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PubMed2001

Comprehensive identification of conditionally essential genes in mycobacteria.

An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a mariner-based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette-Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.

Base Sequence↗
PubMed1993

Identification of an essential gene, l(3)73Ai, with a dominant temperature-sensitive lethal allele, encoding a Drosophila proteasome subunit.

Proteasomes are multicatalytic proteinase complexes that function as a major nonlysosomal proteolytic system in all eukaryotes. These particles are made up of 13-15 nonidentical subunits, and they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. Although the biochemical characterization of proteasomes has been quite extensive, and although a number of the genes encoding proteasome subunits have been cloned from various organisms, there is still much to be learned about their function in vivo and what role(s) they might play during development. Here, we report the identification of the l(3)73Ai1 allele of Drosophila melanogaster as a dominant temperature-sensitive lethal mutation in a gene encoding a component of the proteasome, thus opening the way for future genetic and developmental studies on this important proteolytic system in a higher eukaryote.

Alleles↗
PubMed1996

Evolutionary changes in the expression pattern of a developmentally essential gene in three Drosophila species.

The hypothesis that morphological evolution may largely result from changes in gene regulation rather than gene structure has been difficult to test. Morphological differences among insects are often apparent in the cuticle structures produced. The dopa decarboxylase (Ddc) and alpha-methyldopa hypersensitive (amd) genes arose from an ancient gene duplication. In Drosophila, they have evolved nonoverlapping functions, including the production of distinct types of cuticle, and for Ddc, the production of the neurotransmitters, dopamine and serotonin. The amd gene is particularly active in the production of specialized flexible cuticles in the developing embryo. We have compared the pattern of amd expression in three Drosophila species. Several regions of expression conserved in all three species but, surprisingly, a unique domain of expression is found in Drosophila simulans that does occur in the closely related (2-5 million years) Drosophila melanogaster or in the more remote (40-50 million years) Drosophila virilis. The "sudden" appearance of a completely new and robust domain of expression provides a glimpse of evolutionary variation resulting from changes in regulation of structural gene expression.

Animals↗
PubMed1997

P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E-87F.

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover approximately 25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven "super-contigs" that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the alphagamma element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.

Alleles↗
PubMed1997

P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: mutations affecting embryonic PNS development.

To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens.

Alleles↗
PubMed2003

A P-element insertion screen identified mutations in 455 novel essential genes in Drosophila.

With the completion of the nucleotide sequences of several complex eukaryotic genomes, tens of thousands of genes have been predicted. However, this information has to be correlated with the functions of those genes to enhance our understanding of biology and to improve human health care. The Drosophila transposon P-element-induced mutations are very useful for directly connecting gene products to their biological function. We designed an efficient transposon P-element-mediated gene disruption procedure and performed genetic screening for single P-element insertion mutations, enabling us to recover 2500 lethal mutations. Among these, 2355 are second chromosome mutations. Sequences flanking >2300 insertions that identify 850 different genes or ESTs (783 genes on the second chromosome and 67 genes on the third chromosome) have been determined. Among these, 455 correspond to genes for which no lethal mutation has yet been reported. The Drosophila genome is thought to contain approximately 3600 vital genes; 1400 are localized on the second chromosome. Our mutation collection represents approximately 56% of the second chromosome vital genes and approximately 24% of the total vital Drosophila genes.

Animals↗
PubMed2003

The Drosophila melanogaster importin alpha3 locus encodes an essential gene required for the development of both larval and adult tissues.

The nuclear transport of classical nuclear localization signal (cNLS)-containing proteins is mediated by the cNLS receptor importin alpha. The conventional importin alpha gene family in metazoan animals is composed of three clades that are conserved between flies and mammals and are referred to here as alpha1, alpha2, and alpha3. In contrast, plants and fungi contain only alpha1 genes. In this study we report that Drosophila importin alpha3 is required for the development of both larval and adult tissues. Importin alpha3 mutant flies die around the transition from first to second instar larvae, and homozygous importin alpha3 mutant eyes are defective. The transition to second instar larvae was rescued with importin alpha1, alpha2, or alpha3 transgenes, indicating that Importin alpha3 is normally required at this stage for an activity shared by all three importin alpha's. In contrast, an alpha3-specific biochemical activity(s) of Importin alpha3 is probably required for development to adults and photoreceptor cell development, since only an importin alpha3 transgene rescued these processes. These results are consistent with the view that the importin alpha's have both overlapping and distinct functions and that their role in animal development involves the spatial and temporal control of their expression.

Alleles↗
PubMed2025

A widespread protein misfolding mechanism is differentially rescued by chaperones based on gene essentiality.

Protein misfolding involving changes in non-covalent lasso entanglement (NCLE) status has been proposed based on simulations and biochemical assays of a small number of proteins. Here, we detect hallmarks of these misfolded states across hundreds of proteins by integrating E. coli proteome-wide limited-proteolysis mass spectrometry with structural datasets of protein native structures. Proteins containing native NCLEs are twice as likely to misfold, predominantly in regions where these NCLEs naturally occur. Surprisingly, the chaperones DnaK and GroEL do not typically correct this misfolding, except in the case of essential proteins. Statistical analysis links this differential rescue activity to weaker loop-closing contacts in the NCLEs of essential proteins, suggesting misfolding involving these loops is easier to rectify by chaperones. Molecular simulations indicate a mechanism where premature NCLE loop closure, prior to proper placement of the threading segment, leads to persistent misfolded states. This mechanism explains why, in the mass spectrometry data, proteins with NCLEs are more likely to misfold and misfold in NCLE regions. These results suggest widespread NCLE misfolding, that such misfolded states in non-essential proteins can bypass the refolding action of chaperones, and that some protein sequences may have evolved to allow chaperone rescue from this class of misfolding.

Journal Article↗
PubMed1991

Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum.

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.

Amino Acid Sequence↗
PubMed1995

glmS of Thermus thermophilus HB8: an essential gene for cell-wall synthesis identified immediately upstream of the S-layer gene.

A 30 kbp chromosomal region containing the S-layer gene (slpA) from Thermus thermophilus HB8 was cloned from a lambda phage gene library. DNA sequence analysis of the region upstream to the slpA gene revealed the presence of an open reading frame (ORF) which coded for a 604-amino-acid protein highly homologous to the glucosamine-6-P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin. The identification of this ORF as the glucosamine-6-P synthase gene from T. thermophilus (glmSth) has been carried out using three different strategies: (i) complementation of an Escherichia coli glmS mutant; (ii) in vivo insertional inactivation of the gene; and (iii) in vitro synthesis of glucosamine-6-P at 60 degrees C by a cytoplasmic extract of an overproducing E. coli strain. The glmSth gene is transcribed divergently from slpA in a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3' extreme. As the products of both the glmSth and the slpA genes are main components of the cell envelope of T. thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their coordinate expression.

Amino Acid Sequence↗
PubMed1995

Genes Essential for Amber Disease in Grass Grubs Are Located on the Large Plasmid Found in Serratia entomophila and Serratia proteamaculans.

The bacteria Serratia entomophila and S. proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Disease symptoms include rapid cessation of feeding and amber coloration of larvae. A 105-kb plasmid (designated pADAP) has consistently been found only in pathogenic isolates of both species. Investigations into the involvement of pADAP in amber disease have been hindered by the lack of both a selectable marker on the plasmid and a reliable transposon delivery system. Kanamycin-resistant transposon insertions into three cloned HindIII fragments (9.5, 9.6, and 10.6 kb) were isolated and introduced into pADAP by shuttle mutagenesis. Inserts into the 9.5-and 9.6-kb HindIII fragments on pADAP did not alter disease-causing ability. When plasmids with inserts into the 9.6-kb region were conjugated into plasmid-minus, nonpathogenic isolates of S. entomophila and S. proteamaculans, all of them became pathogenic. Transposon insertions into two regions of the 10.6-kb HindIII fragment continued to cause cessation of feeding but failed to produce amber coloration. Further analysis of a mutant from each amber-minus region (pADK-10 and pADK-13) demonstrated that the antifeeding effect was produced only at dosages higher than that of the wild-type strain. Complementation with the wild-type HindIII fragment restored full-blown disease properties for pADK-13, but not for pADK-10.

Journal Article↗
PubMed2005

Pathogenicity and immunogenicity of a vaccine strain of Listeria monocytogenes that relies on a suicide plasmid to supply an essential gene product.

Listeria monocytogenes is a bacterial pathogen that elicits a strong cellular immune response and thus has potential use as a vaccine vector. An attenuated strain, L. monocytogenes dal dat, produced by deletion of two genes (dal and dat) used for d-alanine synthesis, induces cytotoxic T lymphocytes and protective immunity in mice following infection in the presence of d-alanine. In order to obviate the dependence of L. monocytogenes dal dat on supplemental d-alanine yet retain its attenuation and immunogenicity, we explored mechanisms to allow transient endogenous synthesis of the amino acid. Here, we report on a derivative strain, L. monocytogenes dal dat/pRRR, that expresses a dal gene and synthesizes d-alanine under highly selective conditions. We constructed the suicide plasmid pRRR carrying a dal gene surrounded by two res1 sites and a resolvase gene, tnpR, which acts at the res1 sites. The resolvase gene is regulated by a promoter activated upon exposure to host cell cytosol. L. monocytogenes dal dat/pRRR was thus able to grow in liquid culture and to infect host cells without d-alanine supplementation. However, after infection of these cells, resolvase-mediated excision of the dal gene resulted in strong down-regulation of racemase expression. As a result, this system allowed only transient growth of L. monocytogenes dal dat/pRRR in infected cells and survival in animals for only 2 to 3 days. Nevertheless, mice immunized with L. monocytogenes dal dat/pRRR generated listeriolysin O-specific effector and memory CD8(+) T cells and were protected against lethal challenge by wild-type Listeria. This vector may be an attractive vaccine candidate for the induction of protective cellular immune responses.

Alanine Racemase↗
PubMed1983

Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA.

Plasmid RK2 encodes several kil determinants whose lethal action on Escherichia coli host cells is prevented by RK2 kor genes. Here we show that the mini-RK2 plasmid, pRK248, specifies a kilB component (kilB1) in the region of the replication gene trfA. kilB1 is different from trfA and is completely encoded within the pRK248 HaeII A fragment. Transformation of E. coli cells with hybrid plasmids containing the cloned kilB1 determinant is very inefficient and results in the selection of variant kil- plasmids, many of which show genetic and physical evidence of deletions. If another pRK248 gene (korB1) is present in the cells, kilB1+ plasmids can be established at high efficiency and without any detectable changes. KorB1 is encoded by the trfB region of pRK248 because recombinant plasmids with this region are able to control kilB1 in trans. These results substantiate our earlier explanation for the structure of pRK248 and for the perplexing requirement of the trfB region in this plasmid.

DNA Restriction Enzymes↗
PubMed1985

Identification of five new essential genes involved in the synthesis of a secreted protein in Escherichia coli.

To define additional components of the export machinery of Escherichia coli, I have isolated extragenic suppressors of a mutant [secA(Ts)] that is temperature sensitive for growth and secretion at 37 degrees C. Suppressors that restored growth at 37 degrees C, but that rendered the cell cold sensitive for growth at 28 degrees C, were obtained. The suppressor mutations fall into at least seven loci, two of which (prlA and secC) have been previously implicated in protein secretion. The five remaining loci (ssaD, ssaE, ssaF, ssaG, and ssaH) have been mapped by P1 transduction and appear to define new genes in E. coli. All of the suppressor mutations allow both enhanced growth and protein secretion of the secA(Ts) mutant at 37 degrees C, but not 42 degrees C, indicating a continued requirement for SecA protein. Strains carrying solely the cold-sensitive mutations show reduced levels of certain periplasmic proteins when grown at low temperatures. In at least one case, that of maltose-binding protein, this defect is at the level of synthesis of the protein. Since mutants in any of seven genes as well as secA amber mutants halt or reduce the synthesis of an exported protein, it appears that E. coli may possess a general and complex mechanism for coupling protein synthesis and secretion.

ATP-Binding Cassette Transporters↗
PubMed1987

Initial catabolism of aromatic biogenic amines by Pseudomonas aeruginosa PAO: pathway description, mapping of mutations, and cloning of essential genes.

Pseudomonas aeruginosa PAO1 was able to utilize several aromatic biogenic amines as sole sources of carbon or nitrogen. These included the phenethylamines tyramine and dopamine and the phenethanolamines octopamine, synephrine, and norepinephrine. Initial catabolism of the phenethylamines was mediated by a membrane-bound tyramine dehydrogenase which produced 4-hydroxyphenylacetaldehyde (4HPAL) with tyramine as the substrate. The enzyme was induced by growth with both classes of amines. Initial catabolism of octopamine (except when present as the sole source of carbon and nitrogen) was mediated by a soluble enzyme with activity against the phenethanolamines but not against tyramine or dopamine. The product of the reaction with octopamine as substrate was also 4HPAL. Addition of NAD to reaction mixtures yielded 4-hydroxyphenylacetic acid and NADH. These activities, octopamine hydrolyase and 4-HPAL dehydrogenase (measured as a combined activity, OCAH-4HPALDH), were only induced by growth with phenethanolamines. However, the combined activities were not observed in extracts from cells grown with octopamine as the sole source of carbon and nitrogen, suggesting that an alternate pathway is used under this growth condition. Two independently isolated mutant strains were unable to utilize tyramine as a sole source of carbon or nitrogen. These mutants were also unable to utilize dopamine but grew at wild-type rates on the phenethanolamines. The mutations were mapped at about 70 min on the PAO1 chromosome with the chromosome-mobilizing plasmid R68.45, and both were linked to the catA1, mtu-9002, tyu-9009, and puuE mutations. DNA complementing both of the mutations was cloned on a single BamHI fragment approximately 13.8 kilobase pairs in length. Analysis of a subcloned fragment showed that the two mutations were in different genes.

Biogenic Amines↗
PubMed1989

Nucleotide sequence and expression of cheF, an essential gene for chemotaxis in Bacillus subtilis.

The cheF gene, which is involved in chemotaxis in Bacillus subtilis, has been cloned, expressed, and sequenced. This gene is contained in a 0.7-kilobase PstI DNA fragment that was isolated from a lambda Charon 4A B. subtilis chromosomal DNA library. This fragment was subcloned into the expression vector pSI-1 and shown to complement the cheF mutation both for chemotaxis and for methanol production in response to the addition of attractants. Plasmid-encoded DNA expression in B. subtilis maxicells indicated that a membrane-associated polypeptide of 20-kilodaltons was expressed from this 0.7-kilobase DNA. The nucleotide sequence of this DNA fragment was determined, and an open reading frame capable of encoding a putative 175-amino-acid protein (Mr 20,002) was identified. In an effort to understand the function of the cheF protein, the dosage of the cheF gene product was varied by altering the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) during growth. In the presence of high concentrations of IPTG, chemotaxis was inhibited and methanol production was impaired.

Amino Acid Sequence↗
PubMed1989

pepM is an essential gene in Salmonella typhimurium.

The pepM gene of Salmonella typhimurium codes for a methionine-specific aminopeptidase that removes N-terminal methionine residues from proteins. This gene was inactivated in vitro by the insertion of a DNA fragment coding for kanamycin resistance. The inactivated gene could not replace the wild-type chromosomal pepM gene unless another functional copy was present in the cell. The lethal effect of the pepM insertion was not a result of polarity on any gene downstream, nor was it affected by the presence or absence of other peptidases.

Aminopeptidases↗