We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

/pdf/construction-of-escherichia-coli-k-12-in-frame-single-gene-4c68fx6pb1.pdf

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

KEGG(Kyoto Encyclopedia of Genes and Genomes)〔和文〕 (特集 ゲノム医学の現在と未来--基礎と臨床) -- (データベース)

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

https://www.ibric.org/myboard/down.php?Board=exp_qna&filename=pBBR1MCS.pdf&id=20940&fidx=1

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.

We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

/pdf/comprehensive-transposon-mutant-library-of-pseudomonas-438d5m2ydm.pdf

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

We constructed a derivative of transposon Tn5 that permits the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. Such a hybrid gives alkaline phosphatase activity if the protein fused to alkaline phosphatase contributes sequences that promote export and thus compensate for the missing alkaline phosphatase signal peptide. Fusions to both a secreted periplasmic protein and a complex cytoplasmic membrane protein led to alkaline phosphatase activity. TnphoA fusions should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and transmembrane proteins.

https://www.pnas.org/content/pnas/82/23/8129.full.pdf

TnphoA: a transposon probe for protein export signals.

A set of 30 mutants exhibiting reduced production of the phenazine poison pyocyanin were isolated following transposon mutagenesis of Pseudomonas aeruginosa PAO1. The mutants could be subdivided into those with defects in the primary phenazine biosynthetic pathway and those with more pleiotropic defects. The largest set of pleiotropic mutations blocked the production of the extracellular Pseudomonas quinolone signal (PQS), a molecule required for the synthesis of secondary metabolites and extracellular enzymes. Most of these pqs mutations affected genes which appear to encode PQS biosynthetic functions, although a transcriptional regulator and an apparent response effector were also represented. Two of the genes required for PQS synthesis (phnA and phnB) had previously been assumed to encode phenazine biosynthetic functions. The transcription of one of the genes required for PQS synthesis (PA2587/pqsH) was regulated by the LasI/R quorum-sensing system, thereby linking quorum sensing and PQS regulation. Others of the pleiotropic phenazine-minus mutations appear to inactivate novel components of the quorum-sensing regulatory network, including one regulator (np20) previously shown to be required for virulence in neutropenic mice.

/pdf/functions-required-for-extracellular-quinolone-signaling-by-48dnbu5gio.pdf

Functions Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans . Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase ( hcnC ) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant ( egl-9 ) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning.

The opportunistic pathogenic bacterium Pseudomonas
 aeruginosa uses quorum-sensing signaling systems as global
 regulators of virulence genes. There are two quorum-sensing signal
 receptor and signal generator pairs, LasR–LasI and RhlR–RhlI. The
 recently completed P. aeruginosa genome-sequencing
 project revealed a gene coding for a homolog of the signal receptors,
 LasR and RhlR. Here we describe a role for this gene, which we call
 qscR . The qscR gene product governs the
 timing of quorum-sensing-controlled gene expression and it dampens
 virulence in an insect model. We present evidence that suggests the
 primary role of QscR is repression of lasI . A
 qscR mutant produces the LasI-generated signal
 prematurely, and this results in premature transcription of a number of
 quorum-sensing-regulated genes. When fed to Drosophila
 melanogaster , the qscR mutant kills the animals
 more rapidly than the parental P. aeruginosa . The
 repression of lasI by QscR could serve to ensure that
 quorum-sensing-controlled genes are not activated in environments where
 they are not useful.

https://www.pnas.org/content/pnas/98/5/2752.full.pdf

Colin Manoil

Papers

Comprehensive transposon mutant library of Pseudomonas aeruginosa.

TnphoA: a transposon probe for protein export signals.

Functions Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning.

QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa