René De Mot

Flagella-Driven Chemotaxis Towards Exudate Components Is an Important Trait for Tomato Root Colonization by Pseudomonas fluorescens

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.

The C‐terminal sequence conservation between OmpA‐related outer membrane proteins and MotB suggests a common function in both Gram‐positive and Gram‐negative bacteria, possibly in the interaction of these domains with peptidoglycan

The major outer membrane protein OprF from Pseudomonas species displays strong homology to several outer membrane proteins from unrelated species, including OmpA from enteric bacteria (De Mot et ai, 1992, Mol Gen Genef 231: 489-493). However, this homology is confined to the respective C-terminal regions (about 100-140 residues), and there is no obvious similarity between the A/-terminal regions. Such remarkable intergeneric sequence conservation presumably reflects a similar, but as yet unidentified, function of this domain. For both OmpA of Escherichia coii, and OprF of Pseudomonas aeruginosa, a structural role in stabilizing the outer membrane has been proposed (Gotoh et ai, 1989, J Bacterioi 171: 983-990; Woodruff and Hancock, 1989, J Bacterioi 171: 3304-3309). In addition, pore-forming activity has been demonstrated for both proteins (Nikaido et ai., 1991, J Bioi Chem 266: 770-779; Sugawara and Nikaido, 1992, J Bioi Chem 267: 2507-2511). The extended C-terminal homology is also found in lipoproteins that are tightly, but non-covalently bound to peptidogiycan. These peptidoglycan-associated lipoproteins (PALs) are important structural elements for the cell envelope (Lazzaroni and Portalier, 1992, Mol Microbiol 6: 735-742). The functions of the other outer membrane proteins in this family are poorly characterized. However, it is noteworthy that for several of them strong, non-covalent association with peptidogiycan has been described (Lugtenberg and van Alphen, 1983, Biochim Biophys Acta 737: 51-115; Hancock ef ai, 1990, Moi Microbioi 4: 1069-1075), although the protein domains Interacting with the peptidogiycan layer remain to be identified.

The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus

BACKGROUND The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type). RESULTS We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp. strain NI86/21. The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05% SDS. Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced. Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes. CONCLUSIONS 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.

Proteasomes and other self-compartmentalizing proteases in prokaryotes

The proteasome represents the major non-lysosomal proteolytic system in eukaryotes. It confines proteolytic activity to an inner compartment that is accessible to unfolded proteins only. The strategy of controlling intracellular breakdown of proteins by self-compartmentalization is also used by different types of prokaryotic energy-dependent proteases. Genomic sequencing data reveal that various combinations of these energy-dependent proteases occur in prokaryotic cells from different lineages.

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

SUMMARY A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms.

MS/MS networking guided analysis of molecule and gene cluster families

Significance The paper introduces the concepts of molecular families (MFs) and gene cluster families (GCFs). We define MFs as structurally related molecules based on their mass spectral fragmentation patterns, whereas GCFs are biosynthetic gene clusters that show similar gene cluster organization with a high degree of sequence similarity. We use MS/MS networking as a tool to map the molecular network of more than 60 organisms, most of which are unsequenced, and locate their nonribosomal peptide MFs. These MFs from unsequenced organisms are then connected to GCFs of publicly available genome sequences of closely related organisms. 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 20779T. 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.

Degradation of Pesticides by Actinomycetes

Actinomycetes have considerable potential for the biotransformation and biodegra-dation of pesticides. Members of this group of Gram-positive bacteria have been found to degrade pesticides with widely different chemical structures, including organochlorines, s-triazines, triazinones, carbamates, organophosphates, organophosphonates, acetanilides, and sulfonylureas. A limited number of these xenobiotic pesticides can be mineralized by single isolates, but often consortia of bacteria are required for complete degradation. Cometabolism of pesticides is frequently observed within this group of bacteria. When compared with pesticide degradation by Gram-negative bacteria, much less information about molecular mechanisms involved in biotransformations of pesticides by actinomycetes is available. Progress in this area has been seriously hampered by a lack of suitable molecular genetic tools for most representatives of this major group of soil bacteria. Overcoming this constraint would enable a better exploitation...

Bacteria killing their own kind: novel bacteriocins of Pseudomonas and other γ-proteobacteria

Abstract Pseudomonas aeruginosa strains frequently produce proteinaceous, narrow-spectrum antibacterial bacteriocins known as pyocins. The majority of these compounds exert their toxicity through non-specific DNA degradation inside sensitive cells. Here, we describe the identification of novel bacteriocins of different types in related bacteria inhabiting diverse niches. The abundance of bacteriocin genes in bacteria such as Pseudomonas , Klebsiella and Photorhabdus suggests that these systems play an important role in the competition between rival bacteria. The chimeric nature of these proteins implies that extensive domain swapping has contributed to the diversification of bacteriocins in γ-proteobacteria.

A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes

The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications.

The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri

The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.