Matthias S. Ullrich

Reversible, metal-free, heterolytic activation of H2 at room temperature.

The combination of the borane B(p-C(6)F(4)H)(3) and (o-C(6)H(4)Me)(3)P activates H(2) to give [(o-C(6)H(4)Me)(3)PH][HB(p-C(6)F(4)H)(3)]. This salt when placed under vacuum releases H(2) at room temperature.

The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora.

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.

Characterization of the genes controlling the biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic and coronamic acid.

Pseudomonas syringae pv. glycinea PG4180 produces a chlorosis-inducing phytotoxin, coronatine (COR), which consists of a polyketide component, coronafacic acid (CFA), which is coupled via amide bond formation to coronamic acid (CMA), an ethylcyelopropyl amino acid (aa) derived from isoleucine. P. syringae pv. syringae strains PS51 and PS61, which do not synthesize coronafacoyl compounds (conjugates between CFA and aa), acquired the ability to produce CFA and COR when transformed with p4180A, a 90-kb indigenous plasmid in PG4180. Tn5 mutagenesis indicated that the COR biosynthetic genes in PG4180 are clustered within a 30-kb region on p4180A. The phenotype of selected COR-defective mutants was determined by supplying them with CFA and CMA and by complementation studies with cloned DNA from the COR biosynthetic cluster. Using this approach, the regions encoding CFA and CMA synthesis and coupling activity were localized to the 24-, 12.5- and 2.3-kb regions of the cluster, respectively. Mutants in a 6-kb region required the addition of both CFA and CMA for COR synthesis, which may indicate a regulatory role for this part of the cluster. PS51 and PS61 transconjugants containing cloned DNA from the coupling region produced COR when supplied with CFA and CMA, indicating that coupling activity was cloned and expressed in bacteria lacking the COR biosynthetic cluster.

NorM, an Erwinia amylovora Multidrug Efflux Pump Involved in In Vitro Competition with Other Epiphytic Bacteria

ABSTRACT Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18°C than at 28°C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.

Resistance to ultraviolet light in Pseudomonas syringae: sequence and functional analysis of the plasmid-encoded rulAB genes

The indigenous plasmids, pPSR1 and pPSR5, were each shown to confer resistance to ultraviolet light (UV) in Pseudomonas syringae (Ps) pv. syringae FF5. The UV-resistance (UVR) determinant was subcloned from a cosmid library of pPSR1, and sequence analysis revealed the presence of two ORFs, designated rulAB which are homologous to the Escherichia coli umuDC mutagenic DNA repair systems and other plasmid-encoded UVR operons. Amino acid (aa) alignments indicated that RulAB are most closely related to the RumAB proteins from plasmid R391, sharing 40.5% and 48.6% aa identity with RumA and RumB, respectively. UV sensitivity assays with the cloned rulAB genes indicated that the expression of UVR in Ps required a functional recA gene.

Interactions of Pseudomonas syringae pv. glycinea with host and nonhost plants in relation to temperature and phytotoxin synthesis.

Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coronatine (COR) in a temperature-dependent manner. COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18 degrees C whereas no detectable synthesis occurs at 28 degrees C. We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively. After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18 degrees C prior to inoculation but not when derived from cultures grown at 28 degrees C. The disease outcome was quantified by determination of bacterial populations in planta. The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings. In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature. However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18 degrees C, compared with parallels incubated at 28 degrees C. CFA biosynthesis by PG4180.N9 was optimal at 18 degrees C and negligible at 28 degrees C. The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR. Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFAs putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.

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.

Molecular characterization of field isolates of Pseudomonas syringae pv. glycinea differing in coronatine production

Coronatine-producing and non-producing strains of Pseudomonas syringae pv. glycinea have been examined. We found a connection between copper resistance and synthesis of coronatine. Published data implied that these properties may be encoded on different plasmids. Production of coronatine and copper resistance were also found to be correlated for pv. glycinea in 19 field-isolates from leaf spots of plants in a soybean field and in 28 strains of a bacterial culture collection. Genomic diversity within pv. glycinea was investigated by plasmid profiling, DNA hybridization studies and PCR analysis. All strains unable to produce coronatine (cor-) were sensitive to copper ions and showed no homology to DNA from plasmid pSAY1, which carries a gene cluster for steps in coronatine production. In addition, cor- strains could be distinguished from coronatine-producing strains by a single unique band when amplified by random primer PCR. Plasmid profiles of strains isolated from field-populations during 1983, 1985 and 1990 showed that coronatine-producing and non-producing strains were present. The plasmid patterns also varied in 28 strains examined from a culture collection. No correlation between plasmid patterns and race specificity was observed. Cosmid pSAY1 proved to be an effective probe for detection of the coronatine synthesis genes and also revealed polymorphisms in coronatine producing strains of pv. glycinea.

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.