RESUMEN
The expression of a new acylamidase gene from R. erythropolis 37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4'-nitroacetanilide, N-isopropylacrylamide, N'N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrilhydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system.
Asunto(s)
Amidohidrolasas , Proteínas Bacterianas , Expresión Génica , Plásmidos/genética , Rhodococcus , Amidohidrolasas/biosíntesis , Amidohidrolasas/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Rhodococcus/enzimología , Rhodococcus/genética , Especificidad por SustratoRESUMEN
The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.
Asunto(s)
Amidohidrolasas/genética , Escherichia coli/genética , Rhodococcus/genética , Secuencia de Aminoácidos , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Rhodococcus/enzimología , Homología de Secuencia de AminoácidoRESUMEN
The amidase gene from Rhodococcus rhodochrous M18 was cloned by PCR amplification with primers developed by use of peptide amino acid sequences obtained after treating amidase with trypsin. Nucleotide sequence analysis of this gene revealed high homology with aliphatic amidases from R. erythropolis R312 and Pseudomonas aeruginosa. Considering the substrate specificity and the results of DNA analysis, amidase from R. rhodochrous M8 was assigned to the group of aliphatic amidases preferentially hydrolyzing short-chain aliphatic amides. The amidase gene was expressed in cells of Escherichia coli from the self promoter and from the lac promoter. To clone a fragment of R. rhodochrous M8 chromosome (approximately 9 kb), containing the entire structural gene and its flanking regions, plasmid pRY1 that can be integrated into the chromosome via homology regions was used. No sequences of the nitrile hydratase gene, the second key gene of nitrile degradation in strain R. rhodochrous M8, were detected. Thus, genes encoding amidase and nitrile hydratase in strain R. rhodochrous M8 are not organized into a single operon despite their common regulation.
Asunto(s)
Amidohidrolasas/genética , Genes Bacterianos , Rhodococcus/enzimología , Amidohidrolasas/biosíntesis , Amidohidrolasas/clasificación , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Operón/genética , Filogenia , Regiones Promotoras Genéticas , Rhodococcus/genéticaRESUMEN
Nucleotide sequence of cryptic plasmid pN30 from a Rhodococcus erythropolis 30 soil isolate was determined. Plasmid DNA consists of 5403 nucleotide pairs and contains about 62% GC pairs, which is typical of Rhodococcus DNA. No significant homology was determined between the pN30 DNA sequence and those of known plasmids. Computer-aided analysis of pN30 sequence revealed open reading frames that encode proteins strongly homologous to replicative proteins encoded by small cryptic plasmids of different actinomycetes.
Asunto(s)
ADN Bacteriano/genética , Sistemas de Lectura Abierta/genética , Plásmidos/genética , Rhodococcus/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
New phages K104 and B26, which are relative to F116L by a number of biological characters, appeared to show general transducing activity. Phage K104 transduces all tested markers with higher frequency than the phage B26. Linkage of the bacterial markers pair ilv202--met28 durspectively. When recipient bacteria lysogenic for phages K104 and B26 are used, frequencies of transduction by phage F116L are decreased. In the presence of F116L prophage the frequency of transduction by phage B26 is 10-fold increased. Phages B26 and F116L do not grow on bacteria lysogenic for these phages. Phage F116L does not grow on the lawn of bacteria, lysogenic for phage K104, while phage B26 grows on the same lawn with the efficiency of plating about 10(-2).
Asunto(s)
Bacteriófagos/genética , Pseudomonas aeruginosa/genética , Transducción Genética , Bacteriófagos/inmunología , Marcadores Genéticos , LisogeniaRESUMEN
To reveal possible differences in adsorptional specificities of transposable phages of Pseudomonas aeruginosa and to study the genetical control of this character, we isolated a group of phage-resistant P. aeruginosa mutants using some temperate and virulent phages. The study of resistance of the mutants to all the phages permitted us to find some types of mutants and to build a formal scheme of distribution of adsorptional receptors on the surface of P. aeurginosa cell. According to the results obtained, there are two main "receptor chains", where the receptors for all phages under study are grouped. For the majority of phages, just a single adsorptional receptor is obligatory, and at least two essential receptors are needed for adsorption of virulent phage E79. Two receptors were found also for another virulent phage, phi 11, one of them only being essential. Transposable phages can be grouped into three types, according to their adsorptional specificities. No correlations of adsorptional specificity types and all other characteristics of transposable phages studied (including the sub-groups of transposable phages belonging to different DNA homology types) were found. Genes of natural transposable phages controlling the differences in adsorptional specificities revealed can recombine in phage crosses.
Asunto(s)
Alelos , Bacteriófagos/genética , Genes Bacterianos , Genes Virales , Pseudomonas aeruginosa/genética , Adsorción , Mutación , Plásmidos , Recombinación GenéticaRESUMEN
Catechol 2,3-dioxygenase gene (xylE) was cloned into the Pseudomonas aeruginosa miniphage D3112 delta Hcts. Miniphage provides an effective transduction of gene xylE and its transposition into different replicons. It can be used as an integrative vector in the P. aeruginosa cells. Besides, gene xylE is rather convenient genetic marker for identification of miniphage both in Excherichia coli and Pseudomonas species.
Asunto(s)
Bacteriófagos/genética , Dioxigenasas , Vectores Genéticos , Pseudomonas aeruginosa/genética , Integración Viral/genética , Catecol 2,3-Dioxigenasa , Clonación Molecular , Escherichia coli/genética , Marcadores Genéticos , Oxigenasas/genética , Transducción GenéticaRESUMEN
Conjugative transfer of RP4 plasmid into Pseudomonas aeruginosa PAO at 30 degrees C results in inhibition of growth of plasmid-containing cells. No inhibition of growth of exconjugants takes place under special conditions (usage of minimal M9 medium or incubation at 42 degrees C) and when cells have specific mutations. These mutations were designated as rpm (RP4 maintenance). Some of them render cells resistance to temperate phages of P. aeruginosa. The frequency of transfer of RP4 into PAO1rpm, in comparison with that of transfer into PAO1rpm+, is increased 7-10 times and is equal 0.8-0.9 per a donor cell.
Asunto(s)
Conjugación Genética , Plásmidos , Pseudomonas aeruginosa/genética , Antibacterianos/farmacología , Conjugación Genética/efectos de los fármacos , Medios de Cultivo/farmacología , Escherichia coli/genética , Mutación , Plásmidos/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , TemperaturaRESUMEN
It is found that bacteriophages B3 and B39 specific for Pseudomonas aeruginosa have the same genome structure as previously described phage D3112. On the right (S) end of their genomes a variable non-phage DNA is located (approximately 0.9-2.5 kilobases for different phages). It is probable that this variable DNa has its origin from different regions of bacterial chromosome. In genome of one of the phages, B3 phage, such variable DNA (not more than 150 base pairs) was found on the left end of DNA molecule. Isolation of a viable B3XD3112 recombinant phage and analysis of its genome with restriction technique and with studies of homo- and heteroduplex molecules had confirmed genetical relationship of B3 and D3112. Some essential non-homology of B3 and D3112 DNAs have been found on the right ends of genomes of the phages.
Asunto(s)
Bacteriófagos/genética , ADN Viral , Genes Virales , Pseudomonas aeruginosa/genética , Enzimas de Restricción del ADN , Microscopía Electrónica , Mutación , Ácidos Nucleicos Heterodúplex/análisis , Recombinación Genética , Especificidad de la EspecieRESUMEN
Using a large group of newly isolated deletion mutants of prophage D3112 the location of all known mutations of D3112 phage was more precisely defined. The mutations affecting establishment of lysogenic state were mapped in two regions of the genome- 0-1.3 and 29-30 kb. The replicative A gene is mapped between 1.3 and 4.9 kb, the second replicative B gene being situated on the right of the A gene, between 4.9 and 9.4 kb. The C gene which is responsible for positive regulation of phage late genes' expression is mapped within the 9-12 kb region. It is suggested that promoter of the gene C is situated within the same interval. Mutations were isolated in the Lys gene which is responsible for host cell lysis. The gene is located within the interval 14-22 kb of the physical map. The order of morphogenetic genes in the late genome region was also established.
Asunto(s)
Bacteriófagos/genética , Mapeo Cromosómico , Elementos Transponibles de ADN , Deleción Cromosómica , Lisogenia , Mutación , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genéticaRESUMEN
In vivo transcription of Pseudomonas aeruginosa transposable phage D3112 was studied. The 3H-labelled RNA isolated from the lysogenic cells PAO1 (D3112cts) after heat induction was hybridized with D3112 DNA to estimate phage-specific RNA. Two main stages of D3112 transcription, including transcription of the early (first 6-8 min) and the late (after 8th min) genes, were revealed. The transcription rate of D3112 early genes achieves the maximum at 3-5 min, being reduced to the minimum at 6-8 min after heat induction. These data point to the existence of negative regulation of D3112 early genes' transcription. The influence of ts mutations in early A, B and C genes and in late ts25 gene on transcription of D3112 phage was studied. It is shown that the genes A and B have no effect on the late transcription, while gene C regulates transcription of the D3112 late genes.
Asunto(s)
Bacteriófagos/genética , Elementos Transponibles de ADN , Genes Virales , Transcripción Genética , Mutación , Pseudomonas aeruginosa/genética , Proteínas Virales/genéticaRESUMEN
Two derivatives of D3112 prophage with large internal deletions (mini-D3112) have been constructed. Mini-D3112 delta H is about 3.5 kb long, containing the repressor c1 gene. Mini-D3112 delta E is about 12 kb long, contains the c1 gene, several structural genes and replication gene A. These mini-D3112 are unable to replicate. However, they could replicate and maturated in the presence of the helper D3112cts. Mini-D3112 mediate translocation of the gene argH from the chromosome into the R' plasmids, the translocated fragment being sandwiched between two mini-D3112 genomes.
Asunto(s)
Bacteriófagos/genética , Elementos Transponibles de ADN , Genes Virales , Deleción Cromosómica , ADN Viral/genética , Genes , Plásmidos , Pseudomonas aeruginosa , Supresión Genética , Translocación GenéticaRESUMEN
D3112 phage was shown to replicate via the process of coupled replication--transposition: the phage DNA is not excised from the chromosome after prophage induction and new phage copies insert into many different sites. The transposition is controlled by two D3112 early genes--A (mapped in the 1.5-3 kbp region) and B (3-4.5 kbp), and requires intact attL site (involvement of the phage right end attR not studied). D3112 is capable to transpose RP4 plasmid into the chromosome; both the D3112 and RP4 transpositions are rec-independent. The product of the early C gene which is not required for D3112 transposition has pleiotropic effect on the development of D3112 and is necessary for the process of D3112 DNA excision from the chromosome, for cell lysis as well as for mature phage production. We suggest that this gene is responsible for positive regulation of D3112 late genes expression, similar to the C gene of Mu phage or Q gene of lambda. Mutations in four D3112 late genes ts25, ts35, ts73 and ts110 do not affect transposition or excision processes. No detectable (less than 0.02 copies per cell) amount of linear or circular D3112 DNA is formed during the replication--transposition. Hence, in the course of replication and transposition processes D3112 genome has its ends permanently bound covalently to the chromosome. The excision of the D3112 DNA takes place at late stages.
Asunto(s)
Bacteriófagos/genética , Replicación del ADN , Elementos Transponibles de ADN , Genes Virales , Replicación Viral , Bacteriófagos/fisiología , ADN Viral/genética , Plásmidos , Pseudomonas aeruginosa/genética , Recombinación GenéticaRESUMEN
Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization.
Asunto(s)
Bacteriófagos/genética , Elementos Transponibles de ADN , Genes Virales , Lisogenia , Bacteriófagos/crecimiento & desarrollo , Mapeo Cromosómico , Mutación , Fenotipo , Plásmidos , Pseudomonas aeruginosa/genéticaRESUMEN
Several hybrid RP4 plasmids containing the genome of heat-inducible D3112cts15 phage integrated into 2 different sites of RP4 were selected. It was shown that the plasmids RP4::D3112cts15 mobilized the chromosome of Pseudomonas aeruginosa from many sites located in different chromosome regions. Chromosomal recombinants are, formed at frequencies of about 10(-4) per recipient cell. Analysis of coinheritance of unselected markers showed that the majority of recombinants inherited short donor chromosome fragments (about 5 min). R' plasmids can be easily selected by mating with a rec- recipient. For instance, the frequency of selection of R' plasmids containing argH+ locus was about 10(-5) per donor cell. Conjugative transfer of RP4::D3112cts15 into nonlysogenic strains PAO P. aeruginosa results in partial or complete loss of prophage from a hybrid plasmid. The RP4::D3112cts15 plasmids appear to have retained the broad host range of the original RP4 (they are maintained in P. putida and Escherichia coli).
Asunto(s)
Vectores Genéticos , Pseudomonas aeruginosa/genética , Factores R , Bacteriófagos/genética , Clonación Molecular , Escherichia coli/genética , Marcadores GenéticosRESUMEN
The influence of ts mutations in the early and late genes of transposable phage D3112 on phage morphogenesis was studied. The mutations in the early genes A, B and C were shown to suppress morphogenesis of D3112. Six genes (D, E, F, G, H and I), located from 14 to 29 kbp of the phage physical map, control morphogenesis of phage head. Five genes (J, K, L, M and N), clustered in the 29-36 kbp region of the map, control morphogenesis of tail. The similarity of genetic organization of the Escherichia coli transposable phage Mu and the Pseudomonas aeruginosa phage D3112 is discussed.
Asunto(s)
Bacteriófagos/genética , Genes Virales , Mutación , Bacteriófagos/ultraestructura , Microscopía Electrónica , Morfogénesis/genética , Pseudomonas aeruginosa/genéticaRESUMEN
Plasmid DNA transduction with mini-D3112 delta H, deletion derivative of phage D3112, which lost the genes essential for phage growth but retained the sites required for transposition and packaging was studied. Unlike D3112, mini-D3112 delta H element can transduce plasmids and plasmid markers at frequencies of 10(-5)-10(-8) in rec+ cells of Pseudomonas aeruginosa. Plasmids R1162 and R388 of the size smaller than phage genome were transduced intact. Large plasmids, like RP4 and R151, were deleted under transduction. By this way, we isolated deletion derivatives of RP4. The smallest derivative pN2 contained a 4.5 kb fragment of RP4. Unlike the latter, pN2 plasmid had narrow host range and did not maintain in Escherichia coli cells.
Asunto(s)
Bacteriófagos/genética , Plásmidos , Pseudomonas aeruginosa/genética , Transducción Genética , ADN Viral/ultraestructura , Electroforesis en Gel de Agar , Eliminación de Gen , Microscopía Electrónica , Mapeo RestrictivoRESUMEN
A novel, previously unknown cip gene of Pseudomonas aeruginosa phage D3112 was discovered. The cip gene is responsible for inhibiting the intracellular growth of the related heteroimmune phage B39 of P. aeruginosa. It was shown that the cip gene product inhibits replication of the B39 genome, interacting, apparently, with the definite s+ site in the phage B39 genome. Mutants cip- of phage D3112, incapable of inhibiting the growth of phage B39, and B39 mutants, insensitive to the action of the cip gene product of phage D3112, were isolated.
Asunto(s)
Elementos Transponibles de ADN , Fagos Pseudomonas/genética , ADN Viral/biosíntesis , Genes Recesivos , Genes Virales , Lisogenia , Mutación , Fagos Pseudomonas/crecimiento & desarrollo , Activación ViralRESUMEN
Bacterial cells lysogenic for D3112, a transposable Pseudomonas aeruginosa phage restrict the growth of a related heteroimmune B39 phage. The lysogens are divided into two different types PAO(D3112). In the lysogens of the type I the efficiency of B39 growth only decreases slightly, the lysogens of the type II restricting completely the growth of this phage (e.o.p. is less than 10(-7). As shown by the results of Southern hybridization experiments, lysogens of the type I are monolysogens, while those of the type II are double or polylysogens. Restriction of B39 in PAO(D3112) is caused by expression of a locus in the D3112 genome. The locus has been termed as cip (control of interaction of phages). The cip locus was mapped at the interval 1.3-2.45 kb of the D3112 physical map using different deletion derivatives of D3112. Expression of cip only takes place in the prophage state and not during the phage lytic development. When expressed, cip affects the early steps in the growth of B39 lowering the level of integration and transposition processes; the effect is not dependent on the way of initiation of the lytic cycle (through prophage induction or infection).
Asunto(s)
Bacteriófagos/genética , Elementos Transponibles de ADN , Lisogenia , Mapeo Cromosómico , Cromosomas Bacterianos , ADN Viral/genética , Genes Virales , Pseudomonas aeruginosa/genéticaRESUMEN
Expression of transposable phages (TP) of Pseudomonas aeruginosa in the cells of P. putida was studied. The high efficiency of phage lytic development was shown both as a consequence of zygotic induction after transfer of the RP4::TPc+ plasmid into nonlysogenic recipients, and as a result of heat induction of lysogens PpG1 (D3112cts15). The high phage yield (20-25 particles of D3112cts phage per one cell of P. putida) is an evidence for a high level of transposition in the cells of this bacterial species. Plasmids RP4::TP are transferred into cells of PpG1 and PAO1 with similar frequency. However, the efficiency of establishment of the lysogenic state is lower in PpG1. Transposable phages of P. aeruginosa can integrate into the chromosome of PpG1 producing stable inducible lysogens. The presence of RP4 in the P. putida cells is not necessary for expression of transposable phages. The transposable phage D3112cts15 can be used in experiments of interspecies transduction of plasmids and chromosomal genes.