Publications by authors named "Donald A Morrison"

42 Publications

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

Mol Microbiol 2021 Mar 23. Epub 2021 Mar 23.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.
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http://dx.doi.org/10.1111/mmi.14718DOI Listing
March 2021

ComWΔ6 Stimulates Transcription of Pneumococcal Competence Genes .

Front Mol Biosci 2020 6;7:61. Epub 2020 May 6.

Department of Biological Sciences, The University of Illinois at Chicago, Chicago, IL, United States.

The alternative streptococcal σ-factor and master competence regulator, σ, stimulates transcription from competence promoters, As the only known alternative σ-factor in streptococci, σ expression is tightly controlled in each species and has a specific physiological role. Pneumococcal transformation also requires the DNA binding activity of ComW, a known σ activator and stabilizer. Mutations to the housekeeping σ factor, σ, partially alleviate the ComW requirement, suggesting that ComW is a key player in the σ factor swap during the pneumococcal competence response. However, there is no evidence of a direct ComW - RNA polymerase interaction. Furthermore, if and how ComW functions directly at combox promoters is still unknown. Here we report that a DNA-binding ComW variant, ComΔ6, can stimulate transcription from σ promoters .
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http://dx.doi.org/10.3389/fmolb.2020.00061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218084PMC
May 2020

Multiplex gene transfer by genetic transformation between isolated S. pneumoniae cells confined in microfluidic droplets.

Integr Biol (Camb) 2019 12;11(12):415-424

Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA.

Gene exchange via genetic transformation makes major contributions to antibiotic resistance of the human pathogen, Streptococcus pneumoniae (pneumococcus). The transfers begin when a pneumococcal cell, in a transient specialized physiological state called competence, attacks and lyses another cell, takes up fragments of the liberated DNA, and integrates divergent genes into its genome. Recently, it has been demonstrated that the pneumococcal cells can be enclosed in femtoliter-scale droplets for study of the transformation mechanism, offering the ability to characterize individual cell-cell interactions and overcome the limitations of current methods involving bulk mixed cultures. To determine the relevance and reliability of this new method for study of bacterial genetic transformation, we compared recombination events occurring in 44 recombinants recovered after competence-mediated gene exchange between pairs of cells confined in femtoliter-scale droplets vs. those occurring in exchanges in parallel bulk culture mixtures. The pattern of recombination events in both contexts exhibited the hallmarks of the macro-recombination exchanges previously observed within the more complex natural contexts of biofilms and long-term evolution in the human host.
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http://dx.doi.org/10.1093/intbio/zyz036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011181PMC
December 2019

HtrA-mediated selective degradation of DNA uptake apparatus accelerates termination of pneumococcal transformation.

Mol Microbiol 2019 10 29;112(4):1308-1325. Epub 2019 Aug 29.

Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China.

Natural transformation mediates horizontal gene transfer, and thereby promotes exchange of antibiotic resistance and virulence traits among bacteria. Streptococcus pneumoniae, the first known transformable bacterium, rapidly activates and then terminates the transformation state, but it is unclear how the bacterium accomplishes this rapid turn-around at the protein level. This work determined the transcriptomic and proteomic dynamics during the window of pneumococcal transformation. RNA sequencing revealed a nearly uniform temporal pattern of rapid transcriptional activation and subsequent shutdown for the genes encoding transformation proteins. In contrast, mass spectrometry analysis showed that the majority of transformation proteins were substantially preserved beyond the window of transformation. However, ComEA and ComEC, major components of the DNA uptake apparatus for transformation, were completely degraded at the end of transformation. Further mutagenesis screening revealed that the membrane-associated serine protease HtrA mediates selective degradation of ComEA and ComEC, strongly suggesting that breakdown of the DNA uptake apparatus by HtrA is an important mechanism for termination of pneumococcal transformation. Finally, our mutagenesis analysis showed that HtrA inhibits natural transformation of Streptococcus mitis and Streptococcus gordonii. Together, this work has revealed that HtrA regulates the level and duration of natural transformation in multiple streptococcal species.
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http://dx.doi.org/10.1111/mmi.14364DOI Listing
October 2019

The pneumococcal σ activator, ComW, is a DNA-binding protein critical for natural transformation.

J Biol Chem 2019 07 3;294(29):11101-11118. Epub 2019 Jun 3.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607

Natural genetic transformation via horizontal gene transfer enables rapid adaptation to dynamic environments and contributes to both antibiotic resistance and vaccine evasion among bacterial populations. In (pneumococcus), transformation occurs when cells enter competence, a transient state in which cells express the competence master regulator, SigX (σ), an alternative σ factor (σ), and a competence co-regulator, ComW. Together, ComW and σ facilitate expression of the genes required for DNA uptake and genetic recombination. SigX activity depends on ComW, as Δ cells transcribe late genes and transform at levels 10- and 10,000-fold below that of WT cells, respectively. Previous findings suggest that ComW functions during assembly of the RNA polymerase-σ holoenzyme to help promote transcription from σ-targeted promoters. However, it remains unknown how ComW facilitates holoenzyme assembly. As ComW seems to be unique to Gram-positive cocci and has no sequence similarity with known transcriptional activators, here we used Rosetta to generate an model of pneumococcal ComW's 3D-structure. Using this model as a basis for further biochemical, biophysical, and genetic investigations into the molecular features important for its function, we report that ComW is a predicted globular protein and that it interacts with DNA, independently of DNA sequence. We also identified conserved motifs in ComW and show that key residues in these motifs contribute to DNA binding. Lastly, we provide evidence that ComW's DNA-binding activity is important for transformation in pneumococcus. Our findings begin to fill the gaps in understanding how ComW regulates σ activity during bacterial natural transformation.
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http://dx.doi.org/10.1074/jbc.RA119.007571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643036PMC
July 2019

Competence in and Close Commensal Relatives: Mechanisms and Implications.

Front Cell Infect Microbiol 2019 3;9:94. Epub 2019 Apr 3.

Faculty of Dentistry, Institute of Oral Biology, University of Oslo, Oslo, Norway.

The mitis group of streptococci comprises species that are common colonizers of the naso-oral-pharyngeal tract of humans. and are close relatives and share ~60-80% of orthologous genes, but still present striking differences in pathogenic potential toward the human host. has long been recognized as a reservoir of antibiotic resistance genes for , as well as a source for capsule polysaccharide variation, leading to resistance and vaccine escape. Both species share the ability to become naturally competent, and in this context, competence-associated killing mechanisms such as fratricide are thought to play an important role in interspecies gene exchange. Here, we explore the general mechanism of natural genetic transformation in the two species and touch upon the fundamental clinical and evolutionary implications of sharing similar competence, fratricide mechanisms, and a large fraction of their genomic DNA.
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http://dx.doi.org/10.3389/fcimb.2019.00094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456647PMC
December 2019

Competence beyond Genes: Filling in the Details of the Pneumococcal Competence Transcriptome by a Systems Approach.

J Bacteriol 2019 07 10;201(13). Epub 2019 Jun 10.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.

DNA uptake by natural competence is a central process underlying the genetic plasticity, biology, and virulence of the human respiratory opportunistic pathogen A study reported in this issue (J. Slager, R. Aprianto, and J.-W. Veening, J. Bacteriol. 201:e00780-18, https://doi.org/10.1128/JB.00780-18) combined deep-genome annotation and high-resolution transcriptome analyses to considerably extend the previous model of temporal regulation of competence at the operon and component gene levels. That extended study also provides a playbook for updating, refining, and extending genomic data sets and making them publicly available.
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http://dx.doi.org/10.1128/JB.00238-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560134PMC
July 2019

Hidden Gems in the Transcriptome Maps of Competent Streptococci.

Front Mol Biosci 2018 4;5:116. Epub 2019 Jan 4.

Faculty of Dentistry, Institute of Oral Biology, University of Oslo, Oslo, Norway.

Natural transformation is regarded as an important mechanism in bacteria that allows for adaptation to different environmental stressors by ensuring genome plasticity. Since the discovery of this phenomenon in , remarkable progress has been made in the understanding of the molecular mechanisms and pathways coordinating this process. Recently, the advent of high-throughput sequencing allows the posing of questions that address the system at a larger scale but also allow for the creation of high-resolution maps of transcription. Thus, while much is already known about genetic competence in streptococci, recent studies continue to reveal intricate novel regulation pathways and components. In this perspective article, we highlight the use of transcriptional profiling and mapping as a valuable resource in the identification and characterization of "hidden gems" pertinent to the natural transformation system. Such strategies have recently been employed in a variety of different species. In , for example, genome editing combined with the power of promoter mapping and RNA-Seq allowed for the identification of a link between the ComCDE and the ComRS systems, a ComR positive feedback loop mediated by SigX, and the XrpA peptide, encoded within , which inhibits competence. In , a novel member of the competence regulon termed BriC was found to be directly under control of ComE and to promote biofilm formation and nasopharyngeal colonization but not competence. Together these new technologies enable us to discover new links and to revisit old pathways in the compelling study of natural genetic transformation.
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http://dx.doi.org/10.3389/fmolb.2018.00116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328492PMC
January 2019

Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets.

Lab Chip 2019 02;19(4):682-692

Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA.

Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail. To overcome these limitations, we have developed a droplet microfluidic system for isolating individual episodes of bacterial transformation between two confined cells of pneumococcus. By encapsulating the cells in a 10 μm diameter aqueous droplet, we provide an improved experimental model of genetic transformation, as both participating cells can be identified, and the released DNA is spatially restricted near the attacking strain. Specifically, the bacterial cells, one rifampicin (R) resistant, the other novobiocin (N) and spectinomycin (S) resistant were encapsulated in droplets carried by the fluorinated oil FC-40 with 5% surfactant and allowed to carry out competence-specific attack and DNA uptake (and consequently gain antibiotic resistances) within the droplets. The droplets were then broken, and recombinants were recovered by selective plating with antibiotics. The new droplet system encapsulated 2 or more cells in a droplet with a probability up to 71%, supporting gene transfer rates comparable to standard mixtures of unconfined cells. Thus, confinement in droplets allows characterization of natural genetic transformation during a strictly defined interaction between two confined cells.
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http://dx.doi.org/10.1039/c8lc01367eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487891PMC
February 2019

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in Streptococcus pneumoniae.

PLoS Genet 2018 06 13;14(6):e1007410. Epub 2018 Jun 13.

Department of Epidemiology, Harvard TH Chan School of Public Health, Boston, United States of America.

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent 'target' cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact.
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http://dx.doi.org/10.1371/journal.pgen.1007410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016952PMC
June 2018

Flavin Reductase Contributes to Pneumococcal Virulence by Protecting from Oxidative Stress and Mediating Adhesion and Elicits Protection Against Pneumococcal Challenge.

Sci Rep 2018 01 10;8(1):314. Epub 2018 Jan 10.

The Shraga Segal Department of Microbiology and Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Pneumococcal flavin reductase (FlaR) is known to be cell-wall associated and possess age dependent antigenicity in children. This study aimed at characterizing FlaR and elucidating its involvement in pneumococcal physiology and virulence. Bioinformatic analysis of FlaR sequence identified three-conserved cysteine residues, suggesting a transition metal-binding capacity. Recombinant FlaR (rFlaR) bound Fe and exhibited FAD-dependent NADP-reductase activity, which increased in the presence of cysteine or excess Fe and inhibited by divalent-chelating agents. flaR mutant was highly susceptible to HO compared to its wild type (WT) and complemented strains, suggesting a role for FlaR in pneumococcal oxidative stress resistance. Additionally, flaR mutant demonstrated significantly decreased mice mortality following intraperitoneal infection. Interestingly, lack of FlaR did not affect the extent of phagocytosis by primary mouse peritoneal macrophages but reduced adhesion to A549 cells compared to the WT and complemented strains. Noteworthy are the findings that immunization with rFlaR elicited protection in mice against intraperitoneal lethal challenge and anti-FlaR antisera neutralized bacterial virulence. Taken together, FlaR's roles in pneumococcal physiology and virulence, combined with its lack of significant homology to human proteins, point towards rFlaR as a vaccine candidate.
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http://dx.doi.org/10.1038/s41598-017-18645-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762878PMC
January 2018

A positive feedback loop mediated by Sigma X enhances expression of the streptococcal regulator ComR.

Sci Rep 2017 07 20;7(1):5984. Epub 2017 Jul 20.

Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.

Natural transformation is used by bacteria to take up DNA from their surroundings and incorporate it into their genomes. Streptococci do so during a transient period of competence, triggered by pheromones that they produce, secrete and sense under conditions influenced by the environment. In Streptococcus mutans, Streptococcus suis, and species of the bovis, salivarius and pyogenic groups of streptococci, the pheromone XIP is sensed by the intra-cellular regulator ComR, that in turn activates the transcription of comS, encoding the XIP precursor, and of sigX, encoding the only known alternative sigma factor in streptococci. Although induction of comR during competence has been known for more than fifteen years, the mechanism regulating its expression remains unidentified. By a combination of directional RNA-sequencing, optimal competence conditions, stepwise deletions and marker-less genome editing, we found that SigX is the missing link in overproduction of ComR. In the absence of comR induction, both sigX expression and transformation were significantly reduced. Placing comR and comS transcripts under the control of different regulators so as to form two interlocked positive feedback circuits may enable S. mutans to fine-tune the kinetics and magnitude of the competence response according to their need.
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http://dx.doi.org/10.1038/s41598-017-04768-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519730PMC
July 2017

Markerless Genome Editing in Competent Streptococci.

Methods Mol Biol 2017 ;1537:233-247

Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois at Chicago, 845 West Taylor Street, (MC 066), Chicago, IL, 60607, USA.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.
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http://dx.doi.org/10.1007/978-1-4939-6685-1_14DOI Listing
January 2018

Natural Transformation of Oral Streptococci by Use of Synthetic Pheromones.

Methods Mol Biol 2017 ;1537:219-232

Department of Oral Biology, Faculty of Dentistry, University of Oslo, P.O. Box 1052, Blindern, Oslo, 0316, Norway.

The discovery that Streptococcus pneumoniae uses a competence-stimulating peptide (CSP) to induce competence for natural transformation, and that other species of the mitis and the anginosus streptococcal groups use a similar system, has expanded the tools to explore gene function and regulatory pathways in streptococci. Two other classes of pheromones have been discovered since then, comprising the bacteriocin-inducing peptide class found in Streptococcus mutans (also named CSP, although different from the former) and the SigX-inducing peptides (XIP), in the mutans, salivarius, bovis, and pyogenes groups of streptococci. The three classes of peptide pheromones can be ordered from peptide synthesis services at affordable prices, and used in transformation assays to obtain competent cultures consistently at levels usually higher than those achieved during spontaneous competence. In this chapter, we present protocols for natural transformation of oral streptococci that are based on the use of synthetic pheromones, with examples of conditions optimized for transformation of S. mutans and Streptococcus mitis.
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http://dx.doi.org/10.1007/978-1-4939-6685-1_13DOI Listing
January 2018

Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species.

PLoS Pathog 2016 Dec 1;12(12):e1005979. Epub 2016 Dec 1.

Center for Structural Biology, Research Resources Center, University of Illinois at Chicago, Chicago, IL, United States of America.

Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS) that is processed (XIP), secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a 'test-bed' assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP peptides. Together, our results not only provide a model for XIP recognition and specificity, but also allow for the prediction of novel XIP peptides that induce ComR activity.
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http://dx.doi.org/10.1371/journal.ppat.1005979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131902PMC
December 2016

Overcoming the Barrier of Low Efficiency during Genetic Transformation of Streptococcus mitis.

Front Microbiol 2016 5;7:1009. Epub 2016 Jul 5.

Department of Oral Biology, Faculty of Dentistry, University of Oslo Oslo, Norway.

Objective: Streptococcus mitis is a predominant oral colonizer, but difficulties in genetic manipulation of this species have hampered our understanding of the mechanisms it uses for colonization of oral surfaces. The aim of this study was to reveal optimal conditions for natural genetic transformation in S. mitis and illustrate its application in direct genome editing.

Methods: Luciferase reporter assays were used to assess gene expression of the alternative sigma factor (σ(X)) in combination with natural transformation experiments to evaluate the efficiency by which S. mitis activates the competence system and incorporates exogenous DNA. Optimal amounts and sources of donor DNA (chromosomal, amplicon, or replicative plasmid), concentrations of synthetic competence-stimulating peptide, and transformation media were assessed.

Results: A semi-defined medium showed much improved results for response to the competence stimulating peptide when compared to rich media. The use of a donor amplicon with large homology flanking regions also provided higher transformation rates. Overall, an increase of transformation efficiencies from 0.001% or less to over 30% was achieved with the developed protocol. We further describe the construction of a markerless mutant based on this high efficiency strategy.

Conclusion: We optimized competence development in S. mitis, by use of semi-defined medium and appropriate concentrations of synthetic competence factor. Combined with the use of a large amplicon of donor DNA, this method allowed easy and direct editing of the S. mitis genome, broadening the spectrum of possible downstream applications of natural transformation in this species.
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http://dx.doi.org/10.3389/fmicb.2016.01009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932118PMC
July 2016

Competence for Genetic Transformation in Streptococcus pneumoniae: Mutations in σA Bypass the ComW Requirement for Late Gene Expression.

J Bacteriol 2016 09 11;198(17):2370-8. Epub 2016 Aug 11.

Molecular, Cell, and Developmental Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

Unlabelled: Streptococcus pneumoniae is able to integrate exogenous DNA into its genome by natural genetic transformation. Transient accumulation of high levels of the only S. pneumoniae alternative σ factor is insufficient for development of full competence without expression of a second competence-specific protein, ComW. The ΔcomW mutant is 10(4)-fold deficient in the yield of recombinants, 10-fold deficient in the amount of σ(X) activity, and 10-fold deficient in the amount of σ(X) protein. The critical role of ComW during transformation can be partially obviated by σ(A) mutations clustered on surfaces controlling affinity for core RNA polymerase (RNAP). While strains harboring σ(A) mutations in the comW mutant background were transforming at higher rates, the mechanism of transformation restoration was not clear. To investigate the mechanism of transformation restoration, we measured late gene expression in σ(A)* suppressor strains. Restoration of late gene expression was observed in ΔcomW σ(A)* mutants, indicating that a consequence of the σ(A)* mutations is, at least, to restore σ(X) activity. Competence kinetics were normal in ΔcomW σ(A)* strains, indicating that strains with restored competence exhibit the same pattern of transience as wild-type (WT) strains. We also identified a direct interaction between ComW and σ(X) using the yeast two-hybrid (Y2H) assay. Taken together, these data are consistent with the idea that ComW increases σ(X) access to core RNAP, pointing to a direct role of ComW in σ factor exchange during genetic transformation. However, the lack of late gene shutoff in ΔcomW mutants also points to a potential new role for ComW in competence shutoff.

Importance: The sole alternative sigma factor of the streptococci, SigX, regulates development of competence for genetic transformation, a widespread mechanism of adaptation by horizontal gene transfer in this genus. The transient appearance of this sigma factor is strictly controlled at the levels of transcription and stability. This report shows that it is also controlled at the point of its substitution for SigA by a second transient competence-specific protein, ComW.
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http://dx.doi.org/10.1128/JB.00354-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984550PMC
September 2016

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence.

Infect Immun 2016 06 24;84(6):1887-1901. Epub 2016 May 24.

Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing, China

Natural genetic transformation of Streptococcus pneumoniae, an important human pathogen, mediates horizontal gene transfer for the development of drug resistance, modulation of carriage and virulence traits, and evasion of host immunity. Transformation frequency differs greatly among pneumococcal clinical isolates, but the molecular basis and biological importance of this interstrain variability remain unclear. In this study, we characterized the transformation frequency and other associated phenotypes of 208 S. pneumoniae clinical isolates representing at least 30 serotypes. While the vast majority of these isolates (94.7%) were transformable, the transformation frequency differed by up to 5 orders of magnitude between the least and most transformable isolates. The strain-to-strain differences in transformation frequency were observed among many isolates producing the same capsule types, indicating no general association between transformation frequency and serotype. However, a statistically significant association was observed between the levels of transformation and colonization fitness/virulence in the hypertransformable isolates. Although nontransformable mutants of all the selected hypertransformable isolates were significantly attenuated in colonization fitness and virulence in mouse infection models, such mutants of the strains with relatively low transformability had no or marginal fitness phenotypes under the same experimental settings. This finding strongly suggests that the pneumococci with high transformation capability are "addicted" to a "hypertransformable" state for optimal fitness in the human host. This work has thus provided an intriguing hint for further investigation into how the competence system impacts the fitness, virulence, and other transformation-associated traits of this important human pathogen.
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http://dx.doi.org/10.1128/IAI.00097-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907133PMC
June 2016

Competence for genetic transformation in Streptococcus pneumoniae: mutations in σA bypass the comW requirement.

J Bacteriol 2014 Nov 11;196(21):3724-34. Epub 2014 Aug 11.

Molecular, Cell, and Developmental Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

Competence for genetic transformation in the genus Streptococcus depends on an alternative sigma factor, σ(X), for coordinated synthesis of 23 proteins, which together establish the X state by permitting lysis of incompetent streptococci, uptake of DNA fragments, and integration of strands of that DNA into the resident genome. Initiation of transient accumulation of high levels of σ(X) is coordinated between cells by transcription factors linked to peptide pheromone signals. In Streptococcus pneumoniae, elevated σ(X) is insufficient for development of full competence without coexpression of a second competence-specific protein, ComW. ComW, shared by eight species in the Streptococcus mitis and Streptococcus anginosus groups, is regulated by the same pheromone circuit that controls σ(X), but its role in expression of the σ(X) regulon is unknown. Using the strong, but not absolute, dependence of transformation on comW as a selective tool, we collected 27 independent comW bypass mutations and mapped them to 10 single-base transitions, all within rpoD, encoding the primary sigma factor subunit of RNA polymerase, σ(A). Eight mapped to sites in rpoD region 4 that are implicated in interaction with the core β subunit, indicating that ComW may act to facilitate competition of the alternative sigma factor σ(X) for access to core polymerase.
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http://dx.doi.org/10.1128/JB.01933-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248798PMC
November 2014

Control of competence for DNA transformation in streptococcus suis by genetically transferable pherotypes.

PLoS One 2014 26;9(6):e99394. Epub 2014 Jun 26.

Host-Microbe Interactomics, Animal Sciences, Wageningen University, Wageningen, The Netherlands.

Here we show that S. suis, a major bacterial pathogen of pigs and emerging pathogen in humans responds to a peptide pheromone by developing competence for DNA transformation. This species does not fall within any of the phylogenetic clusters of streptococci previously shown to regulate competence via peptide pheromones suggesting that more species of streptococci may be naturally competent. Induction of competence was dependent on ComX, a sigma factor that controls the streptococcal late competence regulon, extracellular addition of a comX-inducing peptide (XIP), and ComR, a regulator of comX. XIP was identified as an N-terminally truncated variant of ComS. Different comS alleles are present among strains of S. suis. These comS alleles are not functionally equivalent and appear to operate in conjuction with a cognate ComR to regulate comX through a conserved comR-box promoter. We demonstrate that these 'pherotypes' can be genetically transferred between strains, suggesting that similar approaches might be used to control competence induction in other lactic acid bacteria that lack ComR/ComS homologues but possess comX and the late competence regulon. The approaches described in this paper to identify and optimize peptide-induced competence may also assist other researchers wishing to identify natural competence in other bacteria. Harnessing natural competence is expected to accelerate genetic research on this and other important streptococcal pathogens and to allow high-throughput mutation approaches to be implemented, opening up new avenues for research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099394PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072589PMC
October 2015

Exit from competence for genetic transformation in Streptococcus pneumoniae is regulated at multiple levels.

PLoS One 2013 22;8(5):e64197. Epub 2013 May 22.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

Development of natural competence in S. pneumoniae entails coordinated expression of two sets of genes. Early gene expression depends on ComE, a response regulator activated by the pheromone CSP (Competence-Stimulating-Peptide). Subsequently, an early gene product (the alternative sigma factor ComX) activates expression of late genes, establishing the competent state. Expression of both sets of genes is transient, rapidly shut off by a mechanism that depends on the late gene, dprA. It has been thought that the rapid shutoff of late gene expression is the combined result of auto-inhibition of ComE and the instability of ComX. However, this explanation seems incomplete, because of evidence for ComX-dependent repressor(s) that might also be important for shutting off the response to CSP and identifying dprA as such a gene. We screened individual late gene mutants to investigate further the roles of ComX-dependent genes in competence termination. A ΔdprA mutant displayed a prolonged late gene expression pattern, whereas mutants lacking cbpD, cibABC, cglEFG, coiA, ssbB, celAB, cclA, cglABCD, cflAB, or radA, exhibited a wild-type temporal expression pattern. Thus, no other gene than dprA was found to be involved in shutoff. DprA limits the amounts of ComX and another early gene product, ComW, by restriction of early gene expression rather than by promoting proteolysis. To ask if DprA also affects late gene expression, we decoupled late gene expression from early gene regulation. Because DprA did not limit ComX activity under these conditions, we also conclude that ComX activity is limited by another mechanism not involving DprA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064197PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661451PMC
January 2014

NADH oxidase functions as an adhesin in Streptococcus pneumoniae and elicits a protective immune response in mice.

PLoS One 2013 8;8(4):e61128. Epub 2013 Apr 8.

Pediatric Infectious Disease Unit, Soroka University Medical Center, Beer Sheva, Israel.

The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX) was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061128PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620118PMC
October 2013

Competence for natural genetic transformation in the Streptococcus bovis group streptococci S. infantarius and S. macedonicus.

J Bacteriol 2013 Jun 29;195(11):2612-20. Epub 2013 Mar 29.

Deptartment of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.

Natural genetic transformation is common among many species of the genus Streptococcus, but it has never, or rarely, been reported for the Streptococcus pyogenes and S. bovis groups of species, even though many streptococcal competence genes and the competence regulators SigX, ComR, and ComS are well conserved in both groups. To explore the incidence of competence in the S. bovis group, 25 isolates of S. infantarius and S. macedonicus were surveyed by employing culture in chemically defined media devoid of peptide nutrients and treatment with synthetic candidate pheromone peptides predicted from the sequence of the gene comS. Approximately half of strains examined were transformable, many transforming at high rates comparable to those for the well-characterized streptococcal natural transformation systems. In S. infantarius, nanomolar amounts of the synthetic pheromone LTAWWGL induced robust but transient competence in high-density cultures, but mutation of the ComRS locus abolished transformation. We conclude that at least these two species of the S. bovis group retain a robust system of natural transformation regulated by a ComRS pheromone circuit and the alternative sigma factor SigX and infer that transformation is even more common among the streptococci than has been recognized. The tools presented here will facilitate targeted genetic manipulation in this group of streptococci.
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http://dx.doi.org/10.1128/JB.00230-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3676052PMC
June 2013

One if by land, two if by sea: signalling to the ranks with CSP and XIP.

Mol Microbiol 2012 Oct 19;86(2):241-5. Epub 2012 Sep 19.

Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, IL 60607, USA.

In many streptococci, quorum sensing utilizes secreted, linear peptides that engage cognate receptors to coordinate gene expression among members of a local population. Streptococcus mutans employs the secreted peptides CSP and XIP to stimulate production of antimicrobial bacteriocins and to induce development of competence for genetic transformation. Recent progress in the field reveals that these pathways not only monitor the presence of signal emitters but also sense environmental factors. Both kinds of information are integrated by regulatory networks that then generate multiple outcomes, even among parallel cells growing in identical conditions. In this issue of Molecular Microbiology, Son and co-workers investigate how two medium types shape cellular responses to CSP and XIP pheromones in individuals across a population. Their findings characterize restrictive properties of media differing in peptidic fragment content and reveal unusual signalling properties that contribute to bimodal responses of gene expression.
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http://dx.doi.org/10.1111/mmi.12029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468940PMC
October 2012

The cryptic competence pathway in Streptococcus pyogenes is controlled by a peptide pheromone.

J Bacteriol 2012 Sep 22;194(17):4589-600. Epub 2012 Jun 22.

Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA.

Horizontal gene transfer is an important means of bacterial evolution that is facilitated by transduction, conjugation, and natural genetic transformation. Transformation occurs after bacterial cells enter a state of competence, where naked DNA is acquired from the extracellular environment. Induction of the competent state relies on signals that activate master regulators, causing the expression of genes involved in DNA uptake, processing, and recombination. All streptococcal species contain the master regulator SigX and SigX-dependent effector genes required for natural genetic transformation; however, not all streptococcal species have been shown to be naturally competent. We recently demonstrated that competence development in Streptococcus mutans requires the type II ComRS quorum-sensing circuit, comprising an Rgg transcriptional activator and a novel peptide pheromone (L. Mashburn-Warren, D. A. Morrison, and M. J. Federle, Mol. Microbiol. 78:589-606, 2010). The type II ComRS system is shared by the pyogenic, mutans, and bovis streptococci, including the clinically relevant pathogen Streptococcus pyogenes. Here, we describe the activation of sigX by a small-peptide pheromone and an Rgg regulator of the type II ComRS class. We confirm previous reports that SigX is functional and able to activate sigX-dependent gene expression within the competence regulon, and that SigX stability is influenced by the cytoplasmic protease ClpP. Genomic analyses of available S. pyogenes genomes revealed the presence of intact genes within the competence regulon. While this is the first report to show natural induction of sigX, S. pyogenes remained nontransformable under laboratory conditions. Using radiolabeled DNA, we demonstrate that transformation is blocked at the stage of DNA uptake.
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http://dx.doi.org/10.1128/JB.00830-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3415498PMC
September 2012

Development of competence for genetic transformation of Streptococcus mutans in a chemically defined medium.

J Bacteriol 2012 Aug 18;194(15):3774-80. Epub 2012 May 18.

Laboratory for Molecular Biology, Department of Biological Sciences, College of Liberal Arts and Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.

Streptococcus mutans develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun. 27:444, 1980). We confirmed a previous report that in this medium comS mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS(11-17)) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that comS(+) (but not comS mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a comS mutation at levels equivalent to 1 μM synthetic ComS(11-17).
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http://dx.doi.org/10.1128/JB.00337-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416567PMC
August 2012

A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator.

Mol Microbiol 2010 Nov 14;78(3):589-606. Epub 2010 Sep 14.

Center for Pharmaceutical Biotechnology, College of Pharmacy, The University of Illinois at Chicago, Chicago, IL 60607, USA.

All streptococcal genomes encode the alternative sigma factor SigX and 21 SigX-dependent proteins required for genetic transformation, yet no pyogenic streptococci are known to develop competence. Resolving this paradox may depend on understanding the regulation of sigX. We report the identification of a regulatory circuit linked to the sigX genes of mutans, pyogenic, and bovis streptococci that uses a novel small, double-tryptophan-containing sigX-inducing peptide (XIP) pheromone. In all three groups, the XIP gene (comS), and sigX have identical, non-canonical promoters consisting of 9 bp inverted repeats separated from a -10 hexamer by 19 bp. comS is adjacent to a gene encoding a putative transcription factor of the Rgg family and is regulated by its product, which we designate ComR. Deletion of comR or comS in Streptococcus mutans abolished transformability, as did deletion of the oligopeptide permease subunit oppD, suggesting that XIP is imported. Providing S. mutans with synthetic fragments of ComS revealed that seven C-terminal residues, including the WW motif, cause robust induction of both sigX and the competent state. We propose that this circuit is the proximal regulator of sigX in S. mutans, and we infer that it controls competence in a parallel way in all pyogenic and bovis streptococci.
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http://dx.doi.org/10.1111/j.1365-2958.2010.07361.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058796PMC
November 2010

A self-deleting Cre-lox-ermAM cassette, Cheshire, for marker-less gene deletion in Streptococcus pneumoniae.

J Microbiol Methods 2009 Dec 20;79(3):353-7. Epub 2009 Oct 20.

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.

Although targeted mutagenesis of Streptococcus pneumoniae is readily accomplished with the aid of natural genetic transformation and chimeric donor DNA constructs assembled in vitro, the drug resistance markers often employed for selection of recombinant products can themselves be undesirable by-products of the genetic manipulation. A new cassette carrying the erythromycin-resistance marker ermAM that can be used as a temporary marker for selection of desired recombinants is described. The cassette may subsequently be removed at will by virtue of an embedded fucose-regulated Cre recombinase gene and terminal lox66 and lox71 Cre recognition sites, with retention of 34bp from the cassette as an inert residual double-mutant lox72 site.
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http://dx.doi.org/10.1016/j.mimet.2009.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2814315PMC
December 2009

spr1630 is responsible for the lethality of clpX mutations in Streptococcus pneumoniae.

J Bacteriol 2009 Aug 22;191(15):4888-95. Epub 2009 May 22.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.

The Clp protease ATPase subunit and chaperone ClpX is dispensable in some bacteria, but it is thought to be essential in others, including streptococci and lactococci. We confirm that clpX is essential in the Rx strain of Streptococcus pneumoniae but show that the requirement for clpX can be relieved by point mutations, frame shifts, or deletion of the gene spr1630, which is found in many isolates of S. pneumoniae. Homologs occur frequently in Staphylococcus aureus as well as in a few strains of Listeria monocytogenes, Lactobacillus johnsonii, and Lactobacillus rhamnosus. In each case, the spr1630 homolog is accompanied by a putative transcriptional regulator with an HTH DNA binding motif. In S. pneumoniae, the spr1630-spr1629 gene pair, accompanied by a RUP element, occurs as an island inserted between the trpA and cclA genes in 15 of 22 sequenced genomes.
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http://dx.doi.org/10.1128/JB.00285-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715704PMC
August 2009

Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.

J Bacteriol 2009 May 13;191(10):3359-66. Epub 2009 Mar 13.

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, USA.

Competence for genetic transformation in Streptococcus pneumoniae is a transient physiological state whose development is coordinated by a peptide pheromone (CSP) and its receptor, which activates transcription of two downstream genes, comX and comW, and 15 other "early" genes. ComX, a transient alternative sigma factor, drives transcription of "late" genes, many of which are essential for transformation. In vivo, ComW both stabilizes ComX against proteolysis by the ClpE-ClpP protease and stimulates its activity. Interestingly, stabilization of ComX by deletion of the gene encoding the ClpP protease did not extend the period of competence. We considered the hypothesis that the rapid decay of competence arises from a rapid loss of ComW and thus of its ComX stimulating activity, so that ComX might persist but lose its transcriptional activity. Western analysis revealed that ComW is indeed a transient protein, which is also stabilized by deletion of the gene encoding the ClpP protease. However, stabilizing both ComX and ComW did not prolong either ComX activity or the period of transformation, indicating that termination of the transcriptional activity of ComX is not dependent on proteolysis of ComW.
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http://dx.doi.org/10.1128/JB.01750-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2687157PMC
May 2009
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