Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5'' coding region of the adenylate cyclase gene.

Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1.     

Fine Structure Genetic and Physical Map of the Phage P22 Tail Protein Gene

Bacteriophage P22 which are incapable of making functional tail protein can be propagated by the addition of purified mature tail protein trimers to either liquid or solidified medium. This unique in vitro complementation condition has allowed us to isolate 74 absolute lethal tail protein mutants of P22 after hydroxylamine mutagenesis. These phage mutants have an absolute requirement for purified P22 tail protein to be present in a soft agar overlay in order to form plaques and do not grow on any nonsense suppressing strains of Salmonella typhimurium. In order to genetically map and physically locate these mutations we have constructed two complementary sets of fine structure deletion mapping strains using a collection of Tn1 insertions in gene 9, the structural gene for the tail protein. Fourteen bacteriophage P22 strains carrying unique Tn1 transposon insertions (Ap phage) in gene 9 have been crossed with Ap phage carrying Tn1 insertions in gene 20. Phage carrying deletions that arose from homologous recombination between the Tn1 elements were isolated as P22 lysogens. The deletion prophage were shown to be missing all genetic information bracketed by the parental Tn1 elements and thus form a set of deletions into gene 9 from the 5' end of the gene. From the frequency of production of these deletion phage the orientation of the Tn1 insertions in gene 9 could be deduced. The genetic end points of the deletions in gene 9 and thus the order of Tn1 insertions were determined by marker rescue experiments using the original Ap phage. The genetic end points of the deletions in gene 20 were determined in similar experiments using nonsense mutations in gene 20. To locate the physical end points of these deletions in gene 9, DNA containing the Tn1 element has been cloned from each of the original Ap phage into plasmids. The precise point of insertion of Tn1 into gene 9 was determined by restriction enzyme mapping and DNA sequencing of the relevant portions of each of these plasmids. In vitro deletion of different 3' gene 9 sequences in the plasmid clones was accomplished through the use of unique restriction endonuclease sites in Tn1. The resulting plasmids form a set of deletions extending into the 3' end of the gene which are complementary compared to the deletion lysogens.(ABSTRACT TRUNCATED AT 400 WORDS)     

A mutation of the wobble nucleotide of a bacteriophage T4 transfer RNA.

Wild-type bacteriophage T7 is not subject to restriction by the Escherichia coli B and K restriction systems, but T7 mutants that are susceptible to such restriction have been isolated. These mutants are all defective in gene 0.3, the first T7 gene to be expressed after infection. The gene 0.3 protein apparently acts to prevent modification as well as restriction, suggesting that it may interact with a component of the host restriction-modification system that is required for both processes. Mutants in which gene 0.3 is completely deleted are only partially modified by growth on hosts with an active restriction-modification system, presumably because the conditions of T7 infection overload the modifying capacity of the cells. This is in contrast to phages such as lambda that are completely modified during growth. Since gene 0.3 is not essential for growth in non-restricting hosts, it has been possible to isolate deletions which extend to the left of gene 0.3 into the region where E. coli RNA polymerase initiates the synthesis of T7 early RNA. Two of the three strong initiators from which E. coli RNA polymerase transcribes the early region can be deleted.In the course of searching for T7 mutants that are unable to overcome restriction, it was discovered that mutants defective in gene 2 are able to plate on E. coli C with essentially normal efficiency, and most gene 7 mutants are able to plate on both C and K strains. It has not been determined why genes 2 and 7 seem to be needed for growth in some E. coli strains but not in others.     

Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline

Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 microg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 microg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.     

Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322

The Tn10 tetracycline resistance gene, tetA, encodes a tetracycline-inducible protein with an apparent Mr of 36 X 10(3). We have determined the nucleotide sequence of the Tn10 tetA gene. The extent of the tetA gene was determined by analysis of amino-terminal and carboxy-terminal deletion mutants. We conclude that a single Tn10 gene, the tetA gene, is sufficient to confer tetracycline resistance. The predicted Mr of the tetA protein is 43.2 X 10(3). The sequence homology between the Tn10 tetA gene and the pBR322 tetracycline resistance determinant (49% nucleotide homology, 44% amino acid homology) indicates that these phenotypically distinct tetracycline-resistance determinants must have evolved from a common ancestral sequence. The markedly hydrophobic character of the predicted amino acid sequences of the Tn10 tetA and pBR322 tet-coded proteins suggests that a substantial portion of these proteins may be embedded within the cytoplasmic membrane.     

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption. I. Isolation and genetic characterization

Nuclear disruption in T4 phage-infected Escherichia coli as well as the morphology of the nuclear regions in uninfected E. coli can be observed by phase microscopy of cells spread on a thin layer of 17.5% gelatin. We have used this procedure to identify for the first time mutants of phage T4 which fail to induce nuclear disruption. The mutant phenotypes have been further characterized by thin-section electron microscopy.Nuclear disruption is not essential for phage growth. Burst-size and growth-rate experiments indicate that the nuclear disruption-deficient (ndd) mutants grow as well as wild-type T4D under the conditions and in the E. coli strains commonly used in our laboratory.Mapping experiments using multiple amber mutants and rII mutants with deletions extending into the D region adjacent to the rIIB gene indicate that the ndd mutations are located in gene D2b.     

Interactions between stressful environment and gene deletions alleviate the expected average loss of fitness in yeast

The conjecture that the deleterious effects of mutations are amplified by stress or interaction with one another remains unsatisfactorily tested. It is now possible to reapproach this problem systematically by using genomic collections of mutants and applying stress-inducing conditions with a well-recognized impact on metabolism. We measured the maximum growth rate of single- and double-gene deletion strains of yeast in several stress-inducing treatments, including poor nutrients, elevated temperature, high salinity, and the addition of caffeine. The negative impact of deletions on the maximum growth rate was relatively smaller in stressful than in favorable conditions. In both benign and harsh environments, double-deletion strains grew on average slightly faster than expected from a multiplicative model of interaction between single growth effects, indicating positive epistasis for the rate of growth. This translates to even higher positive epistasis for fitness defined as the number of progeny. We conclude that the negative impact of metabolic disturbances, regardless of whether they are of environmental or genetic origin, is absolutely and relatively highest when growth is fastest. The effect of further damages tends to be weaker. This results in an average alleviating effect of interactions between stressful environment and gene deletions and among gene deletions.     

Genes aroA and serC of Salmonella typhimurium constitute an operon.

Genetic analysis of aroA554::Tn10 derivatives of two mouse-virulent Salmonella typhimurium strains, "FIRN" and "WRAY," and of a nonreverting derivative of each constructed for use as a live vaccine, showed the site of the insertion among mapped aroA point mutants. The WRAY live-vaccine strain gave no aro+ recombinants in crosses with aroA point mutations to one side of the insertion, indicating a deletion from Tn10 through the sites of these point mutations. The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants; it probably has a deletion or inversion extending from Tn10 into aroA but not as far as the nearest point mutation. Some tetracycline-sensitive mutants of aroA554::Tn10 strains required serine and pyridoxine, indicating loss of serC function, and some that were found to be SerC- did not produce gas from glucose, indicating a loss of pfl function. These results show the gene order pfl-serC-aroA, as in Escherichia coli. Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with Tn10 insertions near aroA (i.e., zbj::Tn10 strains) yielded Aro- SerC- Pfl-, Aro- SerC+ Pfl+, and Aro- SerC- Pfl+ mutants but none which were Aro+ SerC-. All of the mutants are explicable by deletions or inversions extending clockwise from zbj::Tn10 into or through an operon comprising serC (promoter-proximal) and aroA. Such an operon was also shown by the identification of two Tn10 insertions causing phenotype Aro- SerC-, each able to revert to Aro+ SerC+ by precise excision. serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base sequencing of a cloned aroA segment of S. typhimurium (Comai et al., Science 221:370-371, 1983). Both serine and chorismate are precursors of enterochelin; this may be why serC and aroA are in a single operon.     

Metabolic modeling for multi-objective optimization of ethanol production in a Synechocystis mutant

Cyanobacteria have potential to produce drop-in bio-fuels such as ethanol via photoautotrophic metabolism. Although model cyanobacterial strains have been engineered to produce such products, systematic metabolic engineering studies to identify optimal strains for the same have not been performed. In this work, we identify optimal ethanol producing mutants corresponding to appropriate gene deletions that result in a suitable redirection in the carbon flux. In particular, we systematically simulate exhaustive single and double gene deletions considering a genome scale metabolic model of a mutant strain of the unicellular cyanobacterium Synechocystis species strain PCC 6803. Various optimization based metabolic modeling techniques, such as flux balance analysis (FBA), method of minimization of metabolic adjustment (MOMA) and regulatory on/off minimization (ROOM) were used for this analysis. For single gene deletion MOMA simulations, the Pareto front with biomass and ethanol fluxes as the two objectives to be maximized was obtained and analyzed. Points on the Pareto front represent maximal utilization of resources constrained by substrate uptake thereby representing an optimal trade-off between the two fluxes. Pareto analysis was also performed for double gene deletion MOMA and single and double gene deletion ROOM simulations. Based on these analyses, two mutants, with combined gene deletions in ethanol and purine metabolism pathways, were identified as promising candidates for ethanol production. The relevant genes were adk, pta and ackA. An ethanol productivity of approximately 0.15 mmol/(gDW h) was predicted for these mutants which appears to be reasonable based on experimentally reported values in literature for other strains.     

Comparative studies on the biochemical properties of the malic enzymes from Trypanosoma cruzi and Trypanosoma brucei

The construction of engineered bacterial cells with a reduced genome allows the investigation of molecular mechanisms that may be cryptic in wild-type strains and derivatives. Previously, a large-scale combined deletion mutant of Escherichia coli that lacked 29.7% of the parental chromosome was constructed by combining large chromosome deletions. In this work, we improved the system for making markerless-chromosomal deletions and obtained mutants with a genome that lacked up to 38.9% of the parental chromosome. Although the large-scale deletion mutants possessed genes needed for resistance to oxidative stress, including superoxide dismutase, catalase, and RpoS, they were sensitive to menadione, which induces reactive oxygen species during stationary phase. Small genome size did not necessarily correlate with greater sensitivity to menadione as several mutants with large deletions were more resistant to menadione. The sensitivity to menadione depended on whether the mutants were grown aerobically or anaerobically, suggesting that the mechanism governing menadione resistance depended on the oxygen tension of the growth medium. Further analysis of the large-scale deletion mutants should help identify the regulatory networks that are important for cellular defense against oxidative stress.     

Transfer of the delta (argF-lac)U169 mutation between Escherichia coli strains by selection for a closely linked Tn10 insertion

To facilitate construction of mutants harboring delta lac for use in gene fusion studies, strains were constructed that carry the transposon Tn10 next to the well defined lac deletion U169. This deletion can now be moved to other Escherichia coli strains in transductional or conjugational crosses by selecting resistance to tetracycline.     

Comparative genomic hybridization detects secondary chromosomal deletions in Escherichia coli K-12 MG1655 mutants and highlights instability in the flhDC region

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655, we identified unintended secondary genomic deletions in the flhDC region in Δfnr, Δcrp, and ΔcreB mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.     

Bacteriophage T4 transfer RNA : III. Clustering of the genes for the T4 transfer RNA's

Escherichia coli cells infected with phage strains carrying extensive deletions encompassing the gene for the phage Ser-tRNA are missing the phage tRNA's normally present in wild-type infected cells. By DNA-RNA hybridization we have demonstrated that the DNA complementary to the missing tRNA's is also absent in such deletion mutants. Thus the genes for these tRNA's must be clustered in the same region of the genome as the Ser-tRNA gene. Physical mapping of several deletions of the Ser-tRNA and lysozyme genes, by examination of heteroduplex DNA in the electron microscope, has enabled us to locate the cluster, to define its maximum size, and to order a few of the tRNA genes within it. That such deletions can be isolated indicates that the phage-specific tRNA's from this cluster are dispensable.     

Physical analysis of deletion mutations in the ilvGEDA operon of Escherichia coli K-12.

DNA-DNA hybridization of cloned segments of the Escherichia coli K-12 ilvGEDA operon to genomic blots was used to determine the physical dimensions of a series of deletion mutations of the ilvGEDA operon. The smallest mutation resulted from the deletion of approximately 200 base pairs from within ilvD, whereas the largest mutation resulted from the deletion of 17 kilobases including the rep gene. The structure of three of these mutants indicates that formation of the deletions was mediated by Tn5 (or Tn5-131) that is retained in the chromosome. This is the first observation of this type of Tn5-mediated event. Our analysis of the total acetohydroxy acid synthase activity of strains containing deletions of ilvG indicates that the truncated ilvG polypeptide of wild-type E. coli K-12 lacks enzyme activity. The small 200-base-pair deletion of ilvD confirms the presence of a strong polar site 5' to ilvA. The detailed structure of these deletions should prove useful for the investigation of other genes in this region. This genomic analysis demonstrates that the ilv restriction site map that was established previously by the analysis of recombinant bacteriophage and plasmids is identical to that on the genome.     

Inversions and deletions of the Salmonella chromosome generated by the translocatable tetracycline resistance element Tn10

Spontaneous tetraoyoline-sensitive derivatives of a Tn10 insertion in the hisG gene of Salmonella typhimurium were isolated and subjected to genetic analysis. All 123 of the drug-sensitive derivatives characterized have undergone stable alterations in the Tn10 element itself; over half of the derivatives have also undergone major alterations of neighboring regions of the Salmonella chromosome. These chromosomal rearrangements are of two types: inversions and deletions. Any single inversion or deletion affects a contiguous stretch of chromosomal material extending from the site of the original Tn10 element either leftward or rightward.The genetic properties of deletion and inversion derivatives suggest that these chromosomal alterations are promoted by the Tn10 element itself. The role of translocatable elements in promoting chromosomal deletions is well documented; the ability of an element to promote inversions of chromosomal material has not previously been reported. Possible analogies between the 1400-base-pair inverted repetition at the end of Tn10 and the small insertion sequence IS1 predict particular structures for Tn10-promoted deletions. A structural explanation or model for Tn10-promoted inversions is presented. The observation that Tn10 promotes the formation of inversions suggests that such elements could play a previously unanticipated role in promoting chromosomal inversions during evolution of prokaryotic organisms. Generally applicable genetic methods for the identification and characterization of chromosomal inversions are described.     

Intrachromosomal Amplification,Locus Deletion and Point Mutation in the Aquaglyceroporin AQP1 Gene in Antimony Resistant Leishmania (Viannia) guyanensis

Background

Antimony resistance complicates the treatment of infections caused by the parasite Leishmania.

Methodology/Principal Findings

Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.

Conclusions/Significance

This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.     

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator,DR1558

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H₂O₂). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H₂O₂, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.