Agar composition affects in vitro screening of biocontrol activity of antagonistic microorganisms.

Agar-based screening assays are the method of choice when evaluating antagonistic potential of bacterial biocontrol-candidates against pathogens. We showed that when using the same medium, but different agar compositions, the activity of a bacterial antagonist against Agrobacterium was strongly affected. Consequently, results from in vitro screenings should be interpreted cautiously.

Efficacy of hydrogen peroxide treatment for control of hairy root disease caused by rhizogenic agrobacteria.

AIMS: Rhizogenic Agrobacterium strains are the causative agent of hairy root disease (HRD), an increasing problem in the hydroponic cultivation of tomato and cucumber in Europe. A previous study has demonstrated that different lineages of rhizogenic agrobacteria are able to form biofilms. Although hydrogen peroxide (H2 O2 ) is a frequently used biocide in hydroponic systems, until now its effectiveness to remove rhizogenic agrobacteria has not been unequivocally demonstrated. Therefore, the main objective of this study was to assess the efficacy of H2 O2 in controlling Agrobacterium-containing biofilms. METHODS AND RESULTS: Using lab-scale experiments, we found a huge variation between different rhizogenic Agrobacterium strains in EC50 values, ranging from 18·8 to 600 ppm H2 O2 , representing the lowest and highest concentration tested respectively. Using pilot-scale experiments in which different H2 O2 concentrations were tested, treatment with 25 ppm H2 O2 was found to be ineffective. In contrast, treatment with 50 ppm significantly affected a catalase-negative Agrobacterium population, while a catalase-positive population was only marginally affected. For the catalase-positive Agrobacterium population, a treatment of 100 ppm H2 O2 was required to be effective. Finally, H2 O2 treatment of HRD in two commercial greenhouses was monitored, and showed that the H2 O2 concentration decreased considerably towards the end of the irrigation circuits. Further, a clear correlation was found between the actual concentration of H2 O2 and the incidence of HRD. CONCLUSION: We showed that H2 O2 may be effective to reduce biofilm formation by rhizogenic bacteria. Nevertheless, it was clear from our results that the required H2 O2 concentration depends on the particular Agrobacterium strain(s) present in the greenhouse. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that examined the effectiveness of H2 O2 to control HRD in hydroponic systems, and the effect of catalase activity on H2 O2 effectiveness. Our study has direct relevance for the highly intensive horticultural sector.

HylA, an alternative hydrolase for initiation of catabolism of the phenylurea herbicide linuron in Variovorax sp. strains.

Variovorax sp. strain WDL1, which mineralizes the phenylurea herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in Km, temperature optimum, and phenylurea herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.

Identification and characterization of the omaA gene encoding the major outer membrane protein of Azospirillum brasilense.

The major outer membrane protein (MOMP) of Azospirillum brasilense was purified and degenerate oligonucleotides were constructed on the basis of partial internal amino acid sequences. PCR products were obtained using total DNA of A. brasilense as template. One of these, a 766-bp fragment, was DIG-labelled and used in Southern hybridization against A. brasilense DNA and a genomic library of A. brasilense in Escherichia coli. A clone containing a 20-kb EcoRI insert in pLAFR3 was identified by PCR screening. From this insert, an EcoRI-SalI fragment of approximately 3.5-kb was subcloned in pUC19. The gene encoding the A. brasilense MOMP was sequenced and analyzed. The deduced amino acid sequence contains a putative signal peptide of 23 residues, followed by 367 amino acids of the mature protein with a molecular mass of 38,753 Da. The deduced amino acid sequence shows similarity to certain bacterial porins.

Characterization of an OprF-deficient mutant suggests that OprF is an essential protein for Pseudomonas fluorescens strain MF0.

A stable OprF-deficient mutant for Pseudomonas fluorescens strain MF0 was constructed using reverse genetics. This mutant, called MF372, showed a rounded morphology and grew more slowly in minimal medium, but not in rich medium. Contrary to other Pseudomonas strains, the loss of OprF for strain MF0 was accompanied by an altered outer membrane composition. At least three outer membrane proteins were overexpressed, apparently as a consequence of adaptive mutations. The N-terminal sequence of two of them revealed strong similarities with porins of the OprD family from P. aeruginosa. The data presented here shows that OprF may be an essential protein for this P. fluorescens strain.

Sequence analysis of the oxidase/reductase genes upstream of the Rhodococcus erythropolis aldehyde dehydrogenase gene thcA reveals a gene organisation different from Mycobacterium tuberculosis.

The sequence of the DNA region upstream of the thiocarbamate-inducible aldehyde dehydrogenase gene thcA of Rhodococcus erythropolis NI86/21 was determined. Most of the predicted ORFs are related to various oxidases/reductases, including short-chain oxidases/reductases, GMC oxidoreductases, alpha-hydroxy acid oxidases (subfamily 1 flavin oxidases/dehydrogenases), and subfamily 2 flavin oxidases/dehydrogenases. One ORF is related to enzymes involved in biosynthesis of PQQ or molybdopterin cofactors. In addition, a putative member of the TetR family of regulatory proteins was identified. The substantial sequence divergence from functionally characterized enzymes precludes a reliable prediction about the probable function of these proteins at this stage. In Mycobacterium tuberculosis H37Rv, most of these ORFs have homologs that are also clustered in the genome, but some striking differences in gene organization were observed between Rhodococcus and Mycobacterium.

Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens.

The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In P. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF. Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of the P. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes in P. aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P. aeruginosa clinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.

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.

Degradation of pesticides by actinomycetes.

Actinomycetes have considerable potential for the biotransformation and biodegradation 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 of the biodegradation and biotransformation abilities of actinomycetes for applications such as bioremediation and construction of transgenic herbicide-resistant crops.

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.

The 20S proteasome of Streptomyces coelicolor.

20S proteasomes were purified from Streptomyces coelicolor A3(2) and shown to be built from one alpha-type subunit (PrcA) and one beta-type subunit (PrcB). The enzyme displayed chymotrypsin-like activity on synthetic substrates and was sensitive to peptide aldehyde and peptide vinyl sulfone inhibitors and to the Streptomyces metabolite lactacystin. Characterization of the structural genes revealed an operon-like gene organization (prcBA) similar to Rhodococcus and Mycobacterium spp. and showed that the beta subunit is encoded with a 53-amino-acid propeptide which is removed during proteasome assembly. The upstream DNA region contains the conserved orf7 and an AAA ATPase gene (arc).

Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis.

A gene encoding a AAA ATPase was discovered in the 5' region of the second operon of 20 S proteasome subunits in the nocardioform actinomycete Rhodococcus erythropolis NI86/21. The gene was cloned and expressed in Escherichia coli. The protein, ARC (AAA ATPase forming Ring-shaped Complexes), is a divergent member of the AAA family. The deduced product of the arc gene is 591 residues long (66 kDa). The purified protein possesses a low, N-ethylmaleimide-sensitive ATPase activity and forms rings of six subunits, arranged symmetrically around a central opening or cavity. Two-dimensional crystals grown on lipid monolayers yielded images of the ATPase molecules in "end-on" orientation at 1.9 nm resolution.

A major outer membrane protein of Rahnella aquatilis functions as a porin and root adhesin.

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60 degrees C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand beta-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.

A self-compartmentalizing protease in Rhodococcus: the 20S proteasome.

The 26S proteasome represents a major, energy-dependent and self-compartmentalizing protease system in eukaryotes. The proteolytic core of this complex, the 20S proteasome, is also ubiquitous in archaea. Although absent from most eubacteria, this multi-subunit protease was recently discovered in Rhodococcus and appears to be confined to actinomycetes. The eubacterial 20S proteasome represents an attractive complementary system to study proteasome assembly, quaternary structure, and catalytic mechanism. In addition, it is likely to contribute substantially to our understanding of the role of various self-compartmentalizing proteases in bacterial cells.

Applied aspects of Rhodococcus genetics.

Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.

Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis.

Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21. Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5' end of integrons was found. Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R. erythropolis SQ1.

New method for RNA isolation from actinomycetes: application to the transcriptional analysis of the alcohol oxidoreductase gene thcE in Rhodococcus and Mycobacterium.

A new protocol for the isolation of RNA from Rhodococcus and other actinomycetes such as Mycobacterium and Amycolatopsis was developed. The method is based on rapid lysis of cells in a high-speed reciprocal shaker using small abrasive particles followed by spin column purification of the lysate. This quick procedure yields RNA preparations suitable for functional studies. This was shown for the thcE gene of R. erythropolis NI86/21, which encodes a N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase. The thcE transcript was detected by Northern hybridization in R. erythropolis, R. fascians, Mycobacterium chlorophenolicum and Mycobacterium smegmatis. The transcriptional start point of the gene was determined by primer extension of the R. erythropolis mRNA.

Thiocarbamate herbicide-inducible nonheme haloperoxidase of Rhodococcus erythropolis NI86/21.

During biodegradation of thiocarbamate herbicides by Rhodococcus erythropolis NI86/21, a protein with an M(r) of 30,000 is induced (I. Nagy, G. Schoofs, F. Compernolle, P. Proost, J. Vanderleyden, and R.De Mot, J. Bacteriol. 177:676-687, 1995). Based on N-terminal sequence data for the protein purified by two-dimensional electrophoresis, the corresponding structural gene, thcF, was cloned and sequenced. The deduced protein sequence of ThcF is homologous to those of nonheme haloperoxidases. A particularly high level of sequence identity (72.6%) was observed for the chloroperoxidase from Pseudomonas pyrrocinia. A polyclonal antibody against the latter enzyme cross-reacted with ThcF either produced by the original Rhodococcus cells or overexpressed heterologously in Escherichia coli. In both thiocarbamate-grown Rhodococcus cells and E. coli cells expressing thcF, the haloperoxidase activity of ThcF was demonstrated. The thiocarbamate-inducible R. erythropolis ThcF protein represents the first (nonheme) haloperoxidase to be identified in a nocardioform actinomycete.