Reassembling a cannon in the DNA defense arsenal: Genetics of StySA, a BREX phage exclusion system in Salmonella lab strains.

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA.

Genome archaeology of two laboratory Salmonella enterica enterica sv Typhimurium.

The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for the study of cell surface antigens, metabolic pathways and restriction-modification (RM) studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625). In the ancestry of LB5000, segments of ∼15 and ∼42 kb were introduced from Salmonella enterica sv Abony 803 into S.

Complete Annotated Genome Sequence of the Salmonella enterica Serovar Typhimurium LT7 Strain STK003, Historically Used in Gene Transfer Studies.

The genome of serovar Typhimurium LT7 comprises a chromosome and two plasmids. One plasmid is very close to pSLT of Typhimurium LT2; the second harbors a shufflon region. Prophage content is distinct: LT7 lacks Fels-1, while Gifsy-1 and Fels-2 show island-like divergence and likely programmed inversion, respectively.

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content.

Plasmid replication-associated single-strand-specific methyltransferases.

Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E.

EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: Characterization in vivo and in vitro with a new approach to cleavage site determination.

Here we characterize the modification-dependent restriction enzyme (MDE) EcoBLMcrX in vivo, in vitro and in its genomic environment. MDE cleavage of modified DNAs protects prokaryote populations from lethal infection by bacteriophage with highly modified DNA, and also stabilizes lineages by reducing gene import when sparse modification occurs in the wrong context. The function and distribution of MDE families are thus important.

Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer.

Bacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins.

Complete Genome Sequence of NEB 5-alpha, a Derivative of Escherichia coli K-12 DH5α.

Escherichia coli K-12 DH5α is one of the most popular and widely available laboratory strains, but, surprisingly, no complete genome sequence has been publicly available. Here, we report the complete, finished sequence of NEB 5-alpha (DH5α fhuA2). It should serve as a useful reference for researchers working with DH5α.

Complete Genome Sequence of Escherichia coli ER1821R, a Laboratory K-12 Derivative Engineered To Be Deficient in All Methylcytosine and Methyladenine Restriction Systems.

We present here the complete genomic sequence of a rifampin-resistant derivative of the Escherichia coli K-12 laboratory strain ER1821, engineered to be deficient in all known restriction systems, making it suitable for generating unbiased libraries from organisms with non-K-12 methylation patterns. The ER1821R genome is most closely related to that of DH1, another popular cloning strain (both derived from MM294), but is deleted for the e14 prophage (McrA(-)) and the immigration control (McrBC(-) EcoKI R(-) M(-) Mrr(-)) loci.

Biosynthesis and Function of Modified Bases in Bacteria and Their Viruses.

Naturally occurring modification of the canonical A, G, C, and T bases can be found in the DNA of cellular organisms and viruses from all domains of life. Bacterial viruses (bacteriophages) are a particularly rich but still underexploited source of such modified variant nucleotides. The modifications conserve the coding and base-pairing functions of DNA, but add regulatory and protective functions.

Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents.

SHuffle strains are genetically engineeredEscherichia colistrains that are capable of oxidizing cysteines within proteins to form disulfide bonds. Here we present the complete genome of both the K-12 and B versions of SHuffle strains along with their parental ancestors. These strains have been of significant use to both the general scientific community and the biotech industry, interested in producing novel disulfide-bonded proteins that were hitherto unable to be expressed in standardE.

Korean Adoptee Identity: Adoptive and Ethnic Identity Profiles of Adopted Korean Americans.

Adopted Korean adolescents face the task of grappling with their identity as Koreans and coming to terms with their adoptive status. In order to explore these dual identities, the authors conducted a person-centered study of the identity profiles of 189 adopted Korean American adolescents. Using cluster analytic procedures, the study examined patterns of commitment to ethnic and adoptive identities, revealing six conceptually unique identity clusters.

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination.

Complete Genome Sequence of ER2796, a DNA Methyltransferase-Deficient Strain of Escherichia coli K-12.

We report the complete sequence of ER2796, a laboratory strain of Escherichia coli K-12 that is completely defective in DNA methylation. Because of its lack of any native methylation, it is extremely useful as a host into which heterologous DNA methyltransferase genes can be cloned and the recognition sequences of their products deduced by Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. The genome was itself sequenced from a long-insert library using the SMRT platform, resulting in a single closed contig devoid of methylated bases.

Iliac crest allograft glenoid reconstruction for recurrent anterior shoulder instability in athletes: Surgical technique and results.

Performing a labral repair alone in patients with recurrent anterior instability and a large glenoid defect has led to poor outcomes. We present a technique involving the use of the iliac crest allograft inserted into the glenoid defect in athletes with recurrent anterior shoulder instability and large bony defects of the glenoid (>25% of glenoid diameter). All athletes with recurrent anterior shoulder instability and a large glenoid defect that underwent open anterior shoulder stabilization and glenoid reconstruction with the iliac crest allograft were followed over a 4-year period.

Highlights of the DNA cutters: a short history of the restriction enzymes.

In the early 1950's, 'host-controlled variation in bacterial viruses' was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems.

Type I restriction enzymes and their relatives.

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences.

The other face of restriction: modification-dependent enzymes.

The 1952 observation of host-induced non-hereditary variation in bacteriophages by Salvador Luria and Mary Human led to the discovery in the 1960s of modifying enzymes that glucosylate hydroxymethylcytosine in T-even phages and of genes encoding corresponding host activities that restrict non-glucosylated phage DNA: rglA and rglB (restricts glucoseless phage). In the 1980's, appreciation of the biological scope of these activities was dramatically expanded with the demonstration that plant and animal DNA was also sensitive to restriction in cloning experiments. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction).

1-year retention rates and performance ratings: comparing associate degree, baccalaureate, and accelerated baccalaureate degree nurses.

The purpose of this study was to examine 1-year retention and managerial performance ratings of newly licensed RNs (NLRNs) according to nursing education program types (associate degree, traditional baccalaureate, and accelerated 2nd degree baccalaureate). Findings revealed retention and performance differences, suggesting the possibility of tradeoffs related to educational program type when selecting NLRNs for open positions.

A versatile element for gene addition in bacterial chromosomes.

The increasing interest in genetic manipulation of bacterial host metabolic pathways for protein or small molecule production has led to a need to add new genes to a chromosome quickly and easily without leaving behind a selectable marker. The present report describes a vector and four-day procedure that enable site-specific chromosomal insertion of cloned genes in a context insulated from external transcription, usable once in a construction series. The use of rhamnose-inducible transcription from rhaBp allows regulation of the inserted genes independently of the commonly used IPTG and arabinose strategies.

Cleavage of a model DNA replication fork by a methyl-specific endonuclease.

Epigenetic DNA methylation is involved in many biological processes. An epigenetic status can be altered by gain or loss of a DNA methyltransferase gene or its activity. Repair of DNA damage can also remove DNA methylation.

Evolution of bacterial phosphoglycerate mutases: non-homologous isofunctional enzymes undergoing gene losses, gains and lateral transfers.

Background: The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.

Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis.

Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (-SCH(3)). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N(6)-isopentenyladenosine (i(6)A) to 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms(2)i(6)A formation (MiaB activity), and yqeV is required for modification of N(6)-threonylcarbamoyladenosine (t(6)A) to 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A) in tRNA.

Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast.

Obtaining sufficient amounts of pure glycoprotein variants to characterize their structures is an important goal in both functional biology and the biotechnology industry. We have developed preparative HIC conditions that resolve glycoform variants on the basis of overall carbohydrate content for a recombinant transferrin-exendin-4 fusion protein. The fusion protein was expressed from the yeast Saccharomyces cerevisiae from high density fermentation and is post-translationally modified with mannose sugars through O-glycosidic linkages.