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.     

Cloning and expression of the erythromycin resistance determinant from Campylobacter jejuni in Escherichia coli

The erythromycin resistance gene (Em^r) from Campylobacter jejuni ABA94 plasmid DNA was cloned into the pUC18 vector and then expressed in Escherichia coli. The location of the Em^r determinant on the chimeric plasmid was determined by restriction endonuclease mapping within a 0.8-kb EcoRI fragment. This fragment then hybridized to the 78-kb plasmid DNA but not to the 3.3-or 12.6-kb plasmid DNA of Campylobacter jejuni ABA94. Em^r in Campylobacter jejuni is therefore probably plasmid-mediated.The authors are with the Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Molecular characterization of unique deletion mutants of the streptococcal plasmid,pAMβ1

pAMβ1 is a 17 × 10⁶ dalton plasmid originally isolated in a strain of Streptococcus faecalis. This plasmid confers constitutively expressed macrolide-lincosamide-streptogramin resistance. Following its introduction in Streptococcus sanguis (Challis) by transformation we have detected a class of pAMβ1 derivatives which carry site-specific deletions. Each of these independently obtained, smaller plasmids has been found to be missing an identical 60% of the pAMβ1 molecule when probed by restriction endonuclease digestion. A typical specific deletion derivative, designated pVA1, is present to the extent of ~10 copies per chromosomal equivalent. It is more stably inherited than pAMβ1 (<8.5% frequency of spontaneous loss) in S. sanguis grown at 37 °C. However, both pVA1 and pAMβ1 appear to be rapidly segregated from S. sanguis cells grown at 42 °C. pVA1 should provide a useful replicon for genetic studies including those aimed at elucidating R plasmid organization, expression, and molecular cloning vector development in the streptococci.     

Marker rescue allows direct selection for recombinant plasmids in streptococci

Summary Resident deletion derivatives (Em^s or Cm^s) of the streptococcal plasmid vector pGB301 rescue antibiotic resistance genes from linearized pGB301 (Em^r, Cm^r) DNA with high frequency. Insertion of passenger DNA next to an antibiotic resistance determinant of pGB301, which is missing on the resident plasmid, forces corescue of these two plasmid domains, thus allowing direct selection for recombinant plasmids.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Conjugative transfer and autonomous replication of a promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803

Summary The promiscuous IncQ plasmid pKT210 (Cm^r, Sm^r) is efficiently transferred by transpecific conjugation from Escherichia coli to the facultatively heterotrophic cyanobacterium Synechocystis PCC6803 when mobilized by a helper plasmid coding for IncP transfer functions. The IncQ plasmid is stably maintained in the cyanobacterium as an autonomously replicating multicopy plasmid with no detectable structural alterations and can be recovered by transformation back to E. coli when using a mcrA mcrB host. Thus, the replicative host-range of IncQ plasmids extends beyond purple bacteria to the distinct procaryotic taxon of cyanobacteria, allowing the use of these small plasmids as convenient cloning vectors in Synechocystis PCC6803 and presumably also in cyanobacteria that are not amenable to genetic transformation. In contrast, an IncQ plasmid bearing the TRP1 gene of Saccharomyces cerevisiae failed to replicate when transferred to that yeast by transformation.     

Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

Specific concerns have been raised that third-generation cephalosporin-resistant (3GC^r) Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COT^r) E. coli, 3GC^r Salmonella enterica, and nalidixic acid-resistant (NAL^r) S. enterica may be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n = 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GC^r Salmonella was detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NAL^r S. enterica was detected on only one hide. 3GC^r E. coli and COT^r E. coli were detected on 100.0% of hides during processing. Concentrations of 3GC^r E. coli and COT^r E. coli on hides were correlated with pre-evisceration carcass contamination. 3GC^r E. coli and COT^r E. coli were each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenic E. coli (ExPEC) virulence-associated markers. Only two COT^r E. coli isolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria.     

An efficient chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli

Chloramphenicol (Cm) was demonstrated to be a suitable selective agent for the plasmid-mediated transformation of haploid and polyploid strains of Saccharomyces cerevisiae.A yeast/Escherichia colishuttle Cm-resistance (Cm^R)marker was constructed by inserting the CAT coding sequence from Tn9,and its associated bacterial ribosome-binding site, between a modified yeast ADC1 promoter and CYC1 terminator. When present on a 2 μm-based replicating plasmid, this marker transformed yeast as efficiently as the auxotrophic markers TRP1 and LEU2. When included in an integrating vector, single-copy transformants were formed as efficiently as with LEU2 and HIS3. Industrial yeast strains were transformed with both the replicating and integrating plasmids. The Cm^R marker could also efficiently transform E. coli. This versatile and efficient performance is currently unique for a yeast dominant marker.     

Construction of Broad Host Range Cloning Vectors for Gram-negative Bacteria

A new cloning vector, pMFY31, has been constructed based on the high-copy-number, broad-host-range plasmid RSF1010. The plasmid has a size of 13.2 kb and carries the Ap^r, Cm^r, and Tc^r genes. It contains unique PstI, EcoRI, HindIII, BamHI, and SalI sites, all of which are located within the antibiotic resistance genes, therefore all sites are applicable to insertional inactivation. We also constructed pMFY40, a 11.6 kb derivative of pMFY31, by the elimination of the Cm^r gene. Plasmid pMFY31 has been efficiently introduced into a Pseudomonas putida strain not only by plasmid-DNA transformation but also by conjugal co-transfer with the helper plasmid, and was maintained stably in the strain.     

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range¹. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (Φ) C31^2,3. The ΦC31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)⁴. This recombination is stable and does not revert⁵. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli ß-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene⁶ may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob⁷. The expression of the ΦC31 integrase gene (cloned from pHS62⁸) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK7813⁹.A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.     

Direct plasmid transfer from replica-plated E. coli colonies to competent B. subtilis cells. Identification of an E. coli clone carrying the hisH and tyrA genes of B. subtilis

Summary We have cloned the hisH tyrA wild-type genes of Bacillus subtilis with the aid of the chimeric plasmid pBJ194, which replicates both in B. subtilis and Escherichia coli. Primary cloning was done in E. coli. The original E. coli clone, carrying the recombinant plasmid (pGR1) which complements hisH tyrA mutants of B. subtilis, was selected directly from a mixture of plated E. coli clones by replicaplating these clones onto minimal agar plates without tyrosine spread just before with competent B. subtilis cells. After overnight incubation clusters of small colonies had developed exclusively in the E. coli [pGR1] colony prints.The Tyr⁺ minicolonies were shown to be B. subtilis carrying pGR1 because (i) their appearance depended linearly on the number of B. subtilis cells plated, (ii) they produced extracellular protease and amylase and (iii) plasmids could be reisolated from the minicolonies and used to transform B. subtilis recE4 tyrA1 both to Cm^r and Tyr⁺.Plasmid pGR1 transfer through replica plating was compared with plasmid transfer in liquid. Both systems depended on transformable B. subtilis strains and were sensitive to DNAseI. However, whereas integration of the tyrA ⁺ gene into the chromosome and concomittant loss of plasmids occurred frequently during regular plasmid transformation of Rec⁺ B. subtilis, this was a rare event during plasmid transfer through replica plating.     

Identification and Characterization of a Shuttle Plasmid with Antibiotic Resistance Gene from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

While studying antibiotic-resistant plasmids from multi-drug-resistant nosocomial Staphylococcus aureus strains, we isolated a small (2.889 kb) chloramphenicol-resistant (Cm^r) plasmid, which was designated as pMC524/MBM. The molecular size of pMC524/MBM was close to that of pC194 (2.910 kb), a well-known Cm^r staphylococcal plasmid. Unlike pC194, this plasmid can replicate and express itself efficiently and stably in Escherichia coli. However, Cm is needed for stable maintenance of pMC524/MBM in different hosts. In this study, the nucleotide sequences of these two plasmids were compared after sequencing of pMC524/MBM [EMBL Accession No. AJ312056 SAU312056]. Although these two plasmids have striking nucleotide sequence homology, the Plus Origin, Minus Origin, the replication protein (Rep), and the chloramphenicol acetyl transferase (Cat) have considerable variations. Possibly, these changes have modulated pMC524/MBM into an efficient shuttle-plasmid. Received: 8 April 2002 / Accepted: 27 July 2002     

DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in Pneumococcus

Genes for chloramphenicol resistance (Cm^r) and tetracycline resistance (Tc^r), which are present as heterologous insertions in the chromosomes of some clinical isolates of Streptococcus pneumoniae (pneumococcus) and derivative strains, were transferred at a low frequency to other pneumococci by a DNase-resistant filter mating process that resembles conjugation. Cotransfer of Cm^r and Tc^r was the most common event. Tetracycline resistance was transferred alone from one Tc^r strain or rarely from Cm^rTc^r donors, whereas Cm^r was never transferred alone. Neither the donor strains nor the transconjugants contained detectable plasmids. Transconjugants acted as donors for transformation and for filter mating and had properties similar to those of the parent strain. The presence of the conjugative plasmid pIP501 in the donor did not appear to influence the transfer properties of the Cm^r or Tc^r determinants. No transfer of Cm^r or Tc^r toStreptococcus faecalis JH2-2 was observed.     

Tn5-Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi

Escherichia coli (2492/pJB4JI) matings with Erwinia chrysanthemi produced kanamycin resistant (Km^r) transconjugants, a majority of which were gentamicin sensitive (Gm^s). A small proportion (about 0.8%) of the Km^r Gm^s clones were either auxotrophic or failed to catabolize galacturonate (Gtu⁻). The R plasmid (pJB4JI) DNA was detected in the parent E. coli strain and in a Km^r Gm^r transconjugant, but not in Km^r Gm^sE. chrysanthemi strains carrying Tn5-induced mutations. In Hfr crosses, Km^r (Tn5) was found linked with most mutations. A majority (>95%) of prototrophic recombinants were Km^s, except for Leu⁺ and Arg⁺ recombinants which were 30 to 50% Km^s. Spontaneous revertants were obtained for all markers except car, gtu, lys, thr, and trp. Prototrophic revertants, with the exception of Met⁺, Leu⁺, or His⁺ clones, were Km^s. We conclude from both genetic and physical data that Tn5 transposed from pJB4JI into different sites on the chromosome of E. chrysanthemi.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation

Summary The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.Abbreviations used Ap^r resistance to ampicillin - Cm^r resistance to chloramphenicol - R/M restriction and modification - Tc^r resistance to tetracycline     

Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius

The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.