A pseudomonas syringae pv. tomato DC3000 Hrp (Type III secretion) deletion mutant expressing the Hrp system of bean pathogen P. syringae pv. syringae 61 retains normal host specificity for tomato

The plant pathogenic species Pseudomonas syringae is divided into numerous pathovars based on host specificity. For example, P. syringae pv. tomato DC3000 is pathogenic on tomato and Arabidopsis, whereas P. syringae pv. syringae 61 is pathogenic on bean. The ability of P. syringae strains to elicit the hypersensitive response (HR) in non-hosts or be pathogenic (or parasitic) in hosts is dependent on the Hrp (type III secretion) system and effector proteins this system is thought to inject into plant cells. To test the role of the Hrp system in determining host range, the hrp/hrc gene cluster (hrpK through hrpR) was deleted from DC3000 and complemented in trans with the orthologous cluster from strain 61. Mutant CUCPB5114 expressing the bean pathogen Hrp system on plasmid pCPP2071 retained the ability of wild-type DC3000 to elicit the HR in bean, to grow and cause bacterial speck in tomato, and to elicit a cultivar-specific (gene-for-gene) HR in tomato plants carrying the Pto resistance gene. However, the symptoms produced in compatible tomato plants involved markedly reduced chlorosis, and CUCPB5114(pCPP2071) did not grow or produce symptoms in Arabidopsis Col-0 although it was weakly virulent in NahG Arabidopsis. A hypersensitive-like collapse was produced by CUCPB5114(pCPP2071) in Arabidopsis Col-0 at 1 x 10(7) CFU/ml, but only if the bacteria also expressed AvrB, which is recognized by the RPM1 resistance gene in Col-0 and confers incompatibility. These observations support the concept that the P. syringae effector proteins, rather than secretion system components, are the primary determinants of host range at both the species and cultivar levels of host specificity.     

Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities.

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.     

Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.     

Rough‐and‐tumble play as a window on animal communication

Rough‐and‐tumble play (RT) is a widespread phenomenon in mammals. Since it involves competition, whereby one animal attempts to gain advantage over another, RT runs the risk of escalation to serious fighting. Competition is typically curtailed by some degree of cooperation and different signals help negotiate potential mishaps during RT. This review provides a framework for such signals, showing that they range along two dimensions: one from signals borrowed from other functional contexts to those that are unique to play, and the other from purely emotional expressions to highly cognitive (intentional) constructions. Some animal taxa have exaggerated the emotional and cognitive interplay aspects of play signals, yielding admixtures of communication that have led to complex forms of RT. This complexity has been further exaggerated in some lineages by the development of specific novel gestures that can be used to negotiate playful mood and entice reluctant partners. Play‐derived gestures may provide new mechanisms by which more sophisticated communication forms can evolve. Therefore, RT and playful communication provide a window into the study of social cognition, emotional regulation and the evolution of communication systems.     

The effect of some biphenyl solubilizing and suspending agents on biphenyl 4-hydroxylase of rabbit liver microsomes

The effects of ethanol, acetone, dimethylsulfoxide (DMSO), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monolaurate (Tween 20), Triton X-100, and carboxymethyl cellulose (CMC) on the kinetics of biphenyl 4-hydroxylase of rabbit liver microsomes were investigated in an attempt to find a substrate-solubilizing or suspending agent (carrier) which was itself a non-effector of the mixed-function oxidase. The effects of these carriers on the activities of NADPH-cytochrome P-450 reductase, NADPH-cytochrome c reductase, and cytochrome P-450 content were also investigated.Ethanol and DMSO inhibited biphenyl 4-hydroxylase and NADPH-cytochrome P-450 reductase. Acetone inhibited the hydroxylase uncompetitively at concentrations which appeared to stimulate NADPH-cytochrome P-450 reductase. All of the detergents inhibited biphenyl 4-hydroxylase although only Triton X-100 markedly affected the reduction of cytochrome P-450. The interaction of Tween 80 with the hydroxylase gave rise to non-linear Lineweaver-Burk plots although at high concentrations of biphenyl or low concentrations of the detergent the inhibition appeared to be competitive.Biphenyl caused a 2–3-fold stimulation of NADPH-cytochrome P-450 reductase, but in the presence of Tween 80 the stimulation was absent. Since V of biphenyl 4-hydroxylase in the presence of Tween 80 was not significantly different from V in its absence it would appear that the reduction of cytochrome P-450 was not ratelimiting.Of all the carriers studied only CMC was without effect on all aspects of microsomal electron transport investigated. As far as biphenyl 4-hydroxylase is concerned, CMC appears to be the most suitable substrate carrier.     

The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner.

We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways from Pseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22 degrees C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel the P. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name of hrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.