Metabolite Profiling Uncovers Plasmid-Induced Cobalt Limitation under Methylotrophic Growth Conditions

BACKGROUND:The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases. METHODOLOGY/PRINCIPAL FINDINGS:We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B(12)-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import. CONCLUSIONS/SIGNIFICANCE:This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress

Flower morphology, nectar features, and hummingbird visitation to Palicourea crocea (Rubiaceae) in the Upper Paraná River floodplain, Brazil

We investigated flower morphology, nectar features, and hummingbird visitation to Palicourea crocea (Rubiaceae), a common ornithophilous shrub found in the riparian forest understory in the Upper Paraná River floodplain, Brazil. Flowers are distylous and the style-stamen dimorphism is accompanied by other intermorph dimorphisms in corolla length, anther length, and stigma lobe length and form. We did not observe strict reciprocity in the positioning of stigma and anthers between floral morphs. Flowering occurred during the rainy season, October to December. Nectar standing crop per flowerwas relatively constant throughout the day, which apparently resulted in hummingbirds visiting the plant throughout the day. Energetic content of the nectar in each flower (66.5J) and that required daily by hummingbird visitors (up to 30kJ) would oblige visits to hundreds of flowers each day, and thus movements between plants that should result in pollen flow. Three hummingbird species visited the flowers: the Gilded Sapphire (Hylocharis chrysura), the Black-throated Mango (Anthracothorax nigricollis), and the Glittering-bellied Emerald (Chlorostilbon aureoventris). The frequency of hummingbird visitation, nectar features, and the scarcity of other hummingbird-visited flowers in the study area, indicate that P. crocea is an important nectar resource for short-billed hummingbirds in the study site.<br>Investigamos a morfologia floral, as características do néctar e a visita de beija-flores a Palicourea crocea (Rubiaceae), uma espécie ornitófila arbustiva comumente encontrada no sub-bosque da vegetação ripária na planície de inundação do Alto Rio Paraná, Brasil. As flores são distílicas, sendo o dimorfismo estilete-estames acompanhado por outras variações morfológicas no comprimento da corola, altura das anteras, comprimento das anteras e comprimento e forma das papilas estigmáticas. Não foi observada reciprocidade estrita na posição dos estigmas e anteras entre flores longistilas e brevistilas. A floração da espécie ocorreu durante a estação chuvosa, de outubro a dezembro. A disponibilidade de néctar foi relativamente constante ao longo do dia, o que aparentemente possibilitou aos beija-flores visitar a planta o dia todo. O conteúdo energético de néctar por flor (66,5J) e a demanda diária de energia dos beija-flores visitantes(superior a 30kJ) os obrigaria a visitar diariamente centenas de flores da espécie, o que pode aumentar o fluxo de pólen. Três espécies de beija-flores foram observadas: beija-flor-dourado (Hylocharis chrysura), beija-flor-de-colete-preto ( Anthracothorax nigricollis) e esmeralda-de-bico-vermelho ( Chlorostilbon aureoventris). A freqüência de visita dos beija-flores, as características do néctar e a baixa disponibilidade de outras flores visitadas por beija-flores na área de estudo indicam que P. crocea é uma fonte de néctar importante para beija-flores de bico-curto no local