The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the oilier (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they arc the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38447/1/240290305_ftp.pd

The in vitro synthesis and processing of the branched-chain amino acid binding proteins

The synthesis of the leucine-specific and LIV-binding proteins was examined in vitro in a coupled transcription/translation system using the hybrid plasmids pOX7 and pOX13 as templates. Plasmid pOX7 contains the livK gene coding for the leucine-specific binding protein, and pOX13 contains the liv J gene coding for the LIV-binding protein. Both binding proteins were synthesized in vitro as precursor forms with molecular weights approximately 2,500 greater than their respective mature forms. Conversion of the precursor forms to their mature forms occurred during post-translational incubation following synthesis in the presence of membrane. The precursor of the LIV-binding protein was processed more rapidly than the leucine-specific binding protein precursor. Processing activity could be removed from the in vitro synthesis system by centrifugation, suggesting that the processing activity was membrane associated. Restoration of post-translational processing activity was achieved by adding inside-out membrane vesicles to membrane-depleted reaction mixtures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38208/1/400140305_ftp.pd

Regulation of high-affinity leucine transport in escherichia coli

Leucine is transported into E coli by two osmotic shock-sensitive, high-affinity systems (LIV-I and leucine-specific systems) and one membrane bound, low-affinity system (LIV-II). Expression of the high-affinity transport systems is altered by mutations in liv R and 1st R , genes for negatively acting regulatory elements, and by mutations in rho , the gene for transcription termination. All four genes for high-affinity leucine transport ( livJ, livK, livH , and livG ) are closely linked and have been cloned on a plasmid vector, pOX1. Several subcloned fragments of this plasmid have been prepared and used in complementation and regulation studies. The results of these studies suggest that livJ and livK are separated by approximately one kilobase and give a gene order of livJ–livK–livH. livJ and livK appear to be regulated in an interdependent fashion; livK is expressed maximally when the livJ gene is inactivated by mutation or deletion. The results support the existence of separate promoters for the livJ and livK genes. The effects of mutations in the rho and livR genes are additive on one another and therefore appear to be involved in independent regulatory mechanisms. Mutations in the rho gene affect both the LIV-I and leucinespecific transport systems by increasing the expression of livJ and livK , genes for the LIV-specific and leucine-specific binding proteins, respectively.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38209/1/400140410_ftp.pd

Complex exon-intron marking by histone modifications is not determined solely by nucleosome distribution

It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (“exon-intron marking”), linking chromatin structure and function to co-transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing

Complete Structural Model of Escherichia coli RNA Polymerase from a Hybrid Approach

A combination of structural approaches yields a complete atomic model of the highly biochemically characterized Escherichia coli RNA polymerase, enabling fuller exploitation of E. coli as a model for understanding transcription

1-Phenylthiocyclopropyltriphenylphosphonium fluoborate: A new synthon for cyclopentanone synthesis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22209/1/0000641.pd

P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

<div><p>Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders <i>E</i>. <i>coli</i> more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene <i>ref</i>. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to <i>E</i>. <i>coli</i> in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens <i>Neisseria gonorrhoeae</i> and <i>Staphylococcus aureus</i> serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.</p></div

Ref expression causes cell filamentation independent of the <i>E</i>. <i>coli</i> SOS response.

<p><i>A</i>. Microscopy of cells expressing Ref or RefΔC110. WT <i>E</i>. <i>coli</i> strains EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), <i>ΔrecA</i> strains EAR64 (EV), EAR65 (pRef), EAR74 (pRefΔC110), <i>sulA</i>^<i>-</i> strains EAR77 (EV), EAR78 (pRef), EAR79 (pRefΔC110), and <i>sulA</i>^<i>-</i> <i>lexA3</i> strains EAR69 (EV), EAR70 (pRef), and EAR75 (pRefΔC110) were grown to 1x10⁸ CFU/mL, treated with 1% arabinose, outgrown for 4 hours, and images were obtained as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.g004" target="_blank">Fig 4C</a>. Scale bar = 10 μm, representative images shown. B. Quantification of cell length data from (A) was obtained using the MicrobeTracker plugin for MatLab. Each counted cell is represented by a single data point with the average and standard deviation for the data shown. An average of 90 cells (range: 39–188) were counted for each condition. ** = p-value <0.0001 when compared to EV in same background, * = p-value <0.05 when compared to same vector in WT background. C. Cell survival after expression of Ref. WT <i>E</i>. <i>coli</i> strains EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), <i>ΔrecA</i> strains EAR64 (EV), EAR65 (pRef), EAR74 (pRefΔC110) were treated as in (A). Cells were plated for viability and the average and standard deviation of at least three biological replicates for each condition are reported (error bars small and not visible in some cases). Significant p-values are noted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.s002" target="_blank">S1 Table</a>.</p

Phage-antibiotic synergy with bacteriophage P1 and ciprofloxacin.

<p>A. Survival of P1 lysogens after treatment with 8 ng/mL ciprofloxacin. Log phase cultures of <i>E</i>. <i>coli</i> MG1655, EAR2 (P1<i>ref+)</i> or EAW195 (P1<i>Δref</i>), were adjusted to 5x10⁵ CFU/mL and were treated with water (open markers) or 8 ng/mL ciprofloxacin (filled markers) with six hours outgrowth. The average CFU/mL and standard deviation of three biological replicates is reported (error bars are present, but smaller than markers in most cases). * = p-value<0.0001, when compared to MG1655 treated with 8 ng/mL ciprofloxacin. B. Optical density of P1 lysogens treated with ciprofloxacin. Log phase cultures of above strains were treated with water (open markers) or 4 ng/mL ciprofloxacin (filled markers) with outgrowth at 30°C. The average OD₅₉₅ and standard deviation of five biological and two technical replicates for each condition is reported. C. Optical density of P1 lysogens induced with a temperature shift. Log phase cultures of strains in (B) were shifted from 30°C to 42°C to induce temperature-sensitive phage lysis and optical density was tracked as in (B). The average and standard deviation of three biological and three technical replicates is reported.</p