Systematic Dissection and Trajectory-Scanning Mutagenesis of the Molecular Interface That Ensures Specificity of Two-Component Signaling Pathways

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed

PMAP, PMAPS: DNA physical map constructing programs.

Computer programs are described, which facilitate the construction of the restriction site (physical) map of DNA molecules. By knowing the length of each fragment and its degree of error in the single and the double restriction enzyme digestions, the programs give all the possibilities for the physical map. This method is applicable to linear DNA molecules. Several examples are presented which indicate the high efficiency of the programs in constructing restriction site maps for the 62 Kb chromosome of bacteriophage 16-3. We have constructed complex maps (i.e. EcoRI map with 16 and EcoRV with 11 fragments)

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes

Study of Erwinia amylovora colonization and migration on blossoms of susceptible and tolerant apple cultivars

The stigmata of detached flowers of susceptible and tolerant apple cultivars were inoculated with about 104 gfp labeled Erwinia amylovora . There were no apparent differences in the colonization, multiplication and survival of the bacteria on the stigmatic surface of the culivars. Bacteria were washed down to the hypanthium surface 24 hours after inoculation. The visual symptoms of the infection were the discoloration and shrinkage of the floral parts. The gradual browning associated with the infection appeared first on the surface of the hypanthium followed by the discoloration of the style. The color of the filaments turned into brown only 120 hours after the inoculation. Bacterial cells were not detected in the tissues of the styles and filaments. The traits of the hypanthium surface are of prominent importance in the progression of the infection. The wrinkled surface, the convex shape of the outer epidermal cell walls with thin cuticle and the sunken stomata helped to preserve a water film for a longer period providing medium for the motility of the bacteria in the susceptible cultivar. Bacteria were restricted to small water droplets on the flat and waxy surface of the hypanthium of the tolerant cultivar and only a few were able to enter the tissues. Large bacterium aggregations were detected in the intercellular spaces of the parenchyma of the susceptible cultivar 48 hours after the inoculation. In the next period the Erwinia amylovora cells gradually invaded the intercellulars of the hypanthium wall, the wall of the ovary and the pedicel. Low level of bacterium aggregation was found in the intercellulars of the tolerant cultivars. It is suggested that the progression of the infection was inhibited also by physiological factors

Study of Erwinia amylovora colonization and migration on blossoms of susceptible and tolerant apple cultivars

The stigmata of detached flowers of susceptible and tolerant apple cultivars were inoculated with about 104 gfp labeled Erwinia amylovora . There were no apparent differences in the colonization, multiplication and survival of the bacteria on the stigmatic surface of the culivars. Bacteria were washed down to the hypanthium surface 24 hours after inoculation. The visual symptoms of the infection were the discoloration and shrinkage of the floral parts. The gradual browning associated with the infection appeared first on the surface of the hypanthium followed by the discoloration of the style. The color of the filaments turned into brown only 120 hours after the inoculation. Bacterial cells were not detected in the tissues of the styles and filaments. The traits of the hypanthium surface are of prominent importance in the progression of the infection. The wrinkled surface, the convex shape of the outer epidermal cell walls with thin cuticle and the sunken stomata helped to preserve a water film for a longer period providing medium for the motility of the bacteria in the susceptible cultivar. Bacteria were restricted to small water droplets on the flat and waxy surface of the hypanthium of the tolerant cultivar and only a few were able to enter the tissues. Large bacterium aggregations were detected in the intercellular spaces of the parenchyma of the susceptible cultivar 48 hours after the inoculation. In the next period the Erwinia amylovora cells gradually invaded the intercellulars of the hypanthium wall, the wall of the ovary and the pedicel. Low level of bacterium aggregation was found in the intercellulars of the tolerant cultivars. It is suggested that the progression of the infection was inhibited also by physiological factors

Some structural characteristics of the flowers of apple cultivars with different susceptibilty to fire blight

Several morphological characters of the hypanthium (size and form of the surface, the shape of the hypanthium) and anatomical traits (number and pattern of stomata) of apple cultivars (Malus domestica L.) with different susceptibility to fire blight were studied. The size of hypantium surface was calculated by modelling the hypanthium with a truncated cone. Three types of hypanthium surface form have been revealed: straight, convex and a complex "shouldered" one. The angle between the style and the wall of the hypanthium was narrow or wide. The stomata on the hypanthium surface can be arranged in a zone in the middle third of the hypanthium or dispersed more or less evenly. The number of stomata/flower substantially differed among the cultivars examined. The highest stomata number was detected in the flowers of the tolerant cultivar (Freedom) No single characteristics of the hypanthium could convincingly be correlated with susceptibility to fire blight. We suggest, however, that combination of morphological properties that sustain moist environment in the hypanthium contribute to susceptibility

Some structural characteristics of the flowers of apple cultivars with different susceptibilty to fire blight

Several morphological characters of the hypanthium (size and form of the surface, the shape of the hypanthium) and anatomical traits (number and pattern of stomata) of apple cultivars (Malus domestica L.) with different susceptibility to fire blight were studied. The size of hypantium surface was calculated by modelling the hypanthium with a truncated cone. Three types of hypanthium surface form have been revealed: straight, convex and a complex "shouldered" one. The angle between the style and the wall of the hypanthium was narrow or wide. The stomata on the hypanthium surface can be arranged in a zone in the middle third of the hypanthium or dispersed more or less evenly. The number of stomata/flower substantially differed among the cultivars examined. The highest stomata number was detected in the flowers of the tolerant cultivar (Freedom) No single characteristics of the hypanthium could convincingly be correlated with susceptibility to fire blight. We suggest, however, that combination of morphological properties that sustain moist environment in the hypanthium contribute to susceptibility