Isolation and characterization of multiflagellate mutants of Pseudomonas aeruginosa.

Multitrichously polar flagellated mutants were isolated from a monotrichously flagellated strain of Pseudomonas aeruginosa. The ability of the mutant cells to swarm in semisolid media at given gel strengths was increased by the multiflagellation. Observations of the mutant cells by electron microscopy revealed that the number of flagella produced per cell cycle was increased. F116 phage-mediated transduction showed that the multiflagellation occurred by a single mutation and that the mutation sites were linked to a fla cluster of this organism.     

Isolation of Tyr- mutants of facultative methylotrophic Pseudomonas sp. M

A method for isolation of Tyr- mutants of facultative methylotrophic bacteria Pseudomonas sp. M which possess two tyrosine synthesis pathways is presented. The method is based on the two-step blocking of the tyrosine synthesis: the first step of the supplementary pathway of synthesis from phenylalanine, the second being the main pathway from 4-hydroxyphenylpyruvate.     

Isolation of alginate-producing mutants of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas mendocina

Spontaneous alginate-producing (muc) variants were isolated from strains of Pseudomonas fluorescens, P. putida and P. mendocina at a frequency of 1 in 10(8) by selecting for carbenicillin resistance. The infrared spectrum of the bacterial exopolysaccharide was typical of an acetylated alginate similar to that previously described in Azotobacter vinelandii and in mucoid variants of P. aeruginosa. Mucoid variants were not isolated from P. stutzeri, P. pseudoalcaligenes, P. testosteroni, P. diminuta, P. acidovorans, P. cepacia or P. maltophilia.     

Isolation of glutamate-inserting ochre suppressor mutants of Salmonella typhimurium and Escherichia coli.

Glutamate-inserting ochre suppressors have been identified among late-arising, spontaneous revertants of a hisG428 mutant of Salmonella typhimurium and an argE3 mutant of Escherichia coli. The S. typhimurium suppressors mapped in the tRNA2(Glu) gene gltU at 82 min; those in E. coli were found to be in tRNA2(Glu) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min.     

Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO.

Two independently derived, exotoxin A-deficient (Tox- phenotype), nitroso-guanidine-induced mutants of Pseudomonas aeruginosa PAO1 were isolated by using sensitive immunological assays. One mutant, designated PAOT10, was detected as a colony which failed to produce a halo of immunoprecipitation in an antiserum-agar assay. The other mutant (PAOT20) was independently isolated and was detected by a negative reaction in a staphylococcal coagglutination assay with protein A-containing staphylococci and affinity-purified antibodies. Both mutants produced parental levels of extracellular protein. However, whereas the qualitative and quantitative compositions of proteins produced by PAOT20 were indistinguishable from those of the parental strain as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of extracellular protease, there were marked differences between PAOT10 and the parental strain. The mutation in PAOT10 (tox-1) as mapped by linkage analysis was located between trp-6 and proA. In contrast, linkage analysis and cotransduction placed the mutation in PAOT20 (tox-2), very near trp-6. Data are presented which suggest that tox-1 and tox-2 are regulatory loci.     

Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa.

Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC1 and PC2, were selected by the swarm plate method after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were fully motile but incapable of swarming, suggesting that they had a defect in the intracellular signalling pathway. Computer-assisted capillary assays confirmed that they failed to show behavioral responses to chemical stimuli, including peptone, methyl thiocyanate, and phosphate. Two chemotaxis genes were cloned by phenotypic complementation of PC1 and PC2. From nucleotide sequence analysis, one gene was found to encode a putative polypeptide that was homologous to the enteric CheZ protein, while the other gene was cheY, which had been previously reported (M. N. Starnbach and S. Lory, Mol. Microbiol. 6:459-469, 1992). Deletion and complementation analysis showed that PC1 was a cheY mutant, whereas PC2 had a double mutation in the cheY and cheZ genes. A chromosomal cheZ mutant, constructed by inserting a kanamycin resistance gene cassette into the wild-type gene, changed its swimming direction much more frequently than did wild-type strain PAO1. In contrast, cheY mutants were found to rarely reverse their swimming directions.     

Isolation and characterization of TMPD-oxidase mutants of Pseudomonas aeruginosa

Mutants showing negative oxidase-reaction have been isolated from Pseudomonas aeruginosa following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine. These mutants were compared to the wild type cells with respect to their respiratory activities and cytochrome contents. They exhibit lower respiration rates and contain much less cytochrome c's which are responsible for their weak or negative oxidase-reaction in these mutants. This is supported in part from an initial linear relationship observed between the measured oxidase activities and the lower cytochrome c contents in these mutants. Further evidence comes from analyzing oxidase-negative cells of P. syringae, in which low cytochrome c contents similar to these oxidase mutants account for negative oxidase activities. Cytochrome o was the sole oxidase found among these mutants as well as in the wild type cell, suggesting that cytochrome c+o complex is responsible for the tetramethyl-p-phenylenediamine-oxidase activity in these mutants as the case in the wild-type cells. From the spectral characteristics it seems that all mutants contain about the same amount and type of terminal oxidase as that of the wild-type cells. The mutation occurred which altered the oxidase activities in these mutants appears to affect cytochrome c gene(s), but not the terminal oxidase gene(s).Abbreviations TMPD Tetramethyl-p-phenylenediamine - MD minimal Davis     

Isolation and characterization of Pseudomonas aeruginosa mutants blocked in the synthesis of pyoverdin.

We have isolated and characterized by chemical and enzymatic analyses three distinct types of pyoverdin-defective (pvd) mutants of Pseudomonas aeruginosa PAO1. The pvd-1 mutant is an L-N5-hydroxyornithine (L-N5-OH-Orn) auxotroph unable to hydroxylate L-ornithine (L-Orn) in a cell-free system and requiring L-N5-OH-Orn for pyoverdin production. The other two types of mutants appear to be blocked in further steps of the biosynthetic pathway leading to pyoverdin, namely, the acylation of L-N5-OH-Orn (pvd-2) and chromophore synthesis (pvd-3). The different pvd mutations were all found to be located in the catA1 region at 47 min of the genetic map of P. aeruginosa PAO1.     

Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO.

Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the 11-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc- mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc- mutants but exhibited wild-type inducible expression.     

Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.     

Amber suppressor mutations in Pseudomonas acidovorans.

Almost 50% of the clones of Pseudomonas acidovorans(pLM2) selected for resistance to tetracycline supported the growth of an amber mutant of bacteriophage PRD1.     

Isolation and characterization of Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis.

Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis were isolated after chemical mutagenesis by plating on a siderophore detection medium. Like the wild type, these mutants incorporated 7-[14C]salicylic acid into pyochelin, demonstrating that salicylic acid is an intermediate in the biosynthesis pathway of pyochelin.     

Isolation and characterization of cAMP suppressor mutants of Escherichia coli K12

Summary We have isolated spontaneous and chemically induced revertants of cya mutant strains of Escherichia coli. Three different classes of revertants were obtained. One class consisted of primary site revertants; a second class was pseudorevertants that had phenotypically reverted to wild type but retaining the original cya mutation and the third class of revertants, designated csm, were pseudorevertants hypersensitive to exogenous cAMP. Transductional analysis of the csm mutation indicated the mechanism of suppression in these strains was intergenic. The csm mutation and hypersensitivity to cAMP map in or near the crp gene. Growth of the csm strains of PTS (phosphoenolpyruvate phosphotransferase system) and non-PTS substrates was inhibited by 5 mM cAMP. The csm strains were found to accumulate toxic levels of methylglyocal when grown on non-PTS substrates in the presence of exogenous cAMP. All csm strains were sensitive to catabolite repression mediated by -methylglucoside. Revertants selected as resistant to cAMP fell into four major classes that could be distinguished by their fermentation patterns in the presence and absence of cAMP as well as by their growth response to streptomycin in the presence of cAMP.Paper No. 6623 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650, USA     

Isolation and characterization of Pseudomonas aeruginosa bacteriophage phikF77 mutants]

It was found that phage phi kF77 is resistant to all known Pseudomonas aeruginosa restriction systems. Three types of mutants (dc-) which were unable to grow on different restrictive strains were isolated. All of them belong to one complementation group. Some of these mutations affected also the number of nicks in phage phi kF77 DNA and increased phage resistance to temperature treatment. It may be supposed that genes responsible for antirestriction mechanisms and introduction of nicks into DNA are connected in definite way.     

Isolation and characterization of toluene-sensitive mutants from Pseudomonas putida IH-2000

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P_ow 2.8) but they could grow in medium overlaid with organic solvents having a log P_ow value higher than that of toluene such as p-xylene (log P_ow 3.1), cyclohexane (log P_ow 3.4) and n-hexane (log P_ow 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region containing the cyoB- and cyoC-like genes of strain IH-2000. ORFs 3–7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear.     

Mutations to nonsense codons in human genetic disease: implications for gene therapy by nonsense suppressor tRNAs.

Nonsense suppressor tRNAs have been suggested as potential agents for human somatic gene therapy. Recent work from this laboratory has described significant effects of 3' codon context on the efficiency of human nonsense suppressors. A rapid increase in the number of reports of human diseases caused by nonsense codons, prompted us to determine how the spectrum of mutation to either UAG, UAA or UGA codons and their respective 3' contexts, might effect the efficiency of human suppressor tRNAs employed for purposes of gene therapy. This paper presents a survey of 179 events of mutations to nonsense codons which cause human germline or somatic disease. The analysis revealed a ratio of approximately 1:2:3 for mutation to UAA, UAG and UGA respectively. This pattern is similar, but not identical, to that of naturally occurring stop codons. The 3' contexts of new mutations to stop were also analysed. Once again, the pattern was similar to the contexts surrounding natural termination signals. These results imply there will be little difference in the sensitivity of nonsense mutations and natural stop codons to suppression by nonsense suppressor tRNAs. Analysis of the codons altered by nonsense mutations suggests that efforts to design human UAG suppressor tRNAs charged with Trp, Gln, and Glu; UAA suppressors charged with Gln and Glu, and UGA suppressors which insert Arg, would be an essential step in the development of suppressor tRNAs as agents of human somatic gene therapy.     

Isolation and analysis of Pseudomonas aeruginosa PAO mutants resistant to nonlysogenic bacteriophages

Nonlysogenizing Pseudomonas aeruginosa PAO bacteriophages were studied. According to morphology of the plaques, they were distributed into three groups: phi k, phi m and phi mn. The mutants of P. aeruginosa PAO resistant to these bacteriophages were selected. On the basis of cris-cross resistance analysis of the mutants, a formal scheme of the receptor sites on the P. aeruginosa PAO bacterial cell surface is drawn. It is shown that bacteriophages phi k and phi m use different receptors for their adsorption. The receptors of phi m and phi mn phages are specifically interconnected. Thus, the receptor for phi k phages is connected with the receptor for phage phi 11. It appears that the receptor for bacteriophage E79 is identical to those of phi m phages. The phi m receptor is of a composite structure: it includes two different receptors used by phi mn phages.