Characteristics of Insertional Mutants of Pseudomonas syringae with Reduced Epiphytic Fitness

Random Tn5 mutagenesis was used to identify genes ir. Pseudomonas syringae which contribute to epiphytic fitness. Mutants were selected on the basis of deficiencies in epiphytic growth or survival on plants rather than deficiencies in predetermined phenotypes exhibited in culture. A sample freezing procedure was used to measure the population sizes of 5,300 mutants of P. syringae exposed to alternating wet and dry conditions on bean leaves in growth chambers. Eighty-two mutants exhibited reduced population sizes. Of these mutants, over half exhibited a reduced ability to survive the stresses associated with dry leaves, while others grew more slowly or attained reduced stationary-phase population sizes on leaves. While some epiphytic fitness mutants were altered in phenotypes that could be measured in culture, many mutants were not altered in any in vitro phenotype examined. Only three of the epiphytic fitness mutants were auxotrophs, and none had catabolic deficiencies for any of 31 organic compounds tested. Other mutants that exhibited reductions in one or more of the following were identified: motility, osmotolerance, desiccation tolerance, growth rate in batch culture, and extracellular polysaccharide production. All of the mutants retained the abilities to produce disease symptoms on the compatible host plant, bean, to incite a hypersensitive response on the non-host plant, tobacco, and to produce a fluorescent pyoverdine siderophore.     

Survival, Growth, and Localization of Epiphytic Fitness Mutants of Pseudomonas syringae on Leaves

Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated onto and into plant leaves. For example, while none showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular spaces of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parental strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sites protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possibly several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes.     

Comparison of the Behavior of Epiphytic Fitness Mutants of Pseudomonas syringae under Controlled and Field Conditions

The epiphytic fitness of four Tn5 mutants of Pseudomonas syringae that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established, demonstrating that their fitness was reduced more under field conditions than in the laboratory. Under both conditions, the four mutants exhibited distinctive behaviors. One mutant exhibited particularly large population decreases and short half-lives following inoculation but grew epiphytically at near-wild-type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by B728a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of P. syringae, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes.     

A Novel Positive Selection for Identifying Cold-Sensitive Myosin II Mutants in Dictyostelium

We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.     

A Novel Screening Method for Isolating Exopolysaccharide-Deficient Mutants

A screening method based on differential staining of the wild type and exopolysaccharide-deficient mutants of Rhizobium (Sinorhizobium) meliloti by the lipophilic dye Sudan Black B is described. Mutants defective in the production of either succinoglycan or EPS II (galactoglucan) were isolated by using this method, which might also prove useful for isolating exopolysaccharide-defective derivatives of other bacteria.     

Isolation of Streptomyces rimosus Mutants with Reduced Actinophage Susceptibility

The infection of Streptomyces rimosus by the virulent actinophage RP1 was partially characterized. RP1 infection of the host cells results in a dramatic decrease in viable cell count, followed by reduced antibiotic production. Phage-resistant mutants were isolated after mutagenic treatment and RP1 selective pressure. Characterization of the isolated mutants has revealed that RP1 infection had no influence on their growth and antibiotic production. However, multiplication of the phage particles in the lawns of resistant mutants was detected. Since these strains differ from the wild type in RP1 relative efficiency of plating, plaque morphology, and the time necessary for plaque appearance, they are considered to be semiresistant mutants. The propagation of RP1 on semiresistant strains is characterized by lower adsorption of phage particles and longer latent and rise periods. As a consequence, the multiplication of the phage is slower than that of their host, which consequently reduces the ratio of phage to its host, thus diluting out the phage.     

Histidine Mutants Requiring Adenine: Selection of Mutants with Reduced hisG Expression in SALMONELLA TYPHIMURIUM

A method is described for the selection of Salmonella typhimurium mutants with reduced levels of hisG enzyme activity. This method is based on the fact that the hisG enzyme catalyzes the consumption of ATP in the first step of histidine biosynthesis. Normally, this reaction is closely regulated, both by feedback inhibition and by repression of the operon. However, conditions can be set up that result in the uncontrolled use of adenine in histidine biosynthesis. Cells grown under these conditions become phenotypic adenine auxotrophs. Some revertant clones that no longer require adenine contain mutations in hisG, hisE, or the his-control region. The hisG mutations are of all types (nonsense, frameshift, missense, deletion and leaky types), and they map throughout the hisG gene.     

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.     

Horizontal Transfer of Bacterial Symbionts: Heritability and Fitness Effects in a Novel Aphid Host

Members of several bacterial lineages are known only as symbionts of insects and move among hosts through maternal transmission. Such vertical transfer promotes strong fidelity within these associations, favoring the evolution of microbially mediated effects that improve host fitness. However, phylogenetic evidence indicates occasional horizontal transfer among different insect species, suggesting that some microbial symbionts retain a generalized ability to infect multiple hosts. Here we examine the abilities of three vertically transmitted bacteria from the Gammaproteobacteria to infect and spread within a novel host species, the pea aphid, Acyrthosiphon pisum. Using microinjection, we transferred symbionts from three species of natural aphid hosts into a common host background, comparing transmission efficiencies between novel symbionts and those naturally infecting A. pisum. We also examined the fitness effects of two novel symbionts to determine whether they should persist under natural selection acting at the host level. Our results reveal that these heritable bacteria vary in their capacities to utilize A. pisum as a host. One of three novel symbionts failed to undergo efficient maternal transmission in A. pisum, and one of the two efficiently transmitted bacteria depressed aphid growth rates. Although these findings reveal that negative fitness effects and low transmission efficiency can prevent the establishment of a new infection following horizontal transmission, they also indicate that some symbionts can overcome these obstacles, accounting for their widespread distributions across aphids and related insects.     

A Novel Prioritization Method in Identifying Recurrent Venous Thromboembolism-Related Genes

Identifying the genes involved in venous thromboembolism (VTE) recurrence is important not only for understanding the pathogenesis but also for discovering the therapeutic targets. We proposed a novel prioritization method called Function-Interaction-Pearson (FIP) by creating gene-disease similarity scores to prioritize candidate genes underling VTE. The scores were calculated by integrating and optimizing three types of resources including gene expression, gene ontology and protein-protein interaction. As a result, 124 out of top 200 prioritized candidate genes had been confirmed in literature, among which there were 34 antithrombotic drug targets. Compared with two well-known gene prioritization tools Endeavour and ToppNet, FIP was shown to have better performance. The approach provides a valuable alternative for drug targets discovery and disease therapy.     

Modified Penicillin Enrichment Procedure for the Selection of Bacterial Mutants

Penicillin enrichment and selection of biochemical mutants was performed on agar plates. With this technique, there is an optimum penicillin exposure time for the greatest yield of a particular auxotroph.     

Characterization of Triosephosphate Isomerase Mutants with Reduced Enzyme Activity in Mus Musculus

Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse.     

How Fitness Reduced,Antimicrobial Resistant Bacteria Survive and Spread: A Multiple Pig - Multiple Bacterial Strain Model

More than 30% of E. coli strains sampled from pig farms in Denmark over the last five years were resistant to the commonly used antimicrobial tetracycline. This raises a number of questions: How is this high level sustained if resistant bacteria have reduced growth rates? Given that there are multiple susceptible and resistant bacterial strains in the pig intestines, how can we describe their coexistence? To what extent does the composition of these multiple strains in individual pigs influence the total bacterial population of the pig pen? What happens to a complex population when antimicrobials are used? To investigate these questions, we created a model where multiple strains of bacteria coexist in the intestines of pigs sharing a pen, and explored the parameter limits of a stable system; both with and without an antimicrobial treatment. The approach taken is a deterministic bacterial population model with stochastic elements of bacterial distributions and transmission. The rates that govern the model are process-oriented to represent growth, excretion, and uptake from environment, independent of herd and meta-population structures. Furthermore, an entry barrier and elimination process for the individual strains in each pig were implemented. We demonstrate how competitive growth between multiple bacterial strains in individual pigs, and the transmission between pigs in a pen allow for strains of antimicrobial resistant bacteria to persist in a pig population to different extents, and how quickly they can become dominant if antimicrobial treatment is initiated. The level of spread depends in a non-linear way of the parameters that govern excretion and uptake. Furthermore, the sampling of initial distributions of strains and stochastic transmission events give rise to large variation in how homogenous and how resistant the bacterial population becomes. Most important: resistant bacteria are demonstrated to survive with a disadvantage in growth rate of well over 10%.     

降氰菌突变株处理合成氨企业含氰废水的研究

从合成氨废水污染的土壤和排水沟底泥中经过多次富集、分离,筛选出了具有较高耐氰和降氰能力的野生型菌株,经过紫外诱变得到2株具有高效降氰能力的突变株HN-1和HN-4。利用接触氧化法进行合成氨企业含氰废水的降氰处理时加入突变株HN-1和HN-4,处理效果大大增强,在水力停留时间(HRT)2 h的情况下,氰的降解率可达90%以上。     

Leaf-Atmosphere NH3 Exchange in Barley Mutants with Reduced Activities of Glutamine Synthetase

Mutants of barley (Hordeum vulgare L. cv Maris Mink) with 47 or 66% of the glutamine synthetase (GS) activity of the wild type were used for studies of NH3 exchange with the atmosphere. Under normal light and temperature conditions, tissue NH4+ concentrations were higher in the two mutants compared with wild-type plants, and this was accompanied by higher NH3 emission from the leaves. The emission of NH3 increased with increasing leaf temperatures in both wild-type and mutant plants, but the increase was much more pronounced in the mutants. Similar results were found when the light intensity (photosynthetic photon flux density) was increased. Compensation points for NH3 were estimated by exposing intact shoots to 10 nmol NH3 mol-1 air under conditions with increasing temperatures until the plants started to emit NH3. Referenced to 25[deg]C, the compensation points were 5.0 nmol mol-1 for wild-type plants, 8.3 nmol mol-1 for 47% GS mutants, and 11.8 nmol mol-1 for 66% GS mutants. Compensation points for NH3 in single, nonsenescent leaves were estimated on the basis of apoplastic pH and NH4+ concentrations. These values were 0.75, 3.46, and 7.72 nmol mol-1 for wild type, 47% GS mutants, and 66% GS mutants, respectively. The 66% GS mutant always showed higher tissue NH4+ concentrations, NH3 emission rates, and NH3 compensation points compared with the 47% GS mutant, indicating that NH4+ release was curtailed by some kind of compensatory mechanism in plants with only 47% GS activity.