Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using phi29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial genome and evaluation of the MDA product were performed using cyanobacterium Synechocystis sp. strain PCC6803. An 8-h MDA reaction yielded a sufficient quantity of DNA from an initial amount of 0.4 ng, which is equivalent to approximately 10(5) cells. Uniform amplification of genes randomly selected from the cyanobacterial genome was confirmed by real-time polymerase chain reaction. The metagenome from bacteria associated with scleractinian corals was used for whole-genome amplification using phi29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Data Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that alpha-Proteobacteria was the dominant group in Pocillopora damicornis. This study demonstrates that MDA techniques are efficient for genome wide investigation to understand the actual microbial diversity in limited bacterial samples.
Iron uptake systems were identified by global expression profiling of Magnetospirillum magneticum AMB-1. feo, tpd, and ftr, which encode ferrous iron transporters, were up-regulated under iron-rich conditions. The concomitant rapid iron uptake and magnetite formation suggest that these uptake systems serve as iron supply lines for magnetosome synthesis.Iron is crucial in microbial metabolism. It exists in two redox states: the reduced Fe 2ϩ soluble ferrous state and the oxidized Fe 3ϩ ferric form, which is extremely insoluble (1). In magnetotactic bacteria, the formation of highly organized membranebound intracellular magnetites (Fe 3 O 4 ) or greigites (Fe 3 S 4 ) requires the acquisition of a large amount of iron several orders of magnitude greater than that required by Escherichia coli (5). Because of the large amount of iron to be transported, complex iron uptake systems that differ from known transport mechanisms in other microorganisms are expected to be operating in magnetotactic bacteria. Exceptional progress has been made in the isolation and characterization of functional genes and proteins involved in magnetosome synthesis (2,11,13,21,24), but at present, specific iron transport systems remain unidentified in magnetotactic bacteria.Here we report the identification of iron uptake systems by global expression profiling of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 grown under iron-rich and iron-deficient conditions. Our results indicate that despite the unusual high-iron requirement of M. magneticum AMB-1, it utilizes robust but simple iron uptake systems similar to those of other gram-negative bacteria. This robust ferrous iron uptake suggests a significant contribution to magnetite synthesis. This study is the first to identify specific iron uptake systems in the complex iron metabolism of magnetotactic bacteria. The data presented here may facilitate future studies on the mechanism of magnetosome formation.To monitor iron uptake and magnetite formation, M. magneticum AMB-1 (ATCC 700264) was grown at 25°C under microaerobic conditions by sparging argon gas for 10 min into 500 ml of MSGM medium as previously described (5), with various iron concentrations of 0.1 to 300 M. All iron measurements were performed by atomic absorbance spectrophotometry. Extracellular iron concentrations were measured at different time points in cell-free culture supernatants. For intracellular iron measurements, cells were disrupted by lysozyme treatment (20) and ultracentrifuged at 100,000 ϫ g to separate the insoluble (magnetites) and soluble iron fractions.Iron was rapidly taken up in iron-rich cultures, and a corresponding increase of intracellular iron was observed within 10 min (Fig. 1A). Up to 70% of the initial iron concentration of the medium was taken up, and intracellular iron increased to 5,000 nmol/10 9 cells after 60 min. Insoluble iron in the cytoplasm, which mostly included magnetites, also increased within 10 min (Fig. 1B). These data indicate that the external iron was rapidly assimilate...
Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1 is elicited by sufficient iron rather than by iron starvation. In order to clarify this unusual pattern, siderophore production was monitored in parallel to iron assimilation using the chrome azurol sulfonate assay and the ferrozine method respectively. Iron concentration lowered approximately five times less than its initial concentration only within 4 h post-inoculation, rendering the medium iron deficient. A concentration of at least 6 WM Fe 3þ is required to initiate siderophore production. The propensity of M. magneticum AMB-1 for the assimilation of large amounts of iron accounts for the rapid depletion of iron in the medium, thereby triggering siderophore excretion. M. magneticum AMB-1 produces both hydroxamate and catechol siderophores.
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