 75.6-kb incp-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pdk1 were highly similar to thos ...	2010	20581207
high stability and fast recovery of expression of the tol plasmid-carried toluene catabolism genes of pseudomonas putida mt-2 under conditions of oxygen limitation and oscillation.	pseudomonas putida mt-2 harbors the tol plasmid (pwwo), which contains the genes encoding the enzymes necessary to degrade toluene aerobically. the xyl genes are clustered in the upper operon and encode the enzymes of the upper pathway that degrade toluene to benzoate, while the genes encoding the enzymes of the lower pathway (meta-cleavage pathway) that are necessary for the conversion of benzoate to tricarboxylic acid cycle intermediates, are encoded in a separate operon. in this study, the ef ...	2010	20709833
tol plasmid carriage enhances biofilm formation and increases extracellular dna content in pseudomonas putida kt2440.	adherent growth of pseudomonas putida kt2440 with and without the tol plasmid (pwwo) at the solid-liquid and air-liquid interface was examined. we compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal laser scanning microscopy. the tol-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. microscopy using fluorescent nuc ...	2010	20846143
construction and characterization of escherichia coli whole-cell biosensors for toluene and related compounds.	the xylr regulatory protein is a transcriptional activator from the tol plasmid of pseudomonas putida mt-2 that is involved in the toluene and benzene degradation pathway. here we describe the construction and laboratory characterization of recombinant biosensors (pglpx plasmids) based on xylr and its cognate promoter (pu). in the pglpx plasmid, the reporter luc gene is under the control of the pu promoter. we evaluated the ability of two distinct nucleotide sequences to function as sd elements ...	2010	20872219
an individual-based approach to explain plasmid invasion in bacterial populations.	we present an individual-based experimental framework to identify and estimate the main parameters governing bacterial conjugation at the individual cell scale. from this analysis, we have established that transient periods of unregulated plasmid transfer within newly formed transconjugant cells, together with contact mechanics arising from cellular growth and division, are the two main processes determining the emergent inability of the pww0 tol plasmid to fully invade spatially structured pseu ...	2010	21091520
cooperative amino acid changes shift the response of the σ⁵⁴-dependent regulator xylr from natural m-xylene towards xenobiotic 2,4-dinitrotoluene.	xylr is a σ⁵⁴-dependent transcriptional factor of pseudomonas putida that activates the pu promoter of the tol plasmid upon binding its natural effector, m-xylene. the search for mutants of the signal-sensing module of xylr that respond to the xenobiotic compound 2,4-dinitrotoluene recurrently yields protein variants with a broad effector range. these mutants had amino acid changes not only in the effector recognition moiety (a module), but also in the inter-domain b linker of the protein. a ran ...	2011	21205010
The logic layout of the TOL network of Pseudomonas putida pWW0 plasmid stems from a metabolic amplifier motif (MAM) that optimizes biodegradation of m-xylene.	ABSTRACT: BACKGROUND: The genetic network of the TOL plasmid pWW0 of the soil bacterium Pseudomonas putida mt-2 for catabolism of m-xylene is an archetypal model for environmental biodegradation of aromatic pollutants. Although nearly every metabolic and transcriptional component of this regulatory system is known to an extraordinary molecular detail, the complexity of its architecture is still perplexing. To gain an insight into the inner layout of this network a logic model of the TOL system ...	2011	22078029
An efficient design strategy for a whole-cell biosensor based on engineered ribosome binding sequences.	In prokaryotes, the ribosome binding sequence (RBS), located in the 5' untranslated region (5' UTR) of an mRNA, plays a critical role in enhancing mRNA translation and stability. To evaluate the effect of the RBS on the sensitivity and signal intensity of an environmental whole-cell biosensor, three Escherichia coli-based biosensors that respond to benzene, toluene, ethylbenzene, and the xylenes (BTEX) were constructed; the three biosensors have the same Pu promoter and xylR regulator from the P ...	2011	21947012
genetic bioaugmentation as an effective method for in situ bioremediation: functionality of catabolic plasmids following conjugal transfers.	genetic bioaugmentation is an in situ bioremediation method that stimulates horizontal transfer of catabolic plasmids between exogenous donor cells and indigenous bacteria to increase the biodegradation potential of contaminants. a critical outcome of genetic bioaugmentation is the expression of an active catabolic phenotype upon plasmid conjugation. using a pww0-derivative tol plasmid, we showed that certain genetic characteristics of the recipient bacteria, including genomic guanine-cytosine ( ...	2012	22705839
increasing signal specificity of the tol network of pseudomonas putida mt-2 by rewiring the connectivity of the master regulator xylr.	prokaryotic transcription factors (tfs) that bind small xenobiotic molecules (e.g., tfs that drive genes that respond to environmental pollutants) often display a promiscuous effector profile for analogs of the bona fide chemical signals. xylr, the master tf for expression of the m-xylene biodegradation operons encoded in the tol plasmid pww0 of pseudomonas putida, responds not only to the aromatic compound but also, albeit to a lesser extent, to many other aromatic compounds, such as 3-methylbe ...	2012	23071444
impacts of organic carbon availability and recipient bacteria characteristics on the potential for tol plasmid genetic bioaugmentation in soil slurries.	the effectiveness of genetic bioaugmentation relies on efficient plasmid transfer between donor and recipient cells as well as the plasmid's phenotype in the recipient cell. in the present study, the effects of varying organic carbon substrates, initial recipient-to-donor cell density ratios, and mixtures of known recipient bacterial strains on the conjugation and function of a tol plasmid were tested in sterile soil slurry batch reactors. the presence of soil organic carbon was sufficient in en ...	2012	22743182
stochasticity of tol plasmid catabolic promoters sets a bimodal expression regime in pseudomonas putida mt-2 exposed to m-xylene.	the expression dynamics of the catabolic promoters of the tol plasmid pww0 has been examined in single cells of pseudomonas putida mt-2 exposed to m-xylene. to this end, we employed an á la carte bi-cistronic gfp-lacz reporter system for generating monocopy transcriptional fusions to each of the four promoters (pr, ps, pu and pm) of the regulatory network. whereas expression of xylr (the master regulatory gene of the tol system) behaved in a unimodal fashion, the activation of pu and pm displaye ...	2012	22845424
functionality of the tol plasmid under varying environmental conditions following conjugal transfer.	conjugation of catabolic plasmids in contaminated environments is a naturally occurring horizontal gene transfer phenomenon, which could be utilized in genetic bioaugmentation. the potentially important parameters for genetic bioaugmentation include gene regulation of transferred catabolic plasmids that may be controlled by the genetic characteristics of transconjugants as well as environmental conditions that may alter the expression of the contaminant-degrading phenotype. this study showed tha ...	2013	22367613
accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in pseudomonas putida kt2440.	accumulation of inorganic polyphosphate (polyp), a persistent trait throughout the whole tree of life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. the share of polyp in the tolerance of the soil bacterium pseudomonas putida kt2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations.	2013	23687963
complete nucleotide sequence of the self-transmissible tol plasmid pd2rt provides new insight into arrangement of toluene catabolic plasmids.	in the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pd2rt of pseudomonas migulae strain d2rt isolated from baltic sea water. the pd2rt is 129,894 base pairs in size with an average g+c content of 53.75%. a total of 135 open reading frames (orfs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. orfs encoding proteins with putative functions in stress response, tran ...	2013	24095800
the three-species consortium of genetically improved strains cupriavidus necator rw112, burkholderia xenovorans rw118, and pseudomonas pseudoalcaligenes rw120 grows with technical polychlorobiphenyl, aroclor 1242.	burkholderia xenovorans lb400, cupriavidus necator h850, and pseudomonas pseudoalcaligenes kf707 are bacterial strains able to mineralize biphenyl and to co-oxidize many of its halogenated derivatives (pcbs). only strain lb400 also mineralizes a few mono- and dichlorobiphenyls, due to the presence of a functioning chlorocatechol pathway. here, we used a tn5-based minitransposon shuttle system to chromosomically introduce genes tcbrcdef, encoding the chlorocatechol pathway into kf707, and genes c ...	2013	23658554
deepening tol and tou catabolic pathways of pseudomonas sp. ox1: cloning, sequencing and characterization of the lower pathways.	pseudomonas sp. ox1 is able to metabolize toluene and o-xylene through the tou catabolic pathway, whereas its mutant m1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the tol catabolic pathway. here we report the complete nucleotide sequence of the phe lower operon of the tou catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the tol catabolic pathway. dna sequence analysis shows the gene order within the operons to b ...	2013	23009925
a spatiotemporal view of plasmid loss in biofilms and planktonic cultures.	this commentary by madsen, burmølle, and sørensen discusses the article non-invasive in situ monitoring and quantification of tol plasmid segregational loss within pseudomonas putida biofilms by ma, katzenmeyer, and bryers. (2013. biotechnol bioeng. 110(11):2949-2958. doi: 10.1002/bit.24953).	2013	24014288
non-invasive in situ monitoring and quantification of tol plasmid segregational loss within pseudomonas putida biofilms.	methods for the detection of plasmid loss in natural environments have typically relied on replica plating, selective markers and pcr. however, these traditional methods have the limitations of low sensitivity, underestimation of specific cell populations, and lack of insightful data for non-homogeneous environments. we have developed a non-invasive microscopic analytical method to quantify local plasmid segregational loss from a bacterial population within a developing biofilm. the probability ...	2013	23633286
a second chromosomal copy of the cata gene endows pseudomonas putida mt-2 with an enzymatic safety valve for excess of catechol.	pseudomonas putida mt-2 harbours two different routes for catabolism of catechol, namely one meta pathway encoded by the xyl genes of the tol plasmid pww0 and one ortho pathway determined by the chromosomal ben and cat genes. p. putida mt-2 has a second chromosomal copy of the cata gene (named cata2) located downstream of the ben operon that encodes an additional catechol-1,2-dioxygenase. the metabolic and regulatory phenotypes of strains lacking one enzyme, the other and both of them in cells w ...	2014	24341396
the pww0 plasmid imposes a stochastic expression regime to the chromosomal ortho pathway for benzoate metabolism in pseudomonas putida.	environmental plasmids often expand the metabolic repertoire of bacteria that carry them, but they also interfere with the biochemical and genetic network of the host. the pww0 plasmid born by pseudomonas putida mt-2 encodes the tol pathway for degradation of toluene/m-xylene through production of intermediate compounds benzoate/3-methylbenzoate. these can be also recognized as substrates by the chromosomally encoded ben and cat gene products, thereby creating a manifest regulatory and biochemic ...	2014	25848660
widening functional boundaries of the σ(54) promoter pu of pseudomonas putida by defeating extant physiological constraints.	the extant layout of the σ(54) promoter pu, harboured by the catabolic tol plasmid, pww0, of pseudomonas putida is one of the most complex instances of endogenous and exogenous signal integration known in the prokaryotic domain. in this regulatory system, all signal inputs are eventually translated into occupation of the promoter sequence by either of two necessary components: the m-xylene responsive transcriptional factor xylr and the σ(54) containing form of rna polymerase. modelling of these ...	2015	25560994
freeing pseudomonas putida kt2440 of its proviral load strengthens endurance to environmental stresses.	2.6% of the genome of the soil bacterium pseudomonas putida kt2440 encodes phage-related functions, but the burden of such opportunistic dna on the host physiology is unknown. each of the four apparently complete prophages borne by this strain was tested for stability, spontaneous excision and ability to cause lysis under various stressing conditions. while prophages p3 (pp2266-pp2297) and p4 (pp1532-1584) were discharged from the genome at a detectable rate, their induction failed otherwise to ...	2015	24762028
chemical reactivity drives spatiotemporal organisation of bacterial metabolism.	in this review, we examine how bacterial metabolism is shaped by chemical constraints acting on the material and dynamic layout of enzymatic networks and beyond. these are moulded not only for optimisation of given metabolic objectives (e.g. synthesis of a particular amino acid or nucleotide) but also for curbing the detrimental reactivity of chemical intermediates. besides substrate channelling, toxicity is avoided by barriers to free diffusion (i.e. compartments) that separate otherwise incomp ...	2015	25227915
pyridine nucleotide transhydrogenases enable redox balance of pseudomonas putida during biodegradation of aromatic compounds.	the metabolic versatility of the soil bacterium pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. biodegradation of aromatics occurs via the pathway encoded in the archetypal tol plasmid pww0, yet the effect of running such oxidative route on redox balance against the background metabolism of p. putida remains unexplored. to answer this question, the activity of pyridine nucleotide transhydrogenases (that ca ...	2016	27348295
transcriptional kinetics of the cross-talk between the ortho-cleavage and tol pathways of toluene biodegradation in pseudomonas putida mt-2.	the tol plasmid promoters are activated by toluene leading to gene expression responsible for the degradation of the environmental signal. benzoate is formed as an intermediate, activating the benr protein of the chromosomal ortho-cleavage pathway that up-regulates the chromosomal pbena promoter and the tol pm promoter resulting in cross-talk between the two networks. herein, the transcriptional kinetics of the pbenr and pbena promoters in conjunction with tol promoters was monitored by real-tim ...	2016	27046069
high-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of pseudomonas putida mt-2.	pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the tol plasmid pww0 along with other chromosomally encoded activities. tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. all xyl sequences were transcribed in response to aromatic substrates and the 3'-ter ...	2016	26373670
the rna chaperone hfq enables the environmental stress tolerance super-phenotype of pseudomonas putida.	the natural physiological regime of the soil bacterium pseudomonas putida involves incessant exposure to endogenous metabolic conflicts and environmental physicochemical insults. yet, the role of assisted small rna-mrna pairing in the stress tolerance super-phenotype that is the trademark of this bacterium has not been accredited. we have thoroughly explored the physiological consequences -in particular those related to exogenous stress - of deleting the hfq gene of p. putida, which encodes the ...	2016	26373442
the impact of succinate trace on pww0 and ortho-cleavage pathway transcription in pseudomonas putida mt-2 during toluene biodegradation.	toluene is a pollutant catabolised through the interconnected pww0 (tol) and ortho-cleavage pathways of pseudomonas putida mt-2, while upon succinate and toluene mixtures introduction in batch cultures grown on m9 medium, succinate was previously reported as non-repressing. the effect of a 40 times lower succinate concentration, as compared to literature values, was explored through systematic real-time qpcr monitoring of transcriptional kinetics of the key tol pu, pm and ortho-cleavage pbenr, p ...	2017	28347959
the xyls/pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.	the xyls/pm regulator/promoter system originating from the pseudomonas putida tol plasmid pww0 is widely used for regulated low- and high-level recombinant expression of genes and gene clusters in escherichia coli and other bacteria. induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose-dependent manner and are typically not metabolized by the host cells. combinatorial mutagenesis and selection using th ...	2017	28276630