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1.  CRISPR adaptation biases explain preference for acquisition of foreign DNA 
Nature  2015;520(7548):505-510.
In the process of CRISPR adaptation, short pieces of DNA (“spacers”) are acquired from foreign elements and integrated into the CRISPR array. It so far remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of “self” is mediated by the RecBCD dsDNA break repair complex. Our results suggest that in E. coli, acquisition of new spacers depends on RecBCD-mediated processing of dsDNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers from both high copy plasmids and phages.
PMCID: PMC4561520  PMID: 25874675
2.  Different approaches for using bacteriophages against antibiotic-resistant bacteria 
Bacteriophage  2014;4:e28491.
Bacterial resistance to antibiotics is an emerging threat requiring urgent solutions. Ever since their discovery, lytic bacteriophages have been suggested as therapeutic agents, but their application faces various obstacles: sequestration of the phage by the spleen and liver, antibodies against the phage, narrow host range, poor accessibility to the infected tissue, and bacterial resistance. Variations on bacteriophage use have been suggested, such as temperate phages as gene-delivery vehicles into pathogens. This approach, which is proposed to sensitize pathogens residing on hospital surfaces and medical personnel's skin, and its prospects are described in this addendum. Furthermore, phage-encoded products have been proposed as weapons against antibiotic resistance in bacteria. We describe a new phage protein which was identified during basic research into T7 bacteriophages. This protein may serendipitously prove useful for treating antibiotic-resistant pathogens. We believe that further basic research will lead to novel strategies in the fight against antibiotic-resistant bacteria.
PMCID: PMC3956485  PMID: 24653944
temperate bacteriophage; sensitizing gene; lysin; host takeover; bacterial division
3.  Translation- and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae 
Molecular Biology of the Cell  2013;24(19):3069-3084.
Although mRNAs encoding secreted and membrane proteins are believed to associate with the ER only upon translation, they access the membrane independently of both translational control and the signal recognition particle. Thus, alternate paths exist for RNA delivery to and retention at the ER.
mRNAs encoding secreted/membrane proteins (mSMPs) are believed to reach the endoplasmic reticulum (ER) in a translation-dependent manner to confer protein translocation. Evidence exists, however, for translation- and signal recognition particle (SRP)–independent mRNA localization to the ER, suggesting that there are alternate paths for RNA delivery. We localized endogenously expressed mSMPs in yeast using an aptamer-based RNA-tagging procedure and fluorescence microscopy. Unlike mRNAs encoding polarity and secretion factors that colocalize with cortical ER at the bud tip, mSMPs and mRNAs encoding soluble, nonsecreted, nonpolarized proteins localized mainly to ER peripheral to the nucleus (nER). Synthetic nontranslatable uracil-rich mRNAs were also demonstrated to colocalize with nER in yeast. This mRNA–ER association was verified by subcellular fractionation and reverse transcription-PCR, single-molecule fluorescence in situ hybridization, and was not inhibited upon SRP inactivation. To better understand mSMP targeting, we examined aptamer-tagged USE1, which encodes a tail-anchored membrane protein, and SUC2, which encodes a soluble secreted enzyme. USE1 and SUC2 mRNA targeting was not abolished by the inhibition of translation or removal of elements involved in translational control. Overall we show that mSMP targeting to the ER is both translation- and SRP-independent, and regulated by cis elements contained within the message and trans-acting RNA-binding proteins (e.g., She2, Puf2).
PMCID: PMC3784381  PMID: 23904265
4.  Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli 
Nucleic Acids Research  2012;40(12):5569-5576.
The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.
PMCID: PMC3384332  PMID: 22402487
5.  Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed 
mBio  2010;1(2):e00020-10.
The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed.
The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited in vitro studies, it is believed that the E. coli SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several in vivo studies have suggested otherwise. To address this issue further in vivo, we utilized unbalanced conditions under which E. coli simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the E. coli SRP is capable of regulating membrane protein synthesis and demonstrate that the M domain of Ffh mediates this activity. These results thus open the way for mechanistic characterization of this quality control process in bacteria.
PMCID: PMC2921155  PMID: 20714446
6.  Membrane Protein Biogenesis in Ffh- or FtsY-Depleted Escherichia coli 
PLoS ONE  2010;5(2):e9130.
The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically.
Methodology/Principal Findings
We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization.
Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.
PMCID: PMC2817740  PMID: 20161748
7.  Restoration of Gene Function by Homologous Recombination: from PCR to Gene Expression in One Step 
Applied and Environmental Microbiology  2004;70(12):7156-7160.
We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete λPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage λ Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding β-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.
PMCID: PMC535207  PMID: 15574912

Results 1-7 (7)