Derepression of Uridine Diphosphate-Glucose Pyrophosphorylase (galU) in capR(lon), capS, and capT Mutants and Studies on the galU Repressor

Mutation of the capR(lon), capS, or capT genes in Escherichia coli K-12 causes overproduction of capsular polysaccharide leading to a mucoid phenotype. Several of the enzymes involved in capsular polysaccharide synthesis are derepressed in cap mutants. Previously it was shown that uridine diphosphate-glucose (UDPG) pyrophosphorylase, an enzyme involved in the synthesis of three of the nucleotide sugar precursors of the capsule, is derepressed in capR mutants. The control of galU, the gene which codes for UDPG pyrophosphorylase, is described in this study. In addition, it has been found that the enzyme is also derepressed in capS and capT mutants. The effect of galU gene dosage in cap mutants and the wild-type strain (all lysogenic for 80) was studied by infecting them with the purified transducing phage 80dgalU. The level of UDPG pyrophosphorylase increased in proportion to the number of galU copies added. The rate of enzyme synthesis in the mutants was about sixfold higher than in the wild type per galU gene added for multiplicities of infection from one to twenty. Thus, all the galU copies added to the wild-type lysogen were repressed. We obtain greater than 20 galU copies per cell by infecting the nonlysogenic strain which allows multiplication of 80dgalU. With some number of galU copies greater than 20, the rate of UDPG pyrophosphorylase synthesis in the wild type approaches the mutant rate of synthesis. The results suggest that there may indeed be a galU repressor pool in the cell which can be completely titrated. This pool must be composed of more than 20 galU repressor molecules. Since the capR, capS, and capT gene products or combinations thereof are known to control other widely separated operons of the cell besides the galU gene, it is postulated that the galU repressor may be capable of binding other operators. This would account for the relatively large pool of galU repressors per cell.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

Telomeric gene deletion and intrachromosomal amplification in antimony‐resistant Leishmania

Antimonials are still the mainstay of treatment against leishmaniasis but drug resistance is increasing. We carried out short read next‐generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony‐resistant mutants. Copy number variations were consistently detected with both NGS and CGH. A major attribute of antimony resistance was a novel terminal deletion of variable length (67 kb to 204 kb) of the polyploid chromosome 31 in the three mutants. Terminal deletions in two mutants occurred at the level of inverted repeated sequences. The AQP1 gene coding for an aquaglyceroporin was part of the deleted region and its transfection into resistant mutants reverted resistance to SbIII. We also highlighted an intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded for ascorbate‐dependent peroxidase (APX) and glucose‐6‐phosphate dehydrogenase (G6PDH). Overexpression of these genes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS). Generation of a G6PDH null mutant in one revertant exhibited SbIII sensitivity and a decreased protection of ROS. Our genomic analyses and functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania.     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii

Summary Mutations at seven recombinationally distinct chloroplast loci confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii. Assays of polynucleotide-directed amino acid incorporation by ribosomes reconstituted from mutant and wild type subunits demonstrate that streptomycin, neamine/kanamycin and spectinomycin resistance mutations specifically affect the small ribosomal subunit, whereas mutations to erythromycin resistance affect the large subunit. Although in each case the subunit site of antibiotic resistance is the same as that observed in analogous mutations in Escherichia coli, the number of loci conferring resistance to a given antibiotic differs in the two organisms. We have previously shown that streptomycin resistance mutations in Chlamydomonas map at five discrete loci (one nuclear and four chloroplast), and that mutations to neamine/kanamycin and spectinomycin resistance appear to define a single chloroplast locus. Results presented here confirm our previous report that all chloroplast erythromycin resistance mutations isolated to date fall into two recombinationally distinct loci, and indicate that mutants at one of these loci may be further divided on the basis of their level of cross resistance to other macrolide antibiotics.     

Involvement of lipopolysaccharide in the secretion of Escherichia coli α haemolysin and Erwinia chrysanthemi proteases

The presence of the α-haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram-negative envelope (owing to its large size) (M_r 1400). The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non-haemolytic mutants. In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained. However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis. Mutations in rfaH and galU strongly reduced α-haemolysin secretion as weli as the secretion of Erwinia chrysanthemi proteases in E. coli without affecting their synthesis. These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion. These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope.     

Instability of artificially circularized chromosomes of Streptomyces lividans

The chromosomes of Streptomyces species are linear molecules, containing long terminal inverted repeats and covalently bound terminal proteins. These chromosomes undergo spontaneous deletions of the terminal sequences at high frequencies and become circularized in several cases examined. Artificial circularization of the Streptomyces lividans chromosome was also achieved by targeted recombination in vivo, in which the terminal inverted repeats of the chromosome were connected by a kanamycin resistance gene (aphII ). Under kanamycin selection, the circularized chromosomes harboured tandem amplifications of a 20.2 kb sequence that included the aphII gene flanked by direct repeats and deletions nearby. On release from kanamycin selection, the aphII amplifications and the neighbouring sequences were deleted from the chromosomes, rendering all the cultures kanamycin sensitive. The chloramphenicol resistance gene, which was prone to deletion in wild-type S. lividans, became much more stable in the kanamycin-sensitive derivatives. These results indicate that the telomeres and/or certain terminal sequences may be involved in the structural instability of Streptomyces chromosomes.     

Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation

Haemophilus parasuis, belonging to the family Pasteurellaceae, is the causative agent of Glässer’s disease leading to serious economic losses. In this study, a successive markerless mutation system for H. parasuis using two sequential steps of natural transformation was developed. By the first homologous recombination, the target genes were replaced by a cassette carrying kanamycin resistance gene and sacB (which confers sensitivity to sucrose) gene using kanamycin selection, followed by the second reconstruction to remove the selection cassette, with application of sucrose to further screen unmarked mutants. To improve DNA transformation frequency, several parameters have been analyzed further in this work. With this method, two unmarked deletions in one strain have been generated successfully. It is demonstrated that this system can be employed to construct multi-gene scarless deletions, which is of great help for developing live attenuated vaccines for H. parasuis.     

Synergistic effect of TRM2/RNC1 and EXO1 in DNA double-strand break repair in Saccharomyces cerevisiae

In our recently published study, we provided in vitro as well as in vivo data demonstrating the involvement of TRM2/RNC1 in homologous recombination based repair (HRR) of DNA double strand breaks (DSBs), in support of such claims reported earlier. To further validate its role in DNA DSB processing, our present study revealed that the trm2 single mutant displays higher sensitivity to persistent induction of specific DSBs at the MAT locus by HO-endonuclease with higher sterility rate among the survivors compared to wild type (wt) or exo1 single mutants. Intriguingly, both sensitivity and sterility rate increased dramatically in trm2exo1 double mutants lacking both endo-exonucleases with a progressively increased sterility rate in trm2exo1 double mutants with short-induction periods, reaching a very high level of sterility with persistent DSB inductions. Mutation analysis of the mating type (MAT) locus among the sterile survivors with persistent HO-induction in trm2 and exo1 single mutants as well as in trm2exo1 double mutants revealed a similar small insertions and deletions events, characteristic of non-homologous end joining (NHEJ) that might have occurred due to the lack of proper processing function in these mutants. In addition, trm2ku80 and trm2rad52 double mutants also displayed significantly higher sterility with persistent DSB induction compared to ku80 and rad52 single mutants, respectively, exhibiting a mutation spectra that shifted from base substitution (in ku80 and rad52 single mutants) to small insertions and deletions in the double mutants (in trm2ku80 and trm2rad52 mutants). These data indicate a defective processing in absence of TRM2, with a synergistic effect of TRM2, and EXO1 in such processing.     

Negative control of the galactose operon in E. coli

Summary Non-inducible mutants have been isolated which synthesize the three galactose enzymes with the basal rate both in the absence and in the presence of inducers. These mutations are closely linked to the lysA gene, as are the constitutive mutations in the regulator gene first described by Buttin (1963).The non-inducible mutants are Gal ^– on EMB gal plates. Revertants to the Gal ⁺ phenotpye are constitutive. Heterozygotes have been prepared at the locus of the regulator gene (galR), abd dominance studies involving the different alleles at this locus have been carried out. The non-inducible mutations are dominant over the wildtype, and this in turn is dominant over constitutive mutations in the galR gene.Starting from the non-inducible mutations, deletions have been isolated, which extend from the galR gene into the lysA gene. These are constitutive.The behavior of the non-inducible mutations and of the deletions are strong arguments for negative control of the galactose operon.     

Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium

We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli. The results of transient GUS (β-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus. Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-deficient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait. Received: 26 October 1998 / Accepted: 8 January 1999     

Genetic properties of arsenate sensitive mutants of Bacillus subtilis 168

Summary Arsenate sensitive mutants were isolated from Bacillus subtilis strain 168 after treatment with N-methyl-N-nitro-N-nitrosoguanidine or ethyl methane sulfonate. Though all mutants are phenotypically identical, a high proportion (40%) of the induced mutations are of a multisite nature as they do not revert spontaneously and are poorly transformable to arsenate resistance with wild type DNA. On the basis of transformation efficiency, UV inactivation kinetics and cotransduction frequency of outside markers, four independently isolated multisite arsenate sensitive mutations are characterized as resulting from large deletions of homogenous size (24000±6000 base pairs). The arsenate resistance locus was mapped between phe and aroD on the B. subtilis chromosome by PBS1 mediated transduction. Mechanisms for the formation of such chromosomal deletions are discussed.Part of the dissertation of Alice Adams Lindahl, presented to New York University in partial fulfillment of requirements for the Ph. D. degree. A preliminary report of this work was presented at the NATO International Symposium, Mol, Belgium, August 1970.     

Relationship between Spontaneous Aminoglycoside Resistance in Escherichia coli and a Decrease in Oligopeptide Binding Protein

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of Escherichia coli were investigated. Among 20 colonies obtained from 10⁸ cells cultured in the presence of 20 μg of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the oppA gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the oppA gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.     

Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA

Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l^–1 kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.Communicated by C.F. Quiros     

Searching for tagged male-sterile mutants of arabidopsis

Although many male-sterile mutants have been identified inArbidopsis thaliana, few of the corresponding genes have been cloned. In order to facilitate cloning of a male sterility gene, 23 of Feldmann's T-DNA-generated, reduced-fertility lines were screened to identify a tagged male-sterile mutation. Malesterile mutants were identified, as well as mutants that were both male and female sterile. Segregation of the kanamycin marker gene in the progeny of 15 of these lines was studied. Forty percent had functional T-DNAs (encoding resistance to kanamycin) inserted at a single locus, the remainder segregating for two or more functional T-DNA inserts. Linkage between T-DNA inserts and mutant phenotype was tested for six lines. In three of these lines, mutations were not linked to a T-DNA insert. In three lines, the mutation segregated with a T-DNA insert.     

Isolation and characterization of rice mutants compromised in<Emphasis Type="Italic"> Xa21</Emphasis>-mediated resistance to<Emphasis Type="Italic"> X. oryzae</Emphasis> pv.<Emphasis Type="Italic"> oryzae</Emphasis>

The rice gene, Xa21, confers resistance to diverse races of Xanthomonas oryzae pv. oryzae (Xoo) and encodes a receptor-like kinase with leucine-rich repeats in the extra-cellular domain. To identify genes essential for the function of the Xa21 gene, 4,500 IRBB21 (Xa21 isogenic line in IR24 background) mutants, induced by diepoxybutane and fast neutrons, were screened against Philippine race six (PR6) Xoo for a change from resistance to susceptibility. From two greenhouse screens, 23 mutants were identified that had changed from resistant to fully (6) or partially (17) susceptible to PR6. All fully susceptible mutants carried rearrangements at the Xa21 locus as detected by PCR and Southern hybridization. For the partially susceptible mutants, no changes were detected at the Xa21 locus based on Southern and PCR analyses. However, two of these mutants were confirmed via genetic analysis to have mutations at the Xa21 locus. Partially susceptible mutants exhibited variation in level of susceptibility to different Xoo strains, suggesting that they may carry different mutations required for the Xa21-mediated resistance. The mutants identified in this study provide useful materials for dissecting the Xa21-mediated resistance pathway in rice.Communicated by D.J. Mackill     

Mutations determining generalized resistance to aminoglycoside antibiotics in Escherichia coli.

Summary Mutations conferring resistance to low levels of kanamycin in Escherichia coli have been mapped at 3 locations: the unc locus (min. 83), a locus we have designated, kanA (min. 72), close to strA (rpsL), and a locus at min. 86.5 previously discovered by Plate (1976) that we have designated ecfB. The unc and ecfB mutations are associated with defects in energy metabolism, while mutations at kanA may be in the gene coding for ribosomal protein S12 (rpsL). The three types of mutations cause cross resistance to a number of different aminoglycoside antibiotics and the effects of the mutations are cumulative in combination.     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.