Characteristics of the R-plasmid pM3 (IncP-9) of a broad circle of hosts]

A new broad host range plasmid pM3 (IncP-9) was found in a facultative methylotrophic bacteria Pseudomonas putida and described. The pM3 plasmid is characterized by thermo-instability in Enterobacteriaceae family of bacteria at 36 degrees C or higher temperatures. It is also unable to be inherited as an autonomous element in the obligate methylotrophic bacteria Methylobacillus M75. The peculiarities of plasmid inheritance make possible to use it as a tool for genetic research, for instance, to construct the donor strains in Methylobacillus M75 able to mobilize the chromosomal genes for conjugational transfer in isogenic systems of crosses.     

Molecular relationships between pseudomonas INC P-9 degradative plasmids TOL, NAH, and SAL

We have examined the extent to which the degradative plasmids SAL, NAH, and TOL of the Inc P-9 incompatibility group share common DNA sequences. The homology we observe using 32P-labeled SAL and NAH DNA probes can be assigned to six regions of the TOL (pWWO) restriction endonuclease cleavage map. At least three of these regions are probably related to transfer and replication functions, whereas a fourth region is related to the common metacleavage pathway. Restriction endonuclease maps of the SAL and NAH plasmids are derived and the relationships between these plasmids discussed.     

Application of pM3, a broad host range IncP-9 plasmid, for genetic analysis in Enterobacteriaceae

The possibility of using a transposon-carrying variant of broad host range plasmid pM3 (IncP-9) as a universal vector for transposon mutagenesis and as a chromosome-mobilizing factor was demonstrated in bacteria of the family Enterobacteriaceae.     

Homology of plasmids with a wide range of hosts

According to blotting hybridization and heteroduplex analysis, plasmids R751, R906 and RP4 of Inc Pi group have continuous regions of homology. These homologous regions were mapped on the R751 and RP4-derived pRP401 deletion mutant DNAs. The plasmid pRP401 (m.w. 21.9 kg) retains the broad host range property and has two regions of intensive homology with other Inc P-1 plasmid DNAs. These regions are localized at 8.2-12.0 kb and 13.9-21.9 kb of the physical map of pRP401 plasmid. Homologous regions of pRP401 DNA include at least the replication genes (oriV, trfA, trfB) as well as genes kilB, korA, korB and probably kilC. The data strongly point out that the broad host range plasmids have the same principle of structural and functional organization.     

Plasmid P85 from Azospirillum brasilense SP245: study of the circle of possible hosts and incompatibility with plasmids from Azospirillum brasilense SP7]

The possibility of the stable inheritance of the plasmid p85 mobilized derivatives from Azospirillum brasilense Sp245 in the cells of the bacterial genera Rizobiaceae (Agrobacterium tumfaciens) and Pseudomonadaceae (Pseudomonas putida) has been shown. The plasmid p85 participates in coding for the physiologically active products (the plant hormones). It is not inherited by the Escherichia coli strains. For the first time the incompatibility of azospirillium plasmids has been demonstrated on the example of the plasmid p85 from Azospirillum brasilense Sp245 and the plasmid p115 from Azospirillum brasilense Sp7.     

Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9

A new xylanase gene, xynAS9, was cloned from Streptomyces sp. S9, which was isolated from Turpan Basin, China. The full-length gene consists of 1,395 bp and encodes 465 amino acids including 38 residues of a putative signal peptide. The overall amino acid sequence shares the highest identity (50.8%) with a putative endo-1,4-beta-xylanase from Streptomyces avermitilis of the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.5 and 60 degrees C, respectively. The enzyme showed broad temperature adaptability, retaining more than 65% of the maximum activity when assayed at 50-80 degrees C. The enzyme also had good thermal and pH stability. The K (m) values for oat spelt xylan and birchwood xylan substrates were 2.85 and 2.43 mg ml(-1), with the V (max) values of 772.20 and 490.87 mumol min(-1) mg(-1), respectively. The hydrolysis products of xylan were mainly xylose and xylobiose. These favorable properties should make XynAS9 a good candidate in various industrial applications.     

Application of pM3, a Broad Host Range IncP-9 Plasmid, for Genetic Analysis in Enterobacteriaceae

The possibility of using a transposon-carrying variant of broad host range plasmid pM3 (IncP-9) as a universal vector for transposon mutagenesis and as a chromosome-mobilizing factor was demonstrated in bacteria of the family Enterobacteriaceae.     

Cloning and expression of amino acid transporters from broad bean

This work describes the isolation of a full-length (VfAAP2) and three partial amino acid transporter genes (VfAAPa, VfAAPb, VfAAPc) from broad bean (Vicia faba L.). The function of VfAAP2 was tested by heterologous expression in a yeast mutant deficient in proline uptake. VfAAP2 mediates proton-dependent proline uptake with an apparent K_m of about 1 mM. Analysis of substrate specificity by competition experiments showed that aromatic amino acids, neutral aliphatic acids and L-citrulline are the best competitors, whereas basic amino acids do not compete with proline. Northern analysis indicates that all VfAAPs exhibit different patterns of expression. VfAAP2 is most strongly expressed in the stem and at a lower level in sink leaves and pods. VfAAPa, VfAAPb and VfAAPc are most strongly expressed in the flowers, but their expression in the other organs varies.     

The paradoxical rarity of a fruit fly fungus attacking a broad range of hosts

Understanding the factors that determine the realized and potential distribution of a species requires knowledge of abiotic, physiological, limitations as well as ecological interactions. Fungi of the order Laboulbeniales specialize on arthropods and are typically thought to be highly specialized on a single species or closely related group of species. Because infections are almost exclusively transmitted through direct contact between the hosts, the host ecology, to a large extent, determines the distribution and occurrence of the fungus. We examined ~20,000 fruit flies (Diptera: Dacinae) collected in Malaysia, Sulawesi, Australia, and the Solomon Islands between 2017 and 2019 for fungal infections and found 197 infected flies across eight different Bactrocera species. Morphology and 1,363 bps of small subunit (18S) DNA sequences both support that the infections are from a single polyphagous fungal species Stigmatomyces dacinus—a known ectoparasite of these fruit flies. This leads to the question: why is S. dacinus rare, when its hosts are widespread and abundant? In addition, the hosts are all Bactrocera, a genus with ~480 species, but 37 Bactrocera species found sympatric with the hosts were never infected. Host‐selection does not appear to be phylogenetically correlated. These results suggest a hidden complexity in how different, but closely related, host species vary in their susceptibility, which somehow limits the abundance and dispersal capability of the fungus.     

Structural-functional organization of R-plasmid pBS222 with a broad range of bacterial hosts

The broad host-range plasmid pBS222 is compatible with broad host-range plasmids of all known incompatibility groups and codes for tetracycline resistance. pBS222 is efficiently mobilized by Inc P-1 plasmid RP4 and is also capable of conjugal transfer with low efficiency to different gramnegative microorganisms. The size of the plasmid (17.2 Kb) has been determined and its physical map has been constructed. The plasmid harbours the unique sites for restriction endonucleases BglII, HindIII, HpaI, KpnI, SmaI and XbaI cleawage. The plasmid derivatives pBS352-pBS355 have been obtained that carry kan- and cam-determinants in addition to tet-gene. Plasmid pBS355 has been used to clone EcoRI-fragments of phage lambda DNA. The plasmid pBS222 regions essential for replication and maintenance have been localized by DNA hybridization analysis of its mini-derivatives pBS356 and 357. pBS222 is a convenient model for investigations of the plasmid replication and maintenance mechanisms in different bacterial hosts as well as for the construction of broad host-range vectors.     

Cloning and localization of the replication and mobilization regions in the D-plasmid of Pseudomonas putida pBS286 (IncP-9) with a broad host range

Rep-mob loci of naphthalene degradative plasmid pBS286 (IncP-9) have been cloned on the Escherichia coli vectors pUC19 and pUBR322. These loci confer to recombinant plasmids pBS952 and pBS953 the ability for effective mobilization by RP4 (IncP-1) and F plasmid, as well as constant maintenance in various gram-negative bacteria. Localization of cloned sequences in the restriction fragments of conservative part of the pBS286 genome was established. The data obtained correlate with the analysis of plasmids pBS950 and pBS951 which are spontaneous mini-derivatives of pBS286 and pBS292 (delta NPL1::Tn1/Tra+ Nah-) plasmids formed during transformation of E. coli HB101 cells. Plasmids pBS952 and pBS953 retain the incompatibility properties of parental IncP-9 replicon. These recombinant derivatives can be used for construction of bhr vectors with required properties and compatible with bhr vectors constructed on the basis of plasmids from the IncP-1 and IncP-4 groups.     

Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.     

Inheritance of biodegradation plasmids in the cells of homo- and heterologous hosts

A systematic analysis of the inheritance of D plasmids of the IncP-9 group (α-, β-, γ-, δ-, ?-, ζ-, η-, and θ-subgroups), IncP-7, as well as of those of undefined systematic affiliation in the cells of homologous (Pseudomonas putida) and heterologous (Escherichia coli) hosts was performed for the first time. For this purpose, mini-Tn5 transposons determining resistance to kanamycin (or streptomycin) were introduced into all the D plasmids under study. It has been established that all IncP-9 plasmids can be transmitted to the cells of a heterologous host E. coli (with the exception of plasmid pSVS15 from gq-subgroup). IncP-7 plasmids and those of undefined systematic affiliation do not possess this property and can be transmitted and stably inherited only in P. putida. The distinctive feature of most IncP-9 plasmids (α-, β-, δ-, ?-, and ζ-subgroups) is strict dependence of their inheritance on the temperature factor. At 37°C, the plasmids of δ-, ζ-, and θ-subgroups are unstable in P. putida cells, while in E. coli nearly all plasmids of this systematic group are unstable. The exceptions are the plasmids of η- and γ-subgroups. Inheritance of these plasmids does not depend on temperature. At 28°C and 37°C, the η plasmid is not maintained stably (inheritance stability is 2%), while the γ-plasmid has almost 100% stability.     

Analysis of the conjugation system of plasmid pBS1001 with a broad range of hosts]

Tn5-induced tra mutations were localized on the physical map of a broad host range plasmid pBS1001. Mutations were united into three clusters covering 25% of plasmid DNA. They were distributed in 7 groups by complementation analysis. It was shown that coexistence of tra mutants of pBS1001 and RP4 within the same cell did not restore conjugation properties of both plasmids. High frequency mobilization of some known vectors by pBS1001 was demonstrated.     

Distal 9q trisomy phenotype in a patient with a supernumerary rearranged chromosome [t(X:9)] (author's transl)]

A child with clinical features associated a trisomy for the distal part of 9q was shown to have the following abnormal chromosome complement : 47,XY,+t)X;9) (Xpter yields Xq24:9q31 yields 9qter), inv 9(p11q13), var 14 (14pQFQ34).     

Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4

A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.