Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Alexander, Dylan C.; Devlin, D.J.; Hewitt, Duane D.; Horan, Ann C.; Hosted, Thomas J.

doi:10.1099/mic.0.26318-0

Cited by 11 publications

(7 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Southern blots were performed as described previously (Alexander et al, 2003). To demonstrate transfer of plasmids pSLV45.680 and pSLV45.681 from donor strains SPW680 and SPW681, a Southern blot was prepared from parental, donor, recipient and exconjugant chromosomal DNA digested with BamHI.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Bacterial and derived strains used in this study are listed in Table 1. Actinomycete culture conditions, conjugation protocols and molecular techniques were performed as described previously (Alexander et al, 2003).Preparation of DNA inserts for PFGE analysis. Mycelial cells were prepared for PFGE analysis according to the method of Beyazova & Lechevalier (1993).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45

Hosted¹,

Wang²,

Horan³

2004

Microbiology

Self Cite

View full text Add to dashboard Cite

Streptomyces lavendulae IMRU 3455 contains two large linear plasmids designated pSLV45 (45 kb) and pSLV195 (195 kb). A cosmid, pSPRX604, containing 42 kb from pSLV45 was cloned and sequenced. pSLV45 was tagged with a hygromycin-resistance marker by homologous recombination to generate the derivatives pSLV45.680 and pSLV45.681. An apramycin-resistance marker was introduced into S. lavendulae IMRU 467 using the pSPR910 integration vector to yield the recipient strain SPW910. The self-transmissible nature of pSLV45 was determined by transfer of pSLV45.680 and pSLV45.681 from the donor strains SPW680 and SPW681 into the recipient strain SPW910. Southern analysis indicated the presence of hygromycin-and pSLV45-hybridizing sequences within SPW910 exconjugants. PFGE analysis confirmed pSLV45.680 and pSLV45.681 were transferred intact and formed freely replicating linear plasmids. Sequence analysis of pSPRX604 revealed genes predicted to be involved in plasmid transfer, partitioning and regulation. The transfer of the linear plasmid pSLV45 from S. lavendulae IMRU 3455 into S. lavendulae IMRU 467 may allow the development of pSLV45 as an actinomycete-to-actinomycete conjugative shuttle vector. INTRODUCTIONLinear plasmids with 59 terminally attached proteins (TPs) and terminal inverted repeats (TIRs) have been found in bacteria, plants, filamentous fungi and yeasts, and often within actinomycetes (Griffiths, 1995;Hinnebusch & Tilly, 1993;Meinhardt et al., 1990;Rohe et al., 1992). Actinomycete linear plasmids pSLA1 (17 kb) and pSLA2 (17 kb) were first detected in the lankacidin-producer Streptomyces sp. 7434-AN4 and in Streptomyces rochei, respectively (Hayakawa et al., 1979;Hirochika & Sakaguchi, 1982;Hirochika et al., 1984). Both plasmids were shown to contain TIRs and TPs (Hirochika & Sakaguchi, 1982;Hirochika et al., 1984). With the advent of PFGE analysis, other actinomycete linear plasmids have been identified including Streptomyces lividans SLP2 (50 kb), Streptomyces clavuligerus pSCL1 (12 kb), Streptomyces avermitilis pSA1 and pSA2 (100 kb and 250 kb), Streptomyces rimosus pZG101 (387 kb), Streptomyces lasaliensis pKSL (520 kb) and Streptomyces coelicolor SCP1 (363 kb) Evans et al., 1994;Gravius et al., 1994;Keen et al., 1988;Kinashi & Shimaji, 1987;Kinashi & Shimaji-Murayama, 1991;Kinashi et al., 1994). All these plasmids were found to contain TIRs and TPs. In addition, both pSLA2 and SLP2 were shown to contain an internal origin of replication for bi-directional replication of DNA (Chang & Cohen, 1994;Chang et al., 1996;Huang et al., 2003).Unlike the chromosomes of most bacteria, which are circular in nature, the chromosomes of actinomycetes are linear (Lezhava et al., 1995;Lin et al., 1993). This was first demonstrated in S. lividans and has now been established for several other actinomycetes including S. coelicolor, Streptomyces griseus and Saccharopolyspora erythraea (formerly Streptomyces erythraeus) (Lezhava et al., 1995;Lin et al., 1993;Reeves et al., 1998). All actinomycete chromosomes characterized to da...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45

Hosted¹,

Wang²,

Horan³

2004

Microbiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Phage pMLP1 was found to be present in Micromonospora carbonacea var. africana ATCC 39149 as a replicative form as well as an integrative form, and plasmid derivatives containing the site-specific att/int functions of pMLP1 were found to be able to integrate genes into the chromosome (1). None of the Micromonospora phages, however, has been developed into a gene cloning vector.…”

mentioning

confidence: 99%

Isolation and Characterization of Micromonospora Phage ΦHAU8 and Development into a Phasmid

Zhou

Deng

2004

Appl Environ Microbiol

View full text Add to dashboard Cite

⌽HAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The ⌽HAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. ⌽HAU8 was most stable at 4°C in Difco nutrient broth within a pH range of 6 to 12. ⌽HAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca 2؉ and 30 mM Mg 2؉ . A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that ⌽HAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.The genus Micromonospora has a complex life cycle, which includes differentiation into substrate mycelia, aerial hyphae, and spores (20). Production of many hydrolytic enzymes, such as chitinases (6) and cellulases (14), allows Micromonospora strains to play an active role in the degradation of organic matter in their natural habitats. They are also known as producers of a wide variety of antibiotics, among which aminoglycosides are the most abundant (26).In spite of their commercial importance and ability to produce and modify a large variety of antibiotics, reports on the genetics of Micromonospora strains are scarce. This is due in part to the lack of tools, techniques, and systems for the study of this genus. The advanced Streptomyces gene cloning methods cannot be applied to Micromonospora because Streptomyces vectors do not usually transform members of this genus satisfactorily.In addition to plasmid-based vectors, phages have proven to be useful tools for studying the biology of their hosts, as they can affect the metabolism of the hosts (22) and can be used in the transfer of genetic information by both transduction and transfection (9). A temperate phage could provide the basis of a cloning system similar to that of the Streptomyces-specific ⌽C31 derivatives (4, 18, 27) or the phage R4-derived cosmid (16,19).A number of Micromonospora-specific actinophages have been reported, including the lytic phages ⌽UW21 and ⌽UW51 (10, 11), the temperate phage MP⌽WR-1 (25), and phages with undetermined infection cycles and specificities (3). Several other lytic Micromonospora phages have been used to screen for the presence of restriction enzymes (15). Phage pMLP1 was found to be present in Micromonospora carbonacea var. africana ATCC 39149 as a replicative form as well as an integrative form, and plasmid derivatives containing the site-specific att/int functions of pMLP1 were found to be able to integrate genes into the chromosome (1). None of the Micromonospora phages, however, has been developed into a gene cloning vector.Here we describe a temperate phage (⌽HAU8) that is capable of infecting and transfecting Micromonospora sp. strain 40027 (13), a producer of fortimicin A, which exhibits potent, broad-spectrum anti...

show abstract

“…During a standard fermentation, multiple A54145 factors are produced as the result of natural variation at position 12 (3mGlu or Glu), at position 13 (Ile or Val) and at the lipid tail attached to the peptide core. The A54145 factors A, A 1 , and D (collectively designated the A-core) have the identical peptide containing Glu 12 and Ile 13 but have different lipid tails, whereas factors B, B 1 , and E (the B-core) contain 3mGlu 12 and Ile 13 . During fermentation of S. fradiae, factor A accumulates as a major component but plateaus early, and factor B 1 accumulates preferentially late in the fermentation (11,12).…”

mentioning

confidence: 99%

Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster

Alexander

Rock

et al. 2010

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the C31 or BT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu 12 ), at the expense of factors containing 3-methyl-glutamic acid (3mGlu 12 ). This provided a practical route to produce high levels of pure Glu 12 -containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.

show abstract

Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Cited by 11 publications

References 50 publications

Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45

Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45

Isolation and Characterization of Micromonospora Phage ΦHAU8 and Development into a Phasmid

Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster

Contact Info

Product

Resources

About