Search

Addressing the ecological and evolutionary processes underlying biodiversity patterns is essential to identify the mechanisms shaping community structure and function. In bacteria, the formation of new ecologically distinct populations (ecotypes) is proposed as one of the main drivers of diversification. New ecotypes arise when mutations in key functional genes or acquisition of new metabolic pathways by horizontal gene transfer allow the population to exploit new resources, permitting their coexistence with the parental population. We previously reported the presence of microcystin-producing organisms of the Microcystis aeruginosa complex (toxic MAC) through an 800-km environmental gradient ranging from freshwater to estuarine-marine waters in South America. We hypothesize that the success of toxic MAC in such a gradient is due to the existence of very closely related populations that are ecologically distinct (ecotypes), each specialized to a specific arrangement of environmental variables. Here, we analyzed toxic MAC genetic diversity through quantitative PCR (qPCR) and high-resolution melting analysis (HRMA) of a functional gene (mcyJ, microcystin synthetase cluster). We explored the variability of the mcyJ gene along the environmental gradient by multivariate classification and regression trees (mCART). Six groups of mcyJ genotypes were distinguished and associated with different combinations of water temperature, conductivity, and turbidity. We propose that each mcyJ variant associated with a defined environmental condition is an ecotype (or species) whose relative abundances vary according to their fitness in the local environment. This mechanism would explain the success of toxic MAC in such a wide array of environmental conditions. IMPORTANCE Organisms of the Microcystis aeruginosa complex form harmful algal blooms (HABs) in nutrient-rich water bodies worldwide. MAC HABs are difficult to manage owing to the production of potent toxins (microcystins) that resist water treatment. In addition, the role of microcystins in the ecology of MAC organisms is still elusive, meaning that the environmental conditions driving the toxicity of the bloom are not clear. Furthermore, the lack of coherence between morphology-based and genomic-based species classification makes it difficult to draw sound conclusions about when and where each member species of the MAC will dominate the bloom. Here, we propose that the diversification process and success of toxic MAC in a wide range of water bodies involves the generation of ecotypes, each specialized in a particular niche, whose relative abundance varies according to its fitness in the local environment. This knowledge can improve the generation of accurate prediction models of MAC growth and toxicity, helping to prevent human and animal intoxication.

To improve the enzymatic hydrolysis (saccharification) of lignocellulosic biomass by Trichoderma reesei , a set of genes encoding putative polysaccharide-degrading enzymes were selected from the coprophilic fungus Podospora anserina using comparative genomics. Five hemicellulase-encoding genes were successfully cloned and expressed as secreted functional proteins in the yeast Pichia pastoris . These novel fungal CAZymes belonging to different glycoside hydrolase families ( Pa Man5A and Pa Man26A mannanases, Pa Xyn11A xylanase, and Pa Abf51A and Pa Abf62A arabinofuranosidases) were able to break down their predicted cognate substrates. Although Pa Man5A and Pa Man26A displayed similar specificities toward a range of mannan substrates, they differed in their end products, suggesting differences in substrate binding. The N-terminal CBM35 module of Pa Man26A displayed dual binding specificity toward xylan and mannan. Pa Xyn11A harboring a C-terminal CBM1 module efficiently degraded wheat arabinoxylan, releasing mainly xylobiose as end product. Pa Abf51A and Pa Abf62A arabinose-debranching enzymes exhibited differences in activity toward arabinose-containing substrates. Further investigation of the contribution made by each P. anserina auxiliary enzyme to the saccharification of wheat straw and spruce demonstrated that the endo-acting hemicellulases ( Pa Xyn11A, Pa Man5A, and Pa Man26A) individually supplemented the secretome of the industrial T. reesei CL847 strain. The most striking effect was obtained with Pa Man5A that improved the release of total sugars by 28% and of glucose by 18%, using spruce as lignocellulosic substrate.

Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic- and oxidative-stress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions.

Among the lactic acid bacteria (LAB) present in the oenological microbial ecosystem, Oenococcus oeni , an acidophilic lactic acid bacterium, is essential during winemaking. It outclasses all other bacterial species during malolactic fermentation (MLF). Oenological performances, such as malic acid degradation rate and sensorial impact, vary significantly according to the strain. The genetic diversity of the O. oeni species was evaluated using a multilocus sequence typing (MLST) scheme. Seven housekeeping genes were sequenced for a collection of 258 strains that had been isolated all over the world (particularly Burgundy, Champagne, and Aquitaine, France, Chile, South Africa, and Italy) and in several wine types (red wines, white wines, and champagne) and cider. The allelic diversity was high, with an average of 20.7 alleles per locus, many of them being rare alleles. The collection comprised 127 sequence types, suggesting an important genotypic diversity. The neighbor-joining phylogenetic tree constructed from the concatenated sequence of the seven housekeeping genes showed two major phylogenetic groups, named A and B. One unique strain isolated from cider composed a third group, rooting the phylogenetic tree. However, all other strains isolated from cider were in group B. Eight phylogenetic subgroups were statistically differentiated and could be delineated by the analysis of only 32 mutations instead of the 600 mutations observed in the concatenated sequence of the seven housekeeping genes. Interestingly, in group A, several phylogenetic subgroups were composed mostly of strains coming from a precise geographic origin. Three subgroups were identified, composed of strains from Chile, South Africa, and eastern France.

The mechanisms of interaction between Lactococcus lactis and the food pathogen Staphylococcus aureus are of crucial importance, as one major role of lactic acid bacteria (LAB) in fermented foods is to inhibit undesirable and pathogenic flora. It was never questioned if the presence of a pathogen can actively modify the gene expression patterns of LAB in a shared environment. In this study, transcriptome and biochemical analyses were combined to assess the dynamic response of L. lactis in a mixed culture with S. aureus . The presence of S. aureus hardly affected the growth of L. lactis but dramatically modified its gene expression profile. The main effect was related to earlier carbon limitation and a concomitantly lower growth rate in the mixed culture due to the consumption of glucose by both species. More specific responses involved diverse cellular functions. Genes associated with amino acid metabolism, ion transport, oxygen response, menaquinone metabolism, and cell surface and phage expression were differentially expressed in the mixed culture. This study led to new insights into possible mechanisms of interaction between L. lactis and S. aureus . Moreover, new and unexpected effects of L. lactis on the virulence of S. aureus were discovered, as described elsewhere (S. Even, C. Charlier, S. Nouaille, N. L. Ben Zakour, M. Cretenet, F. J. Cousin, M. Gautier, M. Cocaign-Bousquet, P. Loubière, and Y. Le Loir, Appl. Environ. Microbiol. 75:4459-4472, 2009).

Oenococcus oeni is the acidophilic lactic acid bacterial species most frequently associated with malolactic fermentation of wine. Since the description of the species (formerly Leuconostoc oenos ), characterization of indigenous strains and industrially produced cultures by diverse typing methods has led to divergent conclusions concerning the genetic diversity of strains. In the present study, a multilocus sequence typing (MLST) scheme based on the analysis of eight housekeeping genes was developed and tested on a collection of 43 strains of diverse origins. The eight targeted loci were successfully amplified and sequenced for all isolates. Only three to 11 different alleles were detected for these genes. The average nucleotide diversity also was rather limited (0.0011 to 0.0370). Despite this limited allelic diversity, the combination of alleles of each strain disclosed 34 different sequence types, which denoted a significant genotypic diversity. A phylogenetic analysis of the concatenated sequences showed that all strains form two well distinct groups of 28 and 15 strains. Interestingly, the same groups were defined by pulsed-field gel electrophoresis, although this method targets different genetic variations. A minimum spanning tree analysis disclosed very few and small clonal complexes. In agreement, statistical analyses of MLST data suggest that recombination events were important during O. oeni evolution and contributed to the wide dissemination of alleles among strains. Taken together, our results showed that MLST is more efficient than pulsed-field gel electrophoresis for typing O. oeni strains, and they provided a picture of the O. oeni population that explains some conflicting results previously obtained.

We have shown that the plant pathogen Alternaria brassicicola exhibited very high susceptibility to ambruticin VS4 and to a lesser extent to the phenylpyrrole fungicide fludioxonil. These compounds are both derived from natural bacterial metabolites with antifungal properties and are thought to exert their toxicity by interfering with osmoregulation in filamentous fungi. Disruption of the osmosensor group III histidine kinase gene Ab NIK1 (for A. brassicola NIK1) resulted in high levels of resistance to ambruticin and fludioxonil, while a mutant isolate characterized by a single-amino-acid substitution in the HAMP domain of the kinase only exhibited moderate resistance. Moreover, the natural resistance of Saccharomyces cerevisiae to these antifungal molecules switched to sensitivity in strains expressing AbNIK1p. We also showed that exposure to fludioxonil and ambruticin resulted in abnormal phosphorylation of a Hog1-like mitogen-activated protein kinase (MAPK) in A. brassicicola . Parallel experiments carried out with wild-type and mutant isolates of Neurospora crassa revealed that, in this species, ambruticin susceptibility was dependent on the OS1-RRG1 branch of the phosphorelay pathway downstream of the OS2 MAPK cascade but independent of the yeast Skn7-like response regulator RRG2. These results show that the ability to synthesize a functional group III histidine kinase is a prerequisite for the expression of ambruticin and phenylpyrrole susceptibility in A. brassicicola and N. crassa and that, at least in the latter species, improper activation of the high-osmolarity glycerol-related pathway could explain their fungicidal properties.

The symbiosis between plant roots and arbuscular mycorrhizal (AM) fungi has been shown to affect both the diversity and productivity of agricultural communities. In this study, we characterized the AM fungal communities of Solanum tuberosum L. (potato) roots and of the bulk soil in two nearby areas of northern Italy, in order to verify if land use practices had selected any particular AM fungus with specificity to potato plants. The AM fungal large-subunit (LSU) rRNA genes were subjected to nested PCR, cloning, sequencing, and phylogenetic analyses. One hundred eighty-three LSU rRNA sequences were analyzed, and eight monophyletic ribotypes, belonging to Glomus groups A and B, were identified. AM fungal communities differed between bulk soil and potato roots, as one AM fungal ribotype, corresponding to Glomus intraradices , was much more frequent in potato roots than in soils (accounting for more than 90% of sequences from potato samples and less than 10% of sequences from soil samples). A semiquantitative heminested PCR with specific primers was used to confirm and quantify the AM fungal abundance observed by cloning. Overall results concerning the biodiversity of AM fungal communities in roots and in bulk soils from the two studied areas suggested that potato roots were preferentially colonized by one AM fungal species, G. intraradices .

“Ropiness” is a bacterial alteration in wines, beers, and ciders, caused by β-glucan-synthesizing pediococci. A single glucosyltransferase, Gtf, controls ropy polysaccharide synthesis. In this study, we show that the correspondinggtfgene is also present on the chromosomes of several strains ofOenococcus oeniisolated from nonropy wines.gtfis surrounded by mobile elements that may be implicated in its integration into the chromosome ofO. oeni. gtfis expressed in all thegtf+strains, and β-glucan is detected in the majority of these strains. Part of this β-glucan accumulates around the cells forming a capsule, while the other part is liberated into the medium together with heteropolysaccharides. Most of the time, this polymer excretion does not lead to ropiness in a model medium. In addition, we show that wild or recombinant bacterial strains harboring a functionalgtfgene (gtf+) are more resistant to several stresses occurring in wine (alcohol, pH, and SO2) and exhibit increased adhesion capacities compared to theirgtfmutant variants.

Bacteria of the genus Methylobacterium are widespread in the environment, but their ecological role in ecosystems, such as the plant phyllosphere, is not very well understood. To gain better insight into the distribution of different Methylobacterium species in diverse ecosystems, a rapid and specific cultivation-independent method for detection of these organisms and analysis of their community structure is needed. Therefore, 16S rRNA gene-targeted primers specific for this genus were designed and evaluated. These primers were used in PCR in combination with a reverse primer that binds to the tRNA Ala gene, which is located upstream of the 23S rRNA gene in the 16S-23S intergenic spacer (IGS). PCR products that were of different lengths were obtained due to the length heterogeneity of the IGS of different Methylobacterium species. This length variation allowed generation of fingerprints of Methylobacterium communities in environmental samples by automated ribosomal intergenic spacer analysis. The Methylobacterium communities on leaves of different plant species in a natural field were compared using this method. The new method allows rapid comparisons of Methylobacterium communities and is thus a useful tool to study Methylobacterium communities in different ecosystems.

We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.

Dissimilatory nitrate reduction is catalyzed by a membrane-bound and a periplasmic nitrate reductase. We set up a real-time PCR assay to quantify these two enzymes, using the narG and napA genes, encoding the catalytic subunits of the two types of nitrate reductases, as molecular markers. The narG and napA gene copy numbers in DNA extracted from 18 different environments showed high variations, with most numbers ranging from 2 × 10 2 to 6.8 × 10 4 copies per ng of DNA. This study provides evidence that, in soil samples, the number of proteobacteria carrying the napA gene is often as high as that of proteobacteria carrying the narG gene. The high correlation observed between narG and napA gene copy numbers in soils suggests that the ecological roles of the corresponding enzymes might be linked.

Using a modified random arbitrarily primed PCR approach, the operon encoding the Enterococcus faecalis JH2-2 CroRS two-component regulatory system was shown to be repressed during stationary phase, and a CroRS-regulated operon ( glnQHMP ) was identified. Gel retardation assays showed that the CroR regulator binds specifically to the glnQHMP promoter.

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. ASaccharomyces cerevisiaewine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutatedHXT3allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

IS Xax1 is a novel insertion sequence belonging to the IS 256 and Mutator families. Dot blot, Southern blot, and PCR analyses revealed that IS Xax1 is restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and X. axonopodis pv. vesicatoria strains. Directed AFLP also showed that a high degree of polymorphism is associated with IS Xax1 insertion in these strains.

A normal distribution and a mixture model of two normal distributions in a Bayesian approach using prevalence and concentration data were used to establish the distribution of contamination of the food-borne pathogenic bacteria Listeria monocytogenes in unprocessed and minimally processed fresh vegetables. A total of 165 prevalence studies, including 15 studies with concentration data, were taken from the scientific literature and from technical reports and used for statistical analysis. The predicted mean of the normal distribution of the logarithms of viable L. monocytogenes per gram of fresh vegetables was −2.63 log viable L. monocytogenes organisms/g, and its standard deviation was 1.48 log viable L. monocytogenes organisms/g. These values were determined by considering one contaminated sample in prevalence studies in which samples are in fact negative. This deliberate overestimation is necessary to complete calculations. With the mixture model, the predicted mean of the distribution of the logarithm of viable L. monocytogenes per gram of fresh vegetables was −3.38 log viable L. monocytogenes organisms/g and its standard deviation was 1.46 log viable L. monocytogenes organisms/g. The probabilities of fresh unprocessed and minimally processed vegetables being contaminated with concentrations higher than 1, 2, and 3 log viable L. monocytogenes organisms/g were 1.44, 0.63, and 0.17%, respectively. Introducing a sensitivity rate of 80 or 95% in the mixture model had a small effect on the estimation of the contamination. In contrast, introducing a low sensitivity rate (40%) resulted in marked differences, especially for high percentiles. There was a significantly lower estimation of contamination in the papers and reports of 2000 to 2005 than in those of 1988 to 1999 and a lower estimation of contamination of leafy salads than that of sprouts and other vegetables. The interest of the mixture model for the estimation of microbial contamination is discussed.

Lactobacillus bulgaricus is a lactic acid bacteria (LAB) that, through the production of lactic acid, gradually acidifies its environment during growth. In the course of this process, L. bulgaricus acquires an improved tolerance to acidity. A survey of the recently established genome sequence shows that this bacterium possesses few of the pH control functions that have been described in other LAB and raises the question of what other mechanisms could be involved in its adaptation to the decreasing environmental pH. In some bacteria other than LAB, ion transport systems have been implicated in acid adaptation. We therefore studied the expression of this type of transport system during acid adaptation in L. bulgaricus by reverse transcription and real-time quantitative PCR and mapped transcription start sites. Intriguingly, the most significantly induced were three ATPases carrying the CPX signature of heavy-metal transporters. Protein homology and the presence of a conserved sequence motif in the promoter regions of the genes encoding these proteins strongly suggest that they are involved in copper homeostasis. Induction of this system is thought to assist in avoiding indirect damage that could result from medium acidification.

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10 5 to 8.9 × 10 5 copies per nanogram of DNA but smaller amounts of narG , nirK , and nosZ target molecules. Thus, numbers of narG , nirK , nirS , and nosZ copies per nanogram of DNA ranged from 2.1 × 10 3 to 2.6 × 10 4 , 7.4 × 10 2 to 1.4 × 10 3 , 2.5 × 10 2 to 6.4 × 10 3 , and 1.2 × 10 3 to 5.5 × 10 3 , respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.

The gastrointestinal tract (GIT) of ruminants is the main reservoir of enterohemorrhagic Escherichia coli , which is responsible for food-borne infections in humans that can lead to severe kidney disease. Characterization of biotic and abiotic factors that influence the carriage of these pathogens by the ruminant would help in the development of ecological strategies to reduce their survival in the GIT and to decrease the risk of contamination of animal products. We found that growth of E. coli O157:H7 in rumen fluid was inhibited by the autochthonous microflora. Growth was also reduced when rumen fluid came from sheep fed a mixed diet composed of 50% wheat and 50% hay, as opposed to a 100% hay diet. In fecal suspensions, E. coli O157:H7 growth was not suppressed by the autochthonous flora. However, a probiotic strain of Lactobacillus acidophilus inhibited E. coli O157:H7 growth in fecal suspensions. The inhibitory effect was dose dependent. These lactic acid bacteria could be a relevant tool for controlling O157:H7 development in the terminal part of the ruminant GIT, which has been shown to be the main site of colonization by these pathogenic bacteria.

The Bacillus cereus group consists of gram-positive spore-forming bacteria with an impact on human activities due to their pathogenic properties. Strains capable of producing large amounts of entomopathogenic toxins are designated Bacillus thuringiensis and are commonly used as effective biopesticides. Strains producing emetic toxins or enterotoxins and causing food poisoning following the ingestion of contaminated food are usually classified as Bacillus cereus. Strains of the closely related species Bacillus anthracis cause a lethal disease (anthrax). The recently identified species Bacillus weihenstephanensis (20) is characterized by an ability to grow at low temperatures (below 8°C). Bacillus mycoides forms colonies with a specific form due to rhizoidal growth. Several strain classification studies have shown that it is difficult to separate B. cereus and B. thuringiensis into two clusters (2, 3, 6, 13-16, 25, 32). This suggests that it is not possible to distinguish between these two species on the basis of neutral genome sequences that are not related to the production of entomopathogenic toxins. This situation may arise from intensive exchange of genetic material between the two species in their natural habitat. Formally, B. thuringiensis may be defined as phenotypically similar to B. cereus but with a naturally acquired ability to synthesize large amounts of entomopathogenic toxin, forming parasporal crystals visible under the microscope in sporulating cells. A collection of B. thuringiensis and B. cereus strains was recently isolated from the soil of a forest near Versailles (close to Paris, France) (33). The strains of this collection were classified as B. cereus or B. thuringiensis based on their ability to produce parasporal toxin. Multiple-locus enzyme electrophoresis (MLEE) showed that the populations of B. cereus and B. thuringiensis living in the same soil were genetically distinct and diverged to a greater extent than strains of the same species isolated from geographically different locations. This suggests that ecological separation, which is probably the main force behind separation and cohesion for bacterial genetic clustering, is stronger than the potential of these bacteria to exchange genetic material. In this case, genetic exchange is mediated by plasmids encoding the crystal proteins, enabling the bacterium to occupy the ecological niche of an insect pathogen. We addressed similar questions concerning the fine genetic structure of this collection of strains using a different experimental approach, because the MLEE method is insufficiently precise for studies of the fine genetic structure of bacterial populations. Furthermore, MLEE generates data that cannot easily be compared with data from independent studies and data for other strain collections. We therefore chose to use the multiple-locus sequence typing (MLST) approach (22). This approach involves sequencing short (300- to 700-bp) regions of several selected genes randomly distributed over the bacterial chromosome. The genes are usually chosen such that structural changes reflect vertical bacterial evolution, with no major influence of specific niche-related selection (housekeeping genes). We investigated the following questions: (i) whether toxin-producing and non-toxin-producing populations in the same soil are different enough genetically for formal clustering into two separate clusters; (ii) whether the strains of the Bacillus cereus group collected from small samples of soil presented genetic evidence for active sexual exchange within local populations; (iii) what was the natural potential of different populations to display genetic homogeneity. We tackled these questions by studying natural populations of these bacteria isolated from different samples and under different conditions.

Leptosphaeria maculans is the most ubiquitous fungal pathogen of Brassica crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of L. maculans in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m 2 field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All L. maculans field populations had a high level of gene diversity ( H = 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m 2 field plots. Population differentiation among the four field populations was low ( G ST < 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of L. maculans suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.

Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in Aspergillus niger . FLX construct is composed of the sequences encoding the feruloyl esterase A (FAEA) fused to the endoxylanase B (XYNB) of A. niger . A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immunodetection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physicochemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a K d of 9.9 × 10 −8 M for the dissociation constant and 0.98 μmol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an A. niger -derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.

Lignans are dietary diphenolic compounds which require activation by intestinal bacteria to exert possible beneficial health effects. The intestinal ecosystem plays a crucial role in lignan metabolism, but the organisms involved are poorly described. To characterize the bacterial communities responsible for secoisolariciresinol (SECO) activation, i.e., the communities that produce the enterolignans enterodiol (ED) and enterolactone (EL), a study with 24 human subjects was undertaken. SECO activation was detected in all tested fecal samples. The intestinal bacteria involved in ED production were part of the dominant microbiota (6 × 10 8 CFU g −1 ), as revealed by most-probable-number enumerations. Conversely, organisms that catalyzed the formation of EL occurred at a mean concentration of approximately 3 × 10 5 CFU g −1 . Women tended to have higher concentrations of both ED- and EL-producing organisms than men. Significantly larger amounts of EL were produced by fecal dilutions from individuals with moderate to high concentrations of EL-producing bacteria. Two organisms able to demethylate and dehydroxylate SECO were isolated from human feces. Based on 16S rRNA gene sequence analyses, they were named Peptostreptococcus productus SECO-Mt75m3 and Eggerthella lenta SECO-Mt75m2. A new 16S rRNA-targeted oligonucleotide probe specific for P. productus and related species was designed and further used in fluorescent in situ hybridization experiments, along with five additional group-specific probes. Significantly higher proportions of P. productus and related species ( P = 0.012), as well as bacteria belonging to the Atopobium group ( P = 0.035), were typical of individuals with moderate to high concentrations of EL-producing communities.

2-Amino-3-methylimidazo[4,5- f ]quinoline (IQ) is a mutagenic/carcinogenic compound formed from meat and fish during cooking. Following ingestion, IQ is metabolized mainly by liver xenobiotic-metabolizing enzymes, but intestinal bacteria may also contribute to its biotransformation. The aim of this study was to investigate the metabolism of IQ by the human intestinal microbiota. Following incubation of IQ (200 μM) under anoxic conditions with 100-fold dilutions of stools freshly collected from three healthy volunteers, we quantified residual IQ by high-pressure liquid chromatography (HPLC) analysis and characterized the production of IQ metabolites by in situ 1 H nuclear magnetic resonance ( 1 H-NMR) spectroscopic analysis of crude incubation media. In addition, we looked for IQ-degrading bacteria by screening collection strains and by isolating new strains from the cecal contents of human-microbiota-associated rats gavaged with IQ on a regular basis. HPLC and 1 H-NMR analyses showed that the three human microbiota degraded IQ with different efficiencies (range, 50 to 91% after 72 h of incubation) and converted it into a unique derivative, namely, 7-hydroxy-IQ. We found 10 bacterial strains that were able to perform this reaction: Bacteroides thetaiotaomicron ( n = 2), Clostridium clostridiiforme ( n = 3), Clostridium perfringens ( n = 1), and Escherichia coli ( n = 4). On the whole, our results indicate that bacteria belonging to the predominant communities of the human intestine are able to produce 7-hydroxy-IQ from IQ. They also suggest interindividual differences in the ability to perform this reaction. Whether it is a metabolic activation is still a matter of debate, since 7-hydroxy-IQ has been shown to be a direct-acting mutagen in the Ames assay but not carcinogenic in laboratory rodents.

Numerous industrial bacteria generate hydrogen peroxide (H 2 O 2 ), which may inhibit the growth of other bacteria in mixed ecosystems. We isolated sp ontaneous ox idative-stress-resistant (SpOx) Lactococcus lactis mutants by using a natural selection method with milk-adapted strains on dairy culture medium containing H 2 O 2 . Three SpOx mutants displayed greater H 2 O 2 resistance. One of them, SpOx3, demonstrated better behavior in different oxidative-stress situations: (i) higher long-term survival upon aeration in LM17 and milk and (ii) the ability to grow with H 2 O 2 -producing Lactobacillus delbrueckii subsp. delbrueckii strains. Furthermore, the transit kinetics of the SpOx3 mutant in the digestive tract of a human flora-associated mouse model was not affected.

Viral inactivation and adhesion-aggregation in water are often studied as separate phenomena. When the focus is placed on viral adhesion-aggregation, inactivation is neglected because the phenomena under investigation occur over a short period measured in days. When viral inactivation is studied, adhesion-aggregation phenomena are considered to be negligible because viral survival is traced over several days or months. In the present work, we took a global approach, examining the relative contributions of each of these processes in a complex system composed of groundwater, Poliovirus 1 , and a hydrophobic container (polypropylene) maintained in a dark environment at 20°C. We demonstrated that infectious viral load fell off 2.8 log 10 during the first 20 days. During this time, adhesion was far from negligible because it accounted for most of the decline, 1.5 log 10 . Adhesion was undoubtedly favored by the presence of divalent ions in the groundwater. After 20 days, aggregation may also have been the cause of 0.66 to 0.92 log 10 of viral loss. Finally, viral inactivation was quantitatively the lowest phenomena because it only explained 0.38 to 0.64 log 10 of the viral loss. This study thus clearly demonstrated that estimates of viral survival in a given system must always take into account adhesion-aggregation phenomena which may be responsible for the majority of viral loss in the aqueous phase. Adhesion and aggregation are reversible processes which may lead to an underestimation of viral load in certain studies.

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain ofSaccharomyces cerevisiaeand a natural hybrid strain ofS. cerevisiaeandSaccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. TheS. cerevisiae-S. uvarumhybrid strain produced large amounts of acetaldehyde for which theS. cerevisiaestrain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media byS. cerevisiae.

Byssochlamys species are responsible for spoilage and degradation of fruits and silages and can also produce the mycotoxin patulin. We analyzed secondary metabolite production by Byssochlamys nivea . Mycophenolic acid and its precursors, 5-methylorsellinic acid and 5,7-dihydroxy-4-methylphthalide, were identified in all of the B. nivea strains that we examined.

The aim of the study was to evaluate the presence of pathogenic viruses in the Moselle River and to compare the usefulness of thermotolerant coliforms and somatic coliphages as tools for river water quality assessment in terms of viral contamination. Thermotolerant coliforms and somatic coliphages were enumerated by standardized methods in 170 samples of river water drawn from five sampling sites along the Moselle River (eastern France). BGM cell culture and integrated cell culture-reverse transcription-PCR DNA enzyme immunoassay were used to determine the presence of pathogenic viral genome ( Enterovirus and Norovirus genogroup II [GGII]) and infectious Enterovirus spp. in 90 1-liter samples. No infectious Enterovirus spp. were isolated, but Enterovirus and Norovirus GGII genomes were detected in 38% of the samples. Norovirus GGII genome was mostly detected in winter, whereas Enterovirus genome was mostly detected in summer and fall. Somatic coliphages appeared to be less sensitive to higher river water temperature than thermotolerant coliforms. Furthermore, the number of river water samples positive for pathogenic viral genome increased with increasing concentration of somatic coliphages, whereas coliform concentration was unrelated to viral genome contamination. Consequently somatic coliphages, which are less sensitive to environmental factors than thermotolerant coliforms in river water, would provide a promising tool for assessment of river water quality in terms of fecal and viral pollution.

Appropriate interpretation of a positive reverse transcription-PCR is an important issue for virus-related health hazard assessment because viral genomes and infectious viruses exhibit different behavior patterns in water. In this context, using Poliovirus 1 and Feline calicivirus f9 as examples of enteric viruses, first we demonstrated that the stability of infectious viruses is greatly affected by the temperature of mineral water (10, 20, and 35°C) and that, in contrast, temperature has little effect on the corresponding genomes. Second, we demonstrated that infectious particles are degraded more rapidly than viral genomes at all temperatures studied. At 35°C, Poliovirus 1 infectivity was reduced 4 logs after only 19 days, while an equivalent reduction would have taken 75 years (according to the model applied) for the viral genome. Contradictory conclusions can also be drawn concerning the sensitivity of viral serotypes depending on whether the infectious virus or the viral genome is considered. The Feline calicivirus f9 genome is more resistant than the Poliovirus 1 genome, whereas the opposite is true for the corresponding infectious viruses. Thus, we concluded that a positive test for a viral genome in mineral water must be interpreted with utmost caution because of the lack of a correlation between the presence of viral genomes and viral infectivity. Detection of viral genomes may be necessary to identify infectious risk for the human population, but it cannot be considered sufficient.

This study investigated genetic polymorphism on a local scale in Puccinia striiformis f. sp. tritici populations during natural epidemics, in four fields located in northern France and sampled in 1998 or 1999. Two hundred and forty-seven isolates were analyzed for their amplified fragment length polymorphism (AFLP) pattern through four primer combinations, and 194 of them were also tested for their virulence factors. Only one or two pathotypes were found in each field, and all isolates had virulence v17, matching the recently introduced Yr17 resistance gene. Polymorphism on a field scale was low. Although 67 loci were polymorphic, 77% of the isolates had the same AFLP pattern, all other patterns being rare or unique. Analyses of the genetic distance between AFLP patterns based on the Jaccard index allowed us to define 12 groups, but a bootstrap analysis showed that all isolates could be assigned to a single clonal lineage. This leads us to conclude that P. striiformis f. sp. tritici populations are clonal on a field scale in northern France.

Aquatic hyphomycetes strongly contribute to organic matter dynamics in streams, but their abilities to colonize leaf litter buried in streambed sediments remain unexplored. Here, we conducted field and laboratory experiments (slow-filtration columns and stream-simulating microcosms) to test the following hypotheses: (i) that the hyporheic habitat acting as a physical sieve for spores filters out unsuccessful strategists from a potential species pool, (ii) that decreased pore size in sediments reduces species dispersal efficiency in the interstitial water, and (iii) that the physicochemical conditions prevailing in the hyporheic habitat will influence fungal community structure. Our field study showed that spore abundance and species diversity were consistently reduced in the interstitial water compared with surface water within three differing streams. Significant differences occurred among aquatic hyphomycetes, with dispersal efficiency of filiform-spore species being much higher than those with compact or branched/tetraradiate spores. This pattern was remarkably consistent with those found in laboratory experiments that tested the influence of sediment pore size on spore dispersal in microcosms. Furthermore, leaves inoculated in a stream and incubated in slow-filtration columns exhibited a fungal assemblage dominated by only two species, while five species were codominant on leaves from the stream-simulating microcosms. Results of this study highlight that the hyporheic zone exerts two types of selection pressure on the aquatic hyphomycete community, a physiological stress and a physical screening of the benthic spore pool, both leading to drastic changes in the structure of fungal community.

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides , and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi.

The genus Xenorhabdus consists of the specific bacterial symbionts of the entomopathogenic nematodes of the family Steinernematidae (40) and was separated from the genus Photorhabdus (11), which contains the symbionts of the entomopathogenic nematodes of the family Heterorhabditidae. Both genera are entomopathogenic gram-negative bacteria belonging to the Enterobacteriaceae. The nematodes carry their bacterial symbionts monoxenically in a special vesicle of the infective stage (L3 juveniles) in Steinernematidae (8) and throughout the whole intestine of Heterorhabditidae (20). These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, which is killed, probably via a combination of toxin action and septicemia. The bacterial symbionts also contribute to the symbiotic relationship by establishing and maintaining suitable conditions for nematode reproduction (31). Recently, isolation of some Photorhabdus strains from infected humans in Australia and the United States was reported (21, 30), and the strains from the United States were classified as Photorhabdus asymbiotica (23). The form of the bacterium that is normally isolated from symbiotic infective-stage nematodes is referred to as phase I. Like many pathogenic bacteria, Xenorhabdus and Photorhabdus strains spontaneously produce colonial variants which have been called phase II variants (10). The two variants of the bacteria have generally been shown to be equally pathogenic for the larvae of the greater wax moth, Galleria mellonella (3). However, Volgyi et al. (42) described for the first time a phase II variant that showed reduced virulence in the tobacco hornworm, Manduca sexta. Xenorhabdus nematophila and Photorhabdus luminescens are highly pathogenic to insects, and 50% insect mortality has been reported with direct infection with fewer than 20 bacteria per larva (5). The bacterial factors involved in killing of the insect or in overcoming the insect immune reactions are still under investigation. Following invasion of the insect host by the nematodes, both bacteria produce potential virulence factors, including lipase, protease, lecithinase, and lipopolysaccharides (LPSs), in the hemocoel (for a review, see reference 24). It was shown that purified LPS, Photorhabdus protease fractions, or Xenorhabdus lecithinase isomers showed no toxic effect following injection into insect hemocoel (12, 16, 39). Recently, a novel toxin complex with both oral and injectable activities against a wide range of insects was identified in a supernatant of P. luminescens (13). Purified toxin complex a (Tca) has specific effects on the midgut epithelium of the insect (9). In order to study Xenorhabdus and Photorhabdus virulence in insects, a genetic approach was also used. Avirulent mutants of X. nematophila have been isolated by transposon mutagenesis (Tn5). These mutants were pleiotropic, but all five mutants that tested as avirulent in G. mellonella were nonmotile and partially impaired in blood hemolysis (43). It was also shown that a homoserine lactone autoinducer restored virulence to one avirulent X. nematophila strain and stimulated the level of bacterial lipase activity (17). Recently we reported that flhDC, the flagellar master operon of X. nematophila, controls flagellin expression. Furthermore we revealed that lipolytic and extracellular hemolysin activity is flhD dependent. We also showed that the flhD null mutant displayed an attenuated virulence phenotype in the common cutworm, Spodoptera littoralis, compared to the wild-type strain (25). The recently published partial genome sequence of P. luminescens (22) revealed a diverse array of genes that putatively encodes potential virulence factors. These factors include exoenzymes (proteases, lipases, and chitinases), a type III secretion system (Yop homolog), and several classes of toxins (insecticidal toxin complex, Rtx-like toxins, and hemolysin and cytotoxin homologs) (22). Until now, studies examining hemolytic activity of both genera have not been reported. Cytolysins are proteins which cause lysis of red blood cells (RBC) as well as nucleated cell types by hydrolysis (lipases, phospholipases, or proteases) or by forming pores in the plasma membrane. Surfactants may also cause cytolysis by solubilization of the target cell membrane. Bacterial cytolysins are usually recognized as hemolysin on blood agar where a transparent zone appears around colonies. The production by a few strains of Xenorhabdus and Photorhabdus of hemolysin has been detected on agar supplemented with sheep blood (6, 21, 25). Apart from their phoretic location inside infective juvenile nematodes, these bacteria are only observed in insect hemolymph, where they enter their growth cycle. Here the bacteria are in contact with hemocytes which achieve defense reactions in insects. Some of these cells are immunocompetent cells able to engulf (phagocytosis) or to isolate and kill (nodule formation) bacteria. We hypothesize that hemolytic activities could target the immunocompetent cells in insect hemolymph. In this study, we report that different cytolytic activities were found in supernatants of Xenorhabdus whereas none was detected in supernatants of various strains of Photorhabdus. We have studied the kinetics of the production of cytolytic activities over the course of in vitro bacterial growth. We also provide evidence on the characteristics and on the specificity of each of these cytolytic activities against mammalian RBC and insect hemocyte types.

The similarities and differences in the structures of thenifHgene pools of six different soils (Montrond, LCSA-p, Vernon, Dombes, LCSA-c, and Thysse Kaymor) and five soil fractions extracted from LCSA-c were studied. Bacterial DNA was directly extracted from the soils, and a region of thenifHgene was amplified by PCR and analyzed by restriction. Soils were selected on the basis of differences in soil management, plant cover, and major physicochemical properties. Microenvironments differed on the basis of the sizes of the constituent particles and the organic carbon and clay contents. Restriction profiles were subjected to principal-component analysis. We showed that the composition of the diazotrophic communities varied both on a large scale (among soils) and on a microscale (among microenvironments in LCSA-c soil). Soil management seemed to be the major parameter influencing differences in thenifHgene pool structure among soils by controlling inorganic nitrogen content and its variation. However, physicochemical parameters (texture and total C and N contents) were found to correlate with differences amongnifHgene pools on a microscale. We hypothesize that the observednifHgenetic structures resulted from the adaptation to fluctuating conditions (cultivated soil, forest soil, coarse fractions) or constant conditions (permanent pasture soil, fine fractions). We attempted to identify a specific band within the profile of the clay fraction by cloning and sequencing it and comparing it with the gene databases. Unexpectedly, thenifHsequences of the dominant bacteria were most similar to sequences of unidentified marine eubacteria.

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules.

Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal. The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones. The sequences were the intergenic spacer between the rrs and rrl genes and the first 130 nucleotides of the rrl gene. Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes ( Clostridium -like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to β-proteobacteria ( Ralstonia -like genera). Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities. These studies confirmed the contribution of these populations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking.

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Fourteen bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from soils obtained from two farms in Canada and two farms in France. These strains were indistinguishable from each other based on repetitive extragenic palindromic PCR genomic fingerprinting performed with primers ERIC1R, ERIC2, and BOXA1R. Based on 16S rRNA sequence analysis of one representative isolate, strain C147, the isolates belong to the genus Pseudaminobacter in the family Rhizobiaceae . Strain C147 did not form nodules on the legumes alfalfa ( Medicago sativa L.), birdsfoot trefoil ( Lotus corniculatus L.), red clover ( Trifolium pratense L.), chickpea ( Cicer arietinum L.), and soybean ( Glycine max L.). A number of chloro-substituted s -triazine herbicides were degraded, but methylthio-substituted s -triazine herbicides were not degraded. Based on metabolite identification data, the fact that oxygen was not required, and hybridization of genomic DNA to the atzABC genes, atrazine degradation occurred via a series of hydrolytic reactions initiated by dechlorination and followed by dealkylation. Most strains could mineralize [ ring -U- 14 C]atrazine, and those that could not mineralize atrazine lacked atzB or atzBC . The atzABC genes, which were plasmid borne in every atrazine-degrading isolate examined, were unstable and were not always clustered together on the same plasmid. Loss of atzB was accompanied by loss of a copy of IS 1071 . Our results indicate that an atrazine-degrading Pseudaminobacter sp. with remarkably little diversity is widely distributed in agricultural soils and that genes of the atrazine degradation pathway carried by independent isolates of this organism are not clustered, can be independently lost, and may be associated with a catabolic transposon. We propose that the widespread distribution of the atrazine-degrading Pseudaminobacter sp. in agricultural soils exposed to atrazine is due to the characteristic ability of this organism to utilize alkylamines, and therefore atrazine, as sole sources of carbon when the atzABC genes are acquired.

An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification and characterization of extracellular polysaccharides produced by 1- and 4-day biofilms of 10 bacterial strains isolated from food industry premises. Peroxidase-labeled concanavalin A (ConA) and wheat germ agglutinin (WGA) were used, as they specifically bind to saccharide residues most frequently encountered in biofilms matrices: d -glucose or d -mannose for ConA and N -acetyl- d -glucosamine or N -acetylneuraminic acid for WGA. The ELLA applied to 1- and 4-day biofilms colonizing wells of microtiter plates was able to detect that for Stenotrophomonas maltophilia and to a lesser extent Staphylococcus sciuri , the increase in production of exopolysaccharides over time was not the same for sugars binding with ConA and those binding with WGA. Differences in extracellular polysaccharides produced were observed among strains belonging to the same species. These results demonstrate that ELLA is a useful tool not only for rapid characterization of biofilm extracellular polysaccharides but also, in studies of individual strains, for detection of changes over time in the proportion of the exopolysaccharidic component within the polymeric matrix.

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the Alu I restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.

The effects of citric acid-modified pH (pH 2.5, 2.75, 3, 3.5, 4, 4.5, 5, and 5.5) and a 30% glucose–70% sucrose mixture (300, 400, 500, 600, 700, 800, 875, and 900 g/liter) on an osmophilic yeast, Zygosaccharomyces rouxii , were determined by using synthetic medium. One hundred experiments were carried out; 50-ml culture flasks were inoculated with 10 3 CFU ml −1 by using a collection strain and a wild-type strain cocktail. The biomass was measured by counting cell colonies, and growth curves were fitted by using a Baranyi equation. The growth rate decreased linearly with sugar concentration, while the effect of pH was nonlinear. Indeed, the optimal pH range was found to be pH 3.5 to 5, and pH 2.5 resulted in a 30% reduction in the growth rate. Finally, we evaluated the performance of two nonlinear predictive models developed previously to describe bacterial contamination. Equations derived from the Rosso and Ratkowsky models gave similar results; however, the model that included dimensionless terms based on the Ratkowsky equation was preferred because it contained fewer estimated parameters and also because biological interpretation of the results was easier.

In lactococci, transamination is the first step of the enzymatic conversion of aromatic and branched-chain amino acids to aroma compounds. In previous work we purified and biochemically characterized the major aromatic aminotransferase (AraT) of a Lactococcus lactis subsp. cremoris strain. Here we characterized the corresponding gene and evaluated the role of AraT in the biosynthesis of amino acids and in the conversion of amino acids to aroma compounds. Amino acid sequence homologies with other aminotransferases showed that the enzyme belongs to a new subclass of the aminotransferase I subfamily γ; AraT is the best-characterized representative of this new aromatic-amino-acid-specific subclass. We demonstrated that AraT plays a major role in the conversion of aromatic amino acids to aroma compounds, since gene inactivation almost completely prevented the degradation of these amino acids. It is also highly involved in methionine and leucine conversion. AraT also has a major physiological role in the biosynthesis of phenylalanine and tyrosine, since gene inactivation weakly slowed down growth on medium without phenylalanine and highly affected growth on every medium without tyrosine. However, another biosynthesis aromatic aminotransferase is induced in the absence of phenylalanine in the culture medium.

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria , that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies.

From a hybridoma cell line (2A10) producing an immunoglobulin G1 directed against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable-fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin. The scFv gene was cloned and expressed in Escherichia coli . The expressed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward its phytoplasmal antigen, since enzyme-linked immunosorbent assay or immunofluorescence (IF) detection of the stolbur phytoplasma antigen by the scFv was identical to that of the native immunoglobulin. The scFv gene was then cloned in plasmid pBG-dAb-BIN of Agrobacterium tumefaciens to transform tobacco plants. The transformed plants were screened by PCR and Northern blotting for the presence and expression of the transgene, respectively, and by IF for expression of the scFv. One transgenic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma. When grafted on a stolbur phytoplasma-infected tobacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died.

A human trial was carried out to assess the ileal and fecal survival ofLactobacillus caseiDN-114 001 ingested in fermented milk. Survival rates were up to 51.2% in the ileum and 28.4% in the feces. The probiotic bacterium has the capacity to survive during its transit through the human gut.

Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture. The wild-type and mutant strains exhibited the same behavior. Oxidative metabolism was observed when the substrate uptake rate (qS) was low. Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1. When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway. The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain).

The influence of the type of cell envelope-located proteinase (PI versus PIII) on the associative growth of Lactococcus lactis in milk was studied. Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study. An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the PI-type proteinase strain, whereas that of the PIII-type proteinase strain remained unaffected. The reduction in proteolytic activity of the PI-type proteinase strain (presumably resulting from an inhibition of the synthesis of the enzyme) due to the peptides released by the PIII-type proteinase was found to be partly responsible for this interaction. Extension of the study to wild-type proteinase-positive L. lactis strains showed a systematic imbalance of the mixture of the two strains in favor of the PIII-type proteinase strain.

Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/micrograms of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional beta-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.

Thirteen bacterial strains of Xenorhabdus and 14 strains of Photorhabdus originating from a wide range of geographical and nematode host sources were typed by analyzing 16S rRNA gene (rDNA) restriction patterns obtained after digestion of PCR-amplified 16S rDNAs. Eight tetrameric restriction endonucleases were examined. A total of 17 genotypes were identified, forming two heterogeneous main clusters after analysis by the unweighted pair-group method using arithmetic averages: group I included all Xenorhabdus species and strains, symbionts of Steinernema, whereas group II encompassed the Photorhabdus strains, symbionts of Heterorhabditis. To identify the four valid species of Xenorhabdus and unclassified strains and all the genotypes of Photorhabdus luminescens, three restriction enzymes are required: CfoI, AluI, and HaeIII. Our results, in substantial agreement with DNA-DNA pairing and 16S rDNA sequence data, indicate that amplified 16S rDNA restriction analysis is a simple and accurate tool for identifying entomopathogenic nematode bacterial symbionts.