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SUMMARY: Roper's technique for the production in filamentous fungi of strains with heterozygous diploid nuclei in their hyphae (Roper, 1952) has been applied successfully to Aspergillus niger, in which a sexual cycle does not occur. The diploids, heterozygous for known markers, give origin to new strains, most still diploid, homozygous for some or all of the markers and therefore associating or recombining in all possible ways the properties of the two strains from which the diploid was formed. Genetic recombination has thus been achieved in a filamentous fungus without a normal sexual cycle. Imperfect fungi are now open to genetic investigation.Deliberate 'breeding ' of strains has become a practical proposition in industrial fermentations based on these fungi.

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The immune-modulating properties of certain bifidobacterial strains, such as Bifidobacterium longum subsp. longum 35624 (B. longum 35624), have been well described, although the strain-specific molecular characteristics associated with such immuneregulatory activity are not well defined. It has previously been demonstrated that B. longum 35624 produces a cell surface exopolysaccharide (sEPS), and in this study, we investigated the role played by this exopolysaccharide in influencing the host immune response. B. longum 35624 induced relatively low levels of cytokine secretion from human dendritic cells, whereas an isogenic exopolysaccharide-negative mutant derivative (termed sEPS neg ) induced vastly more cytokines, including interleukin-17 (IL-17), and this response was reversed when exopolysaccharide production was restored in sEPS neg by genetic complementation. Administration of B. longum 35624 to mice of the T cell transfer colitis model prevented disease symptoms, whereas sEPS neg did not protect against the development of colitis, with associated enhanced recruitment of IL-17 ؉ lymphocytes to the gut. Moreover, intranasal administration of sEPS neg also resulted in enhanced recruitment of IL-17 ؉ lymphocytes to the murine lung. These data demonstrate that the particular exopolysaccharide produced by B. longum 35624 plays an essential role in dampening proinflammatory host responses to the strain and that loss of exopolysaccharide production results in the induction of local T H 17 responses. IMPORTANCEParticular gut commensals, such as B. longum 35624, are known to contribute positively to the development of mucosal immune cells, resulting in protection from inflammatory diseases. However, the molecular basis and mechanisms for these commensalhost interactions are poorly described. In this report, an exopolysaccharide was shown to be decisive in influencing the immune response to the bacterium. We generated an isogenic mutant unable to produce exopolysaccharide and observed that this mutation caused a dramatic change in the response of human immune cells in vitro. In addition, the use of mouse models confirmed that lack of exopolysaccharide production induces inflammatory responses to the bacterium. These results implicate the surfaceassociated exopolysaccharide of the B. longum 35624 cell envelope in the prevention of aberrant inflammatory responses. T he gut microbiota contributes significantly to host health via multiple mechanisms, including the digestion of foods, competitive exclusion of pathogens, enhancement of epithelial cell differentiation, and promotion of mucosa-associated lymphoid tissue proliferation (1, 2). Furthermore, accumulating evidence suggests that the composition and metabolic activity of the gut microbiota has profound effects on proinflammatory activity and the induction of immune tolerance within mucosal tissue (3-5). Certain microbes induce regulatory responses, while others induce effector responses, resulting in the case of healthy individuals in a balanced homeostatic...

Previous studies had shown that haploid strains ofAspergillus nidulanswhich have a chromosome segment in duplicate are unstable at mitosis. Through the study of various haploid and diploid strains, with and without translocations and with balanced and unbalanced genomes, it has been shown: (1) that imbalance of chromosome segments is responsible for instability, and (2) that the chromosomal deletions produced are confined solely or largely to the segments which provoke instability.The term ‘mitotic non-conformity’ has been proposed for this instability phenomenon. An explanation for it has been sought in terms of attachment sites, limited in number and specific for chromosome segments, at which replication is initiated.

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

Strains of Aspergillus nidulans with a duplicate chromosome segment are mitotically unstable; in addition to phenotypically improved variants, arising following deletions in either duplicate segment, they give morphologically deteriorated types, some with enhanced stability. In one isolate, deterioration and increased instability were determined by mutation in a duplicate segment; a more stable derivative no longer had this mutation but had one in another linkage group. Another variant, too unstable for analysis, gave derivatives whose single, new mutations were in different linkage groups. It is proposed that deterioration and increased instability result from tandem duplications on either duplicate segment; transposition of these to non-duplicated regions reduces instability. Another 17 variants had a single new mutation each; mutations, possibly clustered, occurred in all linkage groups. In these strains perhaps transposition preceded analysis. Deteriorated variants gave lineages of types with morphological changes caused by further, superimposed mutations. This continued instability is explained as interaction, in fidelity of replication, of non-homologous chromosome segments.Instability in A. nidulans stems from chromosome imbalance. As imbalance is known or suspected in other cases of instability it may be possible to show common mechanisms for apparently diverse phenomena.

Interactions between the host and the microbiota are thought to significantly influence immunological tolerance mechanisms at mucosal sites. We recently described that the loss of an exopolysaccharide (EPS) from Bifidobacterium longum 35624™ eliminated its protective effects in colitis and respiratory allergy murine models. Our goal was to investigate the immune response to purified EPS from B. longum 35624, determine if it has protective effects within the lung and identify the protective mechanisms. Isolated EPS from B. longum 35624 cultures was used for in vitro, ex vivo and in vivo studies. Human monocyte-derived dendritic cells (MDDCs) were used to investigate in vitro immunological responses to EPS. Cytokine secretion, expression of surface markers and signalling pathways were examined. The ovalbumin (OVA) respiratory allergy murine model was used to evaluate the in vivo immunomodulatory potential of EPS. In addition, interleukin (IL)-10 knockout (KO) mice and anti-Toll-like receptor (TLR)-2 blocking antibody were used to examine the underlying protective mechanisms of intranasal EPS administration. Stimulation of human MDDCs with EPS resulted in IL-10 secretion, but not proinflammatory cytokines. IL-10 secretion was TLR-2-dependent. Eosinophil recruitment to the lungs was significantly decreased by EPS intranasal exposure, which was associated with decreased expression of the Th2-associated markers C-C motif chemokine 11 (CCL11), C-C chemokine receptor type 3 (CCR3), IL-4 and IL-13. TLR-2-mediated IL-10 secretion was shown to be required for the reduction in eosinophils and Th2 cytokines. EPS-treatment reduced eosinophil recruitment within the lung in a respiratory inflammation mouse model, which is both TLR-2 and IL-10 mediated. EPS can be considered as a novel molecule potentially reducing the severity of chronic eosinophil-related airway disorders.