Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans

Summary Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408. 1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea

Summary pSE211 fromSaccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination betweenattP andattB, the plasmid and chromosomal attachment sites. Integration depends on the presence ofint, an open reading frame (ORF) that lies adjacent toattP and encodes the putative integrase. Immediately upstream ofint liesxis (formerly calledorf2) which encodes a basic protein that is thought to exhibit DNA binding.xis andint were cloned in various combinations in pUC18 and expressed constitutively inEscherichia coli from thelac promoter.attP andattB were cloned inStreptomyces orE. coli plasmids containing kanamycin resistance (Km^R) or chloramphenicol resistance (Cm^R) markers. Stable Km^R Cm^R cointegrates formed byattP ×attB orattP ×attP recombination (integration) were obtained inE. coli hosts that expressedint. Co-integrates were not found in hosts expressingint+xis. Excision (intraplasmidatt site recombination) was examined by constructing plasmids carryingattL andattR or twoattP sites separating Cm^R from Km^R and by following segregation of the markers in various hosts. BothattL ×attR andattP ×attP excision depended on bothxis andint inE. coli. pSE211att site integration and excision were not affected by a deletion inhimA, the gene encoding a subunit of integration host factor.     

Stability of plasmid pA387 derivatives in Amylcolatopsis mediterranei producing rifamycin

Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability.     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G+C codon optimized integrase for site-specific integration

pMR2, an 11.1 kb plasmid was isolated from Micromonospora rosaria SCC2095, NRRL3718, and its complete nucleotide sequence determined. Analysis revealed 13 ORFs including homologs of a KorSA regulatory protein and TraB plasmid transfer protein found on other actinomycete plasmids. pMR2 contains att/int functions consisting of an integrase, an excisionase, and a putative plasmid attachment site (attP). The integrase gene contained a high frequency of codons rarely used in high G+C actinomycete coding regions. The gene was codon optimized for actinomycete codon usage to create the synthetic gene int-OPT. pSPRX740, containing an rpsL promoter and the att/int-OPT region, was introduced into Micromonospora halophytica var. nigra ATCC33088. Analysis of DNA flanking the pSPRX740 integration site confirmed site-specific integration into a tRNA(Phe) gene in the M. halopytica var. nigra chromosome. The pMR2 attP element and chromosomal attachment (attB) site contain a 63 bp region of sequence identity overlapping the 3' end of the tRNA(Phe) gene. Plasmids comprising the site-specific att/int-OPT functions of pMR2 can be used to integrate genes into the chromosome of actinomycetes with an appropriate tRNA gene. The development of an integrative system for Micromonospora will expand our ability to study antibiotic biosynthesis in this important actinomycete genus.     

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG

Different vaccination procedures to increase the mecha nisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2×10⁸ N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG- treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.     

Production of polyhydroxyalkanoates from inexpensive extruded rice bran and starch by Haloferax mediterranei

Low-cost raw materials can be used to reduce significantly the production cost of polyhydroxyalkanoates (PHA). In this study, extruded rice bran (ERB) and extruded cornstarch (ECS) were used as carbon sources to produce PHA by an archaea, Haloferax mediterranei, which cannot use native rice bran or cornstarch as a carbon source. By employing pH-stat control strategy to maintain pH at 6.9–7.1 in a 5-liter jar fermentor using ERB:ECS (1:8 g/g) as the major carbon source, we obtained a cell concentration of 140 g/L, PHA concentration of 77.8 g/L and PHA content of 55.6 wt.% in a repeated fed-batch fermentation. In contrast, when ECS was used as the major carbon source, we obtained 62.6 g/L cell concentration, 24.2 g/L PHA concentration and 38.7 wt.% PHA content. Under a hyper-saline condition and with no nitrogen-limitation restriction, the repeated fed-batch process can be sustained a long time for the mass production of PHA.     

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an α-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50°C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5–3 M NaCl. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Age dependent alterations in phospholipid composition of a saprophytic and a pathogenic strain of Nocardia

Nocardia polychromogenes (saprophytic) and Nocardia asteroides (pathogenic) showed characteristic patterns in changes of cellular lipids during growth. Total lipids and total phospholipids decreased with the age of the culture in the saprophytic strain, whereas in the pathogenic strain total lipids increased throughout the culture period and the total phospholipids decreased in the late stationary phase. The decrease in total phospholipids in saprophytic strain was reflected in the individual phosphatides. In the pathogenic strain, the phosphatidylinositomannoside content doubled in early stationary phase. Differences were observed in fatty acid composition of phosphatides at various stages of growth, but the ratio of saturated to unsaturated fatty acids remained unaltered.     

Properties of a partially purified acid phosphatase from pathogenic Nocardia brasiliensis

Like many other bacteria, Nocardia sp. possess acid phosphatase activity. In N. brasiliensis, a human and animal pathogen, this activity was resolved into two enzyme forms by native gel electrophoresis. One (isozyme I) was partially purified and characterized. It exhibited an estimated molecular weight on SDS-PAGE of 50 kDa, a pH optimum of 5.2, and a Km value of 1.25 mM for p-nitrophenylphosphate. The N. brasiliensis enzyme was stable at 4 °C for at least 24 h, but readily inactivated at 60 °C. Ammonium molybdate, sodium fluoride and L-(+)-tartrate were found to be potent inhibitors of the enzyme. Although its function is presently unknown, by analogy to other bacterial systems it could be envisioned to play an important role in the physiology and pathogenicity of the microorganism.     

Nocardia brasiliensis: in vitro and in vivo growth response to steroid sex hormones

As actinomycetoma is more frequent in males than in females, the possibility that hormones might modify theNocardia brasiliensis growth and the course of experimental actinomycetoma was explored. FiveN. brasiliensis strains were grown on Sabouraud agar containing estradiol, progesterone or testosterone, in 3 different concentrations. Colony diameters were measured weekly for 7 weeks.N. brasiliensis strains were also grown in Sabouraud broth containing hormones. Glucose concentration was measured weekly for 6 weeks. Finally, experimental actinomycetoma was produced in male and female hormone-treated mice. Invasion rate, plantar pad diameter and positive retrocultures were assessed. In vitro experiments showed that progesterone and testosterone inhibitN. brasiliensis growth, manifested by lower colony diameters and greater glucose concentrations. In vivo experiments demonstrated that estradiol limits actinomycetoma development. Progesterone and testosterone induced greater diameters of inoculated plantar pads and greater invasion rates with greater positive culture numbers than estradiol. Results partially explain the resistance of females to actinomycetoma.     

Cytokine production and lymphocyte proliferation in patients with Nocardia brasiliensis actinomycetoma

IFN-γ, TNF-α, IL-4, IL10 and IL-12 concentrations in the supernatant of peripheral blood mononuclear cell (PBMC) cultures and the in vitro proliferation of PBMC were studied in 25 patients with actinomycetoma caused by Nocardia brasiliensisand in 10 healthy controls from endemic zones. Cell cultures were stimulated by a N. brasiliensiscrude cytoplasmic antigen (NB) and five semi-purified protein fractions (NB2, NB4, NB6, NB8, and NB10) separated by isoelectric. Phytohemagglutinin (PHA) and purified protein derivative (PPD) of Mycobacterium tuberculosiswere used as control antigens. Skin tests were performed by injecting 0.1 ml of candidin and PPD intradermally (ID). Patients showed a poor response to tuberculin, while their response to candidin was more than two fold greater than that observed in the controls. Cell proliferation showed no statistically significant differences in either group. IFN-γ production was higher in the healthy controls than in the patients, whereas TNF-α secretion was slightly higher in the patients’ cultures. IL-4 was detected in the patients’ cultures but not in the controls. IL-10 and IL-12 were present at low concentrations in both groups. These results suggest that patients with actinomycetoma show normal antigen recognition, but with low IFN-γ production, and higher concentrations of IL-4, IL-10 and TNF-α in the patients’ PBMC cultures, indicating that they probably have a Th2 type of immune response.     

First human case of nocardiosis caused by Nocardia pseudobrasiliensis in Japan

Nocardia sp. IFM 0896, an actinomycete with biochemical characteristics that differed from Nocardia brasiliensis, was isolated from a 71-year-old Japanese man with a history of tuberculosis and cancer. Although the isolate was tentatively identified as N. brasiliensis, the morphological and physiological characteristics of strain IFM 0896 were different from those of N. brasiliensis IFM 0236^T. The results of 16S rRNA gene sequence phylogenies and PCR-RFLP analysis of a heat shock protein revealed that Nocardia sp. IFM 0896 belongs to the species N. pseudobrasiliensis. This is the first clinical isolation report of N. pseudobrasiliensis in Japan.This revised version was published online in October 2005 with corrections to the Cover Date.     

Immune Response to Nocardia brasiliensis Extracellular Antigens in Patients with Mycetoma

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 × 10⁷ CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-γ, IL-1α, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-γ production in mice, as well as the CFA, were used to stimulate IFN-γ production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-γ production in the infected group. In humans, only the CFA-induced IFN-γ production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.     

Primary subcutaneous Nocardia asteroides infection in a renal allograft recipient

Nocardia asteroides is an important opportunistic pathogen in immunocompromised hosts. The primary infection is usually in the lungs and is followed by dissemination to other parts of the body. Primary subcutaneous infection with Nocardia asteroides has been reported rarely (three reports) and no such case has been reported in a renal transplant recipient. We describe here a case of renal transplant recipient who developed primary subcutaneous infection with Nocardia asteroides within one and half years of the transplantation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Predicted highly expressed genes in Nocardia farcinica and the implication for its primary metabolism and nocardial virulence

Nocardia farcinica is a Gram positive, filamentous bacterium, and is considered an opportunistic pathogen. In this study, the highly expressed genes in N. farcinica were predicted using the codon adaptation index (CAI) as a numerical estimator of gene expressivity. Using ribosomal protein (RP) genes as references, the top ∼ ∼10% of the genes were predicted to be the predicted highly expressed (PHX) genes in N. farcinica using a CAI cutoff of greater than 0.73. Consistent with earlier analysis of Streptomyces genomes, most of the PHX genes in N. farcinica were involved in various ‘house-keeping’ functions important for cell growth. However, 15 genes putatively involved in nocardial virulence were predicted as PHX genes in N. farcinica, which included genes encoding four Mce proteins, cyclopropane fatty acid synthase which is involved in the modification of cell wall which may be important for nocardia virulence, polyketide synthase PKS13 for mycolic acid synthesis and a non-ribosomal peptide synthetase involved in biosynthesis of a mycobactin-related siderophore. In addition, multiple genes involved in defense against reactive oxygen species (ROS) produced by the phagocyte were predicted with high expressivity, which included alkylhydroperoxide reductase (ahpC), catalase (katG), superoxide dismutase (sodF), thioredoxin, thioredoxin reductase, glutathione peroxidase, and peptide methionine sulfoxide reductase, suggesting that combating against ROS is essential for survival of N. farcinica in host cells. The study also showed that the distribution of PHX genes in the N. farcinica circular chromosome was uneven, with more PHX genes located in the regions close to replication initiation site. The results provided the first estimates of global gene expression patterns in N.␣farcinica, which will be useful in guiding experimental design for further investigations. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Microbiological resolution of chiral arylethyl carbinols by Nocardia corallina

Whole cells of Nocardia corallina B-276 oxidized enantioselectively racemates of arylethyl carbinols, at 0.5 mM, to give ketones in yields from 4 to 97% (w/w) and the unreacted alcohols showing enantiomeric excess ranging from 5 to > 99%. The configuration of the resulting alcohol is (R).     

Emulsification using environmental compatible emulsifiers and de-emulsification using D.C. field and immobilized Nocardia amarae

As environmental compatible emulsifiers, various polysaccharides were investigated and de-emulsification methods were studied using filtration, direct current (D.C.) field, and also Nocardia amarae. 0.01% (v/v) of alginic acid, arabic gum, chitosan, and curdlan showed more than about 2 h of emulsion stability in the half-life of emulsion layer by a homogenizer and showed about twice synergic effects with other polysaccharides in emulsification activity at the ratio of 9:1 (v/v). De-emulsification by 110V D.C. field took about 1 h for the separation of oils from 1 l of oil-in-water emulsion in case of 0.01% arabic gum. Oil-water separator was designed using non-woven fabrics in filtration system and Nocardia amarae grown in n-hexadecane or kerosene was immobilized in the non-woven fabrics.     

Isonicotinic acid hydrazide induced changes and inhibition in mycolic acid synthesis in Nocardia and related taxa

The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 g/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C₄₄ or C₄₆) was inhibited more significantly than shorter homologues such as C₃₈ or C₄₀. In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 g/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of -alkyl chain, but to the inhibition of chain elongation of C₂₈ to C₃₂ straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di-mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 g/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C₄₄ or C₄₆ specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C₂₈ or C₃₀.