Vector Systems Allowing Efficient Autonomous or Integrative Gene Cloning in Micromonospora sp. Strain 40027

Abstract

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 ΦC31. 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.

The bacteria in the genus Micromonospora displays a complex life cycle, with differentiation into mycelia and spores (36). They are abundant producers of hydrolytic enzymes, such as chitinases (6) and cellulases (24), and they are also known as producers of a wide variety of antibiotics, among which aminoglycosides are the most abundant species (41). The availability of gene cloning systems for these organisms would be useful in genetic, molecular biological, and biochemical studies of these important organisms with the aim to improve the productivity of useful antibiotics and to produce novel compounds. However, reports on the genetics of Micromonospora are scarce. This is partly due to the lack of tools, techniques, and systems needed for the study of this genus.

Plasmids are widespread in streptomycetes (12). Most of the well-characterized Streptomyces plasmids, either those replicating autonomously, like pIJ101 (18), or those capable of integration into and excision from the host chromosome to become autonomous, like SLP1 (1) and pSAM2 (28, 32), encode plasmid functions essential for conjugal transfer (8).

The integrating plasmids also encode systems for site-specific recombination (3, 4, 29, 31, 32). Relatively few plasmids have been described in detail for members of the actinomycete genus Micromonospora, which are producers of a variety of commercially important antibiotics (41). pMZ1, isolated from Micromonospora zionensis (27), was shown to replicate by the rolling-circle mechanism in Micromonospora melanospora and Streptomyces lividans and is capable of conjugal transfer (40). Only one of the Micromonospora plasmids was successfully used for vector construction and for gene cloning (5, 10). No low-copy-number plasmids capable of autonomous as well as integrative replication in Micromonospora have been reported.

Transformation of protoplasts was successful for a few Micromonospora species, either by a Micromonospora griseorubida-Escherichia coli shuttle cosmid (15) or by the broad-host-range Streptomyces plasmid pIJ702 for Micromonospora rosaria, Micromonospora echinospora, and M. melanospora (19, 20, 25). More recently, a Micromonospora gene cloning system using conjugal transfer from E. coli with pSG5-derived Streptomyces cosmid vector pGM446 (33) and an E. coli plasmid, pTO1, possessing the int gene and attP site from Streptomyces phage ΦC31 (39) has been reported. Nevertheless, a continued effort to isolate naturally occurring plasmids, which could be further candidates for the construction of more useful new cloning vectors for Micromonospora, is necessary, as so-called rare actinomycetes (other than Streptomyces) continue to be screened for pharmacologically active compounds (35, 42).

Micromonospora sp. strain 40027 was isolated from a soil sample in Yunnan, China (21). It is a producer of fortimicin A which exhibits potent, broad-spectrum antibacterial activity against gram-positive and -negative bacteria both in vitro and in vivo (21). Our attempts to apply conventional transformation and conjugation techniques for Streptomyces to Micromonospora sp. strain 40027 with many of the Streptomyces vectors, including pIJ702 (16), pHZ132 (14, 17), and pHZ1358 (38), were not successful. Attempts were therefore made to isolate naturally occurring plasmids for use as parents for the construction of low-copy-number cloning vectors. Here we report the discovery and characterization of a low-copy-number conjugative plasmid, pJTU112, in Micromonospora sp. strain 40027 and its development into autonomously replicating cloning vectors. We also describe the successful utilization of pJTU112-based vectors and another E. coli plasmid (pSET152) for heterologous gene cloning and expression, either in autonomous form or in the integrated state, in Micromonospora sp. strain 40027. In both systems, conjugal transfer was used for the convenient introduction of these vectors into the host strain.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.

The strains and plasmids used are as described in Table 1. Micromonospora strains were grown at 30°C on Bennet medium (1% glucose, 0.1% yeast extract, 0.1% beef extract, 0.2% NZ amine, 1.5% agar [pH 7.3]) for spores and on mycelium medium (26) for growth of mycelium. Streptomyces strains were grown on HAUCM (30) for spores or on YEME medium (17) for preparing mycelium. Luria-Bertani (LB) liquid medium (34) was used for growing E. coli strains.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid	Characteristicsa	Source or reference
Strains
Micromonospora sp.
40027	Wild type	Gift from Zhu Baoquan
LXH20	40027 spontaneously cured of both free and integrated pJTU112	This work
LXH21	40027 spontaneously cured of autonomous form of pJTU112	This work
LXH22	LXH20 derivative carrying an integrated copy of pSET152	This work
S. lividans
1326	Wild type	13
JT46	str-6 pro-2	13
ZX1	str-6 pro-2 dnd	43
S. albus JA3453	Wild-type strain producing oxazolomycin	7
E. coli
DH5α	supE44 hsdR17 recA1	9
ET12567	dam dcm hsdS	22
Plasmids
pJTU112	Wild type	This work
pJTU111	5.96-kb SacI fragment of pHZ199 (Fig. 1) inserted into SacI site of pJTU112	This work
pJTU113	12.7-kb BamHI fragment of pJTU112 inserted into BamHI site of pOJ260	This work
pJTU116	11.5-kb PvuII fragment of pJTU112 inserted into EcoRV site of pOJ260	This work
pJTU117	Same as pJTU116 but with 11.5-kb PvuII fragment inserted in opposite orientation	This work
pJTU119	12-kb EcoRI fragment carrying the dnd gene cluster inserted into the EcoRI site of pJTU111	This work
pSET152	reppucacc(3)IV oriT attΦC31	2
pIJ8600	tipAp expression vector with 2.61-kb DNA fragment carrying tipAp, tsr, and tfd inserted into pSET152; reppucacc(3)IV oriT attΦC31	37
pHZ199	pVE1011 (23) derivative; reppucoriT bla tsr hyg	Z. Hu, unpublished data (see Fig. 2)
pHZ1904	End-filled 8.3-kb BamHI-AseI fragment containing the dnd cluster inserted into the EcoRV site of pSET152	A. Li, unpublished data
pUZ8002	Nontransmissible oriT-mobilizing RK2 derivative dam dcm hsdS Strr Tetr Cmlr Kmr	M. S. B. Paget, J. W. Wilson, and D. H. Figurski, personal communication

^aStr^r, streptomycin resistance gene; Tet^r, tetracycline resistance gene; Cm1^r, chloramphenicol resistance gene; Km^r, kanamycin resistance gene; tsr, thiostrepton resistance gene; hyg, hygromycin resistance gene; bla, β-lactamase gene; acc(3)IV, apramycin resistance gene; oriT, origin of transfer of plasmid RK2; rep^puc, replication origin of pUC18; att^ΦC31, attachment site of Streptomyces phage ΦC31; tipAp, thiostrepton-inducible promoter from S. lividans (11); dnd, a gene cluster from S. lividans 1326 encoding an unknown but site-specific DNA modification that renders DNA susceptible to degradation during agarose gel electrophoresis (Zhou et al., unpublished data).

Conjugation from E. coli to Micromonospora.

Donor cultures of E. coli ET12567/pUZ8002 were prepared by growing an overnight culture at 37°C in LB medium supplemented with kanamycin and chloramphenicol. The bacteria were washed twice and resuspended in the same volume of LB medium. Micromonospora mycelium was grown in mycelium medium (45) for 36 h at 30°C with addition of mannitol (1.5%) before a dense culture was pelleted by centrifugation and then resuspended in LB medium at a concentration of 10⁸ cells per ml. Donors (ca. 2×10⁸ cells) and recipients (freshly well-suspended Micromonospora mycelium which could give rise to ca. 1×10⁷ colonies) were spread on Bennet medium plates and grown overnight at 30°C. The plates were washed with LB and gently scraped with a pipette until the E. coli layer was removed and the Micromonospora mycelium could be seen still attached to the agar. The plates were then dried and covered with 1 ml of LB medium containing the appropriate antibiotic for selection of the donor plasmid and 20 μg of gentamicin per ml to inhibit the growth of E. coli cells. Exconjugants were counted a week later.

General techniques.

Isolation of plasmid and chromosomal DNAs from E. coli and Micromonospora and other DNA manipulations, such as restriction analysis, electrophoresis, ligation of DNA fragments, and preparation and transformation of Streptomyces protoplasts, were as described previously (17). Plasmid curing was done by selecting spontaneous thiostrepton-sensitive colonies arising after nonselective growth, which was confirmed by plasmid isolation as well as by Southern hybridization with ³²P-labeled pJTU112 as a probe. Two-dimensional gel electrophoresis was done as described previously (30), and determination of plasmid copy number was by densitometric scanning of the autoradiography after electrophoresis and Southern hybridizations and was calculated by the ratio of the band intensities between the sums of the restriction fragments corresponding to autonomous and integrated forms of the pJTU112. In the case that a BamHI fragment of the same size (1.4 kb) was shared between autonomous and integrated pJTU112, which comigrates on an agarose gel (Fig. 1), the intensity of the band for strain 40027 cured of autonomous pJTU112 (LXH21; see Table 1) will be proportionally lower for the determination of copy number. For Southern hybridizations, DNA fragments were transferred to Hybond-N nylon membranes (Amersham, Little Chalfont, England) and cross-linked by exposure to 150 mJ of UV light before hybridization with probes labeled with [α-³²P]dCTP, as specified by the manufacturer, at 65°C.

FIG. 1.

Gel electrophoresis and Southern hybridization with ³²P-labeled pJTU112 as a probe to prove that Micromonospora sp. strain 40027 harbors a low-copy-number plasmid, pJTU112, which is amplifiable by the addition of streptomycin to the culture medium and is present both in free and integrated forms. Lanes with lowercase letters are autoradiographs of the lanes with the corresponding uppercase letters. Genomic DNA samples with (lane C) and without (lane B) amplification of pJTU112 by addition of streptomycin were digested with BamHI (lanes E and F, respectively). The obvious copy number amplification of pJTU112 can be seen by direct comparison either between lanes B (b) and C (c) or between lanes e and f. (pJTU112 DNA, not seen in lane B, was detected after hybridization in lane b). Integration into the chromosome (as detected in lanes b and c) mediated by a 12.7-kb region of pJTU112 resulted two relatively more faintly hybridizing BamHI fragments (ca. 9.5 and 6.4 kb, respectively) (arrows), which was further shown by BamHI digestion of LXH21 (lane d), a mutant cured of the autonomously pJTU112. The 1.4-kb band can be generated from either free or integrated pJTU112 (lanes d, e, and f). Lambda DNA digested with HindIII was used as a size standard (lane A). Numbers on the left and right are molecular sizes in kilobases.

RESULTS

Micromonospora sp. strain 40027 contains a plasmid, pJTU112, in both autonomous and integrated forms.

A close inspection revealed the presence of a faint band running in front of the chromosomal DNA in normal agarose gel electrophoresis of Micromonospora sp. strain 40027 total DNA (Fig. 1). Two-dimensional gel electrophoresis after UV irradiation revealed that the plasmid (pJTU112) is a covalently closed circular molecule (not shown). Its copy number was estimated to be 1 to 2 per genome. With E. coli phage λ DNA digested with HindIII as the standard, the estimated size of the total SacI, PstI, or BamHI fragments of pJTU112 was ca. 14.1 kb. Interestingly, when streptomycin was added to a Micromonospora sp. strain 40027 mycelial culture at a final concentration of 5 μg/ml after an initial 36-h incubation before total DNA was isolated at 48 h, at least six times more pJTU112 DNA could be observed (Fig. 1), indicating that streptomycin can dramatically increase the copy number of pJTU112 in Micromonospora sp. strain 40027.

pJTU112 can be easily isolated in quantity by using a standard alkaline lysis procedure (17) after amplification by the addition of streptomycin. When ³²P-labeled pJTU112 was hybridized against a Southern blot of total genomic DNA of Micromonospora sp. strain 40027, strongly hybridizing signals appeared in positions corresponding not only to pJTU112 but also to the chromosome (Fig. 1), suggesting that pJTU112 may exist in an integrated form as well as in the free state. The identities of the free and integrated forms could clearly be distinguished after Micromonospora sp. strain 40027, along with a mutant strain (LXH21) cured of the free form of pJTU112 (see below), was digested with BamHI and included in parallel in the Southern hybridization experiment (Fig. 1), in which two new BamHI fragments generated after attP-attB integration are observed.

The restriction map of pJTU112 is shown in Fig. 2A. There are unique sites for SacI and XhoI; two sites for BamHI, SphI, KpnI, NotI, and PvuII; and five sites for PstI. More than seven sites for SalI and SstII were not mapped. No sites for BglII, EcoRI, EcoRV, HindIII, and XbaI were detected. The relative positions of the indicated sites were mapped by a comparative analysis of the restriction digests of pJTU112 and its derivatives.

FIG. 2.

pJTU112 and its derivatives. (A) Parent pJTU112. (B) Regions of pJTU112 present in its derivatives, shown in linear form as black bars, with dotted lines representing the replaced region of pJTU112 or vectors (pHZ199 or pOJ260 at the bottom); restriction sites used for construction are boxed. pJTU111 is derived by insertion of the larger (5.96-kb) SacI fragment of pHZ199 in the unique SacI site of pJTU112. pJTU113 has a 1.4-kb BamHI fragment of pJTU112 replaced by BamHI-linearized pOJ260. pJTU116 and pJTU117 have a 11.5-kb blunt-end PvuII fragment inserted into a unique blunt-end EcoRV site of pOJ260, in opposite orientations. + and − indicate the replication ability of each pJTU112 derivative in Micromonospora sp. strain 40027.

Construction of pJTU112 derivatives and localization of a 1.4-kb DNA fragment essential for DNA replication.

As the low-copy-number pJTU112 is difficult to isolate from Micromonospora sp. strain 40027, attempts were made to construct recombinant plasmids to allow DNA manipulations to be done in E. coli. In a first attempt, a SacI-linearized E. coli plasmid, pHZ199 (Fig. 2B), was inserted into the corresponding unique site of pJTU112, resulting in pJTU111 (Fig. 2A). A second attempt was to replace the 1.4-kb BamHI fragment of pJTU112 with the BamHI-linearized plasmid pOJ260 (Fig. 2B), resulting in pJTU113 (Fig. 2B). In a similar way, an 11.5-kb PvuII fragment of pJTU112 was inserted into the EcoRV site of pOJ260 in opposite orientations for the construction of pJTU116 (Fig. 2B) and pJTU117. Both pJTU116 and pJTU117 exconjugants could be recovered at similar frequencies, but only pJTU116 was used in later studies.

When the three pJTU112 derivatives were introduced into E. coli ET12567 carrying pUZ8002 and subsequently transferred by conjugation into Micromonospora sp. strain 40027, with selection for thiostrepton- or apramycin-resistant colonies, exconjugants carrying pJTU111 or pJTU116, but not pJTU113, could be obtained. This experiment suggests that the 1.4-kb BamHI fragment is essential for replication of pJTU112 in Micromonospora sp. strain 40027. None of the pJTU112 derivatives, however, could be transferred into S. lividans 1326 or ZX64, Streptomyces albus JA3453, or Streptomyces coelicolor A3 (2) either by plasmid transformation or by conjugation from E. coli ET12567 carrying pUZ8002 or from Micromonospora sp. strain 40027, suggesting that the replicon of pJTU112 is Micromonospora specific, unlike the reported Micromonospora plasmid pMZ1 (40). Both pJTU111 and pJTU116 seemed to be structurally stable, as determined by comparative restriction analysis of the same plasmid isolated from E. coli and Micromonospora sp. strain 40027, respectively, and to be stably inherited in Micromonospora sp. strain 40027 and in E. coli: after one round of nonselective growth, more than 95% of the colonies still carried pJTU111 or pJTU116. Also, the copy number of pJTU111 could be amplified to a similar extent as for the wild-type plasmid, pJTU112, by addition of streptomycin.

pJTU116 (15 kb) is the smallest bifunctional plasmid derived from pJTU112 obtained so far. It has unique sites for BglII, HindIII, XbaI, and EcoRI, all from pOJ260, which should be available for the cloning of foreign DNA, and an oriT for conjugation between E. coli and Micromonospora, which is useful for the introduction of cloned fragments into Micromonospora species. pJTU111 also has a unique site for EcoRI, which was used successfully for cloning.

Elimination of integrated and/or autonomous pJTU112 from Micromonospora sp. strain 40027 and localization of the attP site on pJTU112.

40027/pJTU111 and 40027/pJTU112 were grown, and eight thiostrepton-sensitive colonies arising after one round of nonselective growth of Micromonospora sp. strain 40027 were examined for their plasmid DNA profiles. Six of the eight colonies were found to have lost autonomous pJTU111, as well as pJTU112 (Fig. 3, lane F). This conclusion was supported by the failure to isolate plasmid DNA by the alkaline lysis procedure, as well as by Southern hybridization with ³²P-labeled pJTU112 as a probe (Fig. 3). As shown in Fig. 3, the autonomous form of pJTU112 (lanes F and f) is missing from the strains in lanes B to E, which seem to have retained a copy integrated into the chromosome as shown by the absence of a 0.4-kb PstI fragment and the appearance of two additional but slightly faint bands (of about 2.7 and 7.8 kb) in lanes B to E. Thus, DNA sequences involved in site-specific excision and integration events (including the attP site) seem to lie on the 0.4-kb PstI fragment of pJTU112. Also, DNA flanking the integration site (attB) of the chromosome evidently has little, if any, DNA homology with pJTU112. A representative strain that had spontaneously lost autonomous pJTU112 was named LXH21. Only one out of eight strains (lane A) was spontaneously cured of both free and integrated forms of pJTU112, and this strain was named LXH20.

FIG. 3.

Southern hybridization with ³²P-labeled pJTU112 as a probe shows curing of integrated and/or autonomous pJTU112 from Micromonospora sp. strain 40027 and localization of the attP site on pJTU112. Lanes with lowercase letters are autoradiographs of the lanes with the corresponding uppercase letters. All of the samples were digested with PstI. Four independent isolates of LXH21 (from lanes B [b] to E [e]) have all lost free pJTU112 (lanes F and f) but evidently contain an integrated copy, mediated by site-specific recombination via a 0.4-kb PstI fragment (open arrow) carrying attP, with concurrent generation of 2.7- and 7.8 kb PstI fragments (solid arrows) flanking the integration site (attB) of the chromosome. Neither autonomous nor integrated copies of pJTU112 are present in LXH20 (lanes A and a). Numbers on the right are molecular sizes in kilobases.

Two bifunctional derivatives of pJTU112, pJTU111 and pJTU116, were found to exist in the wild-type strain or in LXH20 only in the autonomous form. This result suggests that either the SacI site (used for pJTU111 construction [Fig. 2B]) or deletion of the 2.6-kb PvuII fragment (for construction of pJTU116 [Fig. 2B]) caused interruption or deletion of functions, such as that of the int gene, essential for integration of pJTU112 into the chromosome.

pSET152 and its derivatives can be introduced by conjugation into Micromonospora sp. strain 40027 via the attP site of the Streptomyces phage ΦC31 and used as integrative vectors.

Conjugal transfer from E. coli ET12567/pUZ8002 into Micromonospora sp. strain 40027 was also tested by using pHZ132, pHZ1358, pPM927, pOJ446, pSET152, and pIJ8600, all of which carry an oriT from E. coli plasmid RK2. No exconjugants could be obtained, however, by using a protocol that was successful for conjugal transfer from E. coli ET12567/pUZ8002 into Streptomyces, which involves a heat shock for spore germination (17). Conjugal transfer was successful only when pSET152, a 5.5-kb E. coli plasmid carrying the attP site of the Streptomyces phage ΦC31, was used as a vector in the donor E. coli cells and the use of heat-shocked Micromonospora spores was replaced by the use of mycelium so as to favor growth of Micromonospora sp. strain 40027 after conjugation. In addition, gentamicin (20 μg/ml) was used to inhibit the growth of E. coli on plates selecting exconjugants, because Micromonospora sp. strain 40027 is sensitive to nalidixic acid. At least a 10-fold increase in conjugation frequency was observed by the addition of mannitol (1.5%) to the mycelial culture of Micromonospora sp. strain 40027. Conjugal transfer with larger pSET152 derivatives, pIJ8600 (an 8.1-kb tipAp expression vector) and pHZ1904 (with an 8.3-kb dnd gene cluster cloned into the EcoRV site of pSET152 [X. Li et al., unpublished data), was also successful, although with a ca. 10-fold-lower frequency.

Each of the potential 40027::pSET152, 40027::pIJ8600, and 40027::pHZ1904 exconjugants was examined for possible integration into the host chromosome by site-specific recombination via the ΦC31 attP site. A 2.32-kb HindIII-XhoI fragment traversing the ΦC31 attP site and flanking part of the int gene recovered from pSET152 (0.78 + 1.54 kb) was used as a probe to hybridize with total DNA digested with BamHI (Fig. 4). Because each donor plasmid (pSET152, pIJ8600, or pHZ1904) contains only one BamHI site, two hybridizing bands would be expected after integration if recombination is indeed mediated via specific attB-attP interaction. As the sizes of the probe regions flanking the two sides of the attP site are approximately 2:1 (0.78 versus 1.54 kb) (Fig. 4), the relative intensities of the two expected hybridizing bands would roughly correspond to such a ratio. This expectation was clearly fulfilled: two hybridizing bands with approximately the expected ratio of hybridizing intensities were observed for all three strains (Fig. 4b). The sizes of the weaker bands in lanes corresponding to pSET152 (Fig. 4b, lane B) and pHZ1904 (Fig. 4b, lane D) integrations are the same (4.3 kb), while in the lane corresponding to pIJ8600 (Fig. 4b, lane C) the band is 0.76 kb larger (5.06 kb). The sizes of the stronger bands after integration by pIJ8600 and pHZ1904 are incrementally 1.85 and 8.3 kb larger than those for pSET152. These sizes agree well with the predicted sizes, as schematically represented in Fig. 4a. The chromosomal attB site seems to lie in a BamHI fragment of ca. 5.1 kb, with ca. 1.8 kb on one side of attB and ca. 3.3 kb on the other (Fig. 4a). This was predicted by the fact that the common more weakly hybridizing band (4.3 kb) (Fig. 4b) between pSET152 and pHZ1904 is ca. 1.8 kb larger than the DNA fragment between attP and BamHI (ca. 2.5 kb) encompassing the 0.78-kb probe (Fig. 4), and the more strongly hybridizing band (6.3 kb) (Fig. 4b) in pSET152 is ca. 3.3 kb larger than the DNA fragment between attP and BamHI (ca. 3 kb) encompassing the 1.54-kb probe (Fig. 4).

FIG. 4.

(a) Schematic representation of the integration of pSET152 and its two derivatives, pIJ8600 and pHZ1904, into the chromosome of Micromonospora sp. strain 40027 via the attP site of the Streptomyces phage ΦC31. The inner, middle, and outer ovals represent pSET152, pIJ8600, and pHZ1904, respectively. The black box traversing the attP site indicates the region used as the hybridization probe for Southern blots in panel b. Hatched boxes indicate extra DNA fragments cloned in pSET152 to give pIJ8600 and pHZ1904 and their relative positions in relation to the unique BamHI site, a key reference point used for size determination. (b) Determinations of the sizes of the two adjacent BamHI fragments by Southern hybridization after integration via attP-attB interaction. Lane A, total DNA of wild-type Micromonospora sp. strain 40027 was digested with BamHI. Lanes B, C, and D, autoradiographs after integration of pSET152, pIJ8600, and pHZ1904, respectively. Sizes of the larger (stronger) hybridizing bands correspond to the sum of the right (larger) arc between attP and the BamHI site of each vector plus a 3.3-kb fragment flanking one side of attB, and the sizes of the smaller (weaker) hybridizing bands correspond to the sum of the left (smaller) arc between attP and the BamHI site of each vector plus the 1.8-kb fragment flanking the other side of attB. Lambda DNA digested with HindIII was used as size standards (lane S). Numbers on the left and right are molecular sizes in kilobases.

pJTU112 is a self-transmissible conjugative plasmid.

The ability of pSET152 to integrate site specifically into the chromosome of Micromonospora sp. strain 40027 enabled us to obtain an LXH20 derivative carrying an integrated copy of pSET152 (LXH22), thus acquiring apramycin resistance. When LXH22 was used as the recipient for pJTU111 from Micromonospora sp. strain 40027 in a conjugation experiment, exconjugants of LXH22 carrying pJTU111 were successfully obtained by selecting apramycin resistance (specified by pSET152 in the recipient strain) and thiostrepton resistance (specified by pJTU111 in the donor strain). The presence of pJTU111 was confirmed by isolating covalently closed circular plasmid DNA, as well as by Southern hybridization from 11 independent exconjugants (not shown). This experiment demonstrated that pJTU111 (and thus its parental plasmid pJTU112) is self-transmissible.

Use of pSET152- and pJTU112-derived vectors for heterologous gene cloning and expression in Micromonospora.

As briefly mentioned above, the dnd gene cluster from S. lividans 1326 (X. Zhou et al. unpublished data), which encodes an unknown but site-specific DNA modification that renders DNA susceptible to degradation during agarose gel electrophoresis (a diagnostic tool for DNA modification [43]), was cloned into pSET152 to yield pHZ1904 (Li et al., unpublished data). After conjugal transfer from E. coli into Micromonospora sp. strain 40027, with selection for apramycin resistance, exconjugants were confirmed by Southern hybridization (Fig. 4). Total DNAs of Micromonospora sp. strain 40027 and its exconjugants carrying pHZ1904 were electrophoresed under conditions that would degrade the DNA of S. lividans JT46 (13) but not its dnd mutant ZX1 (43, 44). The DNAs of several 40027::pHZ1904 exconjugants were degraded like that of S. lividans JT46, while the DNA of the wild-type Micromonospora sp. strain 40027 was as stable as that of S. lividans ZX1 under the same conditions, indicating that the dnd gene cluster from S. lividans 1326 was integrated into Micromonospora sp. strain 40027 and successfully expressed, although the extent of degradation is obviously weaker than that in S. lividans (Fig. 5).

FIG. 5.

Expression of the dnd gene cluster when cloned by using pSET152 as a vector for integration into the chromosome of Micromonospora sp. strain 40027. Stable DNA of the wild-type Micromonospora sp. strain 40027 (lane I) becomes degraded in vitro during electrophoresis with integration of pHZ1904 (Fig. 4) carrying the dnd gene cluster from S. lividans 1326 (40027::pHZ1904) (lanes D to H), under electrophoresis conditions with added ferrous iron. (The DNA samples were proved to be good in normal electrophoresis buffer [not shown].) DNAs of the degraded S. lividans JT46 (dnd⁺) (lane B) and stable ZX1 (dnd mutant) (lane A) were run under the same conditions as controls. Phage λ DNA digested with HindIII was used as a size standard (lane C).

pJTU111 was also used as a vector to clone the same dnd gene cluster. A 12-kb EcoRI fragment carrying the entire dnd gene cluster was cloned into the unique EcoRI site of pJTU111, resulting in pJTU119. Three thiostrepton-resistant Micromonospora sp. strain 40027 exconjugants of pJTU119 after conjugal transfer from E. coli were tested for their Dnd phenotype. Their DNAs showed exactly the same degradation phenotype as was described for the 40027::pHZ1904 exconjugants (not shown), suggesting that the dnd system from S. lividans was also expressed in Micromonospora sp. strain 40027 with pJTU111 as a vector.

DISCUSSION

As abundant producers for many interesting natural products, Micromonospora spp. are a rich source for continued industrial screening programs for new bioactive compounds, such as antibiotics and other extracellular enzymes. Advancing our capacity to use molecular genetics among such less-studied actinomycetes has received relatively little attention. The lack of efficient genetic systems in these bacteria has often hindered the genetic cloning and characterization of the genes involved in their biosynthesis and genetic and biochemical manipulations of these organisms for the overproduction and creation of useful or novel compounds. Discovery of the indigenous, self-transmissible, and low-copy-number plasmid pJTU112 in Micromonospora sp. strain 40027, which produces the broad-spectrum antibiotic compound fortimicin A, provided a good chance for the development of useful genetic tools. The ability of pJTU112 to replicate autonomously or to integrate into the chromosome by site-specific integration, the finding of a 1.4-kb DNA fragment that is essential for pJTU112 replication, and the availability of strains free of integrated and/or autonomous pJTU112 enabled development of several E. coli-Micromonospora shuttle plasmids by combining E. coli plasmids with pJTU112 so that they are capable of replication in both E. coli and Micromonospora hosts and of conjugal transfer from E. coli to Micromonospora. This vector system, plus an additional one employing an E. coli plasmid, pSET152, carrying the attP site of the Streptomyces phage ΦC31 as an integrative vector for efficient gene cloning and manipulation in Micromonospora, seems to hold good promise for the efficient cloning and expression of heterologous genes in Micromonospora and will thus be of obvious interest to those involved in genetic investigations of this remarkable genus of microorganisms.