SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.
To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.
Attempts to inactivate an essential gene in the protozoan parasite Leishmania have often led to the generation of extra copies of the wild-type alleles of the gene. In experiments with Leishmania tarentolae set up to disrupt the gene encoding the J-binding protein 1 (JBP1), a protein binding to the unusual base beta-D-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of approximately 100 kb. These amplicons consist of a long inverted repeat with telomeric repeats at both ends and contain either the two different targeting cassettes used to inactivate JBP1, or one cassette and one JBP1 gene. Each long repeat within the linear amplicons corresponds to sequences covering the JBP1 locus, starting at the telomeres upstream of JBP1 and ending in a approximately 220 bp sequence repeated in an inverted (palindromic) orientation downstream of the JBP1 locus. We propose that these amplicons have arisen by a template switch inside a DNA replication fork involving the inverted DNA repeats and helped by the gene targeting.
Nap1p is identified in mammalian cell extract by its intrinsic activity to facilitate nucleosome assembly in vitro in the physiological ionic condition. The homologous proteins are present in most eukaryotes, and their functional analyses in vitro have suggested that they are necessary to keep proper nucleosome structures in transcription and replication. This protein is also identified for its interaction with Clb2p in vitro. To address the function of Nap1p in vivo, we have surveyed for proteins to interact with Nap1p by two-hybrid system and obtained two genes, NBP1 and NBP2 (Nap1 Binding Protein 1 and 2). NBP1 is an essential gene and encodes a novel protein consisting of 319 amino acids, with a coiled-coil structure in the center of the predicted amino acid sequence. Several A-kinase dependent phosphorylation sites and Cdc28p kinase-dependent sites are also observed. By isolating the temperature-sensitive mutant, we demonstrate that the nuclear division at a non-permissive temperature is delayed and that the population of cells with a large bud carrying a single nucleus with a short spindle are increased. This mutant also confers resistance against benomyl, a microtubule-destabilizing agent. Judging from the green fluorescent protein (GFP) signal fused with Nbp1p, this protein localizes in the nucleus as one or two tiny dots.
To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of Streptococcus pneumoniae, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in Escherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the Bacillus subtilis competence gene comFA. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless lacZ reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.
Little is known about the genes that regulate telomere length diversity between mammalian species. A candidate gene locus was previously mapped to a region on distal mouse Chr 2q. Within this region, we identified a gene similar to the dog-1 DNA helicase-like gene in C. elegans. We cloned this Regulator of telomere length (Rtel) gene and inactivated its expression in mice. Rtel(-/-) mice died between days 10 and 11.5 of gestation with defects in the nervous system, heart, vasculature, and extraembryonic tissues. Rtel(-/-) embryonic stem cells showed telomere loss and displayed many chromosome breaks and fusions upon differentiation in vitro. Crosses of Rtel(+/-) mice with Mus spretus showed that Rtel from the Mus musculus parent is required for telomere elongation of M. spretus chromosomes in F1 cells. We conclude that Rtel is an essential gene that regulates telomere length and prevents genetic instability.
Enterococcus faecalis divIVA (divIVAEf) is an essential gene implicated in cell division and chromosome segregation. This gene was disrupted by insertional inactivation creating E. faecalis JHSR1, which was viable only when a wild-type copy of divIVAEf was expressed in trans, confirming the essentiality of the gene. The absence of DivIVAEf in E. faecalis JHSR1 inhibited proper cell division, which resulted in abnormal cell clusters possessing enlarged cells of altered shape instead of the characteristic diplococcal morphology of enterococci. The lower viability of the divIVAEf mutant is caused by improper nucleoid segregation and impaired septation within the numerous cells generated in each cluster. Overexpression of DivIVAEf in Escherichia coli KJB24 resulted in enlarged cells with disrupted cell division, suggesting that this round E. coli mutant strain could be used as an indicator for functionality of DivIVAEf. A Bacillus subtilis divIVA mutant was not complemented by DivIVAEf, indicating that this protein does not recognize DivIVA-specific target sites in B. subtilis, or that it does not interact with other proteins of the cell division machinery of this micro-organism. DivIVAEf also failed to complement a Streptococcus pneumoniae divIVA mutant, supporting the phylogenetic distance between Enterococcus and Streptococcus. Our results indicate that DivIVA is a species-specific multifunctional protein implicated in cell division and chromosome segregation in E. faecalis.
We isolated 25 independent mutants that are deficient in essential genes located in the metB-malB region of the Escherichia coli chromosome. The mutations were mapped within this region by using several F' factors and were also classified into 11 cistrons by complementation testing. There is clustering of mutations in essential genes within the metB-malB region.
The plo gene, encoding the Arcanobacterium pyogenes cholesterol-dependent cytolysin, pyolysin (PLO), was localized to a 2.7-kb genomic islet of reduced %G+C content and alternate codon usage frequency. This islet, conserved among isolates from diverse hosts and geographical locations, separated the housekeeping genes smc and ftsY, which are found adjacent in many prokaryotes. The ftsY and ffh genes, located downstream of the plo islet, encode components of the signal recognition particle. Mutational analysis suggested that these genes were essential for viability in A. pyogenes. The A. pyogenes ffh gene was unable to complement a conditional ffh mutant of Escherichia coli and its overexpression was toxic in E. coli. Mutagenesis of the islet-encoded orf121 did not affect plo expression, indicating that it may not be involved directly in the regulation of plo expression. Regardless, the presence of the plo gene as part of a genomic islet inserted between genes essential for normal growth may provide selective pressure for the retention of this important virulence factor.
Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.
A PCR-based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w. The ORFs were deleted in two diploid strains, FY1679 and W303. Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD-box RNA helicase, is an essential gene. A systematic phenotypic analysis of the deleted mutants was carried out. Homozygous diploids ybr128cDelta/ybr128cDelta and ybr131wDelta/ybr131wDelta did not sporulate. The ybr131cDelta mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1. ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996). We found that the aberrant morphology characteristic for ybr133cDelta (hsl7Delta) cells was observed in W303 but not in FY1679 genetic background. Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil. The effects of the deletion were reinforced in W303 background. We found no phenotypic effects of the two remaining deletions, ybr137wDelta and ybr138cDelta.
The sgp gene from Streptococcus mutans has been previously isolated, characterized, and demonstrated to encode a G-protein. In order to investigate the function of this gene, a novel antisense RNA strategy was developed. Expression of sgp antisense RNA in Escherichia coli led to transient inhibition of growth. In addition, sgp antisense RNA expression in S. mutans resulted in decreased growth under environmental stress conditions (44 degrees C, acidic pH, and high osmolarity). Therefore, these results suggest that the sgp gene plays a role in modulating the stress responses of S. mutans. This approach could be applicable for investigating the function of essential genes in other organisms for which mutants are not available.
The ability to analyze null alleles of genes can be an important means of studying both a protein's function and its interactions with other proteins involved in a particular process. However, if the gene encodes a protein that is essential to the viability of the cell, such analysis becomes complicated because a complementing copy of the gene must be present in the cell. In order to study the effects caused by the null allele, the complementing copy must be inactivated or lost. We report the development of a system in Escherichia coli which facilitates the manipulation of null alleles of essential genes. It consists of a strain deleted chromosomally for the essential gene and complemented for its function by a wild-type (wt) copy expressed from a plasmid counter-selectable for two markers bracketing the gene. Using this system, we have (i) searched for bypass suppressors of a deletion of the essential secE gene, (ii) ascertained the ability of various mutant secE genes to complement a deletion of the wt copy and (iii) isolated intragenic pseudorevertants of a null missense allele of secE. This methodology should be widely applicable to other cases in which essential genes are to be studied genetically.
The pELF-type linear plasmid is a critical mobile genetic element responsible for the dissemination of various antimicrobial resistance (AMR) genes, most notably vancomycin resistance in Enterococcus faecium, which is a leading cause of hospital outbreaks worldwide. Despite their crucial role in the expansion of AMR, the molecular mechanisms underlying the conjugative transfer of these linear plasmids remain poorly understood. In this study, the transfer (tra) region of pELF2, a representative vanA-harboring linear plasmid was characterized. Transcriptomic data suggested that the FtsK/VirD4-type adenosine triphosphatase is encoded within a multi-gene operon. By developing a genetic manipulation framework for E. faecium, an extensive mutational analysis of the tra region was performed and the following three essential genes were identified: traCB4 (a putative VirB4 analog), traDD4 (a VirD4-like coupling protein), and traGB6 (a putative VirB6 analog). These genes are indispensable for conjugative transfer. Reporter assays experimentally confirmed the presence of a functional promoter upstream of the identified tra genes. We confirmed that these genes are highly conserved among pELF-type plasmid sequences deposited in public database. The study findings revealed that pELF-type plasmids utilize highly minimized conjugation machinery, which is similar to unusual systems previously identified in other gram-positive bacteria, such as Streptomyces. This study provides the first molecular insights into the transmission of these clinically important linear plasmids in enterococci and lays a foundation for understanding the dissemination of resistance determinants mediated by atypical mobile genetic elements.
We report the construction of recombinant plasmids containing the dihydrofolate reductase structural gene (fol) from several trimethoprim-resistant mutants of Escherichia coli. Strains carrying some of these plasmids produced approximately 6% of their soluble cell protein as dihydrofolate reductase and are therefore excellent sources of the purified enzyme for inhibitor binding or mechanistic studies. The nucleotide sequence of the fol region from each of the plasmids was determined. A plasmid derived from a Ki mutant which produced a dihydrofolate reductase with lowered affinity for trimethoprim contained a mutation in the structural gene that altered the sequence of the polypeptide in a conserved region which is adjacent to the dihydrofolate binding site. Two other independently-isolated mutants which overproduced dihydrofolate reductase had a mutation in the -35 region of the fol promoter. One of them, strain RS35, was also temperature-sensitive for growth in minimal medium. This phenotype was shown to be the result of an additional mutation in a locus unlinked to fol by P1 transduction. The fol regions from two temperature-independent revertants of strain RS35 were sequenced. One of these had a mutation within the dihydrofolate reductase structural gene which altered some properties of the enzyme. This confirmed some previous enzymological data which suggested that some revertants of strain RS35 had mutations in fol (Sheldon 1977). These results suggest that dihydrofolate reductase interacts physically with some other essential gene product in E. coli.
Autophagy is a starvation-induced transport pathway delivering parts of the cytosol into the lysosome (vacuole) for degradation. Autophagy significantly differs from other transport pathways by using double membrane layered transport intermediates. Based on the identification of autophagy genes in Saccharomyces cerevisiae, which served as a pacemaker for higher cells, our mechanistic knowledge of autophagy notably increased over the past few years. We here identify AUT8 as a novel gene essential for autophagy by screening a collection of approximately 5000 yeast deletion strains, each containing a defined deletion in an individual gene. This collection is a result of the world-wide Saccharomyces deletion project and covers the non-essential genes of the whole yeast genome. Homozygous aut8 Delta cells are impaired in maturation of proaminopeptidase I, and they fail to undergo the cell differentiation process of sporulation. The essential function of AUT8 for autophagy is further demonstrated by the lack of accumulation of autophagic vesicles in the vacuoles of aut8 Delta cells starved of nitrogen in the presence of the proteinase B inhibitor phenylmethylsulfonyl fluoride.
The embryo essential gene EMB506 plays a crucial role in the transition of the Arabidopsis embryo from radial symmetry to bilateral symmetry just prior to the early heart stage of development. In addition to influencing embryo development EMB506 also affects chloroplast biogenesis. To further investigate the role of EMB506 gene expression in Arabidopsis we have generated green fluorescent protein (GFP) marked emb506 mosaic sectors at temporally defined stages during embryogenesis and additionally during various stages of vegetative growth, in otherwise phenotypically wild-type plants. We confirm the essential requirement for EMB506 gene expression in chloroplast biogenesis as reflected by the decreased chlorophyll content in emb506 mosaic sectors. We also show that the influence of EMB506 gene expression as it impinges on chloroplast biogenesis is first relevant at an intermediate stage in embryogenesis and that the role of EMB506 gene expression in chloroplast biogenesis is distinct from the essential role of EMB506 gene expression during early embryo development. By inducing emb506 mosaicism after the essential requirement for EMB506 gene expression in embryogenesis and also during vegetative growth we reveal that EMB506 gene expression additionally is required for correct cotyledon-, true leaf- and cauline leaf margin development. The strategy that we describe can be tailored to the mosaic analysis of any cloned EMB gene for which a corresponding mutant exists and can be applied to the mosaic analysis of mutant lethal genes in general.
Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.