Characterization of the genes and attachment sites for site-specific integration of plasmid pSE101 in Saccharopolyspora erythraea and Streptomyces lividans

The 11.3 kb plasmid pSE101 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site and into the chromosome of Streptomyces lividans at many sites. Multisite integration in S. lividans was also observed when a 1.9 kb segment of pSE101 containing attP and adjacent plasmid sequence was used to transform a pSE101^– S. lividans host. Nucleotide sequencing of this segment revealed the presence of a complete open reading frame (ORF) designated int, encoding a putative polypeptide of 448 amino acids that shows similarities to site-specific recombinases of the integrase family. Sequencing of the 1.3 kb segment upstream of int revealed the presence of three additional ORFs: the one most distal to int encodes a putative 76 amino acid basic polypeptide analogous to the Xis proteins of a number of bacteriophages. Nucleotide sequencing of attP, and the attB, attL and attR sites from Sac. erythraea revealed a 46 by sequence common to all sites with no duplications of chromosomal sequences in the integrated state. A putative structural gene for a tRNA^Thr was found to overlap the 46 by common sequence at attB. Sequencing of four pSE101 integration sites (attB) and corresponding attL and attR sites in S. lividans showed that the 46 by sequence was present at each attR site, whereas only the first three bases, CTT, were retained at each attL and attB site. A feature common to the four attB sites and to attB is a highly conserved 21 by segment with inverted repeats flanking the CTT sequence. This indicates that crossover at each attB site in S. lividans employed attP and a site within a 5 by sequence in attB and suggests that the secondary structure of the 21 by sequence is important for site-specific integration at attB or attB.     

Characterization of a cryptic plasmid pPZZ84 from Bacillus pumilus

The complete nucleotide sequence of a cryptic plasmid pPZZ84 from Bacillus pumilus strain ZZ84 was determined. Plasmid pPZZ84 is 6817 bp long with GC content of 36.7%. Seven putative open reading frames were identified. ORF7 shows 91% and 90% amino acid identity with rep proteins of pSH1452 and pPL1, respectively, members of rolling-circle replication (RCR) pC194-family. A typical pC194-family double strand origin (dso), a single-stranded origin (sso) and rap (regulator aspartate phosphatase) proteins were also identified in the plasmid. These results imply that pPZZ84 belongs to the Bacillus subtilis species group of small rolling circle (BsSRC) replicating plasmids. The plasmid copy number of pPZZ84 in B. pumilus ZZ84 was estimated to be 46 per cell, more than that of other BsSRC plasmids in their hosts.     

Construction of a bioluminescence reporter plasmid for Francisella tularensis

A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems.     

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70 kb, pRK10 is only 4241 bp long with 53% G + C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5α. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.     

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.     

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.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients

Over 60 Greenland glacial isolates were screened for plasmids and antibiotic resistance/sensitivity as the first step in establishing a genetic system. Sequence analysis of a small, cryptic, 1,950 bp plasmid, p54, from isolate GIC54, related to Arthrobacter agilis, showed a region similar to that found in theta replicating Rhodococcus plasmids. A 6,002 bp shuttle vector, pSVJ21, was constructed by ligating p54 and pUC18 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. Candidate Gram-positive recipients were chosen among glacial isolates based on phylogenetic relatedness, relatively short doubling times at low temperatures, sensitivity to antibiotics, and absence of indigenous plasmids. We developed an electroporation protocol and transformed seven isolates related to members of the Arthrobacter, Microbacterium, Curtobacterium, and Rhodoglobus genera with pSVJ21. Plasmid stability was demonstrated by successive transformation into Escherichia coli and four Gram-positive isolates, growth without antibiotic, and plasmid re-isolation. This shuttle vector and our transformation protocol provide the basis for genetic experiments with different high G+C Gram-positive hosts to study cold adaptation and expression of cold-active enzymes at low temperatures.     

Characterization of imipenem-resistant Serratia marcescens producing IMP-type and TEM-type beta-lactamases encoded on a single plasmid

To evaluate the roles of blaIMP and blaTEM genes in the resistance of Serratia marcescens against beta-lactams and to find the spreading ways of these genes, 19 clinical isolates of imipenem-resistant Serratia marcescens were analyzed. Six strains bore blaIMP and blaTEM genes on a single plasmid, as confirmed by transferring resistance determinants via conjugation and transformation, and by detecting bla genes with PCR analysis. The six strains showed two different genomic patterns on pulsed-field gel electrophoresis. All the transconjugants and transformants gained high-level resistance to ampicillin, cephalexin, cefoxitin and cefotaxime, and showed a reduced susceptibility to imipenem, but maintained full susceptibility to aztreonam. In addition, the expressions of blaIMP and blaTEM genes were constitutive, either in Serratia marcescens clinical isolates or in their transconjugants and transformants. These findings may explain the rapid spread of the above resistance determinants among Enterobacteriaceae via transmissible plasmids in the clinical setting.     

Complete nucleotide sequence of pLD-TEX-KL, a 66-kb plasmid of Legionella dumoffii TEX-KL strain

The complete nucleotide sequence of a large (66 kb) plasmid pLD-TEX-KL of Legionella dumoffii TEX-KL strain was determined. Of the 57 predicted open reading frames (ORFs), 39 (68%) encoded proteins similar to previously known proteins, five (9%) were assigned with putative functions, three (5%) encoded conserved hypothetical proteins, and 10 (18%) had no homology to any genes present in the current open databases. The ORFs with similar functions were organized in a modular structure; thus, transfer region was identified, as well as a putative heavy-metal ion transporter system (hel). The transfer region encoded homologs of the Salmonella entrica serovar Typhi conjugative system components involved in conjugation. In addition, we also found a potential protein that was analogous to the DNA polymerase III epsilon subunit. It is rarely found that plasmid encode the DNA polymerase.     

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

Summary 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.     

Genetic characterization of pAsa6, a new plasmid from Aeromonas salmonicida subsp. salmonicida that encodes a type III effector protein AopH homolog

A new plasmid designated pAsa6 from an Aeromonas salmonicida subsp. salmonicida strain isolated from diseased turbot has been characterized. pAsa6 consists of 18536 bp, has a G+C content of 53.8% and encodes 20 predicted open-reading frames (ORFs). Eight ORFs showed homology to transposases, of which six are complete and two are partial IS sequences. Two ORFs showed homology to replication proteins, and six ORFs showed homology to hypothetical proteins. Two ORFs are truncated homologs of putative A. salmonicida sulfatases. Two genes, aopH and sycH encode homologs of an effector protein for which a role in fish colonization by A. salmonicida has been previously reported, and its chaperone, respectively. The results of filter conjugation experiments suggested that pAsa6 is not mobilizable, as it failed to be conjugally-transferred to several species of marine bacteria tested. All the ORFs of pAsa6 with the exception of four copies of a IS1 transposase gene, have a counterpart in the recently sequenced 155-kb A. salmonicida plasmid pAsa5, suggesting either that pAsa6 is a derivative of pAsa5, or that pAsa5 is the result of the fusion of a pAsa6-like plasmid and a larger plasmid of ca. 135-kb. The pAsa6-encoded repA and aopH genes could be PCR-amplified from strains lacking pAsa6, suggesting presence of a large, possibly pAsa5-like plasmid that was not detected on agarose gels, or the existence of chromosome-integrated plasmid sequences. This study demonstrates that genomic locations for the aopH gene different to pAsa5 or pAsa5-like plasmids exist in A. salmonicida.     

Direct repeat sequences are essential for function of the cis-acting locus of transfer (clt) of Streptomyces phaeochromogenes plasmid pJV1

The functionality of direct and inverted repeat sequences inside the cis acting locus of transfer (clt) of the Streptomyces plasmid pJV1 was determined by testing the effect of different deletions on plasmid transfer. The results show that the single most important element for pJV1 clt function is a series of evenly spaced 9 bp long direct repeats which match the consensus CCGCACA(C/G)(C/G), since their deletion caused a dramatic reduction in plasmid transfer. The presence of these repeats in the absence of any other clt sequences allowed plasmid transfer to occur at a frequency that was at least two orders of magnitude higher than that obtained in the complete absence of clt. A database search revealed regions with a similar organization, and in the same position, in Streptomyces plasmids pSN22 and pSLS, which have transfer proteins homologous to those of pJV1.     

Isolation and characterization of pHW15, a small cryptic plasmid from Rahnella genomospecies 2

A small cryptic plasmid designated pHW15 was isolated from Rahnella genomospecies 2 WMR15 and its complete nucleotide sequence was determined. The plasmid contained 3002 bp with a G+C content of 47.4%. The origin of replication was identified by deletion analysis as a region of about 600 bp. This region had an identity of 70% to the replication origin of the ColE1 plasmid at the nucleotide level. Sequence analysis revealed the typical elements: RNA I, RNA II and their corresponding promoters, a sequence allowing hybridisation of RNA II to the DNA and favouring processing by RNaseH, a single-strand initiation determinant (ssi) that allows initiation of lagging-strand synthesis, and a terH sequence required for termination of lagging-strand synthesis. The plasmid contained three expressed open reading frames, one of which showed homology to a ColE1 plasmid-encoded protein. Furthermore, a multimer resolution site was identified by sequence analysis. Its deletion resulted in formation of plasmid multimers during growth leading to an increased plasmid loss rate.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the phi105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production.