Nocardia Sp Research Articles

Abstract A171: Antitumor effects of intervenolin, a novel natural compound with anti-Helicobacter pylori activity, and its synthetic derivatives on gastric and colorectal cancer cells.

Abstract Tumor tissues are composed of not only tumor cells but also the surrounding stroma. While the stroma consists of various kinds of cells, among them fibroblasts-like stromal cells can regulate the growth and metastasis of tumor cells positively or negatively. Thus, the modulation of the tumor-stromal cell interactions can be a new antitumor strategy. We have been searching compounds that modulate the interactions using coculture system of tumor cells and stromal cells. In the course of our screening, we have isolated a novel compound, intervenolin, from the culture broth of Nocardia sp. ML96-86F2. Intervenolin inhibits the growth of human gastric and colorectal cancer cell lines when cocultured with the respective organ-derived stromal cells more strongly than that of the cancer cells cultured alone. To improve low yield of intervenolin from the bacterial culture and to create more potent compounds, we have synthesized intervenolin and its derivatives. Some of the derivatives also show the same effect as intervenolin in vitro. Furthermore, intervenolin and its derivatives show antitumor effects against xenograft models of human gastric cancer cells and also MDR-expressing colorectal cancer cells in vivo. Furthermore, intervenolin and its derivatives exert selective anti-Helicobacter pylori activity. We are now investigating the mechanism of its action and trying to synthesize more potent its derivatives. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A171. Citation Format: Manabu Kawada, Hiroyuki Inoue, Shun-ichi Ohba, Hikaru Abe, Masaki Hatano, Masahide Amemiya, Chigusa Hayashi, Ihomi Usami, Takumi Watanabe, Masayuki Igarashi, Tohru Masuda, Masakatsu Shibasaki, Akio Nomoto. Antitumor effects of intervenolin, a novel natural compound with anti-Helicobacter pylori activity, and its synthetic derivatives on gastric and colorectal cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A171.

Intervenolin, a new antitumor compound with anti-Helicobacter pylori activity, from Nocardia sp. ML96-86F2

Because stromal cells can regulate the growth and metastasis of tumor cells, a compound that modulates the interaction between the stromal cells and the tumor cells can control the tumor progression. In the course of our screening for such a compound, we have isolated a new compound, intervenolin, from the culture broth of Nocardia sp. ML96-86F2. Intervenolin inhibits the growth of human gastric and colorectal cancer cell lines in the coculture with the respective organ-derived stromal cells more strongly than that of the cancer cells cultured alone. Intervenolin shows antitumor effect against a xenograft model of human colorectal cancer cells in vivo. Furthermore, intervenolin exerts selective anti-Helicobacter pylori effect.

Actinomycetoma-recurrence after amputation

Actinomycetoma is a chronic granulomatous infection of the subcutaneous tissue caused by fungus-like bacteria, that is, Nocardia sp, Actinomyces sp. This infection is common in tropical countries like India, though the disease is worldwide in distribution. A 22-year-old female who presented with multiple fistulas, sinus tracts, and pain and swelling over left foot following trauma 6 years back. A diagnosis of actinomycetoma was made based on clinical and microbiological ground. Two years after amputation there was recurrence and finally took multiple antibiotic therapies but was left without cure. Another case was that of a 37- year-old male who presented with multiple fistulas, sinus tracts, and pain and swelling on the right sole 6 months after prick of bamboo stick. There was a recurrence after amputation. Both the patients were treated with a Wallace regimen (Amikacin with Cotrimoxazole/Minocycline/Ciprofloxacin). Patient showed excellent response with healing of all sinuses after 2 months of therapy.

Microbial stereospecific reduction of 3-quinuclidinone with newly isolated Nocardia sp. and Rhodococcus erythropolis

Abstract Two bacterium strains, Nocardia sp. WY1202 and Rhodococcus erythropolis WY1406, were isolated from soil samples. They catalyzed the asymmetric reduction of 3-quinuclidinone to give enantiomeric pure (R)- and (S)-3-quinuclidinol, respectively. The optimal temperatures for the bioreduction by Nocardia sp. and R. erythropolis were 30 °C and 37 °C, respectively, while both strains showed highest activity at pH 8.0. Without external addition of expensive NADH or NADPH, (R)-3-quinuclidinol and (S)-3-quinuclidinol were obtained with 93% and 92% isolated yield and >99% enantiomeric excess. As such, microbial reduction by Nocardia sp. WY1202 or R. erythropolis WY1406 offers a new stereospecific approach to both antipodes of 3-quinuclidinol of pharmaceutical importance.

Studies on Microbial Degradation of Natural Rubber Using Dilute Solution Viscosity Measurement and Weight Loss Techniques

Dilute solution viscosity, Microbial degradation, Natural rubber, Nocardia sp, Weight loss Reduction of molecular weight of rubber polymers fo r easy absorption of compounding ingredients is critical in every rubber compounding operation. Yet, rubber mastication which is the current practice of achieving this is expensive, requiring high energy and equipment cost. To addres s this problem, microbial degradation of natural rubber (NR) and waste rubber tire (WRT) by Nocardia sp. strain 385A was studied using dilute solution visco sity measurement and weight loss methods. Solutions of NR and WRT in toluene we re inoculated with the microbes (Nocardia sp. strain 385A) and kept in inc ubator. Incubation period varied between 0 to 10 weeks. The results obtained show that NR and WRT were mineralized and degraded by the microbes. Intrinsic viscosity values of both NR and WRT decreased with increasing period of incubat ion, indicating that degradation increases with increase in the incubati on period. For the incubation periods investigated, WRT produced higher intrinsic viscosity values than NR due to the inhibitory effect of additives present in WR T to microbial degradation. Equivalent reduction in molecular weight obtained b y rubber mastication technique was achieved by microbial degradation aft er 10 weeks incubation period. Rubber degrading bacteria can be useful for the disposal of discarded rubber products

Effect of Different Biosynthetic Precursors on the Production of Nargenicin A1 from Metabolically Engineered Nocardia sp. CS682

Nargenicin A1 is a 28-membered polyketide macrolide, with antibacterial activity against methicillin-resistant Staphylococcus aureus, produced by Nocardia sp. CS682. In this study, the production of nargenicin A1 was improved by enhancing the supply of different biosynthetic precursors. In Nocardia sp. CS682 (KCTC11297BP), this improvement was ~4.62-fold with the supplementation of 30 mM methyl oleate, 4.25-fold with supplementation of 15 mM sodium propionate, and 2.81-fold with supplementation of 15 mM sodium acetate. In Nocardia sp. metK18 and Nocardia sp. CS682 expressing S-adenosylmethionine synthetase (MetK), the production of nargenicin A1 was improved by ~5.57-fold by supplementation with 30 mM methyl oleate, 5.01-fold by supplementation with 15 mM sodium propionate, and 3.64-fold by supplementation with 15 mM sodium acetate. Furthermore, supplementing the culture broth of Nocardia sp. ACC18 and Nocardia sp. CS682 expressing the acetyl-CoA carboxylase complex (AccA2 and AccBE) with 30 mM methyl oleate, 15 mM sodium propionate, or 15 mM sodium acetate resulted in ~6.99-, 6.46-, and 5.58-fold increases, respectively, in nargenicin A1 production. Our overall results showed that among the supplements, methyl oleate was the most effective precursor supporting the highest titers of nargenicin A1 in Nocardia sp. CS682, Nocardia sp. metK18, and Nocardia sp. ACC18.

Skin infection and progressive paraparesis

A 35-year-old female agricultural worker presented with a 4 month history of productive cough, fever, and left subcostal pain, followed by progressive paraparesis and urinary incontinence. 10 years previously, she had been treated for a skin infection on her back at another centre for 3 years, without further follow-up. Examination showed more than one irregular scars, hyperpigmented papules, and few skin sinuses with scanty purulent discharge, without erythema, on the middle and left side of the upper back. The patient was afebrile and did not have palpable cervical or supraclavicular lymphadenopathy. We heard no rales, rhonchi, or substantial wheezing. The patient had fl accid paraplegia with complete sensory loss at T4 level and urinary sphincter incontinence. Laboratory tests showed a leucocyte count of 9·1 cells per μL, a normal liver enzyme profi le, a C-reactive protein concentration of 59·1 mg/dL (normal range 0–0·5 mg/dL), and no evidence of venereal disease or HIV. Protein concentration in her cerebrospinal fl uid was 800 mg/dL, glucose was 32 mg/dL, and no cells were present. Cerebrospinal fl uid stains, bacterial culture, and PCR for Mycobacterium tuberculosis were negative. Chest radiography showed a paravertebral mass with a fragmentary pattern of osseous patchy destruction and periosteal reaction (fi gure, A). Spiral CT showed a left paravertebral extensive osteolytic lesion involving vertebral bodies and costovertebral unions (fi gure, B). T2-weighted MRI (fi gure, C–E) showed widespread hyperintense lesions that infi ltrated vertebral bodies without involvement of intersomatic spaces (arrow), pleural thickening, and lung consolidation of the left lower lobe (asterisks). Gram stain and modifi ed acid-fast stain (Kinyoun) of sinus discharge showed variably Gram-positive and faintly acid-fast positive, slender, non-branching fi lamentous bacilli, consistent with a diagnosis of Nocardia sp. Intravenous amikacin, co-trimoxazole, and an orthopaedic corset were indicated. Surgery for debridement was off ered, but the patient did not choose to have the procedure. The patient attended our neurological care centre 2 months after discharge and showed a discrete amelioration of neurological symptoms, but did not return for further follow-up. Initially, diff erential diagnosis included extramedullary neoplasms and granulomatous infections (aspergillosis, histoplasmosis, tuberculosis, chromomycosis, blastomycosis, coccidiodomycosis, botryomycosis, sporotrichosis, frambesia, and syphilis). Gadolinium MRI is especially useful because it could help the practitioner to diff erentiate between vertebral intraosseous abscesses, meningeal involvement, subligamentous spread, and paraspinal abscess.

Degradation of long-chain n-alkanes in soil microcosms by two actinobacteria

The ability of two recently isolated actinobacteria, that degrade medium and long chain n-alkanes in laboratory water medium, was investigated in soil microcosms using different standard soils that were artificially contaminated with n-alkanes of different length (C12- C20- C24- C30). The two strains, identified as Nocardia sp. SoB and Gordonia sp. SoCp, revealed a similar high HC degradation efficiency with an average of 75% alkane degraded after 28 days incubation. A selectivity of bacteria towards n-alkanes of different length was detected as well as a consistent effect of soil texture and other soil physical chemical characteristics on degradation. It was demonstrated the specific aptitude of these selected strains towards specific environmental conditions.

Metabolic Engineering of Nocardia sp. CS682 for Enhanced Production of Nargenicin A1

A number of secondary metabolites having therapeutic importance have been reported from the genus Nocardia. One of the polyketide antibiotic compounds isolated from Nocardia is nargenicin A(1). Recently, nargenicin A(1) has been isolated from Nocardia sp. CS682, a new Nocardia strain isolated from soil in Jeonnam, South Korea. It possesses strong antibacterial activity against methicillin-resistant Staphylococcus aureus. In this study, we applied a metabolic engineering approach based on recombinant DNA technology in order to boost the production of nargenicin A(1) from Nocardia sp. CS682. Initially, we optimized the transformation of this new strain by electroporation method. Heterologous expression of S-adenosylmethionine synthetase (MetK1-sp) in Nocardia sp. CS682 enhanced the production of nargenicin A(1) by about 2.8 times due to transcriptional activation of biosynthetic genes as revealed by reverse transcription polymerase chain reaction analysis. Similarly, expression of acetyl-CoA carboxylase genes improved nargenicin A(1) production by about 3.8 times in Nocardia sp. ACC18 compared to that in Nocardia sp. CS682 and Nocardia sp. NV18 by increasing precursor pool. Thus, enhanced production of nargenicin A(1) from Nocardia sp. CS682 can be achieved by expression of transcriptional activator genes and precursor genes from Streptomyces strains.

Enhanced phytase production from Nocardia sp. MB 36 using agro‐residues as substrates: Potential application for animal feed production

AbstractPhytases have gathered considerable attention in recent years due to their applications in human and animal nutrition, environmental protection and biotechnology. Phytases were isolated and characterized from a variety of bacteria and fungi but rarely from actinomycetes. In the present study, a Nocardia sp. MB 36, isolated from rice mill waste, was found to be a producer of a thermostable and acid/alkalistable phytase. Optimization of phytase production by Plackett–Burman designed experiments indicated that wheat bran, medium pH, FeSO4 and CaCl2 are the most effective fermentation variables that influence the phytase yield under submerged fermentation. Further optimization of these four variables by using Closed‐Composite‐Design of response surface methodology resulted in enhancement of phytase production by 64.14%. This newly isolated phytase from Nocardia sp. has potentially desirable features for animal feed applications.

Biosynthesis of the nargenicin A1 pyrrole moiety from Nocardia sp. CS682

A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from L-proline. Nargenicin A(1), a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A(1) in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A(1) was also derived from L-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A(1) is initiated by NgnN4 that catalyzes ATP-dependent activation of L-proline into L-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert L-proline into pyrrole-2-carboxylic acid moiety.

방선균(Nocardia sp. CS682) 발효물의 급여가 산란계의 생산성, 혈액성상, 면역글로불린 및 소장내 미생물 함량에 미치는 영향

본 연구는 방선균 목 노카르디아 종 CS682 균주의 발효물 CS682와 이를 바탕으로 한 제품 DSC682<TEX>$^{(R)}$</TEX>를 산란계에게 급여 시 산란 생산성, 난품질, 혈액 성상, 소장 내 미생물, 면역성에 미치는 효과를 알아보기 위해 실시하였다. 실험은 Hy-Line Brown<TEX>$^{(R)}$</TEX> 갈색 산란계로 실시하였는데, 실험 1은 86주령, 실험 2는 26주령의 실험계를 2단 4열 2수 케이지에 수용하여 실시하였다. 실험 1은 대조구, 항생제구(avilamycin 6 ppm), CS 682-0.1(CS682 0.1%), CS682-1.0(CS682 1.0% 첨가) 등 4 처리였으며, 5반복, 반복당 24수(12케이지), 총 480수로 실시하였고, 실험2는대조구, 항생제구(avilamycin 6 ppm), DCS682-0.05(DCS682<TEX>$^{(R)}$</TEX> 0.05%), DSC682-0.1(DCS682<TEX>$^{(R)}$</TEX> 0.1%), 그리고 DCS682-0.2(DCS682<TEX>$^{(R)}$</TEX> 0.2% 첨가) 등 5처리였으며 5반복, 반복당 40수(20케이지), 총 1,000수로 실시하였다. 실험 1에서는 산란율, 난중, 연 파란율, 사료 섭취량, 사료 전환율 등 산란 생산성에는 유의한 차이가 없었다. 난품질에서 난각 강도는 항생제구와 CSC682 처리구들이 유의적(p<0.05)으로 높았으며, 난황색은 CSC682-1.0 처리구가 유의적(p<0.05)으로 높았다. 실험 2에서는 산란 생산성에서 사료 섭취량이 DCS682-0.05구가 대조구와 비교하여 유의적(p<0.05)으로 낮았다. 난각색에서 Hunter Lab color L(Lightness) 즉 밝기가 항생제구와 DCS682 처리구들이 유의적(p<0.05)으로 낮았다. 난황색은 DCS682-0.1~0.2구들이 유의적(p<0.05)으로 높았으며, Haugh unit은 항생제구와 DCS682-0.1구가 유의적(p<0.05)으로 높았다. 실험 1과 2에서 혈장 면역글로부린(IgG, IgA) 및 난황 면역그로부린(IgY)의 함량은 처리구간에 유의한 차이가 없었다. 실험1에서 erythrocytes 중 평균 적혈구 용적(MCV)은 대조구가 유의적(p<0.05)으로 높았으며, 평균 적혈구 혈색소량(MCH)은 항생제구가 유의적으로 높았다. 실험 2에서는 leukocytes 중 단핵구(MO)에서 대조구와 DCS682-0.05구가 유의적으로(p<0.05 또는 0.01) 높았다. 실험 1에서 C. perfringens수는 항생제구에서 대조구와 비교하여 유의적으로(p<0.05) 감소하였다. S. typhimurium수는 유의적으로(p<0.05) 차이가 있었는데 항생제구가 가장 낮았고 다음으로 CS682 처리구들이었으며 대조구에서 가장 높았다. Universal bacteria, Lactobacillus 그리고 E. coli의 수는 처리간에 유의적인 차이가 없었다. 실험 2에서는 Lactobacillus 수가 DCS682-0.05와 DCS682-0.1구들에서 유의적으로(p<0.05) 높았고 다음으로 대조구와 DCS682-0.2 첨가구였으며 항생제구가 가장 낮았다. C. perfringens 수는 항생제구에서 대조구와 비교하여 유의적(p<0.05)으로 감소하였다. S. Typhimurium은 처리간에 유의적인(p<0.05) 차이가 있었는데 항생제구가 가장 낮았고 다음으로 DCS682-0.2이었으며, DCS682-0.05와 0.1구들은 대조구보다는 낮았으나 유의한(p<0.05) 차이는 없었다. 결론적으로 CS682 0.1% 또는 DCS682 0.1~0.2% 첨가는 난각 강도, 난황색, 난각색, Haugh unit를 개선시키고, 소장 내유해 미생물을 억제하는 긍정적인 효과를 보였다. This study was conducted to investigate the effects of dietary supplementation of CS682, a fermentation product of Actinomycetae(Nocardia sp. CS682), and its commercial product DSC682<TEX>$^{(R)}$</TEX> on the performance, blood parameters, intestinal microflora, and immune response in laying hens. Hy-Line Brown<TEX>$^{(R)}$</TEX> laying hens were housed in two bird cages. Feeding trial lasted 5 wk under 16.5 h:7.5 h(L:D) lighting regimen. In Exp.1, a total of 480 birds of 86 wk old were assigned to four dietary treatments: Control, Antibiotics (6 ppm avilamycin), CS682-0.1 (CS682 0.1%) and CS682-1.0 (CS682 1.0% supplementation). Each treatment was replicated five times with 24 birds (or 12 cages) per replication. In Exp. 2, a total of 1,000 birds of 26 wk old were assigned to five dietary treatments: Control, Antibiotics (6 ppm avilamycin), DCS682-0.05 (DCS682 0.05%), DCS682-0.1 (DCS682 0.1%), DCS682-0.2 (DCS682 0.2% supplementation). Each treatment was replicated five times with 40 birds (or 20 cages) per replication. In Exp. 1, there were no significant differences among treatments in egg production, egg weight, broken & soft egg production, feed intake, and feed conversion ratio. Also, there were no significant differences among treatments in eggshell thickness, eggshell color and Haugh unit. However, eggshell strength was significantly (p<0.05) greater in CS682 and Antibiotics treatments than Control, and egg yolk color was significantly (p<0.05) higher in CS682-1.0 than Control. In Exp. 2, feed intake was significantly (p<0.05) lower in DSC682-0.05 than Control. Lightness(L) of Hunter Lab color of eggshell of DCS and Antibiotics treatments was significantly (p<0.05) lower than Control. Egg yolk color of DCS 0.1 and 0.2 treatments was significantly (p<0.05) higher than Control. Haugh unit increased significantly (p<0.05) in Antibiotics and DCS682-0.1 treatments. The immunoglobulin levels of plasma (IgG and IgA) and eggyolk (IgY) were not significantly affected by treatments. Antibiotics and CS682 or DCS682 treatments significantly (p<0.05 or 0.01) influenced some of the erythrocytes and leukocytes parameters in blood. In Exp.1, mean corpuscular volume (MCV) decreased by CS682 treatments and mean corpuscular hemoglobin (MCH) was highest in Antibiotics treatments. In Exp.2, the level of monocyte (MO) decreased in DCS682-0.10 and 0.20 treatments. The cfu of C. perfringens and S. typhimurium in small intestinal content were highest in Control and lowest in Antibiotics in both experiments. In Exp. 2, DSC682-0.05 and -0.1 treatments were highest and Antibiotic treatment was lowest in Lactobacilli spp. The results of the present layer experiments indicated that supplementation of 0.1~0.2% CS682 or DCS682 may increase eggshell strength, color of eggshell and eggyolk, Haugh unit, and control harmful intestinal microbes.

방선균(Nocardia sp. CS682) 발효물의 급여가 육계의 생산성, 혈액성상, 면역글로불린 및 소장 내 미생물 함량에 미치는 영향

This study was conducted to investigate the effects of dietary supplementation of CS682, a fermentation product of Actinomycetae Nocardia sp. CS682, and DCS682 , a commercial product, on the performance, blood parameters, small intestinal microflora, and immunoglobulin contents in broilers. In Exp. 1, a total of 240 ROSS broiler chickens of 1d old were assigned to six dietary treatments: Control, Antibiotics (6 ppm avilamycin), CS682-0.25 (CS682 0.25%), CS682-0.50, CS682-0.75 and CS682-1.00. There were significant (p<0.05) differences among treatments in feed conversion. The CS682-0.25 treatment was sig- nificantly (p<0.05) lower than Antibiotics and other CS682 treatments in 0 2 wk feed conversion. The CS682 treatments influenced MCV (mean corpuscular volume) in blood. The cfu of Escherichia coli in small intestinal content was lowest in Antibiotics treatment followed by CS682 treatments and Control. In Exp. 2, a total of 1,000 ROSS broiler chickens of 1 d old were assigned to five dietary treatments: Control, Antibiotics (6 ppm avilamycin), DCS682-0.05 (DCS682 0.05%), DCS682-0.10 and DCS682- 0.20. There were significant differences (p<0.05) among treatments in mortality. The DCS682-0.20 treatment was lower than DCS682-0.10 in 0 3 wk and lower than Control in 0 5 wk mortality. Antibiotics treatment was lowest in all microbial population in small intestinal content. The cfu of E. coli and Salmonella typhimurium of DCS682 treatments were higher than Antibiotics treatment but lower than the Control. The results of present broiler experiments indicated that supplementation of 0.20 0.25% CS682 and DCS682, improve feed conversion, mortality and control harmful intestinal microbes.

Horizontal gene transfer (HGT) as a mechanism of disseminating RDX-degrading activity among Actinomycete bacteria

Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a cyclic nitramine explosive that is a major component in many high-explosive formulations and has been found as a contaminant of soil and groundwater. The RDX-degrading gene locus xplAB, located on pGKT2 in Gordonia sp. KTR9, is highly conserved among isolates from disparate geographical locations suggesting a horizontal gene transfer (HGT) event. It was our goal to determine whether Gordonia sp. KTR9 is capable of transferring pGKT2 and the associated RDX degradation ability to other bacteria. We demonstrate the successful conjugal transfer of pGKT2 from Gordonia sp. KTR9 to Gordonia polyisoprenivorans, Rhodococcus jostii RHA1 and Nocardia sp. TW2. Through growth and RDX degradation studies, it was demonstrated that pGKT2 conferred to transconjugants the ability to degrade and utilize RDX as a nitrogen source. The inhibitory effect of exogenous inorganic nitrogen sources on RDX degradation in transconjugant strains was found to be strain specific. Plasmid pGKT2 can be transferred by conjugation, along with the ability to degrade RDX, to related bacteria, providing evidence of at least one mechanism for the dissemination and persistence of xplAB in the environment. These results provide evidence of one mechanism for the environmental dissemination of xplAB and provide a framework for future field relevant bioremediation practices.

Genome shuffling of marine derived bacterium Nocardia sp. ALAA 2000 for improved ayamycin production

Genome shuffling is a recent development in microbiology. The advantage of this technique is that genetic changes can be made in a microorganism without knowing its genetic background. Genome shuffling was applied to the marine derived bacterium Nocardia sp. ALAA 2000 to achieve rapid improvement of ayamycin production. The initial mutant population was generated by treatment with ethyl methane sulfonate (EMS) combined with UV irradiation of the spores, resulting in an improved population (AL/11, AL/136, AL/213 and AL/277) producing tenfold (150μg/ml) more ayamycin than the original strain. These mutants were used as the starting strains for three rounds of genome shuffling and after each round improved strains were screened and selected based on their ayamycin productivity. The population after three rounds of genome shuffling exhibited an improved ayamycin yield. Strain F3/22 yielded 285μg/ml of ayamycin, which was 19-fold higher than that of the initial strain and 1.9-fold higher than the mutants used as the starting point for genome shuffling. We evaluated the genetic effect of UV+EMS-mutagenesis and three rounds of genome shuffling on the nucleotide sequence by random amplified polymorphic DNA (RAPD) analysis. Many differences were noticed in mutant and recombinant strains compared to the wild type strain. These differences in RAPD profiles confirmed the presence of genetic variations in the Nocardia genome after mutagenesis and genome shuffling.

Initial step in the catabolism of cholesterol by Mycobacterium smegmatis mc2155

The first step in the catabolism of cholesterol, i.e. the transformation of cholesterol into cholestenone, has been investigated in Mycobacterium smegmatis. In silico analysis identified the MSMEG_1604 gene encoding a putative protein similar to the ChoD cholesterol oxidase of M. tuberculosis H37Rv (Rv3409c) and the MSMEG_5228 gene coding for a protein similar to the NAD(P)-dependent cholesterol dehydrogenase/isomerase of Nocardia sp. The expression of the MSMEG_5228 gene was inducible by cholesterol whereas the expression of MSMEG_1604 gene was constitutive. When both genes were expressed in Escherichia coli only the MSMEG_5228 protein was active on cholesterol. The function of ChoD-like MSMEG_1604 protein remains to be elucidated, but it does not appear to play a critical role in the mineralization of cholesterol as a MSMEG_1604(-) mutant was not affected in the production of cholestenone. However, a MSMEG_5228(-) mutant showed a drastic reduction in the synthesis of cholestenone. The finding that this mutant was still able to grow in cholesterol, allowed us to demonstrate that the cholesterol-inducible MSMEG_5233 gene encodes an additional cholesterol dehydrogenase/isomerase similar to the AcmA dehydrogenase of Sterolibacterium denitrificans. The observation that the double MSMEG_5228-5233(-) mutant was able to grow in cholesterol suggests that in addition to these enzymes other dehydrogenase/isomerases can also catalyse the first reaction of the cholesterol degradation pathway in M. smegmatis, which is not the limiting step of the process.

Actinomicetos produtores de inibidores de β-lactamases com atividade antimicrobiana frente a isolados de mastite bovina

Avaliou-se a capacidade de 71 actinomicetos isolados de líquens da região amazônica em produzir inibidores de β-lactamases com atividade antimicrobiana sobre Staphylococcus aureus, resistentes à penicilina, isolados de mastite bovina do estado de Pernambuco. A seleção dos actinomicetos produtores de inibidores de β-lactamases foi realizada pela técnica de bloco de gelose contra Klebsiella pneumoniae ATCC 29665, e os actinomicetos selecionados foram testados frente a 17 linhagens de Staphylococcus aureus resistentes à penicilina. Os melhores produtores de inibidores de β-lactamases foram Streptomyces sp. DPUA 1542 e Nocardia sp. DPUA 1571, os quais foram submetidos ao cultivo submerso para determinação da curva de crescimento, pH e atividade antimicrobiana. Os maiores halos de inibição foram obtidos pelos metabólitos produzidos após 96 horas de cultivo tanto para Nocardia sp. - 13,5 e 12,0mm - como para Streptomyces sp. - 8,0 e 14,0mm - com os testes de difusão nos discos e poços, respectivamente. Os resultados permitiram concluir que os actinomicetos são fonte promissora de inibidores de β-lactamases, com potencial uso no tratamento de mastites bovinas

Impact of soil amendment on phytotoxicity of a 5-month old waste engine oil polluted soil

The present study investigated the effects of soil amendment on the remediation of waste engine oil (WEO) polluted soil, as well as the eventual phytotoxic effects of remediated amended soil on some growth parameters of cowpea. There was significant decrease in heavy metal concentration of the polluted soil more than average, nine months after amendment. There was also total remediation of some PAH compounds (benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benezo(g,h,i)perylene, benzo(k)fluoranthene, chrysene, dibenzo(a,h)anthracene, fluoranthene, fluorene and Indeno(1,2,3-c,d)pyrene). Achromobacter sp, Clostridium sp,Sarcina sp and Micrococcus sp were prevalent bacteria species found in the polluted soils, while prevalent fungi species included Aspergillus niger, Penicillium sp,Geotrichum sp and Trichoderma sp. The actinomycete Nocardia sp was prevalent as well. Ecological risk factor initially posed by the presence of heavy metals in the unamended soil was significantly reduced to safe levels. Phyto assessment of the polluted soil was carried out just before soil was amended with saw dust and results showed that virtually all the cowpea seedlings died within 2 weeks; only those seedlings in unpolluted soils survived. Nine months after soil was amended, all cowpea plants survived up to fruiting. The present study also showed that cowpea was able to bioaccumulate heavy metals into harvestable parts, though bioaccumulation quotients calculated showed that these accumulations were not significant. Key words: Penicillium sp, Geotrichum sp, phytotoxic, fluoranthene, fluorine

ChemInform Abstract: Nothramicin, a New Anthracycline Antibiotic from Nocardia sp. MJ896-43F17.

A new antibiotic designated nothramicin was isolated from the culture broth of Nocardia sp. MJ896-43F17 which was closely related to Nocardia brasiliensis. It was isolated by the Diaion HP-20 column chromatography, butyl acetate extraction and purified by HPLC. It inhibited the growth of mycobacteria at the concentration of 1.56∼25 μg/ml. Nothramicin was revealed to be a new member of anthracycline antibiotics by the various spectroscopies.

Biosynthesis of ursolic acid derivatives by microbial metabolism of ursolic acid with Nocardia sp. strains—Proposal of new biosynthetic pathways

Our studies of the microbial-metabolism of triterpenoid ursolic acid by various Nocardia sp. strains, have led to the proposal of two novel pathways to produce triterpenoid derivatives. Nocardia sp. NRRL 5646, Nocardia sp. 44822 and Nocardia sp. 44000 generated the following ursolic acid derivatives: ursolic acid methyl ester, ursonic acid, ursonic acid methyl ester, 3-oxoursa-1,12-dien-28-oic acid and 3-oxoursa-1,12-dien-28-oic acid methyl ester. Nocardia sp. 45077 synthesized ursonic acid and 3-oxoursa-1,12-dien-28-oic acid while Nocardia sp. 46002 produced only ursonic acid and Nocardia sp. 43069 showed no metabolism at all. The conversion of ursolic acid by Nocardia sp. NRRL 5646 was independent of the medium used for the fermentation. An increase in temperature from 28 °C to 36 °C doubled the reaction rate of the biotransformation. The analysis of ursane metabolites was done by HPLC, while their structures were established using HPLC–APCI pos-MS/MS and HPLC–NMR spectroscopy. The pseudo molecular ion peaks were determined by HPLC–APCI pos-MS and used to measure their molecular weight. The product ion spectra of the metabolites showed the characteristic fragments of Δ 12-oleanes and Δ 12-ursanes indicating that a substitution in ring A or B was responsible for the decrease in molecular weight. Based on these results, two new biosynthetic pathways are proposed. These new pathways can presumably be used as strategic routes for the biotechnological production of triterpenoid derivatives. It is assumed that a 3β-hydroxysteroid dehydrogenase and a 3-ketosteroid-Δ 1-dehydrogenase are involved in the transformation of the steroid.