Molecular cloning of theEscherichia coli gene encoding xylose isomerase

Summary A 2.4 Kb DNA fragment restricted from a Clarke-Carbon ColEl plasmid, pLC32-9, containing the xylose isomerase gene has been inserted into the PstI site of pDB248, a shuttle plasmid between the bacteriumE. coli and the fission yeast,Schizosaccharomyces pombe. This recombinant plasmid, pDB248-XI, can genetically complement xylose isomerase deficientE. coli strains and xylose isomerase gene can be expressed inSchizosaccharomyces pombe.     

Protein synthesis elongation factor EFTu from streptomyces collinus producing kirromycin

Elongation factor EFTu of Streptomyces collinus has been purified. The molecular weight of the factor determined by SDS-polyacrylamide gel electrophoresis is 52,500 (±1,500). The factor shares common immunochemical determinants with EFTu of Streptomyces aureofaciens and Escherichia coli. The factor is not involved in the mechanism of resistance to the drug produced, since translation of poly(U) in the presence of EFTu and ribosomes of S. collinus is sensitive to the kirromycin effect.     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Transcriptional role in DNA replication: degradation of RNA primer during DNA synthesis

DNA synthesis catalyzed in vitro by E. coli DNA polymeraseI in the presence of single stranded fd DNA or poly (dT) as template is stimulated by RNA primers. When poly(dT) fully or partially saturated with polyriboadenylic acid strands is used as template - primer, DNA synthesis proceeds with concomitant degradation of the ribostrands to 5′-adenosine monophosphate. The fragment of DNA polymerase lacking the 5′→3′ exonuclease shows comparable RNA primer dependency but reduced efficiency for the degradation of the RNA primer from the 5′-end.     

Lysine and methionine overproduction by an Escherichia coli strain transformed with Pseudomonas acidovorans DNA

Summary An ethionine resistant, methionine-overproducingEscherichia coli mutant was transformed with plasmids bearing DNA isolated from aPseudomonas acidovorans, lysine-overproducing strain, with a feedback-insensitive aspartokinase. The resulting transformants overproduced both lysine and methionine.     

Molecular cloning and expression of s-(2-aminoethyl)-L-cysteine resistant aspartokinase gene ofCorynebacterium glutamicum

Summary A 2.9 kb DNA fragment encoding s-(2-aminoethyl)-l-cysteine (AEC) resistant aspartokinase ofCorynebacterium glutamicum was cloned on theC. glutamicum/E. coli shuttle vector pECCG117. A recombinant plasmid, designated pAK12, conferred the AEC resistance, the ability to excrete lysine and threonine, and the 3–5 fold increased specific activity of aspartokinase to host strain.     

Characterization of plasmids from plant pathogenic pseudomonads

Physical characterization of the resident plasmids from Pseudomonas tabaci, P. angulata, and P. coronafaciens strains indicated that they harbored five different plasmid DNA species. Two ATCC strains of P. tabaci contained indistinguishable plasmids that we have named pJP1 and pJP2. An isolate of one of these strains contained a spontaneous variant of pJP1, pJP1¹, which contains an insertion of 3.9 Mdal. This 3.9-Mdal region did not hybridize to pJP1 indicating that the region was foreign DNA and not a duplication of a segment of DNA already present in pJP1. Another P. tabaci strain, PT27881, contained a third plasmid species, pJP27, which had few similarities to pJP1 or pJP2, but was indistinguishable from the plasmids from all three P. angulata strains. pJP27 and pJP1 had a small region, 8.8 Mdal, of sequence homology. The one strain of P. coronafaciens examined contained a plasmid, pJP50, which was different from the P. tabaci plasmids, but had the 8.8-Mdal region and additional regions of sequence homology with pJP1 and pJP27 as well as homology with a portion of the pJP1¹ insertion. A fourth strain of P. tabaci, PTBR-2, a pathogen on beans, contained plasmid pBW, the only plasmid that lacked detectable regions of homology with the other plasmids.     

Identification of genes specifying proline biosynthesis in marine bacteriumAlteromonas haloplanktis

Summary A gene bank of total DNA of a marine bacterium,Alteromonas haloplanktis, was constructed on pBR322. Two hybrid plasmids pUS2010 and pUS2011 carrying inserts of 8.2 and 5.7 kb, respectively, were isolated that complemented theproBA deletion inE.coli CSH26. Restriction map of the inserts showed that both plasmids in common carried a 5.7 kb fragment. This restriction fragment thus contains both the genes involved in proline biosynthesis inA.haloplanktis and could be expressed inE.coli.     

Plasmid-mediated bioaugmentation of sequencing batch reactors for enhancement of 2,4-dichlorophenoxyacetic acid removal in wastewater using plasmid pJP4

Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100 % removal against 200 mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0 % on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10 %, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100 % after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500 mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95 %. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65 % on day 76, almost 100 % removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.     

Transformation of Bacillus subtilis by electroporation

Summary Optimal conditions for the transformation of Bacillus subtilis by electroporation were achieved. Frequencies of greater than 10⁵ transformants/&#x03BC;g of plasmid DNA were obtained for a number of strains and plasmids. Increased transformation efficiency of mini-prep DNA from B. subtilis and Escherichia coli was obtained after microdialysis.     

Cloning and expression of anAeromonas hydrophila chitinase gene inescherichia coli

The gene encoding an extracellular chitinase fromAeromonas hydrophila has been cloned and expressed inEscherichia coli. Plasmid pJP2512 contained the smallest DNA insert (3.9 kb) producing chitinase. The chitinase gene is transcribed from its own promoter, producing a protein of M_r 96,000. The chitinase open reading frame, an estimated 2.6 kb in length, has been subcloned to a 3.0 kb fragment; however, this fragment does not carry the functional chitinase promoter. InE. coli the chitinase enzyme is unable to transverse the outer membrane, being secreted across the cytoplasmic membrane and accumulating in the periplasmic space.     

Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria

Plasmid pJP4 encoding the ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (Tfd⁺) was transferred by conjugation from Escherichia coli JMP397 to various lithoautotrophic strains of Alcaligenes eutrophus and to the autotrophic bacterium Pseudomonas oxalaticus. The herbicide-degrading function of the plasmid was phenotypically expressed in all of the recipients. The majority of Tfd⁺ transconjugants also exhibited additional plasmid-encoded properties such as 3-chlorobenzoate degradation, resistance to mercuric ions, and sensitivity to the male-specific bacteriophage PR11. Furthermore, Tfd⁺ transconjugants were able to act as donors of plasmid pJP4. Physical evidence is presented by agarose gel electrophoresis showing that plasmid pJP4 coexisted with the resident plasmids widely distributed in this group of bacteria. However, in some of the hosts plasmid pJP4 was not stably maintained, had a reduced size and tended to form multimers.     

Partial characterization of anEscherichia coll strain harboring a xylanase encoding plasmid

Summary The plasmid pRH271, harboring a xylanase gene cioned fromBacilius subtilis, has been transferred into a mutant ofE. coli SK2284 which allowed the release of part of the xylanase in the culture supernatant. Kinetic parameters of this recombinantE. coll strain were determined in microscale batch culture with and without the selective pressure of antibiotics. No significant difference in µ_max was observed for the nontransformedE. coli strain when compared to the recombinant strain. However, K₅ values for glucose were two times higher in the case of the recombinant strain. Preliminary study of xylanase production in a large batch farmenter was also described.     

Cloning of bacteriophage PM2 DNA in Escherichia coli K12.

Summary DNA fragments of phage PM2 restricted with Hin dIII endonuclease was cloned in the vector pBR 322 in an Escherichia coli K 12 host. The attempt to clone full length PM2 DNA restricted with PstI endonuclease has been unsuccesful. From six randomly chosen recombinant clones DNA was purified and analysed with EcoRI, PstI and HindIII endonucleases. The physical map of three chimeric plasmids was unequivocally established. It was shown, that the whole PM2 genome was cloned, although in separate fragments. However, most of the recombinant clones were instable in the absence of selective pressure.     

A rapid method for the isolation of plasmid DNA from bacteria

Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield.     

Construction of aClostridium acetobutylicum-Escherichia coli shuttle vector

Summary A 4.1-kb cryptic plasmid, designated pCA134, has been isolated fromClostridium species. In order to develop a vector suitable for transforming saccharolytic clostridia three hybrid plasmids were constructed by inserting pCA134 into pHV32 withEcoRI, orBglII andBamHI. The newly constructed plasmids were propagated inEscherichia coli and were used to transformBacillus subtilis andClostridium acetobutylicum. One of them, pCAB32 (10.1 kb), which contains chloramphenicol acetyltransferase gene and an origin of replication derived from pCA134 was introduced intoB.subtilis andC.acetobutylicum as well asE.coli.     

High level extracellular secretion of thermostable α-amylase ofBacillus stearothermophilus inEscherichia coli

Summary Bacillus stearothermophilus BR135 (ATCC 29609)amy gene was cloned in pBR322 from its plasmid DNA and was subcloned in a vector useful both forB. subtilis andE. coli.E.coli HB101 harboring the plasmid pSS099 when grown in L medium in presence of 5. g/ml chloramphenicol produces 70 units/ml of extracellular -amylase. This is nearly twice that ofE.coli cells harboring pSSO76, a plasmid havingamy ofB.stearothermophilus BR135 atHindIII site of pBR322. Characteristically the protein was a 58 kd protein and cross reacted with antiserum developed against purified -amylase of BR135.     

Improved L-lysine production by the amplification of theCorynebacterium glutamicum dapA gene encoding dihydrodipicolinate synthetase inE. coli

Summary ThedapA gene (L-2,3-dihydrodipicolinate synthetase: DHDP synthetase) ofCorynebacterium glutamicum JS231, a lysine overproducer, cloned and subcloned inE. coli/C. glutamicum shuttle vector pECCG117 was used to transformE. coli threonine producer and threonine and lysine coproducer. The plasmid pDHDP5812 carryingdapA gene ofC. glutamicum led to increase in lysine production in theseE. coli strains. Threonine and lysine co-producerE. coli TF1 with pDHDP5812 produced lysine with small amount of threonine. The DHDP synthetase activity ofE. coli TF1 carrying pDHDP5812 showed high resistance toward inhibition by lysine.