Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing disease caused by the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the diffusible antibiotics phenazine-1-carboxylic acid, 2-hydroxyphenazine and pyrrolnitrin (PRN). Because the individual contribution of these antibiotics to PA23 biocontrol has not been defined, mutants deficient in the production of phenazine (PHZ), PRN or both antibiotics were created. Analysis of the PHZ mutant revealed enhanced antifungal activity in vitro and wild-type levels of Sclerotinia disease suppression. Conversely, the PRN- and the PRN/PHZ-deficient strains exhibited decreased antifungal activity in vitro and markedly reduced the ability to control Sclerotinia infection of canola in the greenhouse. These findings suggest that PRN is the primary antibiotic mediating biocontrol of this pathogen. Analysis of prnA-lacZ and phzA-lacZ transcriptional fusions revealed that PRN and PHZ are not subject to autoregulation; moreover, they do not cross-regulate each other. However, HPLC showed a twofold increase in PRN levels in the PHZ(-) background. Finally, PHZ, but not PRN production, is involved in biofilm development in P. chlororaphis PA23.

Vc-NhaD is a Na؉ /H ؉ antiporter from Vibrio cholerae with a sharp maximum of activity at pH ϳ 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ؉ ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.Sodium proton antiporters are universal secondary ion transporters in bacteria. Typically, they expel toxic Na ϩ and Li ϩ ions from the cytoplasm at the expense of the proton motive force, thus playing an important role in cytoplasmic Na ϩ and pH homeostasis and providing energy for Na ϩ symports (for review, see Refs. 1-4 29 -30). This distinguishes Vc-NhaD from other major enterobacterial antiporters. Indeed, Ec-NhaB from E. coli is pHindependent, whereas the activity of Ec-NhaA gradually increases upon pH shift from 7.0 to 8.0, reaching a plateau (9). Curiously, homologous NhaD from Vibrio parahaemolyticus exhibits pH dependence similar to Ec-NhaA rather than Vc-NhaD (31). The Na ϩ /H ϩ antiporters of NhaD type are widely distributed in nature, being found in genomes of pathogenic vibrios, nitrogen-fixing symbionts, magnetotactic cocci and photosynthetic bacteria as well as in higher plants (Fig. 1). In obligate intracellular parasites of Chlamydia genus, NhaD serves as a sole Na ϩ /H ϩ antiporter (32-33). However, very little is known about the molecular mechanisms of cation exchange mediated by these proteins. In the absence of a detailed crystal structure, identification of functionally important residues in antiporters by sitedirected mutagenesis remains one of the most informative approaches. Because Na ϩ /H ϩ antiporters are exchanging cations, negatively charged residues are obvious primary targets for mutagenesis (see for example Refs. 18 and 23). In our previous work we found that mutation of three polar residues, Asp-344, Thr-345 (TMS 3 X and loop IX-X), and Asp-393 (within TMS XI) severely affects the Na ϩ -dependent proton transfer mediated by . In the present study we extended these observations by muta...