Publications

4472

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.

Phylogenetic analysis and phenotypic characterization were used to assign a multicellular magnetotactic prokaryote the name ‘Candidatus Magnetoglobus multicellularis’. ‘Candidatus Magnetoglobus multicellularis' lives in a large hypersaline coastal lagoon from Brazil and has properties that are unique among prokaryotes. It consists of a compact assembly or aggregate of flagellated bacterial cells, highly organized in a sphere, that swim in either helical or straight trajectories. The life cycle of ‘Candidatus Magnetoglobus multicellularis' is completely multicellular, in which one aggregate grows by enlarging the size of its cells and approximately doubling the volume of the whole organism. Cells then divide synchronously, maintaining the spherical arrangement; finally the cells separate into two identical aggregates. Phylogenetic 16S rRNA gene sequence analysis showed that ‘Candidatus Magnetoglobus multicellularis' is related to the dissimilatory sulfate-reducing bacteria within the Deltaproteobacteria and to other previously described, but not yet well characterized, multicellular magnetotactic prokaryotes.

Six strains (CB7T, CB18, CB23, CB26, CB28 and CB35T) were isolated from human faeces. Based on phylogenetic analysis, phenotypic characteristics, cellular fatty acid profiles and menaquinone profiles, these strains could be included within the genusPrevotellaand made up two clusters. 16S rRNA gene sequence analysis indicated that five strains were most closely related toPrevotella veroralis, sharing about 92 % sequence similarity; the remaining strain was most closely related toPrevotella shahii, sharing about 90 % sequence similarity. All six strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. The cellular fatty acid compositions of the six strains differed significantly from those of otherPrevotellaspecies. Five strains (CB7T, CB18, CB23, CB26 and CB28) contained dimethyl acetals and the major menaquinones of these strains were MK-11, MK-12 and MK-13. The major menaquinones of CB35Twere MK-12 and MK-13. Based on phenotypic and phylogenetic findings, two novel species,Prevotella coprisp. nov. andPrevotella stercoreasp. nov., are proposed, representing the two different strain clusters. The DNA G+C contents of strains CB7Tand CB35Twere 45.3 and 48.2 mol%, respectively. The type strains ofP. copriandP. stercoreaare CB7T(=JCM 13464T=DSM 18205T) and CB35T(=JCM 13469T=DSM 18206T), respectively.

Filamentous bacteria frequently occurring in the pelagic zone of natural freshwater lakes and ponds were previously identified as being related to Haliscomenobacter hydrossis based upon their 16S rRNA gene sequences. These bacteria exhibit a specific morphology characterized by the formation of straight, stick-like filaments of variable length (5 to >100 μm) and quite stable, but narrow, width (0.25 to 0.35 μm). Bacteria with these morphological characteristics form a monophyletic but broad phylogenetic group with a maximal divergence of 16S rRNA gene sequences of 12.0 %. This monophyletic group consists of at least three monophyletic subclusters. H. hydrossis is affiliated to one of these subclusters and represents the sole recognized species affiliated to the broad monophyletic group. ‘Candidatus Haliscomenobacter calcifugiens' and ‘Candidatus Aquirestis calciphila’ are uncultured representatives of the other two subclusters and have 16S rRNA gene sequence dissimilarities of 5.4 % and 8.2 %, respectively, with the type strain of H. hydrossis. ‘Candidatus H. calcifugiens' and ‘Candidatus A. calciphila’ have a 16S rRNA gene sequence dissimilarity of 8.5 %. These large ribosomal divergences justify the classification of these environmentally important bacteria as a novel species and a new genus, respectively. Intensive attempts to cultivate these filamentous bacteria have resulted in the establishment of mixed cultures, however, attempts to establish pure cultures have failed.

Huanglongbing (HLB), also known as greening, is one of the most important diseases of citrus worldwide. The causal agent is a gram-negative bacterium known to inhabit the phloem of infected plants. Three different candidate species infect citrus: ‘Candidatus Liberibacter africanus’ found in the African continent; ‘Ca. L. asiaticus’ found in Asia, Brazil, and the United States; and ‘Ca. L. americanus’ found in Brazil. (1). Tobacco is an easily transformable plant species that can be used as an experimental host system to quickly screen for candidate genes useful to control plant pathogens. However, no evidence exists on the ability of this plant species to sustain populations of ‘Ca. L. americanus’. With the purpose of transmitting ‘Ca. L. americanus’ from citrus to tobacco, fragments of healthy stems of Cuscuta spp. (dodder) were used to connect an HLB-infected sweet orange plant to each of 10 healthy plants of Nicotiana tabacum L. cv. Xanthi and allowed to remain connected for 30, 45, and 50 days. Three different HLB-infected orange plants and 30 tobacco plants were used in three independent experiments. Most HLB-exposed Xanthi plants exhibited chlorotic leaves after 50 days of exposure probably because of the parasitic effect of dodder; however, an average of 6, 1, and 3 Xanthi plants exhibited a unique blotchy mottle symptom after 30, 45, and 50 days of exposure, respectively. Symptomatic and asymptomatic leaves were collected and analyzed by PCR. The results consistently confirmed the presence of ‘Ca. L. americanus’ only in symptomatic leaves. Sequencing of the PCR product and comparison to the NCBI database also confirmed the identity of the pathogen as ‘Ca. L. americanus’. Electron microscopy analysis of four symptomatic leaves indicated the presence of bacterium-like bodies with round to elongated bacilliform shapes and surrounded by two membranes. These bodies resembled those already described in HLB-infected citrus in Brazil (1). The evidence presented above confirms the successful transmission of ‘Ca. L. americanus’ from citrus to Xanthi using the parasitic plant Cuscuta spp. Reference: (1) F. A. O. Tanaka et al. Fitopatol. Bras. 31:99, 2006.