A role for a small stable RNA in modulating the activity of DNA-binding proteins

AbstractThe 10Sa RNA, encoded by the E. coli ssrA gene, appears to modulate action of some DNA-binding proteins. When ssrA is inactivated, lacZ expression from the lac operon, as well as galK from a gal operon fused to a phage λ promoter, is reduced from that observed in bacteria wild-type for ssrA. These differences are not observed if the relevant repressor is inactive, suggesting that in the absence of 10Sa RNA binding of Lacl and λ cl repressors is enhanced. Gel mobility shifts show that 10Sa RNA binds these repressors and that an excess of 10Sa RNA competes for binding of λ cl with a DNA fragment containing the OR2 repressor-binding sequence. Similar observations were made in studies of the E. coil LexA repressor and phage P22 C1 transcription activator proteins. These results suggest that direct interaction with 10Sa RNA may explain this modulation of protein-DNA interactions

Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions

BACKGROUND: In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). RESULTS: The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). CONCLUSION: We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales

Identification of a biological role for the Escherichia coli small stable RNA, 10Sa RNA.

This work addresses the role of the Escherichia coli ssrA gene product, 10Sa RNA, a small, stable, relatively abundant RNA. This RNA is conserved among diverse genera. The effect of ssrA mutations on the growth of bacteriophage $\lambda$, hybrid phage formed between $\lambda$ and P22, as well as the expression of the E. coli lac operon is examined. Certain $\lambda$-P22 hybrids, providing they express the P22 C1 protein, fail to grow in E. coli with the sipB391 mutation. The sipB39l mutation is shown to be a large deletion that extends into the 3\sp\prime end of the ssrA gene. This deletion leaves most of ssrA intact, but removes the sequence encoding the 3\sp\prime end of the precursor form of 10Sa RNA. The lack of functional 10Sa RNA appears to be responsible for the inhibition of $\lambda$-P22 growth in E. coli with the sipB391 mutation. Altering the P22 DNA binding protein C1 so that it binds DNA less tightly alleviates the effect of the ssrA mutation. A single base pair mutation in $\lambda imm$P22 that changes both the DNA target of C1, the $-$35 region of the P\sb{\rm RE} promoter, and the amino terminus of the C1 protein allows for phage growth in an ssrA mutant. Although the mutant C1 protein activates the mutant P\sb{\rm RE} promoter, it fails to activate the wild type P\sb{\rm RE} promoter, suggesting that a single base pair mutation changes the specificities of both a protein and its target site. We propose that the inhibition of $\lambda imm$P22 growth is due to a more avid binding of C1 to its target in the absence of 10Sa RNA. The avidity of the phage $\lambda$ cI and E. coli LacI repressors for their cognate operator sites is also increased in E. coli ssrA mutants. Moreover, these repressor proteins bind to 10Sa RNA in vitro, even in the presence of a 100 fold excess of tRNA. These results suggest that RNAs may contribute to regulation of gene expression by modulating protein-DNA interactions.Ph.D.Microbiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104534/1/9527728.pdfDescription of 9527728.pdf : Restricted to UM users only

rCSP Strain Selection and Identification.

<p>(<b>A</b>) Reduced SDS-PAGE analysis of lysate samples prior to purification. Lanes 1–3 represent samples from strain CSP-1, CSP-3, and CSP-2 fermentations, respectively. Lane 4 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert. Arrow denotes rCSP in lysate samples. (<b>B</b>) Reduced SDS-PAGE analysis of purified proteins from a 2-column purification process. Lanes 1–3 represent samples from CSP-1, CSP-2, and CSP-3 productions, respectively. (<b>C</b>) Western blot of non-reduced purified CSP-1, CSP-2, and CSP-3 proteins (lanes 2–4, respectively). Lane 1 represents a sample from a culture of <i>P. fluorescens</i> transformed with plasmid vector containing no insert.</p

Activity of Prime-Boost Regimens in Mice.

<p>C57BL/6 mice (5–7 per cohort) were immunized 3x with 20 µg of rCSP formulated in GLA-SE, 1–2×10¹⁰ v.p. of Ad35CS, or a combination of the two. Spleens and livers were collected 10 days after the last immunization; sera were collected 10 or 14 days after the last immunization. To assess reduction in parasite load in the liver or sterile protection, mice were challenged with <i>Pb</i>-CS(<i>Pf</i>) parasites 3 weeks after the last immunization. (<b>A</b>) Percent inhibition of LS parasite development in immunized mice challenged with <i>Pb</i>-CS(<i>Pf</i>) sporozoites normalized to naïve mice received the challenge, and percent sterile protection in immunized BALB/c mice upon challenge with a fewer number of Pb-CS(Pf) sporozoites are shown. A statistically significant reduction of LS parasites in the livers of mice immunized with all regimens, except Ad35CS 3x and Ad35CS (1x), GLA-SE (2x), was seen upon sporozoites challenge, compared to the level of LS parasites in the livers of negative control mice following sporozoites challenge, based on the mean parasite's 18s rRNA copy number (as noted by asterisks). Three regimens were assessed in two independent studies (GLA-SE 3x; GLA-SE 1x, Ad35CS 2x; and Ad35CS 1x, GLA-SE 2x); for these regimens, average percent inhibition is shown with error bars represented as standard error. Statistical significance with the GLA-SE 3x; GLA-SE 1x, Ad35CS 2x was seen in both studies. (<b>B</b>) ELISA was used to evaluate the level of humoral response in immunized mice to the repeat region of CSP with pooled mouse sera and to whole rCSP with individual mouse sera. Titer was assessed at OD = 1.0 based on 4-parameter logistic curve fits. Average titers are shown with error bars representing standard error of the mean. (<b>C</b>) The level of T-cell response was determined via ELISpot using lymphocytes isolated from the spleens and livers of immunized mice (5 mice per cohort were used for this study) and overlapping peptides (15-mers encompassing the CSP repeat region) for in vitro restimulation. Average numbers of IFN-γ producing cells/10⁶ lymphocytes are shown with error bars representing standard error of the mean. No more than 5 IFN-γ producing cells/10⁶ lymphocytes were counted with cells harvested from naïve mice (data not shown).</p

rCSP/Ad35CS Heterologous Prime-Boost Regimens.

<p>C57BL/6 mice were immunized with rCSP formulated in GLA-SE or Ad35CS according to the regimens shown.</p><p>rCSP/Ad35CS Heterologous Prime-Boost Regimens.</p

Immunogenicity and Efficacy of rCSP Immunization in Mice.

<p>Five C57BL/6 mice per cohort were immunized 3x with 25 µg of rCSP from 3 production strains (CSP-1, CSP-2, and CSP-3) formulated in incomplete Freund's adjuvant. Sera were collected 2 weeks after the last immunization and pooled for analysis by ELISA, IFA, and ISI. Mice were challenged with <i>Pb</i>-CS(<i>Pf</i>) parasites 3 weeks after the last immunization. (<b>A</b>) Pooled sera from mice immunized with the 3 recombinant proteins showed similar ELISA and IFA titers. ELISA titers to a [NANP]₆ peptide were calculated at OD = 1.0 based on 4-parameter logistic curve fits, and IFA titers were determined based on the lowest serum dilution to give sporozoites-specific fluorescence above background level shown by sera from naïve mice. (<b>B</b>) Sera from rCSP-immunized mice reacted to <i>Pb</i>-CS(<i>Pf</i>) sporozoites demonstrated the expected pattern of surface fluorescence by IFA. (<b>C</b>) Sera from rCSP-immunized mice diluted 1∶100 and assayed for the ability to block <i>Pf</i> sporozoite infection of hepatocytes in an ISI assay demonstrated the highest ISI activity for anti-CSP-1 protein sera. The percent inhibition is shown relative to the number of sporozoites after incubating with the sera of naïve mice. Percent inhibition of LS parasite development in rCSP-immunized mice challenged with <i>Pb</i>-CS(<i>Pf</i>) sporozoites normalized to naïve control mice is also shown. The reduction of LS parasites in livers of mice immunized with CSP-1 or CSP-3 proteins was statistically significant based on the mean parasite-specific 18 s rRNA copy number compared the level of LS parasites in livers of naïve and adjuvant-only administered mice (as noted by asterisks).</p