Molecular Biology

ABSTRACT Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as “ Candidatus Cloacamonas acidaminovorans.” “ Candidatus Cloacamonas acidaminovorans” is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.

SUMMARY Phytoplasmas are plant‐pathogenic bacteria that are associated with numerous plant diseases. We have previously reported the complete genomic sequence of Candidatus Phytoplasma asteris, OY strain, OY‐M line, which causes mild symptoms. The phytoplasma genome lacks several important metabolic genes, implying that the consumption of metabolites by phytoplasmas in plants may cause disease symptoms. Here we show that the approximately 30‐kb region including the glycolytic genes was tandemly duplicated in the genome of OY‐W phytoplasma, which causes severe symptoms. Almost duplicated genes became pseudogenes by frameshift and stop‐codon mutations, probably because of their functional redundancy. However, five kinds of genes, including two glycolytic genes, remained full‐length ORFs, suggesting that it is advantageous for the phytoplasma to retain these genes in its lifestyle. In particular, 6‐phosphofructokinase is known as a rate‐limiting enzyme of glycolysis, implying that the different number of glycolytic genes between OY‐W and OY‐M may influence their respective glycolysis activities. We previously reported that the phytoplasma population of OY‐W was higher than that of OY‐M in their infected plants. Taking this result into account, the higher consumption of the carbon source may affect the growth rate of phytoplasmas and also may directly or indirectly cause more severe symptoms.

ABSTRACT “ Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181: 17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c ) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of “ Ca . Chlorothrix halophila,” that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ∼6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “ Ca . Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus .

ABSTRACT The pigment composition of “ Candidatus Chlorothrix halophila,” a filamentous anoxygenic phototrophic bacterium found in Baja California Sur, Mexico, was determined. Previous work showed that bacteriochlorophyll c (BChl c ) was the major pigment in “ Ca . Chlorothrix halophila,” but it was not clear if this bacterium also contains BChl a (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181: 17-25, 2004). Here we show that in addition to BChl c , a small amount of a pigment that is spectrally indistinguishable from BChl a is present in cell extracts of “ Ca . Chlorothrix halophila.” Nevertheless, the BChl a -like pigment from “ Ca . Chlorothrix halophila” has a different molecular weight and a different high-performance liquid chromatography elution time than BChl a from other photosynthetic bacteria. Based on mass spectrometry and other spectroscopic analysis, we determined that the BChl a -like pigment in “ Ca . Chlorothrix halophila” contains a tetrahydrogeranylgeraniol tail rather than the phytol tail that is present in BChl a . The carotenoids and major BChl c homologs in “ Ca . Chlorothrix halophila” were also identified. BChls c were found to be farnesol esterified and geranylgeraniol esterified.

ABSTRACTTo survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned adps(stands forDNA-bindingprotein fromstarved cells) gene of the symbiotic “CandidatusLegionella jeonii” organism (called the X bacterium) (dpsX) that grows inAmoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented thedpsmutant ofEscherichia coliand conferred resistance to hydrogen peroxide. DpsXproteins purified fromE. colitransformed with thedpsXgene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H2O2-mediated damage. The expression of thedpsXgene in “CandidatusLegionella jeonii” was enhanced when the host amoeba was treated with 2 mM H2O2and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion ofLegionellasp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization ofdpsXexpression in theLegionellasp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.