Novel plasmid vectors for gene cloning in Pseudomonas.

Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.     

Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.     

Novel cloning vectors for Bacillus thuringiensis

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Construction of shuttle vectors for cloning inVibrio cholerae andEscherichia coli

Starting from a naturally occurring cryptic plasmid pVC540 ofVibrio cholerae non-OI. strain 1095, a number of plasmid vectors have been constructed for cloning genes inVibrio cholerae by introducing antibiotic resistance markers containing a set of unique cloning sites. The constructs pVC810 and pVE920 have the origins of bothVibrio cholerae andEscherichia coli replicons and are stable in both organisms in the absence of selective pressure. These plasmids can serve as shuttle vectors betweenEscherichia coli andVibrio cholerae. The plasmid vectors reported here along with the demonstration of transformation inVibrio cholerae by plasmid DNA will facilitate genetic analysis of this important human pathogen.     

Construction of shuttle vectors for cloning inEscherichia coli andAcetobacter pasteurianus

New cloning vectors were prepared with the aid of a large plasmid isolated fromAcetobacter pasteurianus and from plasmids pBR322 and pUC4-KAPA. Of the prepared cloning vectors, pACK5 contains a gene coding for kanamycin resistance, pACT7 and pACT71 contain a gene coding for tetracycline resistance and vector pACG3 with a gene coding for both kanamycin and tetracycline resistance. The vectors prepared only contained the beginning of replication from the pAC1 plasmid and possessed the ability to replicate withinE. coli andA. pasteurianus. The vectors are highly stable in both strains and during the 5-d cultivation under nonselective conditions are not eliminated.     

Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression

Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments. pDK3 was used to test the expression in B. fragilis of five foreign tetracycline resistance (TcR) genes. The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes.     

Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors

pSSU1, a native plasmid of Streptococcus suis DAT1, was used to construct pSET-series shuttle vectors. In addition to the replication function of pSSU1, these vectors contain the multiple cloning sites and lacZ' gene from pUC19, which means that X-gal screening can be used to select recombinants in Escherichia coli. pSET1, pSET2, and pSET3 carry cat, spc, and both of these genes, respectively, as selectable markers. These vectors could be introduced into S. suis, E. coli, Salmonella typhimurium, S. pneumoniae, and S. equi ssp. equi by electrotransformation. The recA gene was cloned from S. suis and sequenced, and this information was used in the construction of a recA mutant of S. suis. Transformation frequencies and/or plasmid stability of all pSET vectors tested were decreased in both S. suis and E. coli recA mutants compared with the parental strains. These results suggested that functional RecA protein improved the maintenance of pSET vectors in both S. suis and E. coli. Moreover, cloning of the functional S. suis recA gene into pSET2 and complementation analysis of the recA mutant were successful in S. suis but not in E. coli. These results showed that pSET vectors are useful tools for cloning and analyzing S. suis genes in S. suis strains directly.     

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cm^r, Tc^r and Em^r determinants that are expressed in E. coli. Only the Em^r determinant is expressed in S. sanguis. Both the Cm^r and the Tc^r of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras in to S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345–353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.     

Novel shuttle vectors for improved streptokinase expression in streptococci and bacterial L-forms

Novel shuttle vectors of small size and increased copy number capable of replication in Escherichia coli, L-forms of Proteus mirabilis, and streptococci were constructed from a streptococcal erythromycin-resistant plasmid and an Escherichia coli phasmid. The streptokinase gene, skc, was inserted into one of them, and skc expression was studied in Streptococcus sanguis, Streptococcus lactis, and in an L-form strain (LVI) of Proteus mirabilis. The new streptokinase shuttle plasmid, pMLS10 (7.3 kb), specified higher Skc yields in all hosts when compared to pSM752 constructed previously. In particular Proteus mirabilis LVI(pMLS10) proved to be the most productive host, exhibiting complete secretion of the active protein at yields as high as 24000 unit per ml.     

Novel narrow-host-range vectors for direct cloning of foreign DNA inPseudomonas

Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in aPseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly inPseudomonas, DNA fragments fromAgrobacterium, Pseudomonas, andRhizobium. A clone containingAgrobacterium and Km^R gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of fivePseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA inPseudomonas.     

Development of shuttle vectors for spirochetes

Constructions of Escherichia coli-spirochete shuttle vectors are based on naturally occurring plasmids, broad host range plasmids or bacteriophages. This review primarily focuses on genetic tools for Treponema denticola which is associated with periodontal diseases. The T. pallidum FlaA protein, E. coli beta-galactosidase, and the green fluorescent protein were successfully expressed in T. denticola from a shuttle vector system.     

Improved EBV shuttle vectors

Shuttle vectors based on Epstein-Barr virus (EBV) replicate autonomously in the nuclei of human cells. These vectors represent reasonable models for chromosomes, have low background mutation frequencies, and have been useful for studying induced mutation in human cells. Two improvements in the EBV vector system are discussed. Attempts are described to increase vector copy number per cell by using a limited period of replication driven by the simian virus 40 (SV40) origin of replication. Isolation of human sequences that can replace the viral origin of replication in providing for autonomous replication of the vectors is also described. These improvements are leading toward shuttle vectors that are more efficient and more closely resemble authentic chromosomes.     

Improved cloning vectors for Bifidobacterium spp.

The recombinant plasmids pDLI41, pDGA7 and pDCO7 were constructed by cloning in pDG7, a vector based on Bifidobacterium longum replicon pMB1, the following heterologous genes: Pseudomonas fluorescens lipase, Bacillus licheniformis α-amylase and Streptomyces sp. cholesterol oxidase. The hybrid plasmids efficiently transformed Bifidobacterium belonging to five different species. A novel Escherichia coli-Bifidobacterium set of shuttle vectors based on the replicon pMB1 (pLF5, pCLJ15, pSPEC1) featuring chloramphenicol, erythromycin and spectinomycin resistance genetic determinants as selection marker for bifidobacteria, was developed. The plasmid pTRE3, a derivative of pLF5, was the smallest (2·8 kb) Bifidobacterium vector, possessed a convenient multicloning site and presented high structural and segregational stability.     

Family of shuttle vectors for ruminal Bacteroides

A family of shuttle plasmids was constructed for genetic transformation of Escherichia coli and of ruminal Bacteroides strains AR20 and AR29. Plasmids were based on the replicon from Bacteroides plasmid pBI191 and were designed for studies of chromosomal integration (pBA), for the identification and study of Bacteroides gene promoters (pPPR) and for the expression of heterologous genes in Bacteroides (pBAC). Electroporation efficiency of Bacteroides was up to 10(5) transformants/microg plasmid, depending on the source of the DNA. The largest plasmid, pBA, was maintained at approximately 8 copies per cell in AR20 and did not measurably alter in vitro growth of transformed cells. In the current work, pBA did not integrate into the chromosomes of AR20 or AR29. The ability of plasmid pPPR to select promoter sequences was demonstrated by removal and replacement of promoters that activate the clindamycin resistance gene. The suitability of pBAC for expression of heterologous genes was demonstrated by expression of the Moraxella species fluoroacetate dehalogenase gene H1 to give intracellular activity of 7 nmol fluoride released/min/mg soluble protein in AR20 and 4 nmol/min/mg in AR29. Spontaneous loss of pBAC under non-selective conditions was 0.11-0.165% per generation, significantly less than loss of the native Bacteroides plasmid pBI191, which was lost at 0.53% per generation.