Experimental pathogenicity of isolates of Nocardia asteroides, Nocardia brasiliensis and Nocardia caviae from different sources

The experimental pathogenicity of 14 isolates of Nocardia brasiliensis, 15 of N. asteroides, and 5 of N. caviae was investigated for the white Swiss mice inoculated intraperitoneally and in the foot pad, and for the guinea-pig and the hamster (Mesocricetus auratus) both inoculated intratesticularly. The guinea-pig was remarkably sensitive to N. asteroides, with an apparent relationship between pathogenicity and thermotolerance, confirming previous observations. Mice were in general less susceptible to this species. In both guinea-pigs and hamsters it was possible to observe typical granules with or without clubs. N. caviae was highly pathogenic for the guinea-pig and the hamster but no mycetomas were produced in the mice inoculated in the foot pad. Isolates of N. brasiliensis from natural sources were scarcely virulent for the different animals. Those of human origin produced significant lesions in the mice inoculated intraperitoneally with granules. Foot pad inoculation of mice with N. brasiliensis caused mycetomas in several animals.     

The Nocardia salmonicida clade, including descriptions of Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov.

Large numbers of strains selectively isolated from soil, water and deteriorating vulcanised natural rubber pipe rings were provisionally assigned to the genus Nocardia. Twenty-eight representative isolates were found to have chemical and morphological properties typical of nocardiae. These organisms formed a monophyletic clade in the 16S rDNA tree together with Nocardia salmonicida. Three of the strains, isolates S1, W30 and R89, were distinguished from one another and from representatives of the validly described species of Nocardia using genotypic and phenotypic data. These organisms were considered to merit species status and were named Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov. respectively. Additional comparative studies are needed to resolve the finer taxonomic relationships of the remaining isolates assigned to the Nocardia salmonicida clade and to further unravel the extent of nocardial diversity in artificial and natural ecosystems.     

Identification of nocobactin NA biosynthetic gene clusters in Nocardia farcinica

We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The nbt clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of Nocardia farcinica IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the nbt clusters and the predicted functions of the nbt genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of the nbtA and nbtE genes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of the nbtS gene revealed that this gene encoded a salicylate synthase. These results indicate that the nbt clusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of the nbtA, -G, -H, -S, and -T genes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest that nbt genes are regulated coordinately by IdeR protein in an iron-dependent manner. The ΔnbtE mutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence of N. farcinica.     

Chemotherapeutic sensitivity and viability of Nocardia asteroides and Nocardia brasiliensis in drugs

Procedures for testing drug sensitivity ofNocardia were standardized.Five strains each ofN. asteroides (NA) andN. brasiliensis (NB) isolated from pathogenic materials were tested for drug sensitivity in Sabouraud's glucose agar (SGA), Sabouraud's glucose broth (SGB) and SGB with addition of 10 % horse serum against sulphadiazine (S), penicillin (P), streptomycin (St), chloramphenicol (C), tetracycline (T) and 4-4-diamino diphenyl sulphone (DDS). The viability of strains at minimum inhibitory concentrations (MIC) of different drugs were also tested.Results showed that liquid medium is preferable to solid medium for drug testing as pellicle formation observed on the surface of liquid medium helps in accurate assessment of MIC. Serum addition did not appear to be necessary. Ten days incubation at 37 °C gives optimum results for determination of MIC. Taking the maximum drug concentration attainable in blood during therapy, the strains were classified as sensitive and resistant. All strains were resistant to DDS and P. One strain of NB was sensitive to St. One strain each of NA and NB was borderline sensitive to C. Four out of 5 strains in both species were sensitive to S. All strains were sensitive to T. The organisms remained viable in drugs, thus suggesting the limitations of antinocardial therapy.From Mycology Research Unit, Department of Dermatology, Seth G.S. Medical College and K.E.M. Hospital, Bombay-12.Hon. Professor Dermato-Venereology.Research Fellow.     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

Mitogenic Activity of the Cell Walls of Mycobacteria,Nocardia, Corynebacteria and Anaerobic Coryneforms

The mitogenic activity of the cell walls prepared from Mycobacterium bovis BCG, Nocardia rubra, Corynebacterium diphtheriae PW8, and four species of Propionibacterium, Corynebacterium parvum ATCC 11829, Propionibacterium acnes C7, Propionibacterium granulosum ATCC 25564 and Propionibacterium avidum ATCC 25577, were investigated. These cell walls were active as mitogens on normal spleen cells, anti-θ sera-treated spleen cells, macrophage-depleted spleen cells of C57BL/6J mice and cortisone-treated thymocytes of C57BL/6J mice. It was also shown that these cell walls were mitogenic on spleen cells and macrophage-depleted spleen cells of congenitally athymic (nude) mice. The above results suggest that the cell walls investigated in this study act as mitogens on both thymus-derived lymphocytes (T-cells) and bone marrow-derived lymphocytes (B-cells).     

Cloning and characterization of the replicon of the Nocardia italica plasmid, pNI100

A 19-kb plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading frame (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three consensus sequences and a P-loop-like motif in the Rep protein of plasmid pSG5, a conjugative plasmid involving a rolling-circle replication mechanism, were conserved in those of plasmid pNI100. Phenotypically, a pock structure was produced in the regenerated mycelium by introducing pNI100 DNA into the Streptomyces lividans protoplast. This result strongly suggests that pNI100 is a conjugative plasmid and probably replicates by a rolling-circle replication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was constructed and found to be a useful cloning shuttle vector.     

Mycolic acid analysis in Nocardia species. The mycolic acid compositions of Nocardia asteroides, N. farcinica, and N. nova.

Mycolic acids from twelve Nocardia species were analyzed for structure using capillary gas chromatography and mass spectrometry. This high-resolution procedure permitted good separation of the trimethylsilyl (TMS) ether derivatives of mycolic acid methyl ester according to the total number of carbon and double bonds. The profiles of the mycolic acid molecular species were used as models to illustrate the difference in the structures of each species, even in the case of N. asteroides complex; N. asteroides, N. farcinica and N. nova. Although N. asteroides and N. farcinica had similar lengths of carbon skeleton, i.e., 51.9-53.7 was the average carbon number (Av.Nc.), they had different compositions of unsaturated acids. Mycolic acids from N. asteroides were composed of abundant saturated acids and less than 1% tetraenoic acids; mycolic acids from N. farcinica were composed of unsaturated acids, which were composed of abundant dienoic acids, 2-12% of tetraenoic acids and a trace of pentaenoic acids. In contrast, Av.Nc. of mycolic acids from N. nova were 55.7-56.3, which were relatively longer than those from N. asteroides or N. farcinica. Regarding the characteristics of the structure of alpha-branch, major components were C16:0 and C18:0 for N. asteroides 23206T, and C16:0 and C14:0 for N. farcinica 23157T, respectively. The presence of monounsaturated alpha-branch (C18:1 and C16:1) was characteristic of N. nova.     

Linkage and Segregation of Unselected Markers in Matings of Nocardia erythropolis with Nocardia canicruria

The segregation of unselected genes expressing resistance or susceptibility to acriflavine, erythromycin, streptomycin, and tetracycline was analyzed in selected prototrophic recombinants resulting from matings of Nocardia erythropolis and N. canicruria. The organisms were shown to be functionally haploid and appeared to contain not more than one genome. It was postulated that all observed genes were present in a linear linkage group. The ordering of the genes in N. erythropolis was: tetB10 eryB9 his-3 purA1 acr-2 strA1 (respectively, resistance to tetracycline and erythromycin, deficiency for histidine and for purine, and resistance to acriflavine and streptomycin). The ordering of the genes in N. canicruria was: purB2 tetA9 eryA7 acr-11 strB2 (respectively, deficiency for purine, and resistance to tetracycline, erythromycin, acriflavine, and streptomycin). Excluding the genes for acriflavine resistance, acr-2 and acr-11, resistance loci in N. erythropolis were not allelic to and showed lateral displacement from genes controlling phenotypically similar resistance in N. canicruria. Evidence for some lack of homology between N. erythropolis and N. canicruria genomes was found. Recombination phenomena between the nocardial species was postulated to occur as a result of formation of a heterogenomic zygote in which new combinations were produced. Production of selectable, haploid recombinants was ascribed to subsequent haploidization of the zygote.     

Bioactive molecules from Nocardia: diversity,bioactivities and biosynthesis

Journal of Industrial Microbiology & Biotechnology - Nocardia spp. are catalase positive, aerobic, and non-motile Gram-positive filamentous bacteria. Many Nocarida spp. have been reported as...     

Thiostrepton induced proteins in Streptomyces, Amycolatopsis and Nocardia species

Abstract Thiostrepton-induced proteins have been observed in several actinomycetes, including Streptomyces clavuligerus (15 kDa protein), S. griseus (21 kDa), Nocardia lactamdurans (17 kDa), Amycolatopsis sp 239 (14 and 16 kDa), S. fradie (18 and 20 kDa) and S. coelicolor (17, 19, 30 and 56 kDa). The thiostrepton-induced proteins of S. coelicolor seem to be identical to those reported in S. lividans . The induction of the 18 and 20 kDa proteins of S. fradiae (up to 20% of the total protein in the cells) was dependent upon the concentration of thiostrepton (1 to 50 μg per ml) added to the cultures. The induction of proteins by thiostrepton in N. lactamdurans (and probably in the other actinomycetes as well) is not mediated by the tsr gene of pIJ702, since spontaneous thiostrepton-resistant mutants of N. lactamdurans which do not carry the tsr gene were used for the induction studies. Induction of proteins by thiostrepton may be a protective mechanism to counteract the strong effect of this antibiotic which is known to be produced by several soil microorganisms.     

诺卡氏菌腈水合酶基因在重组毕赤酵母中的表达

为从基因水平上改造腈水合酶,进行了诺卡氏菌腈水合酶基因的外源表达研究。在重组大肠杆菌表达系统内,腈水合酶的α亚基几乎不能正常表达,在重组E. coli BL21(DE3) (pET32aNHBAX)中,腈水合酶活性仅为0.04U/mg。构建重组毕赤酵母表达质粒pPIC3.5kNHBAX,采用电穿孔转化法将其转入宿主菌P. pastoris GS115中,经过菌株培养和腈水合酶的诱导表达,筛选获得了优选菌株P. pastoris NH4。对P. pastoris NH4的细胞培养和腈水合酶的诱导表达条件进行优化,结果表明,重组腈水合酶在毕赤酵母中的表达水平可以达到0.52U/mg,但不能稳定积累。     

Sucrose Lipids of Arthrobacteria,Corynebacteria and Nocardia Grown on Sucrose

Arthrobacter paraffineus KY 4303, when grown on sucrose as the sole carbon source, produced novel glycolipids, either of which was different from trehalose lipid produced from n-alkane by the same microorganism. Two kinds of glycolipids were isolated by chromatography on silicic acid columns. Major components of these lipids were sucrose and α-branched β-hydroxy fatty acid. One of the lipid (SL–1, having high polarity) was identified as 6-O-monofattyacyl glucosly-β-fructoside. Another (SL–2, having low polarity) was partly characterized as sucrose ester of at least two moles of the fatty acid.

Formation of sucrose lipids was also demonstrated in sucrose-grown cells of several microorganisms of Corynebacteria, Nocardia and Brevibacteria, which were isolated as hydrocarbon-utilizing bacteria and could produce a considerable amount of trehalose lipid from n-alkane.