The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.     

Connection of outer membrane protein composition in Yersinia pestis cells with intrinsic plasmids]

A set of isogenic derivatives of Yersinia pestis EV strain was obtained including the variants harbouring the different compositions of Yersinia own plasmids. The protein profiles of outer membranes of the set of strains were defined. The polyacrylamide gel electrophoresis has shown the small 6.1 Md plasmid to code an outer membrane protein with mol mass 29 kDa, different from pesticin I, while the heavy 60.0 Md plasmid encodes the 15-16 kDa polypeptide different from monomers of F1 and T-antigens of plague microbe.     

Plasmids for biodegradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine, and pyridine in strains of Arthrobacter]

Arthrobacter crysallopoietes strain KM-4 degrading 2,6-dimethylpyridine and strain KM-4a degrading both 2,6-dimethylpyridine and pyridine, Arthrobacter sp. KM-4b degrading 2,4-dimethylpyridine were isolated from soil. Arthrobacter crystallopoietes KM-4 and Arthrobacter sp. KM-4b contain 100 Md plasmids pBS320 and pBS323. Arthrobacter crystallopietes KM-4a harbours a 100 Md and 80 Md plasmids. Plasmid curing and conjugation transfer results confirm that these plasmids are involved in degradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine and pyridine. A mutant with lost ability to degrade 2,6-dimethylpyridine was isolated during the growth of strain KM-4 rifR at 42 degrees C. Electrophoretic analysis of the plasmid from temperature sensitive mutant revealed the deletion the size of 26 Md from pBS320 plasmid.     

A transposon-like structure related to the delta-endotoxin gene of Bacillus thuringiensis.

A DNA segment (Th-sequence) has been found in several strains of Bacillus thuringiensis. This Th-sequence [3 megadaltons (Md)] induces adjacent deletions when it is located in the pAM beta 1 plasmid derived from Streptococcus faecalis. Electron microscopic examination of reannealed single strands of one plasmid (pMT9) carrying such a deletion revealed that the Th-sequence corresponds to a single-stranded loop (2.8 Md) bounded by a short double-stranded stem (less than 0.2 Md). Southern blotting experiments established that in B. thuringiensis the Th-sequence was generally located on the large plasmid which also harbours the gene coding for the delta-endotoxin (crystal protein). Hybridization and heteroduplex analysis of the extrachromosomal DNA from the berliner 1715 strain demonstrated that the crystal gene and the Th-sequence are located in close vicinity on a 42-Md plasmid and that they are separated by a 1.3-Md DNA segment. This DNA segment is repeated in inverted orientation, once immediately adjacent to the Th-sequence and once 1.8 Md beyond the crystal gene. A model for the organization of these DNA sequences inside a transposon-like structure is proposed.     

Genetic determination of the vibriocinogenicity trait in Vibrio cholerae of the El Tor biotype

In two different strains of cholera vibrios two recA-dependent plasmids, pVib I (1.9-2.2 Md) and pVib II (5.2-5.8 Md), have been detected. These plasmids determine the synthesis of vibriocin, coagulase and fibrinolysin, which has been established by the cotransformation of the DNA of plasmids pVib I and pBR322 and by the transfer mobilization with the use of plasmid RP4.     

Multiple pathways for toluene degradation in Burkholderia sp. strain JS150.

Burkholderia (Pseudomonas) sp. strain JS150 uses multiple pathways for the metabolism of catechols that result from degradation of aromatic compounds. This suggests that the strain also uses multiple upstream pathways for the initial hydroxylation of aromatic substrates. Two distinct DNA fragments that allowed Pseudomonas aeruginosa PAO1c to grow with benzene as a sole carbon source were cloned from strain JS150. One of the recombinant plasmids containing the initial steps for the degradative pathway contained a 14-kb DNA insert and was designated pRO2016. We have previously shown that the DNA insert originated from a plasmid carried by strain JS150 and contained genes encoding a multicomponent toluene-2-monooxygenase (tbmABCDEF) as well as the cognate regulatory protein (tbmR) that controls expression of the 2-monooxygenase (G. R. Johnson and R. H. Olsen, Appl. Environ. Microbiol. 61:3336-3346, 1995). Subsequently, we have identified an additional region on this DNA fragment that encodes toluene-4-monooxygenase activity. The toluene-4-monooxygenase activity was also regulated by the tbmR gene product. A second DNA fragment that allowed P. aeruginosa to grow with benzene was obtained as a 20-kb insert on a recombinant plasmid designated pRO2015. The DNA insert contained genes encoding toluene-4-monooxygenase activity but no toluene-2-monooxygenase activity. The pRO2015 insert originated from the chromosome of strain JS150, unlike the region cloned in pRO2016. Southern blots and restriction map comparisons showed that the genes for the individual 4-monooxygenases were distinct from one another. Thus, strain JS150 has been shown to have at least three toluene/benzene monooxygenases to initiate toluene metabolism in addition to the toluene dioxygenase reported previously by others.     

Molecular characterization and interstrain variability of pHPS1, a plasmid isolated from the Sydney strain (SS1) of Helicobacter pylori

The 5846-bp circular plasmid pHPS1 of Helicobacter pylori Sydney strain, SS1, was cloned, sequenced, and structurally characterized. The SS1 strain is widely used in animal studies of H. pylori infection. The sequence of pHPS1 revealed three open reading frames (ORFs), all of which are transcribed. Two ORFs encode putative plasmid replication proteins, RepA and RepB, similar to replicases resident on theta plasmids. In contrast, the function of ORF2 remains cryptic due to the absence of sequence similarity with any known protein in sequence databases. In addition, species specificity of these three coding regions was shown using DNA dot blot hybridization in 57 diverse clinical H. pylori isolates and 32 Helicobacter and Campylobacter strains. RepA appears to be the predominant plasmid replication protein of H. pylori and the deduced amino acid sequence was highly conserved (76-96%) in 8 H. pylori isolates, including SS1. RepB was detected in 3 H. pylori isolates examined in this study, 2 of which possess only the repB gene. Analysis of the protein sequences of these two replicases, together with previously characterized H. pylori plasmid replication proteins, supports the formation of a distinct class of H. pylori plasmid proteins. Moreover, comprehensive analysis of the whole genome sequence of H. pylori strain 26695, pHPS1, and other H. pylori plasmid sequences that are available revealed interesting insights as to the occurrence of plasmid-mediated recombination within H. pylori. Common regions between plasmids and chromosome sequences of H. pylori were identified in this study which could only have arisen by genetic recombination, thus providing the first line of evidence, albeit indirectly, of the contribution of H. pylori plasmids in generating an extensive genetic heterogeneity characteristic of this important gastroduodenal pathogen.     

Plasmids associated with a phagelike particle and with a satellite inclusion in Bacillus thuringiensis ssp. israelensis

Variants of Bacillus thuringiensis ssp. israelensis were isolated using a 42 degrees C plasmid curing method. Those that were missing the large toxic crystalline inclusion still possessed in their sporulated cells a small inclusion similar to the satellite inclusion of the wild type. These crystal-minus but satellite-positive variants, like the wild type, produced sporulation-dependent phagelike particles; variants lacking both the crystal and the satellite did not. These latter strains lacked both a 75 and a 68 megadalton plasmid present in the wild type. However, a crystal-positive, satellite-minus strain possessing the 75 but lacking the 68 megadalton plasmid produced the phagelike particles. Examination of recipient strains that were initially devoid of crystal, satellite, and plasmids revealed the satellite and phagelike producing characteristics could have been cotransformed with the 68 megadalton plasmid. Likewise the toxic crystal and phagelike particle producing characters could have been cotransformed into a recipient which had acquired the 75 megadalton plasmids already known to be associated with the synthesis of the crystal inclusion. Thus the gene coding for the satellite inclusion appears to reside in the 68 megadalton plasmid, while that coding for the phagelike particle can reside in either the 68 or 75 megadalton plasmid.     

Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.     

Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry⁻) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized (González and Carlton, 1980). The plasmid patterns of both Cry⁻ and Cry⁺ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry⁻ mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry⁻ mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (˜130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.     

Characterisation of plasmids coding for the restriction endonuclease EcoRI

Summary The properties of two plasmids coding for the EcoRI restriction and modification enzymes are described. Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors. Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes. This plasmid has a molecular weight of 6x10⁶ daltons and is present as approximately 12 copies per chromosome. The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor R1–19. NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1. The molecular weight of NTP14 is 10.7x10⁶ daltons and there are about 14 copies per chromosome. DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1–19. NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol.     

苏云金芽孢杆菌以色列亚种130kd杀蚊蛋白基因的...

The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid was confirmed by southern hybridization using a 18-base oligonucleotide probe. The crystal protein containing the component of 130kd toxic protein was purified. The crystal protein exhibiting the mosquitocidal activity against larvae of Aedes aegypti was shown by bioassay. The purified 75Md plasmid DNA of Bti 4Q5 strain was completely digested with HindIII restriction enzyme, ligated with the vector pUC18 and transformed into the recipient cells of E. coli TG1. From Apr transformants, four clones with HindIII restriction fragment inserts highly homologous to the 18-base oligonucleotide probe were obtained by in situ hybridization and southern hybridization. The 5.2kb HindIII restriction fragment insert was obtained in clone pFH2 and clone pFH4, and 2.3kb HindIII restriction fragment insert in clone pFH1 and pFH3. For pFH2 and pFH4, the 5.2kb fragment was inserted in pUC18 in opposite orientation. It contained 130kd mosquitocidal protein gene (type I) identified by restriction enzyme map analysis. The 2.3kb HindIII fragment insert in other two clones (pFH1 and pFH3) harbored a part of the type II mosquitocidal protein gene which can be used as a probe for cloning of the type II mosquitocidal protein gene.     

Recent aspects of genetic manipulation in Bacillus thuringiensis

The conjugative plasmid pAM beta 1 was transferred from Streptococcus faecalis to several strains of Bacillus thuringiensis by a filter-mating process. From a transconjugant clone of B. thuringiensis a hybrid plasmid resulting from an in vivo insertion into pAM beta 1 of a 3 Md DNA sequence was isolated. This 3 Md DNA molecule (Th sequence) is related to several host plasmids found in different serotypes of B. thuringiensis. A reciprocal conjugation-like process involving the transfer of pAM beta 1 from B. thuringiensis to S. faecalis was also demonstrated. The comparison of the restriction maps of the crystal genes from plasmid and chromosomal origins of different serotypes, six of which having been cloned in E. coli, revealed the existence of two classes of genes which are very similar in the map corresponding to the N-terminal part of the protein, and which differ essentially in the 3' region. The presence of the transposon-like Th sequence was found in several cases associated with the crystal gene in the same host plasmid, and a model for their structural organization is proposed.     

Plasmids of Azospirillum brasilense

The cells from natural isolates of A. Brasilense were found to harbour 1 to 4 plasmids with the molecular masses within the 27-300 Md range. 100 Md plasmids are specific for this bacterial species. Strains isolated from the roots of cereals (wheat, maize, barley) have more heterogeneous plasmid composition as compared to the strains isolated from soil.     

Biocontrol of the sugarcane borer Eldana saccharina by expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA genes in sugarcane-associated bacteria

The cry1Ac7 gene of Bacillus thuringiensis strain 234, showing activity against the sugarcane borer Eldana saccharina, was cloned under the control of the tac promoter. The fusion was introduced into the broad-host-range plasmid pKT240 and the integration vector pJFF350 and without the tac promoter into the broad-host-range plasmids pML122 and pKmM0. These plasmids were introduced into a Pseudomonas fluorescens strain isolated from the phylloplane of sugarcane and the endophytic bacterium Herbaspirillum seropedicae found in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the tac promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nm(r) promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher mortality of larvae than did P. fluorescens 14::tox. An increased toxic effect was observed when P. fluorescens 14::ptac-tox was combined with P. fluorescens carrying the Serratia marcescens chitinase gene chiA, under the control of the tac promoter, integrated into the chromosome.     

Isolation of the R'his plasmids of Vibrio cholerae

V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.     

Regulation of protoxin synthesis in Bacillus thuringiensis.

A derivative of Bacillus thuringiensis subsp. kurstaki (HD-1) formed parasporal inclusions at 25 degrees C, but not at 32 degrees C. This strain differed from the parent only in the loss of a 110-megadalton (Md) plasmid, but plasmid and chromosomal copies of protoxin genes were present in both strains. On the basis of temperature shift experiments, the sensitive period appeared to be during midexponential growth, long before the time of protoxin synthesis at 3 to 4 h after the end of exponential growth. The conditional phenotype could be transferred by cell mating to naturally acrystalliferous Bacillus cereus. In all such cases, a 29-Md protoxin -encoding plasmid was transferred, but this plasmid alone was barely sufficient for protoxin synthesis. Protoxin production increased to detectable levels, but well below those of the parental donor strain, by simultaneous transfer of a 44-Md protoxin -encoding plasmid. Transfer of a 5-Md plasmid with the two larger protoxin -coding plasmids resulted in a protoxin synthesis level approaching that of the donor strain. A role for some of the cryptic plasmids of kurstaki in parasporal body formation was implied. In contrast, a closely related B. thuringiensis strain, HD73 , produced crystals at both 25 and 32 degrees C even when the capacity was transferred on a 50-Md plasmid to B. cereus. The amount of protoxin produced in these B. cereus transcipients , however, was somewhat less than that produced in the parental strain HD73 , implying that catabolic differences, gene dosage, or the presence of a chromosomal gene (or a combination of these) may be necessary for maximum production. A regulatory component of the 29-Md plasmid appeared to be trans-acting and dominant since B. cereus transcipients containing the 29-Md plasmid from kurstaki and the 50-Md plasmid from HD73 produced more protoxin at 25 degrees C than at 30 degrees C. Similar results were obtained when protoxin synthetic capacity was transferred from B. thuringiensis subsp. israelensis to the conditional B. thuringiensis subsp. kurstaki strain.     

Characterization of a new strain of Bacillus thuringiensis, a producer of an endotoxin against coleoptera

A new strain of Bacillus thuringiensis 2-7 was found to belong to the serotype H8. Cells of this strain contained irregular and flat crystalline inclusions and two large plasmids. The gene responsible for crystal formation is most likely located on the large plasmid greater than 105 MDa in size. Comparison of the cry gene of B. thuringiensis 2-7 and the cryIIIA gene of B. thuringiensis subsp. tenebrionis showed that their nucleotide sequences are identical.     

Molecular analysis of pDL10 from Acidianus ambivalens reveals a family of related plasmids from extremely thermophilic and acidophilic archaea.

The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.