Identification of the biosynthetic gene cluster for 3-methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93.

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K(m), 7 mM; k(cat), 85 min(-1)). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

The application of soccer performance testing protocols to the non-elite player.

AIM: The application of performance testing for the evaluation of non-elite soccer players has received little attention. The purpose of this investigation was to use tests developed for elite soccer players to evaluate performance in non-elite soccer players and compare performance test results between elite (literature) and non-elite (data) players. METHODS: Thirteen male soccer players volunteered to participate. The tests included a treadmill VO2max test, 20 m sprint, vertical jump (VJ), 30 s Wingate cycle ergometer test, the Loughborough Intermittent Shuttle Test (LIST), and 2 20-m multi-stage shuttle runs to exhaustion (fatigue test). Actual VO2max (absolute and relative) scores were correlated with the estimated VO2max scores (fatigue test), 20 m sprint, VJ, and 30 s Wingate using a Pearson's product-moment correlation. A paired t-test was conducted on the fatigue test trials. RESULTS: Non-significant relationships were observed between actual VO2max scores and estimated VO2max from the fatigue test (absolute and relative terms). Non-significant relationships were also observed between peak and average power output (Wingate), 20 m sprint, and VJ. Mean heart rates (HRs) throughout the LIST was 165+/-7 bpm, which represented 88% of HRmax. CONCLUSIONS: The results of this study demonstrate that to elicit physiological differences between elite and non-elite players, assessment must include both an aerobic and anaerobic component.

Thermoregulated expression of virulence factors in plant-associated bacteria.

Pathogenic bacteria with habitats inside and outside a given host react to changes in environmental parameters by synthesizing gene products specifically needed during pathogenic or saprophytic growth. Temperature effects have been investigated in detail for pathogens of warm-blooded hosts, and major principles governing the temperature-sensing mechanism have been uncovered. Generally, transcription of virulence genes in these pathogens is induced at higher temperatures (37-41 degrees C), which are typical for body cavities and host tissues. However, effects of temperature on virulence determinants in plant pathogenic bacteria have not been focused on in detail. Interestingly, almost all virulence genes of plant pathogenic bacteria studied with respect to temperature exhibit increased transcription at temperatures well below the respective growth optima. This includes virulence determinants such as those directing bacteria-to-plant gene transfer, plant cell-wall-degrading enzymes, phytotoxins, ice nucleation activity, exopolysaccharide production, and the type III protein secretion machinery. Although many of the studied phytopathogens cause "cold-weather" diseases, the ecological rationale for this phenomenon remains to be studied in detail. This mini-review summarizes our current knowledge on thermoregulation of cellular processes taking place in bacterial phytopathogens in response to temperature changes. Since the temperature range of interest is different from that relevant to pathogens of mammals, one envisions novel principles of thermo-sensing in bacteria interacting with plants.

The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola.

ABSTRACT The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum.

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.

Toxin production by pathovars of Pseudomonas syringae and their antagonistic activities against epiphytic microorganisms.

75 strains of 21 various Pseudomonas syringae (P.) pathovars were investigated in different tests for their toxin production. Data from literature about the production of the known phytotoxins phaseolotoxin (pv. phaseolicola), tabtoxin (pv. coronafaciens, pv. tabaci), coronatine (pv. atropurpurea, pv. glycinea, pv. maculicola, pv. morsprunorum, pv. tomato), and toxins of the lipodepsipeptide group (pv. aptata, pv. atrofaciens, pv. syringae) could be confirmed. Besides, a production of the phytohormone ethylene was detected for P. phaseolicola isolates from kudzu (Pueraria lobata) and for all tested P. glycinea and P. cannabina strains. Strains of P. apii, P. aptata, P. atrofaciens, and P. tomato produced antimetabolic toxins which could be detected with an agar diffusion assay with Escherichia coli as indicator strain. These antimetabolites inhibit a step in the arginine/ornithine biosynthesis. P. maculicola strains caused inhibition zones in this assay which could not be reversed by the tested amino acids. All strains with inhibitory effect against E. coli and Geotrichum candidum were also proved for their antagonistic activity against a selection of typical phyllosphere microorganisms. Most of the phytotoxins possess antimicrobial activity with different spectrum and efficiency. Only the lipodepsipeptide-producers showed antifungal activities. Our results show that the production of toxins is a widespread property among Pseudomonas syringae pathovars, and that some pathovars can produce more than one toxin. This characteristic and the antimicrobial activity of most toxins could be of advantage for the toxin-producing bacteria to adapt to different habitats.

Ethylene Production by Pseudomonas syringae Pathovars In Vitro and In Planta.

Significant amounts of ethylene were produced by Pseudomonas syringae pv. glycinea, pv. phaseolicola (which had been isolated from viny weed Pueraria lobata [Willd.] Ohwi [common name, kudzu]), and pv. pisi in synthetic medium. On the other hand, the bean strains of P. syringae pv. phaseolicola and strains of 17 other pathovars did not produce ethylene. P. syringae pv. glycinea and P. syringae pv. phaseolicola produced nearly identical levels of ethylene (about 5 x 10(sup-7) nl h(sup-1) cell(sup-1)), which were about 10 times higher than the ethylene level of P. syringae pv. pisi. Two 22-bp oligonucleotide primers derived from the ethylene-forming enzyme (efe) gene of P. syringae pv. phaseolicola PK2 were investigated for their ability to detect ethylene-producing P. syringae strains by PCR analysis. PCR amplification with this primer set resulted in a specific 0.99-kb fragment in all ethylene-producing strains with the exception of the P. syringae pv. pisi strains. Therefore, P. syringae pv. pisi may use a different biosynthetic pathway for ethylene production or the sequence of the efe gene is less conserved in this bacterium. P. syringae pv. phaseolicola isolated from kudzu and P. syringae pv. glycinea also produced ethylene in planta. It could be shown that the enhanced ethylene production in diseased tissue was due to the production of ethylene by the inoculated bacteria. Ethylene production in vitro and in planta was strictly growth associated.

[Comparative clinical studies on the use of porous hydroxylapatite (HAP) and decalcified freeze-dried bone (DFB) in the treatment of deep intra-alveolar periodontal bone defects]. / Vergleichende klinische Untersuchungen zur Anwendung von porösem Hydroxylapatit (HAP) und dekalzifiziertem gefriergetrocknetem Knochen (DFB) bei der Behandlung tiefer intraalveolärer periodontaler Knochendefekte.

In 12 cases a systematical periodontal therapy involved surgical treatment (mod. Widman flap operation). 16 intrabony defects with more than 6 mm pocket depth measured at the beginning of periodontal therapy were filled with porous hydroxyl- (Interpore 200) as well as decalcified freeze dried bone grafts. For judgement of the therapeutic success plaque index, sulcus bleeding index, mobility of teeth treated by surgical methods, pocket depth, attachment level, and x-ray documentation were used before the operation and after 6 month maintenance therapy.

[Structural changes in the dental hard substances and jaw bones of animals due to short-duration weightlessness]. / Strukturveränderungen an tierischen Zahnhartsubstanzen und Kieferknochen durch kurzdauernde Schwerelosigkeit.

Molars, incisors and parts of corpus mandibulae from a total of 40 male, 30 female (WISTAR SPF), who were exposed for a short-time microgravity, and of their 90 d old offspring were examined. It was calculated the carbonateapatite content in the teeth and in bone in percent by infrared spectroscopy. In terms of quantitative parameters the substances of the space flight group versus the controls varied clearly. Quality and size of these changes depend from sex, investigated material and time of space flight. It can be concluded that an accommodation to the flight stress is possible without any functional disorders also in the oral system.