The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

In this study, we explored whether SYBR Green-based quantitative real-time PCR (qPCR) could be used to determine the copy number of a plasmid and whether the method was broadly applicable to chromosomally encoded genetic elements often occurring in multiple copies, such as rRNA genes and insertion sequences (IS). Three different template sources (whole cells, total DNA, and restriction-enzyme digested total DNA) derived from the bacterium Comamonas sp. strain JS46 were analyzed by qPCR using primer-pairs targeting plasmid pJS46 and three chromosomally encoded sequences (16S rDNA, ISCsp1, and IS1071). The difference between threshold cycle values, C(T), of amplicons targeting these elements and of an amplicon targeting the single-copy reference element mnbA (chromosomally encoded) was used to establish DeltaC(T). DeltaC(T) values were then used to derive copy number. For pJS46, qPCR analyses of whole cells and total DNA underestimated the copy number of pJS46 approximately 7-fold and approximately 2.5-fold, respectively, whereas copy number values derived from qPCR analyses of digested total DNA were comparable to those derived from Southern blot (SB) analyses. In contrast, for the chromosomally encoded elements, qPCR analyses of all three template sources gave copy number values that were virtually identical to or differed by approximately 2 from copy number values derived by SB analysis. These data indicate that qPCR can be used to estimate the copy number of various genetic elements but that the accuracy of qPCR-derived values is affected by the template source.     

Fitness drift of an atrazine-degrading population under atrazine selection pressure

Pseudomonas sp. ADP harbouring the atrazine catabolic plasmid ADP1 was subcultured in liquid medium containing atrazine as sole source of nitrogen. After approximately 320 generations, a new population evolved which replaced the initial population. This newly evolved population grew faster and degraded atrazine more rapidly than the initial population. Plasmid profiles and Southern blot analyses revealed that the evolved strain, unlike the ancestral strain, presented a tandem duplication of the atzB gene encoding the second enzyme of the atrazine catabolic pathway responsible for the transformation of hydroxyatrazine to N-isopropylammelide. This duplication resulted from a homologous recombination that occurred between two direct repeats of 6.2 kb flanking the atzB gene and constituted by the insertion sequences IS 1071 , IS Pps1 and a pdhL homologous sequence. This study highlights the IS-mediated plasticity of atrazine-degrading potential and demonstrates that insertion sequences not only help to disperse the atrazine-degrading gene but also improve the fitness of the atrazine-degrading population.     

Analysis of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pEST4011 of Achromobacter xylosoxidans subsp. denitrificans strain EST4002

The 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002, isolated in Estonia more than 10years ago, was found to contain the 70kb plasmid pEST4011 that is responsible for the bacterium having had obtained a stable 2,4-D(+) phenotype. The tfd-like genes for 2, 4-D degradation of the strain EST4002 were located on a 10.5kb region of pEST4011, but without functional genes coding for chloromuconate cycloisomerase and chlorodienelactone hydrolase. The latter two genes are probably encoded by homologous, tcb-like genes, located elsewhere on pEST4011. We also present evidence of two copies of insertion element IS1071-like sequences on pEST4011. IS1071 is a class II (Tn3 family) insertion element, associated with different catabolic genes and operons and globally distributed in the recent past. We speculate that this insertion element might have had a role in the formation of plasmid pEST4011. The 28kb plasmid pEST4012 is generated by deletion from pEST4011 when cells of A. xylosoxidans EST4002 are grown in the absence of 2,4-D in growth medium. We propose that this is the result of homologous recombination between the two putative copies of IS1071-like sequences on pEST4011.     

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.     

Large Inversion in Escherichia Coli K-12 1485in between Inversely Oriented Is3 Elements near Lac and Cdd

A companion study has shown that the inversion carried by strain 1485IN has one terminus between lac and proC and the other between his and cdd of the normal strain. Starting with this mapping data, we have done molecular work demonstrating that the inversion occurred by recombination between inversely oriented two IS3 elements, one present near lac and the other near the cdd locus; i.e., the inversion is IN(is3B-is3E). Evidence supporting this conclusion includes: (i) Normal and inversion strains share two short regions with identical restriction maps. One of these regions is near lac and the other near cdd. (ii) IS3 homology was detected in each of the terminus regions of both the normal and inversion strains. (iii) The sequence on one side of the original IS3 element near lac has been exchanged with the sequence on one side of the IS3 near cdd. Whether the inversion has occurred by one event of homologous recombination between the two IS3 elements or has been caused by involvement of IS3 elements on an F factor is discussed. Another rearrangement, probably related to inversion and deletion, was detected between the IS3 and cdd of the inversion strain.     

Joint Distribution of Insertion Elements Is4 and Is 5 in Natural Isolates of ESCHERICHIA COLI

A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurrence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 +/- 0.8; the comparable figure for IS5 was 3.7 +/- 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements. The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.     

On the role of IS1 in the formation of hybrids between the bacteriophage P1 and the R plasmid NR1

Summary The genomes of bacteriophage P1 derivatives carrying drug resistance genes derived from an R plasmid NR1 were analysed by restriction cleavage and be DNA-DNA hybridization. Two representatives of a class of oversized P1CmSmSu phages were identified as P1 carrying the entire r-determinant of NR1 together with its two flanking, directly repeated IS1. In one case the r-determinant insertion is carried at the site of the residential IS1 of P1, in the other case it is transposed into another region of the P1 genome. Models postulate that the first type resulted from reciprocal recombination within IS1 elements and that the formation of the second type of P1-R hybrid depended both on IS1 mediated transposition and reciprocal recombination. Plaque forming P1Cm or P1CmSm phages are explained as IS1 mediated deletion derivatives of P1CmSmSu, although an alternative model postulates that sometimes P1Cm phages might result from two consecutive transposition events of only one IS1 without involving reciprocal recombination. Secondary P1 derivatives carrying only one IS1 at the site of the original r-determinant or of Cm insertions into P1 must have been produced by reciprocal recombination between the two IS1 flanking the insertions. An implication from this study, that any genetic material carried adjacent to an IS1 element may undergo passive transposition, is discussed.     

Evolution of a chlorobenzene degradative pathway among bacteria in a contaminated groundwater mediated by a genomic island in Ralstonia

The genetic structure of two Ralstonia spp., strain JS705 and strain JS745, isolated from the same groundwater aquifer, was characterized with respect to the degradation capacities for toluene and chlorobenzene degradation. Cosmid library construction, cloning, DNA sequencing and mating experiments indicated that the genes for chlorobenzene degradation in strain JS705 were a mosaic of the clc genes, previously described for Pseudomonas sp. strain B13, and a 5 kb fragment identical to strain JS745. The 5 kb fragment identical to both JS705 and JS745 was flanked in JS705 by one complete and one incomplete insertion (IS) element. This suggested involvement of the IS element in mobilizing the genes from JS745 to JS705, although insertional activity of the IS element in its present configuration could not be demonstrated. The complete genetic structure for chlorobenzene degradation in strain JS705 resided on a genomic island very similar to the clc element (Ravatn, R., Studer, S., Springael, D., Zehnder, A.J., van der Meer, J.R. 1998. Chromosomal integration, tandem amplification, and deamplification in Pseudomonas putida F1 of a 105-kilobase genetic element containing the chlorocatechol degradative genes from Pseudomonas sp. strain B13. J Bacteriol 180: 4360-4369). The unique reconstruction of formation of a metabolic pathway through the activity of IS elements and a genomic island in the chlorobenzene-degrading strain JS705 demonstrated how pathway evolution can occur under natural conditions in a few 'steps'.     

High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements

We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon Sulfolobus solfataricus in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10(-4) and 10(-5) per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal pyrEF locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS4-IS5 IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different S. solfataricus strains, no homologous sequences were identified in DNA of other well-characterized strains of the order Sulfolobales.     

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences.

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.     

Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines.     

Insertion sequence inversions mediated by ectopic recombination between terminal inverted repeats

Transposable elements are widely distributed and diverse in both eukaryotes and prokaryotes, as exemplified by DNA transposons. As a result, they represent a considerable source of genomic variation, for example through ectopic (i.e. non-allelic homologous) recombination events between transposable element copies, resulting in genomic rearrangements. Ectopic recombination may also take place between homologous sequences located within transposable element sequences. DNA transposons are typically bounded by terminal inverted repeats (TIRs). Ectopic recombination between TIRs is expected to result in DNA transposon inversions. However, such inversions have barely been documented. In this study, we report natural inversions of the most common prokaryotic DNA transposons: insertion sequences (IS). We identified natural TIR-TIR recombination-mediated inversions in 9% of IS insertion loci investigated in Wolbachia bacteria, which suggests that recombination between IS TIRs may be a quite common, albeit largely overlooked, source of genomic diversity in bacteria. We suggest that inversions may impede IS survival and proliferation in the host genome by altering transpositional activity. They may also alter genomic instability by modulating the outcome of ectopic recombination events between IS copies in various orientations. This study represents the first report of TIR-TIR recombination within bacterial IS elements and it thereby uncovers a novel mechanism of structural variation for this class of prokaryotic transposable elements.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Evolution of a Pathway for Chlorobenzene Metabolism Leads to Natural Attenuation in Contaminated Groundwater

Complete metabolism of chlorinated benzenes is not a feature that is generally found in aerobic bacteria but is thought to be due to a novel recombination of two separate gene clusters. Such a recombination could be responsible for adaptation of a natural microbial community in response to contamination with synthetic chemicals. This hypothesis was tested in a chlorobenzene (CB)-contaminated aquifer. CB-degrading bacteria from a contaminated site were characterized for a number of years by examining a combination of growth characteristics and DNA-DNA hybridization, PCR, and DNA sequence data. The genetic information obtained for the CB pathway of the predominant microorganism, Ralstonia sp. strain JS705, revealed a unique combination of (partially duplicated) genes for chlorocatechol degradation and genes for a benzene-toluene type of aromatic ring dioxygenase. The organism was detected in CB-polluted groundwater by hybridizing colonies cultivated on low-strength heterotrophic media with probes for the CB pathway. Southern hybridizations performed to determine the organization of the CB pathway genes and the 16S ribosomal DNA indicated that CB-degrading organisms isolated from different wells at the site were identical to JS705. Physiological characterization by the Biolog test system revealed some differences. The genes for the aromatic ring dioxygenase and dihydrodiol dehydrogenase of JS705 were detected in toluene and benzene degraders from the same site. Our results suggest that recent horizontal gene transfer and genetic recombination of existing genes between indigenous microorganisms were the mechanisms for evolution of the catabolic pathway. Evolution of the CB pathway seems to have created the capacity for natural attenuation of CB at the contaminated site.     

Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in <Emphasis Type="Italic">Terrabacter</Emphasis> sp. strain DBF63

Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.     

Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae.

A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.