Significance to metallomicsDithiol gliotoxin is a near-terminal biosynthetic intermediate from the gliotoxin biosynthetic pathway in the human pathogen Aspergillus fumigatus. Chemically reduced gliotoxin, dithiol gliotoxin (DTG), is revealed as a biological zinc chelator, and conversely, zinc can relieve the hitherto cryptic fungal autotoxicity of DTG. There is a systems-wide impact of zinc chelation by DTG on the fungal proteome, and we suggest it is DTG, as opposed to gliotoxin, which chelates zinc from metalloproteins. Since gliotoxin can be sequestered by both fungi and bacteria, our findings infer a new avenue to interfere with, and exploit, cellular zinc homeostasis in microorganisms. IntroductionGliotoxin and holomycin are microbial natural products, which are produced by fungal and bacterial spp., respectively (Fig. 1). [1][2][3] Both are low molecular mass metabolites, and each contains a disulphide bridge formed by the action of oxidoreductases, namely GliT and HlmI, on the respective dithiol precursor (Fig. 1). [4][5][6] Disulphide bridge formation is essential for microbial self-protection against these reactive dithiol intermediates, and is a pre-requisite for the Major Facilitator Superfamily transporter GliA-mediated secretion of gliotoxin by Aspergillus fumigatus. 5-8Both metabolites are also present as bis-thiomethylated forms, and gliotoxin bis-thiomethyltransferase GtmA converts dithiol gliotoxin (DTG) to bis-dethiobis(methylthio)gliotoxin (BmGT) in A. fumigatus, whereas the origin of the cognate activity against dithiol holomycin in Streptomyces clavuligeris is unknown (Fig. 1). 9,10Bernardo et al. have shown that upon uptake by eukaryotic cells,
The fimbrial subunit genes of Bacteroides nodosus may be divided into two distinct classes, based on the sequence of the major subunit gene fimA (accompanying paper--Mattick et al., 1991). The genetic organization of the fibrial gene region in these two classes is also distinct. Upstream of fimA in both classes in opposite transcriptional orientation is the gene aroA which encodes amino acid biosynthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase. However, downstream of fimA the two classes are quite different until homology is restored at a bidirectional transcription termination signal separating the fimbrial operon from a gene clpB, which appears to encode the regulatory subunit of an ATP-dependent protease. Between aroA and clpB class I strains contain, apart from fimA, only one other gene (fimB). Sequence and polymerase chain reaction analyses indicate that fimB does not have a separate promoter but rather is co-transcribed with fimA at a level attenuated by the strength of the transcription termination signal in the intergenic region. In class II strains fimA is followed by a more extended region containing three genes, which appear to have the same transcriptional arrangement as fimB. The second of these genes (fimD) may represent a functional analogue of fimB although there is no close sequence homology. The first gene (fimC) has no obvious similarity to either fimB or fimD. Beyond fimD, at the 3' end of the class II-specific region, is a variant fimbrial subunit gene (fimZ) which is virtually identical in serogroups D and H and which appears to represent a duplicate, possibly redundant, gene closely related to the progenitor of the more divergent structural subunit fimA gene found in these strains. Comparisons of the predicted fimZ product with those of fimA in class I and class II strains, as well as of the boundaries of the class-specific regions, suggest that the class II sequences evolved in another type 4 fimbriate species and were subsequently substituted in the B. nodosus genome by lateral transfer. Analysis of the sequences flanking fimA in different strains indicates that recombinational exchange of both fimA and the entire operon has also occurred between strains, and is possibly a mechanism for disseminating structural diversity in the population.
Mycoplasma hyopneumoniae, M. hyorhinis and M. flocculare are commonly isolated from the respiratory tract of pigs and are phylogenetically related. The identification and characterization of antigens specific for M. hyopneumoniae is crucial for the development of serological reagents and for understanding the mechanisms of pathogenicity of this pathogen. Protein and antigen profiles of six strains of M. hyopneumoniae, four strains of M. hyorhinis and a type strain of M. flocculare were compared using SDS-PAGE and immunoblotting. Five strains of M. hyopneumoniae originally isolated from diverse geographical regions produced similar protein and antigen profiles. One strain, C1735/2, produced a unique protein profile and was poorly immunoreactive, suggesting that some strains of M. hyopneumoniae may possess a structurally modified repertoire of antigens. Major M. hyopneumoniae antigens with molecular masses of approximately 36,43,48, 528 76, 78, 80, 82,94, 106,114 and 200 kDa were identified by immunoblotting using hyperimmune pig sera raised against both high and low passage strains of M. hyopneumoniae. Porcine hyperimmune sera raised against the GDL type strain of M. hyorhinis reacted strongly with all M. hyorhinis strains although the profiles displayed considerable variation. Major antigens of molecular mass 42,49, 52,78,80 and 82 kDa were identified in type strains GDL and BTS-7 and field strain 2; however, field strain 1 produced a unique profile. A preparative SDS-PAGE profiling (PPP) technique was developed which enabled quantification of the immunoreactivity of denatured antigens with porcine serum by ELISA. PPP facilitated the rapid identification of species-specif ic and cross-reactive antigens among the three mycoplasma species. PPP studies revealed several strongly immunoreactive M. hyopneumoniae-specif ic antigens of 43,76,94,114 and 200 kDa as well as antigens of molecular mass between 52 and 62 kDa which were not apparent in immunoblotting studies. Rabbit monospecific anti43 kDa serum reacted specifically with a 43 kDa antigen in whole cell lysates of geographically diverse strains of M. hyopneumoniae and failed to cross-react with M. flocculare or M. hyorhinis whole cell lysates. This study has identified a number of M. hyopneumoniaespecific antigens which warrant further investigation to determine their potential as diagnostic reagents and the role they play, if any, in pathogenicity.
A complete medium of defined composition has been developed for quantitative growth of wild-type and auxotrophic mutant strains of Anacystis nidulans. This medium has proved to be more satisfactory than other complex media (for example casein hydrolysate, yeast extract) for both the isolation and the growth of auxotrophs. Rigorous control of the pH of complete and other supplemented media is essential for quantitative growth on agar. Four diagnostic media are described which each contain a different combination of the supplements used in the complete medium and facilitate the identification of the nutritional requirements of mutants. By using these media a number of auxotrophs have been isolated including five with novel phenotypes which require respectively (i) thiamine, (ii) p-aminobenzoic acid, (iii) a combination of pyruvate or acetate plus malate or succinate or fumarate, (iv) serine or glycine and (v) adenine.
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