Interplay between site-specific mutations and cyclic nucleotides in modulating DNA recognition by Escherichia coli cyclic AMP receptor protein

Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate protein compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844-3852]. Cooperativity of cAMP binding to CRP and the apparent DNA binding affinity are perturbed [Lin and Lee (2002) Biochemistry 41, 11857-11867]. The aim of this study is to explore the effects of mutation on the surface chemistry of CRP and to define the consequences of these changes in affecting specific DNA sequence recognition by CRP. Furthermore, the role of the interplay between mutation and specific identity of the bound cyclic nucleotide in this DNA recognition was explored. In the current study, effects of eight site-specific mutations (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) on DNA recognition of four sequences (Class I (site PI of lac), Class II (site PI of gal), and synthetic sequences that are hybrids of Classes I and II sites) modulated by three different cyclic nucleotides (cAMP, cCMP, and cGMP) were investigated. All mutations altered the surface chemistry of CRP as evidenced by the change in elution properties of these proteins from different matrixes. While T127L, S62F, K52N, and H159L exhibited unexpected behavior under combinations of specific experimental conditions, such as the identity of bound cyclic nucleotide and DNA sequence, in general, results showed that the affinities of CRP for DNA were sequence-dependent, increasing in the order of lacgal26 < gal26 < lac26 < gallac26 for all the mutants in the presence of 200 microM cAMP. The apparent association constants significantly increased in the order of no cyclic nucleotide approximately cGMP < cCMP < cAMP for all the examined DNA sequences. Linear correlation between the DeltaG for CRP-DNA complex formation and the cooperativity energy for cAMP binding was observed with gallac26, gal26, and lacgal26; however, the slope of this linear correlation is DNA sequence dependent. Structural information was presented to rationalize the interplay between CRP sequence and cyclic nucleotides in defining the recognition of DNA sequences.     

Tuning the redox and enzymatic activity of glucose oxidase in layered organic films and its application in glucose biosensors

Glucose oxidase was embedded in organic films through a layer-by-layer approach, where the enzyme demonstrated significantly enhanced electron-transfer reactivity and finely tuned enzymatic activity. An unmediated, reagentless glucose biosensor was accordingly prepared with two polyethylenimine/glucose oxidase bilayers-modified pyrolytic graphite electrode. A calibration linear range of glucose was 0.5-8.9 mM with a detection limit of 50 microM and sensitivity of 0.76 microA mM(-1).     

Potential for the Anopheles gambiae Densonucleosis Virus To Act as an “Evolution-Proof” Biopesticide

“Evolution-proof” or “late-life-acting” insecticides (LLAIs) preferentially kill older adult mosquitoes and are of extreme interest to control vector-borne diseases such as malaria. We used quantitative PCR to assess whether the Anopheles gambiae densonucleosis virus (AgDNV) had potential as an LLAI. After infection, AgDNV titers increased modestly during larval development but replicated slower than the host cells, resulting in a significant decrease in the normalized virus titer during larval and pupal development. Normalized virus titers dramatically increased after adult emergence, peaking in 7- to 10-day-old adults. Unlike other DNVs, AgDNV does not significantly replicate in preadult mosquitoes but rather preferentially replicates in older adults. The natural dynamics of AgDNV make it ideal for expression of insect-specific toxin genes as a biological LLAI.Malaria infects several hundred million people and results in over one million deaths annually. Vector control is a major component of current malaria control strategies. However, the evolution of insecticide resistance by Anopheles mosquito vectors has severely hampered control efforts (5, 13, 14). Although novel chemistries are being explored, new insecticides will face similar problems with resistance evolution. Recently, late-life-acting insecticides (LLAIs) have been proposed as novel agents to control vector-borne diseases such as malaria (1, 10). LLAIs selectively kill the older mosquitoes responsible for the bulk of parasite transmission while allowing for reproduction of the younger age classes that contribute to the bulk of evolutionary fitness, i.e., there is an optimal window of time wherein mosquitoes live long enough to reproduce but not long enough to transmit pathogens (8, 9). Reproduction allows for relaxation of evolutionary pressures that select for resistance to the agent. If resistance alleles exert fitness costs, there are theoretical scenarios under which resistance is not expected to evolve, leading some to provocatively term LLAIs as “evolution-proof” (1, 10).LLAI do not have to be conventional chemical pesticides. Like all organisms, mosquitoes can be infected with pathogens of their own (including fungi, bacteria, or viruses) that can be exploited to shorten mosquito life span to control disease (6, 9-12). Some pathogens can be vertically transmitted (9, 11), which not only results in relaxed selection pressure but also allows for their transmission and spread into the vector population.Densonucleosis viruses (or densoviruses [DNVs]) are icosahedral, nonenveloped parvoviruses that have been identified from many invertebrate taxa, including multiple mosquito species (2, 11). Naturally occurring DNVs typically infect mosquitoes during the aquatic larval phase. Infection of young larvae (first or second instar) is generally lethal, resulting in virus amplification and release into the larval environment. Larvae infected at later time points (third or fourth instar) develop into infected adults that inoculate virus vertically and horizontally into the larval environment during oviposition, completing the virus life cycle (2, 11).The Aedes aegypti densovirus (AeDNV) is generally lethal to Ae. aegypti larvae in a dose-dependent manner, and high virus titers in larvae are observed both by quantitative PCR (qPCR) and by expression of foreign transgenes such as green fluorescent protein (GFP) (2, 8, 15). In contrast, the Anopheles gambiae densovirus (AgDNV) is not lethal to An. gambiae larvae (11). Using GFP-transducing virus and epifluorescence microscopy, we have also never seen GFP expression in larvae, pupae, or young adults; GFP is not observable until the adults are ∼1 week postemergence (J. L. Rasgon and X. Ren, unpublished observations) (Fig. ​(Fig.1).1). Based on these observations, we hypothesized that AgDNV has different replication kinetics in An. gambiae than AeDNV has in Ae. aegypti, remaining at relatively low titers in the immature life stages but replicating to high titers after adult emergence.Open in a separate windowFIG. 1.Epifluorescent image (dorsal view) of a 10-day-old adult An. gambiae female infected with GFP-expressing AgDNV (as described in reference 11). GFP is not visible in infected mosquitoes until 7 to 10 days postemergence.     

Fission Yeast Germinal Center (GC) Kinase Ppk11 Interacts with Pmo25 and Plays an Auxiliary Role in Concert with the Morphogenesis Orb6 Network (MOR) in Cell Morphogenesis

How cell morphology and the cell cycle are coordinately regulated is a fundamental subject in cell biology. In fission yeast, 2 germinal center kinases (GCKs), Sid1 and Nak1, play an essential role in septation/cytokinesis and cell separation/cell polarity control, respectively, as components of the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR). Here we show that a third GCK, Ppk11, is also required for efficient cell separation particularly, at a high temperature. Although Ppk11 is not essential for cell division, this kinase plays an auxiliary role in concert with MOR in cell morphogenesis. Ppk11 physically interacts with the MOR component Pmo25 and is localized to the septum, by which Ppk11 is crucial for Pmo25 targeting/accumulation to the septum. The conserved C-terminal WDF motif of Ppk11 is essential for both septum accumulation of Pmo25 and efficient cell separation. In contrast its kinase activity is required only for cell separation. Thus, both interaction of Ppk11 with Pmo25 and Ppk11 kinase activity are critical for efficient cell separation.     

The neogregarine protozoan Farinocystis sp. reduces longevity and fecundity in the West Indian sweet potato weevil, Euscepes postfasciatus (Fairmaire)

The number of West Indian sweet potato weevils, Euscepes postfasciatus, being mass-reared in a facility for use in sterile insect technique (SIT) eradication programs has undergone a drastic reduction. A neogregarine protozoan pathogen Farinocystis sp. (an undescribed species) was detected in vivo in the mass-reared E. postfasciatus. We investigated the effects of this disease on the longevity and fecundity of host weevils and the incubation time of the disease in the host body under mass-rearing conditions. Our results demonstrated that infection by this Farinocystis sp. decreased both longevity and fecundity in E. postfasciatus. In particular, the pathogen severely limited the production of progeny by infected females compared to healthy females. Therefore, we consider this protozoan infection to be the major cause of the decreased E. postfasciatus production in the mass-rearing facility.