Waksman Institute

Piscataway, NJ, United States

Waksman Institute

Piscataway, NJ, United States
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Tikhonov A.,Russian Academy of Sciences | Kazakov T.,Waksman Institute | Semenova E.,Waksman Institute | Serebryakova M.,Federal Agency for Health Care and Social Development | And 8 more authors.
Journal of Biological Chemistry | Year: 2010

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. Inside a sensitive cell, McC is processed by aminopeptidases, which release a nonhydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. The mccABCDE operon is sufficient for McC production and resistance of the producing cell to McC. An additional gene, mccF, which is adjacent to but not part of the mccABCDE operon, also provides resistance to exogenous McC. MccF is similar to Escherichia coli LdcA, an L,D-carboxypeptidase whose substrate is monomeric murotetrapeptide L-Ala-D-Glu-meso-A2pm-D-Ala or its UDP-activated murein precursor. The mechanism by which MccF provides McC resistance remained unknown. Here, we show that MccF detoxifies both intact and processed McC by cleaving an amide bond between the C-terminal aspartate and the nucleotide moiety. MccF also cleaves the same bond in nonhydrolyzable aminoacyl sulfamoyl adenosines containing aspartyl, glutamyl, and, to a lesser extent, seryl aminoacyl moieties but is ineffective against other aminoacyl adenylates. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Westra E.,Wageningen University | van Erp P.,Wageningen University | Kunne T.,Wageningen University | Wong S.,Wageningen University | And 14 more authors.
Molecular Cell | Year: 2012

The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg 2+-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization. © 2012 Elsevier Inc.


Westra E.R.,Wageningen University | Semenova E.,Waksman Institute | Datsenko K.A.,Waksman Institute | Datsenko K.A.,Purdue University | And 6 more authors.
PLoS Genetics | Year: 2013

Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism. © 2013 westra et al.


Pougach K.,Russian Academy of Sciences | Semenova E.,Waksman Institute | Bogdanova E.,Waksman Institute | Datsenko K.A.,Purdue University | And 4 more authors.
Molecular Microbiology | Year: 2010

CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ-phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently 'silent' E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection. © 2010 Blackwell Publishing Ltd.


Novikova M.,Russian Academy of Sciences | Kazakov T.,Russian Academy of Sciences | Vondenhoff G.H.,Waksman Institute | Vondenhoff G.H.,Rega Institute for Medical Research | And 8 more authors.
Journal of Biological Chemistry | Year: 2010

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. McC is excreted from producing cells by the MccC transporter. The residual McC that remainsin the producing cell can be processed by cellular amino-peptidases with the release of a non-hydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. Accumulation of processed McC inside producing cells should therefore lead to translation inhibition and cessation of growth. Here, we show that a product of another gene of the McC biosynthetic cluster, mccE, acetylates processed McC and converts it into a non-toxic compound. MccE also makes Escherichia coli resistant to albomycin, a Trojan horse inhibitor unrelated to McC that, upon processing, gives rise to a serine coupled to a thioxylofuranosyl pyrimidine, an inhibitor of seryl-tRNA synthetase. We speculate that MccE and related cellular acetyl-transferases of the Rim family may detoxify various aminoacyl- nucleotides, either exogenous or those generated inside the cell. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Marcello M.R.,Waksman Institute | Marcello M.R.,Rutgers University | Singaravelu G.,Waksman Institute | Singaravelu G.,Rutgers University | And 2 more authors.
Advances in Experimental Medicine and Biology | Year: 2013

Fertilizationthe fusion of gametes to produce a new organismis the culmination of a multitude of intricately regulated cellular processes. In Caenorhabditis elegans, fertilization is highly efficient. Sperm become fertilization competent after undergoing a maturation process during which they become motile, and the plasma membrane protein composition is reorganized in preparation for interaction with the oocyte. The highly specialized gametes begin their interactions by signaling to one another to ensure that fertilization occurs when they meet. The oocyte releases prostaglandin signals to help guide the sperm to the site of fertilization, and sperm secrete a protein called major sperm protein (MSP) to trigger oocyte maturation and ovulation. Upon meeting one another in the spermatheca, the sperm and oocyte fuse in a specific and tightly regulated process. Recent studies are providing new insights into the molecular basis of this fusion process. After fertilization, the oocyte must quickly transition from the relative quiescence of oogenesis to a phase of rapid development during the cleavage divisions of early embryogenesis. In addition, the fertilized oocyte must prevent other sperm from fusing with it as well as produce an eggshell for protection during external development. This chapter will review the nature and regulation of the various cellular processes of fertilization, including the development of fertilization competence, gamete signaling, sperm-oocyte fusion, the oocyte to embryo transition, and production of an eggshell to protect the developing embryo. © 2013 Springer Science+Business Media New York.


PubMed | Waksman Institute and Rutgers University
Type: | Journal: Chromosoma | Year: 2016

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.


Soutourina O.A.,Institute Pasteur Paris | Soutourina O.A.,University Paris Diderot | Monot M.,Institute Pasteur Paris | Boudry P.,Institute Pasteur Paris | And 13 more authors.
PLoS Genetics | Year: 2013

Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5′-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen. © 2013 Soutourina et al.


PubMed | Urbana University, Rega Institute for Medical Research, Purdue University, Russian Academy of Sciences and 2 more.
Type: Journal Article | Journal: Journal of bacteriology | Year: 2014

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.


Datsenko K.A.,Waksman Institute | Datsenko K.A.,Purdue University | Pougach K.,Waksman Institute | Pougach K.,Russian Academy of Sciences | And 6 more authors.
Nature Communications | Year: 2012

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) is a small RNA-based adaptive prokaryotic immunity system that functions by acquisition of short fragments of DNA (mainly from foreign invaders such as viruses and plasmids) and subsequent destruction of DNA with sequences matching acquired fragments. Some mutations in foreign DNA that affect the match prevent CRISPR/Cas defensive function. Here we show that matching sequences that are no longer able to elicit defense, still guide the CRISPR/Cas acquisition machinery to foreign DNA, thus making the spacer acquisition process adaptive and leading to restoration of CRISPR/Cas-mediated protection. We present evidence suggesting that after initial recognition of partially matching foreign DNA, the CRISPR/Cas acquisition machinery moves along the DNA molecule, occasionally selecting fragments to be incorporated into the CRISPR locus. Our results explain how adaptive CRISPR/Cas immunity becomes specifically directed towards foreign DNA, allowing bacteria to efficiently counter individual viral mutants that avoid CRISPR/Cas defense. © 2012 Macmillan Publishers Limited. All rights reserved.

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