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Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

The allelic variation in four avirulence (Avr) and four extracellular protein (Ecp)-encoding genes of the tomato pathogen Cladosporium fulvum was analyzed for a worldwide collection of strains. The majority of polymorphisms observed in the Avr genes are deletions, point mutations, or insertions of transposon-like elements that are associated with transitions from avirulence to virulence, indicating adaptive evolution of the Avr genes to the cognate C. fulvum resistance genes that are deployed in commercial tomato lines. Large differences in types of polymorphisms between the Avr genes were observed, especially between Avr2 (indels) and Avr4 (amino-acid substitutions), indicating that selection pressure favors different types of adaptation. In contrast, only a limited number of polymorphisms were observed in the Ecp genes, which mostly involved synonymous modifications. A haplotype network based on the polymorphisms observed in the effector genes revealed a complex pattern of evolution marked by reticulations that suggests the occurrence of genetic recombination in this presumed asexual fungus. This, as well as the identification of strains with identical polymorphisms in Avr and Ecp genes but with opposite mating-type genes, suggests that development of complex races can be the combined result of positive selection and genetic recombination.

Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.

Cyclic lipopeptides (CLPs) are versatile molecules produced by a variety of bacterial genera, including plant-associated Pseudomonas spp. CLPs are composed of a fatty acid tail linked to a short oligopeptide, which is cyclized to form a lactone ring between two amino acids in the peptide chain. CLPs are very diverse both structurally and in terms of their biological activity. The structural diversity is due to differences in the length and composition of the fatty acid tail and to variations in the number, type, and configuration of the amino acids in the peptide moiety. CLPs have received considerable attention for their antimicrobial, cytotoxic, and surfactant properties. For plant-pathogenic Pseudomonas spp., CLPs constitute important virulence factors, and pore formation, followed by cell lysis, is their main mode of action. For the antagonistic Pseudomonas sp., CLPs play a key role in antimicrobial activity, motility, and biofilm formation. CLPs are produced via nonribosomal synthesis on large, multifunctional peptide synthetases. Both the structural organization of the CLP synthetic templates and the presence of specific domains and signature sequences within peptide synthetase genes will be described for both pathogenic and antagonistic Pseudomonas spp. Finally, the role of various genes and regulatory mechanisms in CLP production by Pseudomonas spp., including two-component regulation and quorum sensing, will be discussed in detail.

SUMMARY Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated resistance genes of the RLP class are the tomato Cf genes, which confer resistance to the fungal pathogen Cladosporium fulvum. To date, several other RLP genes are known to be implicated in resistance in other plant-pathogen interactions. These include HcrVf2 from apple, Ve1 and Ve2 from tomato, and RPP27 from Arabidopsis, which are involved in resistance to Venturia, Verticillium and Peronospora, respectively. Furthermore, the tomato RLP gene LeEix initiates defence responses upon elicitation with a fungal ethylene-inducing xylanase (EIX) of non-pathogenic Trichoderma. The tomato Cf genes, which are the most intensively studied RLP resistance genes, are usually found in clusters of several homologues. Whereas some of these homologues are functional Cf resistance genes, others have no known function in resistance. Different evolutionary processes contribute to variation in functional Cf genes, and functional as well as non-functional homologues may provide a source for the generation of novel Cf resistance genes. To date, little is known of the proteins that interact with Cf proteins to initiate defence responses. In contrast to the LeEix protein and the corresponding EIX elicitor, for which a direct interaction was found, no direct interaction between Cf proteins and the corresponding C. fulvum elicitors has been demonstrated. Analogous to the CLAVATA signalling complex, which comprises an RLP, a receptor-like kinase (RLK) and a small proteineous ligand, Cf proteins may form a complex with RLKs and thus initiate signalling upon recognition of the corresponding elicitors. The presence of RLP resistance genes in diverse plant species suggests that these genes play an important role in the extracellular recognition of plant pathogens.

SUMMARY Cladosporium fulvum is a fungal pathogen of tomato that grows exclusively in the intercellular spaces of leaves. Ecp2 is one of the elicitor proteins that is secreted by C. fulvum and is specifically recognized by tomato plants containing the resistance gene Cf-Ecp2. Recognition is followed by a hypersensitive response (HR) resulting in resistance. HR-associated recognition of Ecp2 has been observed in Nicotiana paniculata, N. sylvestris, N. tabacum and N. undulata that are non-host plants of C. fulvum. Absence of Ecp2-recognition did not lead to growth of C. fulvum on Nicotiana plants. We show that HR-associated recognition of Ecp2 is mediated by a single dominant gene in N. paniculata. However, based on PCR and hybridization analysis this gene is not homologous to known Cf-genes.

summary A striking feature of all elicitor proteins of Cladosporium fulvum that are specifically recognized by tomato is that they contain an even number of cysteine residues. These cysteine residues are thought to be involved in disulphide bridges. In this study, a mutational analysis of the cysteine residues of ECP1, ECP2 and ECP5 was performed, to examine their role in stability and hypersensitive response-inducing activity of the proteins. We show that not all cysteine residues of the ECPs are critical for the hypersensitive response-inducing activity of the proteins, and we propose that the role of cysteine residues in the ECPs is more complex than anticipated.

The suggested use of tobramycin in selective decontamination of the digestive tract led to the investigation of the part played clinically by ribosomal resistance in Pseudomonas aeruginosa. Examination of 32 tobramycin-resistant isolates, both enzyme and non-enzyme producing, suggests that ribosomal resistance is clinically important.

Strains of Klebsiella pneumoniae isolated from sewage were examined for their resistance spectra and for the presence of R factors. Infectious drug resistance was demonstrated in 104 (80%) of the 130 strains tested. Resistance to chloramphenicol, kanamycin or tetracycline was usually R factor-determined. Resistance to gentamicin was not encountered in this species.

A naturally occurring R factor which carried resistance determinants to tetracycline and chloramphenicol as well as a marker for lactose fermentation was found in a strain of Klebsiella pneumoniae isolated from sewage. The TcCmlac+ factor was transferred by conjugation to E. coli K12. It showed no recombination or stable coexistence with R factors of the fi- class. Studies in recombination-deficient (rec-) strains of E. coli K12 showed that the TcCmlac+ factor is an autonomously replicating plasmid. Preparative ultracentrifugation and electron microscopic studies revealed the presence of more than one molecular species. Average contour lengths for two of these species were 11,20 +/- 1,55 mum and 16,14 +/- 1,45 mum, which correspond to molecular weights of about 23,18 X 10(6) and 34,63 X 10(6) daltons, respectively.