Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg

Summary Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied. In a restriction-deficient but modification-proficient mutant of B. subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E. coli (10⁴ clones per g target DNA). Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb). In the restriction-proficient B. subtilis strain, the class of large inserts was underrepresented. Transformation of B. subtilis with E. coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways. First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host. The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb). Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid. In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0–16 kb), and no structural instability was observed. It is concluded that for shotgun cloning in B. subtilis, the use of restriction-deficient strains is highly preferable. Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction. It is postulated that AsuII sites are additional target sites for BsuM restriction.     

Development of a Host-vector System for Gluconobacter suboxydans

A shuttle vector for Gluconobacter suboxydans and Escherichia coli was constructed by ligation of a cryptic plasmid, pMV201, found in G. suboxydans IFO 3130 to E. coli plasmid pACYC177. The chimeric plasmid named pMGlOl carries the ampicillin resistance gene derived from pACYC177 and transforms G. suboxydans var. α IFO 3254 as well as E. coli. The transformation conditions for G. suboxydansvar. α IFO 3254 were examined using pMGlOl DNA. Competent cells were induced efficiently by treatment with LiCl or RbCl CaCl₂ which induced the competency of Acetobacter was much less effective. Addition of polyethylene glycol enhanced the transformation efficiency significantly. An efficiency of approximately 10² transformants per μg DNA was finally obtained.     

A quantitative analysis of shotgun cloning in Bacillus subtilis protoplasts

Summary We used the Escherichia coli-Bacillus subtilis shuttle vector pHP13, which carries the replication functions of the cryptic B. subtilis plasmid pTA1060, to study the effects of BsuM restriction, plasmid size and DNA concentration on the efficiency of shotgun cloning of heterologous E. coli DNA in B. subtilis protoplasts. In a restriction-deficient strain, clones were obtained with low frequency (19% of the transformants contained a recombinant plasmid) and large inserts (>6 kb) were relatively rare (12% of the clones contained inserts in the range of 6–9 kb). The efficiency of shotgun cloning was severely reduced in restricting protoplasts: the class of large inserts (>6 kb) was under-represented in the clone bank (4% of the clones contained inserts in the range of 6–6.1 kb). Furthermore, BsuM restriction caused structural instability of some recombinant plasmids. Transformation of protoplasts with individual recombinant plasmids showed that plasmid size and transforming activity were negatively correlated. The size effect was most extreme with cut and religated plasmid DNA. The yield of clones was independent of the DNA concentration during transformation. It is therefore unlikely that clones were not detected because of simultaneous uptake of more than one plasmid. It is concluded that shotgun cloning in B. subtilis protoplasts is inferior to that in competent cells.     

Spheroplast Fusion of Acetobacter aceti and Its Application to the Breeding of Strains for Vinegar Production

Spheroplasts of auxotrophic mutants derived from Acetobacter aceti subsp. aceti No. 1023 were efficiently prepared by treatment with lysozyme, using sucrose as an osmotic stabilizer, and regenerated on an agar plate containing sorbitol and gelatin. In addition, spheroplast fusion between the several auxotrophic mutants was achieved in the presence of polyethylene glycol and CaCl₂. The frequency of fusion was found to be about 5 × 10 ⁵. Spheroplast fusion between A. aceti subsp. aceti No. 2 with the ability to grow at high temperature and A. aceti subsp. xylinum NBI1002 with high resistance to acetic acid was also achieved by the same method, with a frequency of 6.0 × 10 ⁶. The fusants showed various degrees of resistance to acetic acid and ability to grow at high temperature. One of the fusants, named No. 116, could produce acetic acid from ethanol continuously under conditions under which both parent strains were unable to grow. This suggests that spheroplast fusion is applicable to the breeding of strains for vinegar production.     

A Host–Vector System for a Cellulose-Producing Acetobacter Strain

An indigenous plasmid, named pAH4, was detected in a cellulose-producing Acetobacter strain. This plasmid, consisting of 4002 bp, contained an AT-rich region and encoded several open reading frames, as deduced by the complete nucleotide sequence. One of the putative open reading frames showed homology with replication proteins of other plasmids. A shuttle vector of Escherichia coli and this strain was constructed by connecting pAH4 to pUC18. Electroporation of the shuttle vector into the strain yielded 1.7 × 10⁵ ampicillin resistant transformants per μg DNA. The shuttle plasmid was very stably maintained in the strain.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Cloning and expression of Bacillus subtilis spore genes

Summary Two spore genes, spoOB and spoIIG have been cloned from the B. subtilis genome library, constructed by ligating Sau3A partially digested DNA to the dephosphorylated pHV33 plasmid vector at its BamH1 site.An hybrid plasmid pGsOB2, carrying a 1.7 Kb insert of B. subtilis DNA amplifiable in E. coli was cloned. This recombinant plasmid was capable of transforming the appropriate B. subtilis Rec⁺ and Rec^- recipients to Spo⁺ at very high efficiency. The pGsOB2 was further subcloned and four hybrid plasmids, pGsOB8, pGsOB9, pGsOB10 and pGsOB11 were selected and their restriction enzyme maps established. The four subcloned hybrid plasmids retained their entire transforming activity in both Rec⁺ and Rec^- recipients although two of them carry the insert in an inverse orientation, indicating thus, that the spoOB gene in these plasmids is being transcribed by the B. subtilis RNA polymerase using an internal promotor of the cloned DNA fragment. The adjacent genes spoIVF and pheA, mapped respectively to the right and left of the spoOB locus, that normally show 90% cotransformation, are absent on the cloned DNA fragments. The cloned hybrid plasmids have been expressed in E. coli minicells and it was shown that the spoOB locus encoded a polypeptide of 24 K.We have also cloned the spoIIG gene in two hybrid plasmids, pGsIIG24 and pGsIIG26, carrying respectively inserts of 2 and 3 Kb. From the transforming activity and the endonuclease cleavage maps it was shown that these two hybrid plasmids do not carry the entire spoIIG locus. The use of these plasmids for further cloning of this gene is discussed.     

Drug resistance in Indian isolates of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Five different strains of enteropathogenic Escherichia coli (EPEC) and one non-pathogenic strain of E. coli were studied for the determination of resistance to various antibiotics by disc inhibition test. Our results demonstrated multiple drug resistance among the selected E. coli isolates with all of them including the non-pathogenic control strain showing high resistance towards ampicillin. Our results from conjugation test clearly showed the presence of most of the antibiotic markers on the transferred plasmids. Simultaneously, the plasmid profile of conjugants also indicated the presence of more than one plasmid along with the expected megaplasmid of ~90 Kb present in them. Thus our results amply demonstrate that these drug markers are associated with these mobile plasmids.     

Transformation of Zymomonas mobilis by a hybrid plasmid

The transformation of Zymomonas mobilis by plasmid DNA was achieved using a modification of the CaCl₂ method for Escherichia coli. The highest frequency of transformation obtained was 5 × 10³ transformants/μg DNA. The success of the method depended upon the use of a plasmid which is a cointegrate between a Z. mobilis cryptic plasmid and an E. coli plasmid carrying two selectable drug resistance markers.     

Cloning of the β-Isopropylmalate Dehydrogenase Gene from Acetobacter aceti and Its Use for Construction of a New Host-vector System for Acetobacter

The β-isopropylmalate dehydrogenase (β-IPMDH) gene of Acetobacter aceti No. 1023, which complemented the leuB mutation of Escherichia coli, was cloned and expressed in E. coli. Plasmids pCAL1 and pCAL4, carrying a 5.44 kilobase pairs (kb) HindIII-fragment in the opposite orientation, conferred the same β-IPMDH activity as that of the wild type strain of E. coli. Restriction mapping and deletion analysis indicated that the β-IPMDH gene was located on a 1.65 kb AatII-HindIII fragment. Plasmids pMVL1 and pMVL2, composing the cloned β-IPMDH gene and plasmid pMV102, a plasmid indigenous to Acetobacter, were constructed as plasmid cloning vectors which allow selection of leu⁺ transformants in an A. aceti leu^- host. The A. aceti leu^- host was obtained through the insertional inactivation occurring as a result of homologous recombination between the chromosome of A. aceti and the cloned β-IPMDH gene, which was disrupted by insertion of the kanamycin resistance gene from pACYC177 into the BamHI site in the AatII-HindIII fragment. This system constitutes a relatively simple technique for gene disruption or replacement in Acetobacter that requires a single transformation.     

Cloning and expression of a glucoamylase gene from Lactobacillus amylovorus ATCC 33621 in Escherichia coli

Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C.     

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.     

Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum

Summary Hybrid plasmids were constructed by combining in vitro the Escherichia coli plasmid pGA22, which carries the genes determining resistance to kanamycin, tetracycline, chloramphenicol and ampicillin, with the cryptic plasmids, pCG1 and pCG2, of Corynebacterium glutamicum. The hybrid plasmids were introduced into C. glutamicum and E. coli and replicated in both hosts. They expressed all the E. coli resistance phenotypes except ampicillin resistance in C. glutamicum. The levels of antibiotic inactivating enzymes encoded on these plasmids were about four to ten times lower in C. glutamicum than in E. coli. Despite the lack of expression of ampicillin resistance, -lactamase activity was detected in C. glutamicum carrying hybrid plasmids.     

Construction of a Chimeric Plasmid in Bacillus subtilis

Staphylococcal plasmids pTP4 (2.7 megadaltons encoding resistance to chloramphenicol) and pTP5 (2.6 megadaltons encoding resistance to tetracycline), which replicate and express resistance in B. subtilis, were found to cut by HindIII endonuclease respectively at a single site and three sites. A chimeric plasmid pTA1245 (4.1 megadaltons) was constructed from pTP4 and pTP5 by HindIII digestion and ligation with E. coli DNA ligase. pTA1245 expresses resistances to chloramphenicol and tetracycline in B. subtilis, and pTA1245 is amplified in the presence of tetracycline. A physical map of pTA1245 was constructed.     

High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors inPseudomonas stutzeri

A number ofEscherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained inPseudomonas stutzeri ATCC 17588. A restrictionless mutant ofP. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With theE. coli cloning vector pHSG298, transformation frequencies of up to 2×10⁷ transformants/g DNA were achieved. This frequency is comparable to that obtained for CaCl₂-mediated transformation ofE. coli; thus, direct cloning of DNA intoP. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G+C genera in cases in whichE. coli is not a suitable heterologous cloning host.     

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

Summary The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and an approximate copy number of 20–50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S. lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

Molecular cloning of tobacco chloroplast ribosomal RNA genes

Summary Tobacco chloroplast ribosomal RNAs were shown to be hybridized with two EcoRI fragments of tobacco chloroplast DNA. These DNA fragments having molecular weights of 1.9x10⁶ and 2.8x10⁶ daltons were cloned using the bacterial plasmid pMB9 as a vector and E. coli HB101 as host bacteria. The recombinant plasmids containing either or both of these fragments were constructed and characterized.Abbreviations rRNA ribosomal RNA - EDTA ethylenediamine tetraacetic acid - SSC 0.15 M NaCl-0.015 M sodium citrate - EcoRI and HindIII restriction endonucleases isolated from E. coli RY13 and Haemophilus influenzae Rd, respectively     

Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli

A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error‐prone PCR or molecular shuffling, and a linear vector backbone was prepared by high‐fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap‐extension PCR with a pair of 5'‐phosphorylated primers. Third, full‐length linear plasmids with phosphorylated 5'‐ends were self‐ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high‐efficiency transformation. Self‐made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10⁵ cfu/µg of the self‐ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6‐phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5‐fold improved catalytic efficiency (k_cat/K_m) on NAD⁺ as compared to the wild‐type. This protocol is DNA‐sequence independent, and does not require restriction enzymes, special E. coli host, or labor‐intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids.