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Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.

Members of the Pseudomonas genus produce diverse secondary metabolites affecting other bacteria, fungi or predating nematodes and protozoa but are also equipped with the capacity to secrete different types of ribosomally encoded toxic peptides and proteins, ranging from small microcins to large tailocins. Studies with the human pathogen Pseudomonas aeruginosa have revealed that effector proteins of type VI secretion systems are part of the antibacterial armamentarium deployed by pseudomonads. A novel class of antibacterial proteins with structural similarity to plant lectins was discovered by studying antagonism among plant-associated Pseudomonas strains. A genomic perspective on pseudomonad bacteriocinogeny shows that the modular architecture of S pyocins of P. aeruginosa is retained in a large diversified group of bacteriocins, most of which target DNA or RNA. Similar modularity is present in as yet poorly characterized Rhs (recombination hot spot) proteins and CDI (contact-dependent inhibition) proteins. Well-delimited domains for receptor recognition or cytotoxicity enable the design of chimeric toxins with novel functionalities, which has been applied successfully for S and R pyocins. Little is known regarding how these antibacterials are released and ultimately reach their targets. Other remaining issues concern the identification of environmental triggers activating these systems and assessment of their ecological impact in niches populated by pseudomonads.

The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779 T . The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family-gene cluster families of hundreds or more diverse organisms in one single MS/MS network.MS/MS molecular networking | mass spectrometry | microbial ecology T ens of thousands of sequenced microbial genomes or rough drafts of genomes are available at this time, and this number is predicted to grow into the millions over the next decades. This wealth of sequence data has the potential to be used for the discovery of small bioactive molecules through genome mining (1-6). Genome mining is a process in which small molecules are discovered by predicting what compound will be genetically encoded based on the sequences of biosynthetic gene clusters. However, the process of mining genetically encoded small molecules is not keeping pace with the rate by which genome sequences are being obtained. In general, genome mining is still done one gene cluster at a time and requires many person-years of effort to annotate a single molecule. The time and significant expertise that current genome mining requires also make genome mining very expensive. In light of this extensive effort and cost, alternative approaches to genome mining and annotating specialized metabolites must be developed that not only take advantage of the sequenced resources available and make it efficient to perform genome mining on a more global scale but also enable the molecular analysis of unsequenced organisms. Such methods will then significantly reduce the cost of genome mining by increasing the speed with which molecules are connected to candidate genes and using resources already available. Here, we put fo...

The genus Pseudomonas hosts an extensive genetic diversity and is one of the largest genera among Gram-negative bacteria. Type strains of Pseudomonas are well known to represent only a small fraction of this diversity and the number of available Pseudomonas genome sequences is increasing rapidly. Consequently, new Pseudomonas species are regularly reported and the number of species within the genus is constantly evolving. In this study, whole genome sequencing enabled us to define 43 new Pseudomonas species and provide an update of the Pseudomonas evolutionary and taxonomic relationships. Phylogenies based on the rpoD gene and whole genome sequences, including, respectively, 316 and 313 type strains of Pseudomonas, revealed sixteen groups of Pseudomonas and, together with the distribution of cyclic lipopeptide biosynthesis gene clusters, enabled the partitioning of the P. putida group into fifteen subgroups. Pairwise average nucleotide identities were calculated between type strains and a selection of 60 genomes of non-type strains of Pseudomonas. Forty-one strains were incorrectly assigned at the species level and among these, 19 strains were shown to represent an additional 13 new Pseudomonas species that remain to be formally classified. This work pinpoints the importance of correct taxonomic assignment and phylogenetic classification in order to perform integrative studies linking genetic diversity, lifestyle, and metabolic potential of Pseudomonas spp.

The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

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Determination of the N-terminal sequences of two EPTC (S-ethyl dipropylcarbamothioate)-induced proteins from thiocarbamate-degradingEvidence for the involvement of this cytochrome P-450 system in EPTC degradation and herbicide biosafening for maize was obtained by complementation experiments using EPTC-negative Rhodococcus erythropolis SQ1 and mutant FAJ2027 as acceptor strains. N dealkylation by cytochrome P-450 and conversion of the released aldehyde into the corresponding carboxylic acid by aldehyde dehydrogenase are proposed as the reactions initiating thiocarbamate catabolism in Rhodococcus sp. strain NI86/21. In addition to the major metabolite N-depropyl EPTC, another degradation product was identified, EPTC-sulfoxide.