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.

In vitro interactions between probiotic bacteria and milk proteins probed by atomic force microscopy.

Interactions between microbial cells and milk proteins are important for cell location into dairy matrices. In this study, interactions between two probiotic strains, Lactobacillus rhamnosus GG and Lactobacillus rhamnosus GR-1, and milk proteins (micellar casein, native and denatured whey proteins) were studied. The bacterial surface characterization was realized with X-ray photoelectron spectroscopy (XPS) to evaluate surface composition (in terms of proteins, polysaccharides and lipid-like compounds) and electrophoretic mobility that provide information on surface charge of both bacteria and proteins along the 3-7 pH range. In addition, atomic force microscopy (AFM) enabled the identification of specific interactions between bacteria and whey proteins, in contrast to the observed nonspecific interactions with micellar casein. These specific events appeared to be more important for the GG strain than for the GR-1 strain, showing that matrix interaction is strain-specific. Furthermore, our study highlighted that in addition to the nature of the strains, many other factors influence the bacterial interaction with dairy matrix including the nature of the proteins and the pH of the media.

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.

Adaptation factors of the probiotic Lactobacillus rhamnosus GG.

Probiotic bacteria are administered as live micro-organisms to provide a health benefit to the host. Knowledge on adaptation factors that promote the survival and persistence of probiotics in the intestine is key to understand and improve their ecological and probiotic performance. Adaptation factors include adhesins, molecules conferring stress tolerance and nutritional versatility, antimicrobial products against competing microbes, and factors promoting resistance against the host immune system. Here, we present an overview of the current knowledge on adaptation factors of probiotic lactobacilli, with focus on the prototypical and widely documented probiotic strain Lactobacillus rhamnosus GG.

Identification and characterization of starter lactic acid bacteria and probiotics from Columbian dairy products.

AIMS: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells. METHODS AND RESULTS: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of approximately 10(7) CFU ml(-1). Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed. CONCLUSIONS: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.

Azospirillum brasilense produces the auxin-like phenylacetic acid by using the key enzyme for indole-3-acetic acid biosynthesis.

An antimicrobial compound was isolated from Azospirillum brasilense culture extracts by high-performance liquid chromatography and further identified by gas chromatography-mass spectrometry as the auxin-like molecule, phenylacetic acid (PAA). PAA synthesis was found to be mediated by the indole-3-pyruvate decarboxylase, previously identified as a key enzyme in indole-3-acetic acid (IAA) production in A. brasilense. In minimal growth medium, PAA biosynthesis by A. brasilense was only observed in the presence of phenylalanine (or precursors thereof). This observation suggests deamination of phenylalanine, decarboxylation of phenylpyruvate, and subsequent oxidation of phenylacetaldehyde as the most likely pathway for PAA synthesis. Expression analysis revealed that transcription of the ipdC gene is upregulated by PAA, as was previously described for IAA and synthetic auxins, indicating a positive feedback regulation. The synthesis of PAA by A. brasilense is discussed in relation to previously reported biocontrol properties of A. brasilense.

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.

Peptide signal molecules and bacteriocins in Gram-negative bacteria: a genome-wide in silico screening for peptides containing a double-glycine leader sequence and their cognate transporters.

Quorum sensing (QS) in Gram-negative bacteria is generally assumed to be mediated by N-acyl-homoserine lactone molecules while Gram-positive bacteria make use of signaling peptides. We analyzed the occurrence in Gram-negative bacteria of peptides and transporters that are involved in quorum sensing in Gram-positive bacteria. Many class II bacteriocins and inducing factors produced by lactic acid bacteria (LAB) and competence stimulating peptides (CSPs) synthesized by streptococci are processed by their cognate ABC-transporters during their secretion. During transport, a conserved leader sequence, termed the double-glycine motif (GG-motif), is cleaved off by the N-terminal domain of the transporter, which belongs to the Peptidase C39 protein family. Several peptides containing a GG-motif were recently described in Gram-negative bacteria (Trends Microbiol 2001;9:164-8). To screen for additional putative GG-motif containing peptides, an in silico strategy based on MEME, HMMER2.2 and Wise2 was designed. Using a curated training set, a motif model of the leader peptide was built and used to screen over 120 fully sequenced bacterial genomes. The screening methodology was applied at the nucleotide level as probably many small peptide genes have not been annotated and may be absent from the non-redundant databases. It was found that 33% of the screened genomes of Gram-negative bacteria contained one or more transporters carrying a Peptidase C39 domain, compared to 44% of the genomes of Gram-positive bacteria. The transporters can be subdivided into four classes on the basis of their domain organization. Genes coding for putative peptides containing 23-142 amino acids and a GG-motif were found in close association with genes coding for Peptidase C39 domain containing proteins. These peptides show structural similarity to bacteriocins and peptide pheromones of Gram-positive bacteria. The possibility of signal transduction based on peptide signaling in Gram-negative bacteria is discussed.