No abstract

For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis-or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when crosslinked show the largest "photoswitch effect," i.e., difference in activity when illuminated with UV vs. blue light, >30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V max rather than K m .azobenzene | DNA cleavage | endonuclease | photoswitch | PvuII P roteins exist in nature whose activity can be controlled by light; perhaps one of the best known examples is rhodopsin, which is regulated by the cis∕trans isomerization of its cofactor retinal. For many biological applications it would be desirable to selectively switch the activity of a protein on and off by light in a similar manner (1). This could be accomplished by the introduction of a photosensitive compound into the protein of interest. Recent developments in photosensitive compounds such as the azobenzene derivatives have made the scenario a reality. Azobenzene can be reversibly isomerized between the extended trans-and the more compact cis-configuration by illumination with UV (trans → cis) or blue-light (cis → trans) as well as by thermal relaxation (cis → trans) (2-4). Four generally applicable approaches have been used to introduce azobenzene groups into peptides or proteins: (i) incorporation during peptide synthesis (5-8), (ii) incorporation during in vitro translation (9, 10), (iii) incorporation in vivo by using an orthogonal tRNA/aminoacyl tRNA synthetase pair specific for phenylalanine-4′-azobenzene (11), and (iv) chemical modification of peptides and proteins (3,12,13). Another more specific approach is to use azobenzenemodified ligands (e.g., inhibitors) for proteins (14,15). Chemical modification, the most widely used of these approaches, can be done with mono-or bifunctional azobenzene derivatives. Modification with monofunctional azobenzene derivatives relies on steric effects (e.g., interference with ligand binding), whereas modification with bifunctional azobenzene derivat...

SummaryType III secretion systems (TTSSs) are essential mediators of the interaction of many Gram-negative bacteria with human, animal or plant hosts. Extensive sequence and functional similarities exist between components of TTSS from bacteria as diverse as animal and plant pathogens. Recent crystal structure determinations of TTSS proteins reveal extensive structural homologies and novel structural motifs and provide a basis on which protein interaction networks start to be drawn within the TTSSs, that are consistent with and help rationalize genetic and biochemical data. Such studies, along with electron microscopy, also established common architectural design and function among the TTSSs of plant and mammalian pathogens, as well as between the TTSS injectisome and the flagellum. Recent comparative genomic analysis, bioinformatic genome mining and genome-wide functional screening have revealed an unsuspected number of newly discovered effectors, especially in plant pathogens and uncovered a wider distribution of TTSS in pathogenic, symbiotic and commensal bacteria. Functional proteomics and analysis further reveals common themes in TTSS effector functions across phylogenetic host and pathogen boundaries. Based on advances in TTSS biology, new diagnostics, crop protection and drug development applications, as well as new cell biology research tools are beginning to emerge.

With the advent of recombinant DNA techniques, the field of molecular plant pathology witnessed dramatic shifts in the 1970s and 1980s. The new and conventional methodologies of bacterial molecular genetics put bacteria center stage. The discovery in the mid-1980s of the hrp/hrc gene cluster and the subsequent demonstration that it encodes a type III secretion system (T3SS) common to Gram negative bacterial phytopathogens, animal pathogens, and plant symbionts was a landmark in molecular plant pathology. Today, T3SS has earned a central role in our understanding of many fundamental aspects of bacterium-plant interactions and has contributed the important concept of interkingdom transfer of effector proteins determining race-cultivar specificity in plant-bacterium pathosystems. Recent developments in genomics, proteomics, and structural biology enable detailed and comprehensive insights into the functional architecture, evolutionary origin, and distribution of T3SS among bacterial pathogens and support current research efforts to discover novel antivirulence drugs.