New insights into indole-3-acetic acid metabolism in Azospirillum brasilense.

AIMS: The aim of this research was to analyse the global indole-3-acetic acid (IAA) metabolism in three commercially used strains of Azospirillum brasilense. METHODS AND RESULTS: Azospirillum brasilense Sp245, Az39 and Cd, containing a plasmid with the ipdC-gusA fusion (pFAJ64), were cultured in minimal medium MMAB with or without 10 mg l-1 of l-trp till exponential or stationary growth phase. The cultures were then split into 10 ml tubes and individually treated with 10 mg ml-1 IAA, IBA or NAA (auxin catabolism and homeostasis); IAPhe, IALeu, IAA-ala, IAA-glucose (IAA conjugate hydrolysis); or l-lys, l-leu, l-ileu, l-phe, l-ala, l-val, l-arg, l-glu, l-his, l-met, l-asp, l-cys, l-ser, l-pro, l-thr and l-trp (regulation of IAA biosynthesis and IAA conjugation). Bacterial growth, IAA production and ipdC expression were evaluated. None of the A. brasilense strains were able to hydrolyse IAA conjugates, catabolize auxins, or conjugate IAA with amino acids or glucose. l-amino acids l-met, l-val, l-cys and l-ser inhibited bacterial growth and decreased IAA biosynthesis. The expression of ipdC and IAA biosynthesis but not bacterial growth was affected by l-leu, l-phe, l-ala, l-ile, l-pro. l-arg, l-glu, l-his, l-lys, l-asp and l-thr did not affect any of the measured parameters. CONCLUSIONS: In this paper, we confirmed that A. brasilense produces IAA only in presence of l-trp is not able to degrade auxins, conjugate IAA with sugars and/or l-amino acids, or hydrolyse such conjugates to release free IAA. Finally, we found that bacterial growth and/or IAA biosynthesis were inhibited by the presence of several l-amino acids probably by diversion of the cellular metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose a renewed model to explain IAA metabolism in A. brasilense, one of the most studied phytostimulatory bacteria.

Repurposing of nucleoside- and nucleobase-derivative drugs as antibiotics and biofilm inhibitors.

There is an urgent need for new antibacterial drugs that are robust against the development of resistance. Drug repurposing is a cost-effective strategy to fast-track the drug development process. Here we examine why the nucleoside and nucleobase analogue drugs in particular present an attractive class for repurposing. Some of these drugs have already been evaluated for their potential as antibacterial agents. In addition to inhibiting bacterial growth and survival, some also act synergistically with antibiotics, and as such can enhance the therapeutic spectrum of currently available antibiotics. Furthermore, nucleoside and nucleobase analogue drugs can inhibit bacterial virulence and biofilm formation. Biofilms are known to impart antibiotic tolerance and are associated with chronic infections. Targeting biofilm formation thus renders pathogens more susceptible to antibiotic treatment and host immune defences. Moreover, specific analogues have properties that make them less susceptible to the development of resistance. Thus, nucleoside and nucleobase analogue drugs ought to be considered as new weapons in our fight against pathogenic bacteria.

A GFP promoter fusion library for the study of Salmonella biofilm formation and the mode of action of biofilm inhibitors.

Salmonella, an important foodborne pathogen, forms biofilms in many different environments. The composition of these biofilms differs depending on the growth conditions, and their development is highly coordinated in time. To develop efficient treatments, it is therefore essential that biofilm formation and its inhibition be understood in different environments and in a time-dependent manner. Many currently used techniques, such as transcriptomics or proteomics, are still expensive and thus limited in their application. Therefore, a GFP-promoter fusion library with 79 important Salmonella biofilm genes was developed (covering among other things matrix production, fimbriae and flagella synthesis, and c-di-GMP regulation). This library is a fast, inexpensive, and easy-to-use tool, and can therefore be conducted in different experimental setups in a time-dependent manner. In this paper, four possible applications are highlighted to illustrate and validate the use of this reporter fusion library.

FISH analysis of Lactobacillus biofilms in the gastrointestinal tract of different hosts.

AIMS: To investigate the spatial organization of endogenous and exogenously applied Lactobacillus communities at specific locations in the adult gastrointestinal tract of different hosts. METHODS AND RESULTS: Samples of the human, murine and avian gastrointestinal tract of subjects that received or not received a Lactobacillus probiotic were analysed by fluorescence in situ hybridization (FISH) with rRNA-targeted probes. High levels of endogenous lactobacilli were observed on the nonsecretory, stratified squamous epithelia present in the forestomach of mice and crop of chickens, respectively. These epithelial associations showed characteristics of bacterial biofilms, i.e. bacteria attached to a surface and embedded in a matrix of extracellular polymeric substances. In other regions of the analysed intestines, lactobacilli seemed to occur mainly as dispersed bacterial cells or as microcolonies. Exogenous administration of a Lactobacillus probiotic did increase the levels of loosely adherent Lactobacillus cells detected. However, the probiotic strains were unable to establish themselves inside the gastrointestinal biofilms. CONCLUSIONS: Gastrointestinal biofilms of lactobacilli occur only in specific niches in certain hosts, such as the murine forestomach and avian crop. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm formation by lactobacilli in specific parts of animal gastrointestinal tracts was documented for the first time by FISH.

Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis.

While some probiotic strains might have adjuvant effects in the therapy for inflammatory bowel diseases (IBD), these effects remain controversial and cannot be generalized. In this study, a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having a drastic modification in its lipoteichoic acid (LTA) molecules, was analysed for its effects in an experimental colitis model. Dextran sulphate sodium (DSS) was used to induce either moderate to severe or mild chronic colitis in mice. Mice received either phosphate-buffered saline (PBS), LGG wild-type or the dltD mutant via the drinking water. Macroscopic parameters, histological abnormalities, cytokine and Toll-like receptor (TLR) expression were analysed to assess disease activity. LGG wild-type did not show efficacy in the different experimental colitis set-ups. This wild-type strain even seemed to exacerbate the severity of colitic parameters in the moderate to severe colitis model compared to untreated mice. In contrast, mice treated with the dltD mutant showed an improvement of some colitic parameters compared to LGG wild-type-treated mice in both experimental models. In addition, treatment with the dltD mutant correlated with a significant down-regulation of Toll-like receptor-2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD.

Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects.

Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum-plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (alpha- and beta-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P(II). The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (gamma-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a P(II)-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O(2) level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.

Processing and export of peptide pheromones and bacteriocins in Gram-negative bacteria.

Cell-density-dependent gene expression is widespread in bacteria and is mediated by extracellular communication molecules. Gram-negative bacteria often use N-acyl homoserine lactones, whereas cell-cell signaling in Gram-positive bacteria is accomplished using post-translationally processed peptide pheromones. In many Gram-positive bacteria, export of these peptides requires the activity of a dedicated ATP-binding cassette (ABC) transporter, which cleaves off a typical leader peptide termed the double-glycine leader sequence concomitant with translocation across the membrane. Inspection of bacterial genome sequences has revealed the presence of similar ABC transporters, as well as genes encoding peptides with double-glycine-type leader sequences in Gram-negative bacteria, and it is suggested that the postulated transported peptides could perform a signaling function.

Cloning and sequence of the Rhizobium leguminosarum biovar phaseoli fixA gene.

We report the identification and cloning of Rhizobium leguminosarum biovar phaseoli fixABCX homologous genes and the complete nucleotide sequence of the fixA gene. The corresponding gene product is highly homologous to the Rhizobium meliloti and Azorhizobium caulinodans FixA proteins. Putative NtrA- and NifA-binding sites are identified in the fixA promoter region.

Identification of a nifW-like gene in Azospirillum brasilense.

A small ORF, 5' upstream of the fixABC operon in Azospirillum brasilense Sp7, has been identified. Sequence comparison shows significant homology to the Azotobacter vinelandii and Azorhizobium caulinodans nifW gene.

Transcriptional analysis of the Azospirillum brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence involved in auxin responsive expression.

Expression of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase, a key enzyme in the production of indole-3-acetic acid (IAA) in this bacterium, is upregulated by IAA. Here, we demonstrate that the ipdC gene is the promoter proximal gene in a bicistronic operon. Database searches revealed that the second gene of this operon, named iaaC, is well conserved evolutionarily and that the encoded protein is homologous to the Escherichia coli protein SCRP-27A, the zebrafish protein ES1, and the human protein KNP-I/GT335 (HES1), all of unknown function and belonging to the DJ-1/PfpI superfamily. In addition to this operon structure, iaaC is also transcribed monocistronically. Mutation analysis of the latter gene indicated that the encoded protein is involved in controlling IAA biosynthesis but not ipdC expression. Besides being upregulated by IAA, expression of the ipdC-iaaC operon is pH dependent and maximal at acidic pH. The ipdC promoter was studied using a combination of deletion analyses and site-directed mutagenesis. A dyadic sequence (ATTGTTTC(GAAT)GAAACAAT), centered at -48 was demonstrated to be responsible for the IAA inducibility. This bacterial auxin-responsive element does not control the pH-dependent expression of ipdC-iaaC.

Nucleotide sequence analysis of four genes, hupC, hupD, hupF and hupG, downstream of the hydrogenase structural genes in Bradyrhizobium japonicum.

The nucleotide sequence of a 2.2 kb region downstream of the hydrogenase structural genes in Bradyrhizobium japonicum was determined. Four genes encoding predicted polypeptides of 27.8 (HupC), 21.4 (HupD), 10.6 (HupF) and 15.8 (HupG) kDa were identified, of which the first three probably belong to the same operon as the hup structural genes, hupS and hupL. HupC is homologous to the hydrophobic polypeptides with four potential transmembrane regions that are encoded by open reading frames following the hydrogenase structural genes in Rhodobacter capsulatus, Escherichia coli, Azotobacter vinelandii, Wolinella succinogenes, Rhizobium leguminosarum and Alcaligenes eutrophus. Also HupD, HupF and HupG are homologous to genes involved in processing, maturation, functioning and regulation of hydrogenase activity in various hydrogen-oxidizing bacteria.

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.

Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in gram-negative bacteria.

The construction of several stable RK2-derived cloning vectors for the analysis of gene expression and function in gram-negative bacteria is reported. Plasmid stability is conferred by the RK2 par locus or by insertion of the spsAB or spsCD symbiotic plasmid stability loci from pNGR234a of Rhizobium sp. NGR234. The vectors carry multiple cloning sites with protection against read-through transcriptional activity of vector sequences. Vector derivatives with the constitutive nptII promoter or a promoterless gusA gene are suitable for the study of gene function or regulation in bacteria.

The nodS gene of Rhizobium tropici strain CIAT899 is necessary for nodulation on Phaseolus vulgaris and on Leucaena leucocephala.

Rhizobium tropici strain CIAT899 induces nitrogen-fixing nodules on the roots of a wide range of tropical legumes, including Phaseolus vulgaris and Leucaena leucocephala. Previously, a DNA region of the CIAT899 pSym plasmid containing the common nodulation genes nodABC and one of the nodD alleles was characterized (P. van Rhijn, B. Feys, C. Verreth, and J. Vanderleyden, J. Bacteriol. 175: 438-447, 1993). As reported here, the region immediately downstream of nodC contains the nodSU genes. The nucleotide sequence of these genes is presented. CIAT899 nodS and nodU mutants were constructed. The nodS mutant was completely deficient in nodulation on the host plants P. vulgaris and L. leucocephala. The nodU mutation caused a decrease in nodulation on Leucaena but resorted no effect on Phaseolus. Introduction of the CIAT899 nodABCSU region in R. etli CE-3, a strain that only nodulates P. vulgaris, caused an extension of the host range of strain CE-3 to L. leucocephala.

Identification and characterization of a Rhizobium leguminosarum bv. phaseoli gene that is important for nodulation competitiveness and shows structural homology to a Rhizobium fredii host-inducible gene.

DNA sequence analysis of a 1.4-kb SalI-HindIII segment located approximately 2 kb upstream of the Rhizobium leguminosarum bv. phaseoli syrM gene revealed the presence of an open reading frame (ORF3) encoding a putative 295-amino acid polypeptide with a molecular mass of 33,401 Da. ORF3 is homologous to a R. fredii host-inducible gene. The proteins encoded by R. l. bv. phaseoli ORF3 and by the R. fredii host-inducible gene share 37% sequence identity. In contrast to the R. fredii host-inducible gene, expression of ORF3 is not induced in the presence of Phaseolus vulgaris root exudates or by specific flavonoids, able to induce nodulation genes in R. l. bv. phaseoli. A R. l. bv. phaseoli ORF3 mutant was constructed by site-directed deletion/replacement mutagenesis. This mutant strain is not affected in symbiotic nitrogen fixation but exhibits a delay in nodulation on Phaseolus vulgaris. Moreover, this mutant was shown to be defective in competition for nodulation.

Isolation and characterization of a pSym locus of Rhizobium sp. BR816 that extends nodulation ability of narrow host range Phaseolus vulgaris symbionts to Leucaena leucocephala.

Introduction of a cosmid library of megaplasmid DNA of Rhizobium sp. BR816, a broad host range Rhizobium strain, into R. etli CE3, a narrow host range bean symbiont, resulted in the isolation of a transconjugant that could effectively nodulate Leucaena leucocephala. Analysis of the corresponding cosmid, pBRF2, revealed the presence of genes required for elicting nitrogen-fixing nodules on L. leucoephala. Subcloning and Tn5 tagging identified a locus responsible for the host range extension. Sequence analysis of this locus revealed an ORF that shows significant identity with NodO of R. leguminosarum bv. viciae.

Three genes encoding for putative methyl- and acetyltransferases map adjacent to the wzm and wzt genes and are essential for O-antigen biosynthesis in Rhizobium etli CE3.

The elucidation of the structure of the O-antigen of Rhizobium etli CE3 predicts that the R. etli CE3 genome must contain genes encoding acetyl- and methyltransferases to confer the corresponding modifications to the O-antigen. We identified three open reading frames (ORFs) upstream of wzm, encoding the membrane component of the O-antigen transporter and located in the lps alpha-region of R. etli CE3. The ORFs encode two putative acetyltransferases with similarity to the CysE-LacA-LpxA-NodL family of acetyltransferases and one putative methyltransferase with sequence motifs common to a wide range of S-adenosyl-L-methionine-dependent methyltransferases. Mutational analysis of the ORFs encoding the putative acetyltransferases and methyltransferase revealed that the acetyl and methyl decorations mediated by these specific enzymes are essential for O-antigen synthesis. Composition analysis and high performance anion exchange chromatography analysis of the lipopolysaccharides (LPSs) of the mutants show that all of these LPSs contain an intact core region and lack the O-antigen polysaccharide. The possible role of these transferases in the decoration of the O-antigen of R. etli is discussed.

Phaseolus vulgaris recognizes Azorhizobium caulinodans Nod factors with a variety of chemical substituents.

Phaseolus vulgaris is a promiscuous host plant that can be nodulated by many different rhizobia representing a wide spectrum of Nod factors. In this study, we introduced the Rhizobium tropici CFN299 Nod factor sulfation genes nodHPQ into Azorhizobium caulinodans. The A. caulinodans transconjugants produce Nod factors that are mostly if not all sulfated and often with an arabinosyl residue as the reducing end glycosylation. Using A. caulinodans mutant strains, affected in reducing end decorations, and their respective transconjugants in a bean nodulation assay, we demonstrated that bean nodule induction efficiency, in decreasing order, is modulated by the Nod factor reducing end decorations fucose, arabinose or sulfate, and hydrogen.