Microbiology (United Kingdom)

Here we tested the hypothesis that species of the soil-inhabiting insect-pathogenic fungusMetarhiziumare not randomly distributed in soils but show plant-rhizosphere-specific associations. We isolatedMetarhiziumfrom plant roots at two sites in Ontario, Canada, sequenced the 5′ EF-1α gene to discernMetarhiziumspecies, and developed an RFLP test for rapid species identification. Results indicated a non-random association of threeMetarhiziumspecies (Metarhizium robertsii,Metarhizium brunneumandMetarhizium guizhouense) with the rhizosphere of certain types of plant species (identified to species and categorized as grasses, wildflowers, shrubs and trees).M. robertsiiwas the only species that was found associated with grass roots, suggesting a possible exclusion ofM. brunneumandM. guizhouense. Supporting this,in vitroexperiments showed thatM. robertsiiconidia germinated significantly better inPanicum virgatum(switchgrass) root exudate than didM. brunneumorM. guizhouense.M. guizhouenseandM. brunneumonly associated with wildflower rhizosphere when co-occurring withM. robertsii. With the exception of these co-occurrences,M. guizhouensewas found to associate exclusively with the rhizosphere of tree species, predominantlyAcer saccharum(sugar maple), whileM. brunneumwas found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species ofMetarhiziumassociate with specific plant types.

A number of physiologically different nitrile-hydrolysing bacteria were isolated from coastal marine sediments in Denmark by enrichment culture. One strain, BL1, identified as Rhodococcus erythropolis, grew on acetonitrile as sole carbon and nitrogen source in a defined medium. Growth occurred between 0 and 8% NaCl with an optimum around 2%, thus reflecting the marine origin of the isolate. Intact cells of R. erythropolis BL1 could hydrolyse a large variety of saturated and unsaturated aliphatic nitriles to their corresponding acids. Benzonitrile and benzylcyanide were not hydrolysed, whereas some aromatic compounds containing a -CN group attached to a C₃ or C₄ aliphatic side chain were accepted as substrates. The substrate spectrum of R. erythropolis BL1 was thus markedly different from those of other Grampositive nitrile-hydrolysing bacteria isolated from non-marine environments. Nitrile hydrolysis during growth and in resting cell suspensions usually occurred without intermediate accumulation of amide outside the cells. Detailed studies, however, showed that nitrile hydrolysis by strain BL1 was due to a nitrile hydratase/amidase enzyme system. Nitrile hydratase activity was found to be inducible whereas amidase activity was constitutive. The amidase activity of cells could, however, be enhanced manyfold by growth in media containing acetamide or acetonitrile. In most cases amides were hydrolysed at a much higher rate than the corresponding nitriles, which explained why amides were rarely detected in the surrounding medium during nitrile hydrolysis. R. erythropolis BL1 exhibited the highest tolerance towards acetonitrile ever reported for a nitrile-hydrolysing bacterium, as demonstrated by its ability to grow exponentially in the presence of 900 mM acetonitrile.

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene andrpoC1phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon–Wiener (J′) indices a high diversity was observed within all species, withPlanktothrix agardhiishowing the lowest diversity values (D=0.83;J′=0.88) andAphanizomenon flos-aquaethe highest ones (D=J′=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.

The intestinal epithelium forms a protective barrier against luminal contents and the external environment, mediated via intercellular tight junctions (TJs). The TJ can be disrupted via cell signalling induced by either enteric pathogens or pro-inflammatory cytokines, thereby contributing to various intestinal disorders ranging from acute infectious diarrhoea to chronic inflammatory bowel diseases. Probiotics, such asLactobacillus rhamnosusGG (LGG), are reported to confer beneficial effects on epithelial cells, including antagonizing infections and reducing overt pro-inflammatory responses, but the underlying mechanisms of these observed effects require further characterization. We hypothesized that probiotics preserve barrier function by interfering with pro-inflammatory cytokine signalling. Caco-2bbe cells were seeded into Transwells to attain polarized monolayers with intercellular TJs. Monolayers were inoculated apically with the probiotic LGG 3 h prior to the addition of IFN-γ(100 ng ml⁻¹) to the basolateral medium overnight. The monolayers were then placed in fresh basal medium±TNF-α(10 ng ml⁻¹) and transepithelial electrical resistance (TER) measurements were taken over the time-course of TNF-αstimulation. To complement the TER findings, cells were processed for zona occludens-1 (ZO-1) immunofluorescence staining. As a measure of TNF-αdownstream signalling, cells were immunofluorescently stained for NF-κB p65 subunit and CXCL-8 mRNA was quantified by qRT-PCR. Basal cell culture medium was collected after overnight TNF-αstimulation to measure secreted chemokines, including CXCL-8 (interleukin-8) and CCL-11 (eotaxin). Following LGG inoculation, IFN-γpriming and 24 h TNF-αstimulation, epithelial cells maintained TER and ZO-1 distribution. LGG diminished the nuclear translocation of p65, demonstrated by both immunofluorescence and CXCL-8 mRNA expression. CXCL-8 and CCL-11 protein levels were decreased in LGG-inoculated, cytokine-challenged cells. These findings indicate that LGG alleviates the effects of pro-inflammatory cytokines on epithelial barrier integrity and inflammation, mediated, at least in part, through inhibition of NF-κB signalling.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated CRISPR-associated sequence (CAS) proteins constitute a novel antiviral defence system that is widespread in prokaryotes. Repeats are separated by spacers, some of them homologous to sequences in mobile genetic elements. Although the whole process involved remains uncharacterized, it is known that new spacers are incorporated into CRISPR loci of the host during a phage challenge, conferring specific resistance against the virus. Moreover, it has been demonstrated that such interference is based on small RNAs carrying a spacer. These RNAs would guide the defence apparatus to foreign molecules carrying sequences that match the spacers. Despite this essential role, the spacer uptake mechanism has not been addressed. A first step forward came from the detection of motifs associated with spacer precursors (proto-spacers) ofStreptococcus thermophilus, revealing a specific recognition of donor sequences in this species. Here we show that the conservation of proto-spacer adjacent motifs (PAMs) is a common theme for the most diverse CRISPR systems. The PAM sequence depends on the CRISPR-CAS variant, implying that there is a CRISPR-type-specific (motif-directed) choice of the spacers, which subsequently determines the interference target. PAMs also direct the orientation of spacers in the repeat arrays. Remarkably, observations based on such polarity argue against a recognition of the spacer precursors on transcript RNA molecules as a general rule.

The cell wall of Gram-positive bacteria has been a subject of detailed chemical study over the past five decades. Outside the cytoplasmic membrane of these organisms the fundamental polymer is peptidoglycan (PG), which is responsible for the maintenance of cell shape and osmotic stability. In addition, typical essential cell wall polymers such as teichoic or teichuronic acids are linked to some of the peptidoglycan chains. In this review these compounds are considered as ‘classical’ cell wall polymers. In the course of recent investigations of bacterial cell surface layers (S-layers) a different class of ‘non-classical’ secondary cell wall polymers (SCWPs) has been identified, which is involved in anchoring of S-layers to the bacterial cell surface. Comparative analyses have shown considerable differences in chemical composition, overall structure and charge behaviour of these SCWPs. This review discusses the progress that has been made in understanding the structural principles of SCWPs, which may have useful applications in S-layer-based ‘supramolecular construction kits' in nanobiotechnology.