DNA looping can enhance lysogenic CI transcription in phage lambda.

The lysogenic state of bacteriophage lambda is maintained by CI repressor, which negatively regulates two promoters to block lytic gene expression. Expression of CI is itself controlled by positive and negative feedback as CI binds to O(R) to regulate the P(RM) promoter. In addition to direct interactions with operator DNA, CI tetramers bound at O(L) and O(R) can come together to form an octamer, looping the DNA that lies between them and allowing O(L) to assist with negative regulation of P(RM). We used a fluorescent reporter protein to measure the CI concentration for a set of constructs that differ in their ability to assume various forms of the looped structure. Based on the observed steady-state fluorescence for these constructs, the presence of O(L) increases P(RM) activation unless both operators can be fully occupied. By calculating the probabilities for the underlying operator configurations present in each construct, two different models for the mechanism of enhanced activation allow us to predict that when the DNA is looped, P(RM) activation can be 2- to 4-fold higher than is possible for unlooped DNA. Based on our results, transcriptional regulation for lambda's lysogenic/lytic switch includes both activation and repression due to DNA looping.

Real-time chemical imaging of bacterial activity in biofilms using open-channel microfluidics and synchrotron FTIR spectromicroscopy.

Real-time chemical imaging of bacterial activities can facilitate a comprehensive understanding of the dynamics of biofilm structures and functions. Synchrotron-radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy can yield high spatial resolution and label-free vibrational signatures of chemical bonds in biomolecules, but the abundance of water in biofilms has hindered SR-FTIR's sensitivity in investigating bacterial activity. We developed a simple open-channel microfluidic system that can circumvent the water-absorption barrier for chemical imaging of the developmental dynamics of bacterial biofilms with a spatial resolution of several micrometers. This system maintains a 10 microm thick laminar-flow-through biofilm system that minimizes both the imaging volume in liquid and the signal interference from geometry-induced fringing. Here we demonstrate the ability of the open-channel microfluidic platform to maintain the functionality of living cells while enabling high-quality SR-FTIR measurements. We include several applications that show how microbes in biofilms adapt to their immediate environments. The ability to directly monitor and map bacterial changes in biofilms can yield significant insight into a wide range of microbial systems, especially when coupled to more sophisticated microfluidic platforms.

Native state energetics of the Src SH2 domain: evidence for a partially structured state in the denatured ensemble.

We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen-deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent approximately 30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.

An Extracellular Protease of Pseudomonas fluorescens Inactivates Antibiotics of Pantoea agglomerans.

ABSTRACT Pseudomonas fluorescens A506 and Pantoea agglomerans strains Eh252 and C9-1 are biological control agents that suppress fire blight, an important disease of pear and apple caused by the bacterium Erwinia amylovora. Pseudomonas fluorescens strain A506 suppresses disease largely through competitive exclusion of E. amylovora on surfaces of blossoms, the primary infection court, whereas Pantoea agglomerans strains Eh252 and C9-1 produce antibiotics that are toxic to E. amylovora. In this study, an extracellular protease produced by A506 is characterized and evaluated for its capacity to inactivate the antibiotics produced by the strains of Pantoea agglomerans. Activity of the extracellular protease was optimal at pH 9 and inhibited by zinc- or calcium-chelators, indicating that the protease is an alkaline metalloprotease. In an agar plate bioassay, partially purified extracellular protease inactivated the antibiotics mccEh252 and herbicolin O, which are produced by Pantoea agglomerans strains Eh252 and C9-1, respectively. Derivatives of A506 deficient in extracellular protease production were obtained by transposon mutagenesis, and the aprX gene encoding the protease was cloned and sequenced. Strain A506 inactivated mccEh252 and herbicolin O in agar plate bioassays, whereas the aprX mutant did not inactivate the antibiotics. Both A506 and the aprX mutant were insensitive to antibiosis by C9-1 and Eh252; thus, the protease was not required to protect A506 from antibiosis. These data highlight a previously unknown role of the extracellular protease produced by Pseudomonas fluorescens A506 in interactions among plant-associated microbes.

A simplified model for lysogenic regulation through DNA looping.

The lysogenic state of bacteriophage lambda has been a key model for understanding gene regulation. A single protein, CI repressor, maintains this state by blocking gene expression from two promoters separated by approximately 2.3 kbp of DNA. CI controls its own expression by positive and negative feedback by binding to OR to regulate the PRM promoter. Not only does CI interact directly with operator DNA, but CI tetramers bound to OL and OR can form an octamer, looping the DNA that lies between them. Previous studies show that OL can assist with negative regulation of PRM, and we recently showed that DNA looping can also enhance looping activation. In this paper we present a new interpretation of our recent experimental data based a new crystal structure of CI. The simpler model suggested by the new structural information predicts that the most common forms of the DNA loop have similar activation behavior, and are enhanced approximately 2.2-fold relative to unlooped activation.