Site-specific integration and excision of pMEA100 in Nocardia mediterranei

Summary Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al. 1985). The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA. After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus. The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing. The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N. mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100. Only one such sequence was found in the chromosome of pMEA100-free N. mediterranei derivatives as suggested by the single integration locus.     

A complex role of <Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis> GlnR in nitrogen metabolism and related antibiotics production

Amycolatopsis, genus of a rare actinomycete, produces many clinically important antibiotics, such as rifamycin and vancomycin. Although GlnR of Amycolatopsis mediterranei is a direct activator of the glnA gene expression, the production of GlnR does not linearly correlate with the expression of glnA under different nitrogen conditions. Moreover, A. mediterranei GlnR apparently inhibits rifamycin biosynthesis in the absence of nitrate but is indispensable for the nitrate-stimulating effect for its production, which leads to the hyper-production of rifamycin. When glnR of A. mediterranei was introduced into its phylogenetically related organism, Streptomyces coelicolor, we found that GlnR widely participated in the host strain’s secondary metabolism, resemblance to the phenotypes of a unique S. coelicolor glnR mutant, FS2. In contrast, absence or increment in copy number of the native S. coelicolor glnR did not result in a detectable pleiotrophic effect. We thus suggest that GlnR is a global regulator with a dual functional impact upon nitrogen metabolism and related antibiotics production.     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

Nocardia gamkensis sp. nov.

A novel actinomycete, strain CZH20^T, was isolated from a soil sample taken from the banks of the Gamka River in the Swartberg Nature Reserve, Western Cape Province, South Africa. Strain CZH20^T was identified as a member of the genus Nocardia by a polyphasic approach. Strain CZH20^T could be differentiated from other members of the genus Nocardia on the basis of physiology and 16S-rRNA gene sequence analysis. It exhibited weak antibiosis against Mycobacterium aurum A+. Organic solvent extracts of the culture filtrate and mycelial mass of CZH20^T exhibited moderate antibiotic activity against Mycobacterium smegmatis LR222 and Mycobacterium tuberculosis H37Rv. The name Nocardia gamkensis is proposed, with the type strain CZH20^T (=DSM 44956^T =NRRL B-24450^T).The GenBank accession number for the 16S-rRNA-gene sequence of Nocardia gamkensis CZH20^T is DQ235272.     

Purification and properties of an acetyl specific carboxylesterase from Nocardia mediterranei

Summary An acetyl specific carboxylesterase has been purified from Nocardia mediterranei. The purified enzyme is homogeneous as shown by SDS polyacrylamide gel electrophoresis. The esterase has a molecular weight of 68,000 and is composed of two identical subunits. The enzyme exhibits optimal activity at pH 7.5 and at 35°C and is stable below 40°C. The enzyme activity is inhibited by several sulfhydryl reagents. The esterase hydrolyzes preferentially acetyl esters. Propionyl esters are cleaved very slowly whereas butyryl esters are no substrates at all. In addition, the esterase shows a pronounced regiospecificity. On the other hand the enantiospecificity is rather low as demonstrated by the hydrolysis of prochiral and racemic substrates.     

Phospholipid and acid composition of Nocardia and nocardoid bacteria as criteria of classification

Phospholipid and acid composition of 5 strains of ‘true’ Nocardia and 4 strains of nocardoid bacteria have been studied. A great homogeneity was found in all the Nocardia species: phospholipids consist of cardiolipin, phosphatidyl ethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Streptomyces (Nocardia) mediterranei did not contain phosphatidylinositol and Oerskovia (Nocardia) turbata had no phosphatidyl ethanolamine. The fatty acid composition of these phospholipids was determined and was found different in Nocardia and nocardoid species. Nocardia were rich in straight chain fatty acids and tuberculostearic acid while the phospholipids of nocardoid bacteria contained greater amounts of branched fatty acids. The fatty acids from acetone soluble lipids consisted of hydroxy and non-hydroxy compounds. Hydroxy acids were found in Nocardia which contained nocardic acids: high MW β-hydroxy α-branched acids and in S. mediterranei which contained β-hydroxy acids with 15–17 carbon atoms. Non-hydroxy acids were essentially palmitic and tuberculostearic acids in Nocardia species while S. mediterranei and O. turbata contained great amounts of iso acids from C₁₄ to C₁₇. Phospholipid and acid composition are discussed as criteria of taxonomic classification of Nocardia and related Actinomycetes.     

Nocardia hydrocarbonoxydans n. spec., ein oligocarbophiler Actinomycet

Zusammenfassung Eine neue Actinomycetenart (Stamm 70), aufgetreten als Luftverunreinigung einer Kieselgelplatte, wird beschrieben.Auf Grund der Fähigkeit, mit aliphatischen Kohlenwasserstoffen aus der Gasphase zu leben, soll die neue Art Nocardia hydrocarbonoxydans genannt werden. Sie ist von anderen, bereits bekannten oligocarbophilen Actinomycetenarten durch ihre Kolonieform, durch die Fähigkeit Hexan, Heptan und Octan zu oxydieren, und durch ihr Unvermögen, auf Glycerin-Kartoffelkeilen, Kartoffelkeilen oder Blut-Agar zu wachsen, wohl unterschieden. Ihre Stellung im System und die Verwandtschaft mit anderen Arten der Gattung Nocardia wird diskutiert.

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Summary A new actinomycete species, and aerial contamination on a silica gel plate, is described. Because of its ability to utilize gaseous aliphatic hydrocarbons (C₆–C₁₄) for growth, Nocardia hydrocarbonoxydans is proposed as the name of the new species. It differs from other oligocarbophilic actinomycetes in its colony form, its ability to oxidize hexane, heptane and octane, and its inability of growing on glycerol potatoe slants, potatoe slants or blood agar. Taxonomical relationships to species of the genus Nocardia are discussed.

     

Denitrifying haloarchaea within the genus Haloferax display divergent respiratory phenotypes,with implications for their release of nitrogenous gases

Haloarchaea are extremophiles, generally thriving at high temperatures and salt concentrations, thus, with limited access to oxygen. As a strategy to maintain a respiratory metabolism, many halophilic archaea are capable of denitrification. Among them are members of the genus Haloferax, which are abundant in saline/hypersaline environments. Three reported haloarchaeal denitrifiers, Haloferax mediterranei, Haloferax denitrificans and Haloferax volcanii, were characterized with respect to their denitrification phenotype. A semi-automatic incubation system was used to monitor the depletion of electron acceptors and accumulation of gaseous intermediates in batch cultures under a range of conditions. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N₂, while the other two released significant amounts of NO and N₂O, which affect tropospheric and stratospheric chemistries respectively. The prevalence and magnitude of hypersaline ecosystems are on the rise due to climate change and anthropogenic activity. Thus, the biology of halophilic denitrifiers is inherently interesting, due to their contribution to the global nitrogen cycle, and potential application in bioremediation. This work is the first detailed physiological study of denitrification in haloarchaea, and as such a seed for our understanding of the drivers of nitrogen turnover in hypersaline systems.     

Identification and Molecular Characterization of Nocardia sp. as a New Causal Agent of Tobacco False Broomrape

Tobacco false broomrape disease is a serious problem in tropical countries. To identify its cause, experiments were conducted in tobacco fields. Six actinomycete strains were isolated from white succulent outgrowths of tobacco roots and their pathogenicity was confirmed by biological testing. Based on phenotypic and 16S rRNA gene sequence BLAST analysis, the strains were identified as members of the genus Nocardia. This association was also confirmed by secA1 gene phylogenetic analysis. This is the first report of Nocardia sp. as the cause of tobacco false broomrape.     

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.     

Nocardia rhamnosiphila sp. nov., isolated from soil

In this study two actinomycete strains were isolated in Cape Town (South Africa), one from a compost heap (strain 202GMO^T) and the other from within the fynbos-rich area surrounded by the horseracing track at Kenilworth Racecourse (strain C2). Based on 16S rRNA gene sequence BLAST analysis, the strains were identified as members of the genus Nocardia. Phylogenetic analysis showed that the strains clustered together and are most closely related to Nocardia flavorosea NRRL B-16176^T, Nocardia testacea JCM 12235^T, Nocardia sienata IFM 10088^T and Nocardia carnea DSM 43397^T. This association was also supported by gyrB based phylogenetic analysis. The results of DNA–DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of both strains 202GMO^T and C2 from related species. However, their high DNA relatedness showed that they belong to the same species. Strain 202GMO^T was selected as the type strain to represent this novel species, for which the name Nocardia rhamnosiphila is proposed (=DSM 45147^T = NRRL B-24637^T).     

Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.     

Enzymatic characterization of Nocardia spp. and related bacteria by API ZYM profile

Characterization of 62 isolates belonging to the genus Nocardia and related bacteria was obtained by using the API ZYM system. The difference in enzymatic profile should allow a relatively efficient, low-cost means to identify aerobic actinomycetes of clinical significance.     

地中海富盐菌中非编码RNA的鉴定与分析

【目的】通过转录组高通量测序技术(即RNA-seq),结合生物信息学分析和分子生物学方法,在组学水平鉴定极端嗜盐菌中可能的非编码RNA(nc RNA)。【方法】将培养至对数中期的地中海富盐菌在不同盐浓度下处理30分钟,提取RNA,进行链特异的转录组测序和5′端区分的转录组测序,通过生物信息学分析在全基因组范围内鉴定nc RNA,预测其转录边界;然后通过Northern blot和环化RNA反转录聚合酶链式反应(CR-RT-PCR)对部分预测的nc RNA进行实验验证。【结果】比较两种RNA-seq技术在不同培养条件下的RNA测序结果和对转录单元的精细分析,共鉴定到105个高可信度的nc RNA,并发现4个在不同盐度下表达差异较大的nc RNA,通过Northern blot和CR-RT-PCR验证了inc RNA1436和inc RNA1903的表达情况、转录本、转录起始位点及终止位点等。【结论】首次在组学水平鉴定了地中海富盐菌中的nc RNA,不同盐浓度刺激下部分nc RNA的转录差异暗示其有可能参与地中海富盐菌对盐胁迫的适应,高可信度nc RNA的组学发现为今后全面开展嗜盐古菌nc RNA的功能机制研究提供了基础数据及重要的切入点。     

Glucose transport into the extremely halophilic archaebacteria

Penetration of glucose into cells of several extremely halophilic archaebacteria of the Halobacterium and Haloferax genera (Halobacterium saccharovorum and Halobacterium salinarium, Haloferax volcanii and Haloferax mediterranei) has been studied. Some characteristics of transport systems of carbohydrate-utilizing halobacteria Halobacterium saccharovorum, Haloferax mediterranei and Haloferax volcanii (pH and temperature optima, stereospecificity, kinetic parameters) have been determined. Inability of H. salinarium cells for active glucose transport has been shown. The dependence of glucose transport on the Na⁺ ions gradient (on the whole cells and membrane vesicles) has been demonstrated. Cells or membrane vesicles of carbohydrate-utilizing halobacteria grown in media containing this sugar indicated the activation of glucose transport, whereas cells grown in media without sugars did not. This fact has allowed us to conclude that corresponding transport systems are inducible.     

Rifamycin: XXXIII. Isolation of Actinophages Active on Streptomyces mediterranei and Characteristics of Phage-Resistant Strains

Five actinophages highly specific for Streptomyces mediterranei were isolated from lysed broth cultures. Studies were performed on the effect of plating conditions on plaque formation. The development of phage-resistant strains of S. mediterranei not only eliminated the phage but also significantly increased rifamycin yields. The phage-resistant cultures proved to be more unstable than the original sensitive strain. Maintenance of the cultures as frozen vegetative mycelium assured culture stability and reproducibility of the results. Strict aseptic precautions throughout the laboratories and fermentation areas did not eliminate the danger of phage infection; effective control was obtained only with the introduction of resistant strains. S. mediterranei phages proved to be highly specific for calcium as an adsorption cofactor; addition of calcium-sequestering agents to sensitive mycelium completely prevented its lysis by the phage. The resistant strains developed were capable of adsorbing the phage and of releasing it without multiplication upon aging of the mycelium. No marked morphological, cultural, or biochemical differences were found among the various phage-resistant strains.     

Nitrate and nitrite removal from salted water by Haloferax mediterranei

Haloferax mediterranei is a denitrifying halophilic archaeon, able to assimilate nitrate or nitrite in the presence of oxygen by the assimilatory nitrate pathway. It can also grow in the presence of high nitrate or nitrite concentrations under anoxic conditions, using both nitrogen species as electron acceptors. In this study, the ability of H. mediterranei to remove high nitrate and nitrite concentrations from culture media has been demonstrated. This suggests that this haloarchaeon could be applied in water bioremediation processes to repair damage caused by anthropogenic activities. This could be beneficial in regions such as Comunidad Valenciana or Murcia (Spain), where the water tables contain high nitrate and nitrite concentrations due to fertiliser addition, and high salt concentrations due to marine intrusions.     

NO3 −/NO2 − assimilation in halophilic archaea: physiological analysis, nasA and nasD expressions

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.     

A halocin-H4 mutant Haloferax mediterranei strain retains the ability to inhibit growth of other halophilic archaea

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype.