The interaction between the acidic transactivation domain of the human tumor suppressor protein p53 (p53TAD) and the 70 kDa subunit of human replication protein A (hRPA70) was investigated using heteronuclear magnetic resonance spectroscopy. A 1H–15N heteronuclear single quantum coherence (HSQC) titration experiment was performed on a 15N-labeled fragment of hRPA70, containing the N-terminal 168 residues (hRPA701–168) and p53TAD. HRPA701–168 residues important for binding were identified and found to be localized to a prominent basic cleft. This binding site overlapped with a previously identified single-stranded DNA-binding site, suggesting that a competitive binding mechanism may regulate the formation of p53TAD–hRPA70 complex. The amide 1H and 15N chemical shifts of an uniformly 15N-labeled sample of p53TAD were also monitored before and after the addition of unlabeled hRPA701–168. In the presence of unlabeled hRPA701–168, resonance lineshapes increased and corresponding intensity reductions were observed for specific p53TAD residues. The largest intensity reductions were observed for p53TAD residues 42–56. Minimal binding was observed between p53TAD and a mutant form of hRPA701–168, where the basic cleft residue R41 was changed to a glutamic acid (R41E), demonstrating that ionic interactions play an important role in specifying the binding interface. The region of p53TAD most affected by binding hRPA701–168 was found to have some residual alpha helical and beta strand structure; however, this structure was not stabilized by binding hRPA701–168. 15N relaxation experiments were performed to monitor changes in backbone dynamics of p53TAD when bound to hRPA701–168. Large changes in both the transverse (R2) and rotating frame (R1ρ) relaxation rates were observed for a subset of the p53TAD residues that had 1H–15N HSQC resonance intensity reductions during the complex formation. The folding of p53TAD upon complex formation is suggested by the pattern of changes observed for both R2 and R1ρ. A model that couples the formation of a weak encounter complex between p53TAD and hRPA701–168 to the folding of p53TAD is discussed in the context of a functional role for the p53–hRPA70 complex in DNA repair.

b CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (⌬C1S), CRISPR2 (⌬C2E), or both CRISPR1؉2 (⌬C1SC2E) or the removal of spacers 2 and 3 (⌬CR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ⌬C1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ⌬C2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one) was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.

Gluconacetobacter xylinus is a plant-associated bacterium best studied for its cellulose production. Bacterial cellulose is important in facilitating plant-microbe interactions but little is known about the effect that exogenous phytohormones have on bacterial cellulose synthesis or the growth of G. xylinus. We characterized the growth, development and effect on pellicle characteristics caused by exogenous indole-3-acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA) and zeatin (Z) over a range of concentrations (1 nM to 100 μM). These phytohormones are plant growth regulators known to be involved plant development including fruit ripening and stress tolerance. Each of these hormones stimulated G. xylinus growth and influenced its pellicle characteristics. Exogenous IAA had the greatest effect on G. xylinus pellicles. Growth in IAA produced thin pellicles with very little cellulose. In general, pellicle wet weight was inversely proportional to the bacterial cellulose yield when cultures were grown in the presence of ABA, suggesting ABA influenced pellicle density and hydration. The crystallinity index, CI (IR) of cellulose produced in the presence of each phytohormone over a variety of concentrations was determined by Fourier transform infrared spectroscopy. The observed effect on cellulose crystallinity was concentration and hormone dependent. GA caused the greatest alterations in crystallinity with the highest CI (IR)=0.94 at 1 μM and the lowest CI (IR)=0.47 at 500 nM. Endogenous production of hormones by G. xylinus was investigated by high performance liquid chromatography of extracts prepared from both cell pellets and culture supernatants. We found G. xylinus synthesized GA, ABA and Z but did not produce IAA.