Tandem chromosomal duplications in Salmonella typhimurium: fusion of histidine genes to novel promoters.

Salmonella strains harboring tandem chromosomal duplications have been identified following selection for expression of a histidine biosynthetic gene whose promoter is deleted. In such strains, tandem duplications fuse the selected his gene to “foreign” regulatory elements, thereby allowing gene expression. Selection is made for hisD⁺ activity in deletion strain hisOG203. Among the revertants, strains harboring tandem chromosomal duplications have been identified by a number of their properties. (1) Their HisD⁺ phenotype is genetically unstable. (2) Such instability is dependent on recombination (recA) activity. (3) Genetic tests demonstrate that these strains are merodiploid for large regions (up to 25%) of the Salmonella genome. (4) Recipient strains that inherit the HisD⁺ phenotype of these duplication-carrying revertants also inherit the donor's merodiploid state. (5) In certain revertants the functional hisD⁺ gene and the sequence which promotes merodiploid transductant formation are linked to chromosomal markers located far from the normal his region.Previous reports have concluded that the instability of strains isolated by this selection is due to translocation of the hisD⁺ gene to an extrachromosomal element (the pi-histidine factor). We believe that in all strains we have tested (33 independent isolates) instability can better be accounted for as due to tandem duplication events which permit expression of hisD. At least two mechanisms are responsible for duplication formation. One mechanism is dependent on recombination function and generates identical revertants having a duplication of 16% of the chromosome. A second mechanism operates independently of recombination activity; individual duplications produced by this process have variable endpoints.     

Duplication processes in Saccharomyces cerevisiae haploid strains

Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.     

Stable production of human gastric lipase by chromosomal integration in the fission yeast Schizosaccharomyces pombe

Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette on a multicopy plasmid. Received: 27 March 1997 / Received revision: 13 August 1997 / Accepted: 25 August 1997     

Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

We describe here the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had a 5′-deleted pyrG upstream of the region targeted for tandem chromosomal duplication and a 3′-deleted pyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under nonselective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method.     

A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae

Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.     

Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium. II. Stimulation of duplication-loss from merodiploids.

Strains of Salmonella typhimurium which contain a duplication of approximately 30% of the genome may be obtained by a simple selective procedure. These strains are highly unstable, losing the duplication when grown on non-selective medium. In this paper we report that treatment of merodiploid bacteria with mutagenic agents stimulates the rate at which haploid segregants are obtained from merodiploid strains. The mutagens which have been tested for this effect are X-rays, ultraviolet light (UV), ethyl methanesulfonate (EMS), and the azaacridine half-mustard ICR-372.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.     

Generation of Tandem Direct Duplications by Reversed-Ends Transposition of Maize Ac Elements

Tandem direct duplications are a common feature of the genomes of eukaryotes ranging from yeast to human, where they comprise a significant fraction of copy number variations. The prevailing model for the formation of tandem direct duplications is non-allelic homologous recombination (NAHR). Here we report the isolation of a series of duplications and reciprocal deletions isolated de novo from a maize allele containing two Class II Ac/Ds transposons. The duplication/deletion structures suggest that they were generated by alternative transposition reactions involving the termini of two nearby transposable elements. The deletion/duplication breakpoint junctions contain 8 bp target site duplications characteristic of Ac/Ds transposition events, confirming their formation directly by an alternative transposition mechanism. Tandem direct duplications and reciprocal deletions were generated at a relatively high frequency (∼0.5 to 1%) in the materials examined here in which transposons are positioned nearby each other in appropriate orientation; frequencies would likely be much lower in other genotypes. To test whether this mechanism may have contributed to maize genome evolution, we analyzed sequences flanking Ac/Ds and other hAT family transposons and identified three small tandem direct duplications with the structural features predicted by the alternative transposition mechanism. Together these results show that some class II transposons are capable of directly inducing tandem sequence duplications, and that this activity has contributed to the evolution of the maize genome.     

On the mechanism of production of tandem genetic duplications in phage lambda

We have asked whether the mechanism by which tandem genetic duplications arise in the chromosome of phage lambda is inter- or intramolecular. Two parental phages carrying genetic markers at opposite ends of the phage chromosome have been grown in mixed infection, and progeny phages carrying newly-arising tandem duplications have been analysed to determine whether they carry the markers in parental or recombinant configuration. Ordinary genetic recombination of the markers has been prevented by mutations in the phage and host. Phages carrying tandem duplications are isolated by use of CsCl density gradients and an Escherichia coli strain that does not plate deletion phages. Of the duplication mutants isolated under these conditions, 13% carry the input markers in recombinant configuration. This suggests that tandem duplications can be produced via an intermolecular route which joins sequences originally present on different DNA molecules.     

Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

Isolation and Characterization of Xylan-Degrading Strains of Butyrivibrio fibrisolvens from a Napier Grass-Fed Anaerobic Digester

Six new xylanolytic bacterial strains have been isolated from a Napier grass-fed anaerobic digester. These strains were identified as Butyrivibrio fibrisolvens and were similar in many respects to ruminal isolates described previously. The new isolates exhibited a high degree of DNA homology with several ruminal strains of B. fibrisolvens. Xylan or xylose was required to induce the production of enzymes for xylan degradation, xylanase and xylosidase. Production of these enzymes was repressed in the presence of glucose. Xylanase activity was predominantly extracellular, while that of xylosidases was cell associated. The new isolates of B. fibrisolvens grew well in defined medium containing xylan as the sole carbon source and did not produce obvious slime or capsular layers. These strains may be useful for future genetic investigations.     

Isolation and characterization of bacteria capable of metabolizing lignin-derived low molecular weight compounds

Lignin, a major component of biomass, composed of homogeneous phenolic monomers and functions as a synthetic precursor in the production of specialty chemicals or polymers. In this study, bacterial strains that metabolize lignin-derived low molecular weight compounds (LLCs) were cultured which are capable of LLC bioconversion. We used an LLC mixture primarily composed of vanillin (VL), syringaldehyde (SA), vanillic acid (VA) and p-hydroxybenzoic acid which were prepared from a commercial alkaline lignin product. Enrichment culture was repeated twice in a medium containing the soil sample, the LLCs and inorganic salts. Three bacterial strains belonging to the genera Pseudomonas, Ochrobactrum, and Klebsiella were isolated. We found that only VL, SA, and VA were metabolized by the Pseudomonas strain, which was then found to grow in a medium with VL or VA as the sole source of carbon and energy. The VL isomers, namely, ovanillin and isovanillin were converted to the corresponding carboxylic acids but were not utilized as carbon sources by Pseudomonas. VL and VA are intermediates in the pathway of bacterial degradation of eugenol via ferulic acid. Several bacterial strains that metabolize VL, eugenol, and ferulic acid have been reported but such strains are rarely isolated from enrichment culture medium containing LLCs, due to insufficient induction by the precursors in the LLC medium. In this study, we demonstrated that the microorganisms involved in the bioconversion of LLCs can be isolated from simple enrichment culture.     

A neutron small-angle scattering study of bovine fibrinogen.

A number of glycyl-tRNA synthetase (glyS) mutants have been isolated as glycine auxotrophs in Salmonella typhimurium. One of the mutants, glyS141, has a glycyl-tRNA synthetase with a K_m for glycine that is 700 times higher than the wild-typeK_m. Prototrophic revertants glyS141 occur at high spontaneous frequencies (>5 × 10^?5). The majority of these revertants contain large tandem duplications including the mutant glyS gene. Some of the duplications cover at least 22% of the chromosome. The duplications overlap with a large duplication isolated previously by a different selection procedure (Straus &; Hoffmann, 1975). Evidence has been obtained which suggests that formation of the duplications may occur by recA-dependent recombination. The Gly⁺ phenotype of revertants carrying the duplications does not appear to be explainable simply by the increased gene dosage of glyS.     

Rearrangement of the Bacterial Chromosome Using Tn10 as a Region of Homology

The transposable tetracycline resistance element, Tn10, can serve as a region of homology to promote rec-dependent deletion, duplication and directed transposition of bacterial genes. Tn10 insertions in regions of the chromosome near the histidine operon (his) have been isolated and characterized in Salmonella typhimurium. When strains are constructed containing two Tn10 insertions flanking the his operon in the same orientation (Tn10-his-Tn10), recombination can occur between Tn10 sequences resulting in the deletion of the intervening his region. The sites of the Tn10 insertions determine the endpoints of the deletion. In crosses designed to construct strains carrying Tn10-his-Tn10, another class of unstable recombinants arises in which the his region exists in tandem duplication, with a Tn10 insertion joining the duplicated copies (his-Tn10-his). The sites of the parental Tn10 insertions mark the endpoints of the duplication. When a strain carrying Tn10-his-Tn10 is used as a donor of his⁺ in conjugation or P22-mediated transduction, recombinants can arise in which the his region has been transposed to the site of any Tn10 insertion, far from the normal location of his in the recipient chromosome. In this manner, the his operon has been moved to the site of a pyrB::Tn10 insertion and has been placed on F'' plasmids. At these new locations, the his⁺ character shows the rec-dependent deletion of his⁺ expected for a Tn10-his-Tn10 duplication. These methods should be generally useful for the manipulation of bacterial genes.     

Acetamide Selection of Kluyveromyces lactis Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.     

An intraspecific gene duplication polymorphism of the urate oxidase gene of Drosophila virilis: a genetic and molecular analysis

Nineteen strains of Drosophila virilis from diverse geographic locations were examined by genetic and molecular analyses, revealing (a) 12 strains with a single copy of the urate oxidase (UO) gene per haploid genome and (b) 7 strains with a tandem duplication of the UO locus. The D. virilis strains with the UO duplication appear to have identical restriction maps of this region, implying either a single origin for the duplication or several similar events occurring at a hot spot. On the basis of the location of the duplication breakpoints and the restriction sites flanking these breakpoints, this duplication probably arose through nonhomologous recombination involving either a breakage and rejoining event or replication slippage. because documented cases of intraspecific gene duplication polymorphism are rare, the D. virilis UO duplication will be useful in identifying the molecular event giving rise to a gene duplication.      

Mitogenomes of two neotropical bird species and the multiple independent origin of mitochondrial gene orders in Passeriformes

At least four mitogenome arrangements occur in Passeriformes and differences among them are derived from an initial tandem duplication involving a segment containing the control region (CR), followed by loss or reduction of some parts of this segment. However, it is still unclear how often duplication events have occurred in this bird order. In this study, the mitogenomes from two species of Neotropical passerines (Sicalis olivascens and Lepidocolaptes angustirostris) with different gene arrangements were first determined. We also estimated how often duplication events occurred in Passeriformes and if the two CR copies demonstrate a pattern of concerted evolution in Sylvioidea. One tissue sample for each species was used to obtain the mitogenomes as a byproduct using next generation sequencing. The evolutionary history of mitogenome rearrangements was reconstructed mapping these characters onto a mitogenome Bayesian phylogenetic tree of Passeriformes. Finally, we performed a Bayesian analysis for both CRs from some Sylvioidea species in order to evaluate the evolutionary process involving these two copies. Both mitogenomes described comprise 2 rRNAs, 22 tRNAs, 13 protein-codon genes and the CR. However, S. olivascens has 16,768 bp showing the ancestral avian arrangement, while L. angustirostris has 16,973 bp and the remnant CR2 arrangement. Both species showed the expected gene order compared to their closest relatives. The ancestral state reconstruction suggesting at least six independent duplication events followed by partial deletions or loss of one copy in some lineages. Our results also provide evidence that both CRs in some Sylvioidea species seem to be maintained in an apparently functional state, perhaps by concerted evolution, and that this mechanism may be important for the evolution of the bird mitogenome.     

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa.     

Improved Method for Detection of Vibrio parahaemolyticus in Seafood

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.