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PubMed2005

Micromonospora mirobrigensis sp. nov.

An actinomycete strain was recovered from a pond where radon is known to be dissolved. A polyphasic study was undertaken to identify the new isolate. The 16S rRNA gene sequence of strain WA201(T) showed closest similarity to the type strains of Micromonospora carbonacea (98.5 %) and Micromonospora matsumotoense (98.1 %). The chemotaxonomic results confirmed the taxonomic position of the isolate in the genus Micromonospora. DNA-DNA relatedness values supported the classification of this isolate as a novel species. A number of physiological and biochemical tests were able to distinguish strain WA201(T) from its closest phylogenetic neighbours. Therefore, it is proposed that isolate WA201(T) (=DSM 44830(T)=LMG 22229(T)) be considered the type strain representing a novel species, Micromonospora mirobrigensis sp. nov.

Bacterial Typing Techniques↗

PubMed2006

Micromonospora coriariae sp. nov., isolated from root nodules of Coriaria myrtifolia.

An actinomycete strain, NAR01(T), was isolated from root nodules of a Coriaria plant. The 16S rRNA gene sequence of strain NAR01(T) showed most similarity to the type strains of Micromonospora endolithica (98.94 %) and Micromonospora chersina (98.4 %). The chemotaxonomic results obtained confirmed the taxonomic position of the isolate within the genus Micromonospora, and revealed differences at the species level. Physiological and biochemical tests showed that strain NAR01(T) could be clearly distinguished from its closest phylogenetic neighbours, while DNA-DNA hybridization results indicated that the isolate represents a novel species. On the basis of these results, strain NAR01(T) (=DSM 44875(T)=LMG 23557(T)) is proposed as the type strain of the novel species Micromonospora coriariae sp. nov.

Bacterial Typing Techniques↗

PubMed1987

Transfection in Micromonospora spp.

The introduction of bacteriophage DNA into Micromonospora protoplasts, resulting in the production of infective viral progeny, is reported. Transfection was affected by several factors. We observed that it reached a maximum when protoplasts from young mycelium (15 h old) were used. Maximum transfection took place when polyethylene glycol (PEG) was added to the mixtures at a final concentration of 20% (vol/vol) and did not occur at PEG concentrations under 10% or over 35%. The addition of positively charged liposomes to the mixtures was essential, since no transfectants were detected in the absence of liposomes at any PEG concentration. When DNA was present in nonlimiting amounts, a maximum efficiency of around 10(-3) to 10(-4) PFU per protoplast was obtained. The efficiency per DNA molecule showed a constant value of around 10(-4) to 10(-5) PFU, but the data suggest that transfection could be achieved by a single DNA molecule. The method proved to be equally efficient for the DNAs of at least five Micromonospora bacteriophages. On the contrary, we failed to transfect five of seven Micromonospora strains. These data suggest that only a minor subpopulation of protoplasts is competent and that the main factors influencing the transfection of Micromonospora protoplasts are neither the characteristics nor the origin of the DNA but the properties and status of the protoplasts.

Bacteriophages↗

PubMed2004

Differentiation of Micromonospora isolates from a coastal sediment in Wales on the basis of Fourier transform infrared spectroscopy, 16S rRNA sequence analysis, and the amplified fragment length polymorphism technique.

A number of actinomycetes isolates were recovered from coastal sediments in Aberystwyth (Wales, United Kingdom) with standard isolation techniques. Most of them were putatively assigned to the genera Streptomyces and Micromonospora on the basis of their morphological characteristics, and there appeared to be no difference whether the isolation media contained distilled water or seawater. A group of 20 Micromonospora isolates was selected to undergo further polyphasic taxonomic investigation. Three approaches were used to analyze the diversity of these isolates, 16S rDNA sequencing, fluorescent amplified fragment length polymorphism (AFLP), and Fourier transform infrared spectroscopy (FT-IR). The 16S rDNA sequence analysis confirmed that all of these isolates should be classified to the genus Micromonospora, and they were analyzed with a group of other Micromonospora 16S rDNA sequences available from the Ribosomal Database Project. The relationships of the 20 isolates were observed after hierarchical clustering, and almost identical clusters were obtained with these three techniques. This has obvious implications for high-throughput screening for novel actinomycetes because FT-IR spectroscopy, which is a rapid and reliable whole-organism fingerprinting method, can be applied as a very useful dereplication tool to indicate which environmental isolates have been cultured previously.

Bacterial Typing Techniques↗

PubMed1978

[Directed isolation of Micromonospora generic cultures from moist soils and silts].

The data on the study of various soils and substrates for isolation of Micromonospora from them are presented. It was shown that Micromonospora predominated in moist soils and especially in such substrates as silts where their content with respect to the all actinomycetous isolates amounted to 88.9 per cent. In the silts the content of Micromonospora amounted to 66.6--83 per cent, in the sopromely its content was 66 per cent, while in the ordinary non-moist soil its content was from 6 to 11 per cent. Predominance of Micromonospora in silts and moist soils makes its directed search possible.

Asia, Eastern↗

PubMed1978

[Data on the conditions for the prolonged storage of Micromonospora generic cultures].

Data on studying various conditions for prolonged storage of Micromonospora are presented. It was shown that Micromonospora were satisfactorily stored under conditions of all maintenance methods used. The optimal methods were the following: storage of Micromonospora on agarized media under a layer of vaseline oil, storage of Micromonospora in the form of a mature submerged culture on liquid media optimal for its growth and development. Stimulating effect of low temperature on the spore germination was shown.

Culture Media↗

PubMed1980

[Micromonospora resistence to definite antibiotics and their ability to produce structurally analogous antibiotics].

Thirty six cultures of Micromonospora freshly isolated from soil samples were studied with respect to their sensitivity to a number of antibiotics of various structures and modes of action. It was found that all of them were highly sensitive to penicillin, ristomycin, tetracycline, rifampicin, streptomycin, olivomycin, carminomycin and dactinomycin. Significant differences in sensitivity of the Micromonospora cultures were revealed only with respect to gentamicin, tobramicin, erythromycin and lincomycin. Seven cultures were resistant to gentamicin and tobramicin and sensitive to all of the other antibiotics. Broad spectrum antibiotics were isolated from these cultures. The study of the antibiotic chemistry showed that they were 2-desoxystreptamine-containing aminoglycosides. Two cultures proved to be resistant to erythromycin and lincomycin. When identified with the use of antibiotic resistant staphylococcal strains, the crude antibiotic substances isolated from these cultures appeared to be not active against staphylococci resistant to erythromycin and lincomycin. By their chromatograpi- behaviour the antibiotics were close to macrolides. Therefore, it was found that production of aminoglycoside and macrolide antibiotics was most characteristic of Micromonospora. A certain correlation between resistance of Micromonospora to these 2 antibiotic groups and capacity for their production was shown.

Anti-Bacterial Agents↗

PubMed1984

Occurrence of gentamicin-producing micromonosporae in Lake Balaton (W. Hungary).

The total number of platable bacteria in the wet mud material characterized by the predominant occurrence of high-magnesian calcite and protodolomite of the Lake Balaton fluctuates between 3.5 X 10(4) and 2.3 X 10(5) g-1. Actinomycetes constitute about 25-30 per cent of this mud-community and not more than half of them belong to the genus Micromonospora. The presence of 10 species of this genus was detected among the isolated and studied strains identified by means of conventional methods and computer aided numerical analysis in which 20 authentic, mostly type strains of Micromonospora spp. were also involved. All of the three species of gentamicin-producing Micromonospora (M. echinospora, M. purpurea and M. sagamiensis) known so far were found in mud samples. These three species form stable but less numerous populations (less than 0.5 per cent of the total platable microflora) in the Balaton's mud, the members of which are capable to produce gentamicins at about 40-50 micrograms ml-1 level at most.

Bacteriological Techniques↗

PubMed1992

Organization and nature of fortimicin A (astromicin) biosynthetic genes studied using a cosmid library of Micromonospora olivasterospora DNA.

The cloning of five DNA segments carrying at least seven genes (fms1, fms3, fms4, fms5, fms7, fms11, and fms12) that participate in fortimicin A (astromicin) biosynthesis was described previously. These DNA fragments were used to screen a cosmid library of genomic DNA in order to examine if these biosynthetic genes are clustered in Micromonospora olivasterospora. One cosmid clone (pGLM990) was obtained, which hybridized to all the probes. Complementation analysis, using mutants blocked at various steps and chimeric plasmids subcloned from pGLM990, showed that three additional genes (fms8, fms10, and fms13) are present in pGLM990. A gene conferring self-resistance to the antibiotic, which was independently cloned in Streptomyces lividans, using the plasmid vector pIJ702 was also found to be linked to the cluster of biosynthetic genes. Thus, at least ten biosynthetic genes and a self-defense gene are clustered in a chromosomal region of about 27 kb in M. olivasterospora. Interestingly, the fms8 gene which participates in the dehydroxylation step of fortimicin A biosynthesis was found to have homology with a neomycin resistance gene nmrA from the neomycin-producing Micromonospora sp. MK50. Studies using a cell-free extract of the fms8 mutant and its parent strain showed that the enzyme encoded by fms8 phosphorylates a biosynthetic precursor, fortimicin KK1, in the presence of ATP. Thus the dehydroxylation reaction is suggested to occur via the phosphorylation of the target hydroxyl group. DNA regions homologous to fms genes were found in Micromonospora sp. SF-2098 and Dactylosporangium matsuzakiense, both producers of fortimicin group antibiotics.

Actinomycetales↗

PubMed1992

Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis.

We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employes the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pMO116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.

Aminoglycosides↗

PubMed1992

A novel, highly efficient gene-cloning system in Micromonospora applied to the genetic analysis of fortimicin biosynthesis.

We have developed a gene-cloning system in Micromonospora olivasterospora, a fortimicin A (astromicin) producer. Plasmids of Micromonospora from two strains of M. olivasterospora were used for construction of the vectors. Two antibiotic-resistance genes, nmrA and nmrB, cloned from a neomycin-producing Micromonospora, were introduced into these plasmids for the selection of transformants. In a new protoplasting protocol for lysozyme-resistant bacteria, protoplasts of M. olivasterospora were found in short-time incubation with lysozyme and transformed efficiently, indicating that the method was suitable to shotgun cloning. Using this system, seven biosynthetic genes for fortimicin A were cloned. Their physical maps revealed that at least four of these genes were clustered. Analysis of a cosmid library of M. olivasterospora showed that eleven biosynthetic genes and a self-defense gene existed in a region of approx. 25 kb of DNA.

Aminoglycosides↗

PubMed2005

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.

Attachment Sites, Microbiological↗

PubMed2003

Vector systems allowing efficient autonomous or integrative gene cloning in Micromonospora sp. strain 40027.

Vector systems allowing autonomous or site-specific integrative gene cloning were developed for Micromonospora sp. strain 40027, a producer of the antibiotic fortimicin A. The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca. 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states. The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium. The integrative system was developed by the use of a pBR322-derived Escherichia coli plasmid vector, pSET152, mediated by the attP site of the Streptomyces phage PhiC31. Both vectors can be transferred by conjugation from E. coli into Micromonospora sp. strain 40027. The heterologous cloning and expression of the dnd gene cluster originating from Streptomyces lividans 1326 into Micromonospora sp. strain 40027 demonstrated the use of the two systems.

Attachment Sites, Microbiological↗

PubMed1988

Temporally regulated tandem promoters in Micromonospora echinospora.

A collection of promoters from the Micromonospora echinospora strain that produces the calichemicin antitumor antibiotics was identified by the use of the promoter-probe vector pIJ486 in Streptomyces lividans. A 0.4-kilobase-pair Micromonospora DNA fragment was found to contain multiple tandem promoters which were characterized by S1 nuclease protection, Northern blotting, and DNA sequence determination. Analysis of RNA isolated from timed Micromonospora cultures revealed two classes of promoters within the 0.4-kilobase-pair fragment. The P2 promoter was maximally active during the exponential phase. In contrast, the P1 promoter cluster, consisting of three closely spaced start sites located 80 base pairs upstream of P2, was maximally active during the stationary phase. Because P1 was strongly induced in synchrony with calichemicin drug production, P1 is of potential utility in expressing cloned genes specifically during the stationary phase.

DNA, Bacterial↗

PubMed1991

A novel, highly efficient gene-cloning system for Micromonospora strains.

A highly efficient gene-cloning system for Micromonospora olivasterospora, a producer of the antibiotic fortimicin A (astromicin), suited to shotgun cloning has been developed. The system is supported by two new advancements accomplished in this study. One is the construction of novel plasmid vectors pMO116, pMO126, pMO133, pMO136, and pMO217, all consisting of replicons from newly found Micromonospora plasmids and selectable markers cloned from a neomycin-producing Micromonospora strain. The other advancement is the establishment of a new protocol for bacterial protoplasting in which some kinds of sugar alcohols are added in precultures. Such sugar alcohols were found to sensitize a wide taxonomical range of bacteria to lysozyme. The system is reproducible and reliable and has a high efficiency of more than 10(6) CFU/micrograms of DNA.

Cloning, Molecular↗

PubMed2005

[Study on cloning of sisomicin-resistant gene (sisR) from Micromonospora inyoensis].

A new sisomicin resistance gene sisR was cloned from sisomicin-producing Micromonospora inyoensis. The sisR fragment was obtained by PCR amplification. The primer pairs were designed based on grm gene sequence from gentamicin-producing Micromonospora purpurea. The template DNA was isolated from Micromonospora inyoensis. A series of different DNA fragments were amplified by PCR, which were sub-cloned to vector pUC19 for further identification. It was found that five specific transformants containing target DNA fragments could resist high concentrations of sisomicin (over 1000 microg/mL sisomicin). One of them designated as sisR, was then sequenced and the alignment among sisR and other related genes showed that sisR gene differs from any known genes. It was concluded that sisR gene is a sequence that has not been reported so far.

Anti-Bacterial Agents↗

PubMed1979

[Formation of broad-spectrum antibiotics by cultures of the genus Micromonospora].

Data on the study of antibiotic production by the representatives of Micromonospora and the use of ion exchange resins for intensification of screening antibiotic-producing organisms among Micromonospora are presented. It was found that out of 172 strains of Micromonospora tested 92 (53.5 per cent) cultures produced antibiotics, 18 of which were active against gramnegative bacteria. The use of carboxylic ion exchange resins at early microbiological stages of the screening provided an increase in the frequency of finding broad spectrum antibiotics from 10.4 to 19.7 per cent.

Adsorption↗

PubMed1983

[Screening of beta-lactamase inhibitors among representatives of the genus Micromonospora and the genus Streptoverticillium].

A total of 123 cultures of the genus Micromonospora and 89 cultures of the genus Streptoverticillium were studied for their capacity to produce beta-lactamase inhibitors. It was shown that streptoverticilla were highly resistant to beta-lactam antibiotics. No organisms producing beta-lactamase inhibitors were detected among the cultures of this genus. As for Micromonospora, 30 cultures (24.4 per cent) of this genus produced inhibitors. They were most frequently isolated from orange and brown cultures of Micromonospora. The inhibitors had no antibacterial activity, inhibited beta-lactamases of gram-negative bacteria and did not inhibit exogenic beta-lactamases of staphylococci. Their inhibitory activity was evident only with the use of isolated enzymes. With the use of intact growing cultures of gram-negative bacteria producing the same enzymes (Pseudomonas, Klebsiella, Enterobacter) no inhibitory activity was observed. Though the inhibitors had some features in common, they did not seem to be similar, since they had different spectra with respect to their effect on various beta-lactamases of gram-negative bacteria.

Anti-Bacterial Agents↗