Identification of “Candidatus Thioturbo danicus,” a Microaerophilic Bacterium That Builds Conspicuous Veils on Sulfidic Sediments

Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the -subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “Candidatus Thioturbo danicus.”     

Pigment analysis of "Candidatus Chlorothrix halophila," a green filamentous anoxygenic phototrophic bacterium

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.     

Ultrastructure of the denitrifying methanotroph "Candidatus Methylomirabilis oxyfera," a novel polygon-shaped bacterium

"Candidatus Methylomirabilis oxyfera" is a newly discovered denitrifying methanotroph that is unrelated to previously known methanotrophs. This bacterium is a member of the NC10 phylum and couples methane oxidation to denitrification through a newly discovered intra-aerobic pathway. In the present study, we report the first ultrastructural study of "Ca. Methylomirabilis oxyfera" using scanning electron microscopy, transmission electron microscopy, and electron tomography in combination with different sample preparation methods. We observed that "Ca. Methylomirabilis oxyfera" cells possess an atypical polygonal shape that is distinct from other bacterial shapes described so far. Also, an additional layer was observed as the outermost sheath, which might represent a (glyco)protein surface layer. Further, intracytoplasmic membranes, which are a common feature among proteobacterial methanotrophs, were never observed under the current growth conditions. Our results indicate that "Ca. Methylomirabilis oxyfera" is ultrastructurally distinct from other bacteria by its atypical cell shape and from the classical proteobacterial methanotrophs by its apparent lack of intracytoplasmic membranes.     

"Candidatus contubernalis alkalaceticum," an obligately syntrophic alkaliphilic bacterium capable of anaerobic acetate oxidation in a coculture with Desulfonatronum cooperativum

From the silty sediments of the Khadyn soda lake (Tuva), a binary sulfidogenic bacterial association capable of syntrophic acetate oxidation at pH 10.0 was isolated. An obligately syntrophic, gram-positive, spore-forming alkaliphilic rod-shaped bacterium performs acetate oxidation in a syntrophic association with a hydrogenotrophic, alkaliphilic sulfate-reducing bacterium; the latter organism was previously isolated and characterized as the new species Desulfonatronum cooperativum. Other sulfate-reducing bacteria of the genera Desulfonatronum and Desulfonatronovibrio can also act as the hydrogenotrophic partner. Apart from acetate, the syntrophic culture can oxidize ethanol, propanol, isopropanol, serine, fructose, and isobutyric acid. Selective amplification of 16S rRNA gene fragments of the acetate-utilizing syntrophic component of the binary culture was performed; it was found to cluster with clones of uncultured gram-positive bacteria within the family Syntrophomonadaceae. The acetate-oxidizing bacterium is thus the first representative of this cluster obtained in a laboratory culture. Based on its phylogenetic position, the new acetate-oxidizing syntrophic bacterium is proposed to be assigned, in a Candidate status, to a new genus and species: "Candidatus Contubernalis alkalaceticum."     

Genome sequence for "Candidatus Mycoplasma haemominutum," a low-pathogenicity hemoplasma species

We present the genome sequence of "Candidatus Mycoplasma haemominutum" strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.     

Diversity of "Candidatus Liberibacter asiaticus," based on the omp gene sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, "Candidatus Liberibacter africanus" in Africa and "Candidatus Liberibacter asiaticus" in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of "Ca. Liberibacter asiaticus" isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species "Ca. Liberibacter asiaticus," even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating "Ca. Liberibacter asiaticus" isolates.     

"Candidatus Endobugula glebosa," a specific bacterial symbiont of the marine bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, "Candidatus Endobugula sertula." "Candidatus E. sertula" has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with "Candidatus E. sertula." In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status "Candidatus Endobugula glebosa" is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

Isolation, free-living capacities, and genome structure of "Candidatus Glomeribacter gigasporarum," the endocellular bacterium of the mycorrhizal fungus Gigaspora margarita

"Candidatus Glomeribacter gigasporarum" is an endocellular beta-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita. We established a protocol to isolate "Ca. Glomeribacter gigasporarum" from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria. They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili. Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks. PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to "Ca. Glomeribacter gigasporarum." In particular, the presence of nif genes could not be detected. Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of "Ca. Glomeribacter gigasporarum" to approximately 1.4 Mb with a ca. 750-kb chromosome and a 600- to 650-kb plasmid. This is the smallest genome known for a beta-proteobacterium. Such small genome sizes are typically found in endocellular bacteria living permanently in their host. Altogether, our data suggest that "Ca. Glomeribacter gigasporarum" is an ancient obligate endocellular bacterium of the AM fungus G. margarita.     

Survey of motile microaerophilic bacterial morphotypes in the oxygen gradient above a marine sulfidic sediment

Enrichment cultures for free-swimming microaerophilic bacteria were prepared from marine sulfidic sediment samples (Niv? Bay, Denmark). We observed nine different morphotypes; three of these morphotypes represented already-described species, i.e., Thiovulum majus, "Candidatus Ovobacter propellens," and an as-yet-unnamed large vibrioid bacterium. In addition, we observed several morphotypes of spirilla and one vibrioid morphotype. A common feature of all investigated bacteria was that they aggregated chemotactically at the oxic-anoxic interface, whereas preferred oxygen concentration were in the range of 1 to 10 muM. The motile behavior and flagellar dynamics are analyzed in detail with an emphasis on spirilla.     

Phylogeny and in situ identification of a morphologically conspicuous bacterium, Candidatus Magnospira bakii, present at very low frequency in activated sludge

A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation-independent molecular and physical methods. The large, corkscrew-shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation. In situ hybridization with the group-specific, rRNA-targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the Cytophaga-Flexibacter group of the Cytophaga-Flavobacterium-Bacteroides phylum. Based on strong morphological resemblance to members of the genus Saprospira, additional 16S rRNA-targeted oligonucleotides with more narrow specificity were designed and evaluated for in situ hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%. In situ hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of Saprospira grandis were indeed retrieved from the corkscrew-shaped filaments. The bacterium is likely to be a member of a genus of which no species has been cultured hitherto. It was consequently tentatively named 'Magnospira bakii' and has the taxonomic rank of Candidatus Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultivation for further molecular analysis.     

Initial characterization of the photosynthetic apparatus of "Candidatus Chlorothrix halophila," a filamentous, anoxygenic photoautotroph

“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.     

"Candidatus hepatoplasma crinochetorum," a new, stalk-forming lineage of Mollicutes colonizing the midgut glands of a terrestrial isopod

Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales. Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name "Candidatus Hepatoplasma crinochetorum" is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P. scaber.     

Intact membrane lipids of "Candidatus Nitrosopumilus maritimus," a cultivated representative of the cosmopolitan mesophilic group I Crenarchaeota

In this study we analyzed the membrane lipid composition of "Candidatus Nitrosopumilus maritimus," the only cultivated representative of the cosmopolitan group I crenarchaeota and the only mesophilic isolate of the phylum Crenarchaeota. The core lipids of "Ca. Nitrosopumilus maritimus" consisted of glycerol dialkyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl moieties. Crenarchaeol, a unique GDGT containing a cyclohexyl moiety in addition to four cyclopentyl moieties, was the most abundant GDGT. This confirms unambiguously that crenarchaeol is synthesized by species belonging to the group I.1a crenarchaeota. Intact polar lipid analysis revealed that the GDGTs have hexose, dihexose, and/or phosphohexose head groups. Similar polar lipids were previously found in deeply buried sediments from the Peru margin, suggesting that they were in part synthesized by group I crenarchaeota.     

Draft genome sequence of "Candidatus Hamiltonella defensa," an endosymbiont of the whitefly Bemisia tabaci

"Candidatus Hamiltonella defensa" is a facultative endosymbiont of the whitefly Bemisia tabaci. Herein, we report the first draft genome sequence of "Candidatus Hamiltonella defensa" from the invasive Mediterranean cryptic species of the B. tabaci complex. The 1.84-Mbp genome sequence comprises 404 contigs and contains 1,806 predicted protein-coding genes.     

一株将甜菊苷转化为甜茶甙的细菌鉴定及转化特性

摘要：【目的】筛选一株对甜菊苷具有特异转化性能的细菌,并对该菌及转化产物进行鉴定,探讨转化酶及酶对甜菊苷的转化特性。【方法】通过16S rDNA序列分析,构建该菌系统进化树,结合菌体形态及菌落特征,确立该菌系统发育学地位。通过高效液相色谱（HPLC）及液质联用（LC－MS）法检测并鉴定转化产物。用菌液直接对甜菊苷进行转化以研究菌的转化能力。用静息细胞、胞外液和胞内液对甜菊糖分别转化法,确定转化酶与菌体的关系,并用该酶液进行转化特性研究。【结果】该菌株与黄杆菌属的16S rDNA序列相似性为99%,结合菌体     

Draft genome sequence of the sulfur-oxidizing bacterium "Candidatus Sulfurovum sediminum" AR, which belongs to the Epsilonproteobacteria

Sulfur-oxidizing bacteria are common microorganisms in a variety of sulfide-rich environments. They play important roles in the global sulfur cycle on earth. Here, we present a high-quality draft genome sequence of a sulfur-oxidizing bacterium, "Candidatus Sulfurovum sediminum" strain AR, which belongs to the class Epsilonproteobacteria and dominated an enrichment culture from a marine sediment collected off Svalbard, within the Arctic Circle. Its genome contains genes for sulfur oxidation and carbon fixation. The size of the draft genome is 2.12 Mb, and the G+C content is 39.4%.     

Tissue localization of the endosymbiotic bacterium "Candidatus Blochmannia floridanus" in adults and larvae of the carpenter ant Camponotus floridanus

The distribution of endosymbiotic bacteria in different tissues of queens, males, and workers of the carpenter ant Camponotus floridanus was investigated by light and electron microscopy and by in situ hybridization. A large number of bacteria could be detected in bacteriocytes within the midguts of workers, young virgin queens, and males. Large amounts of bacteria were also found in the oocytes of workers and queens. In contrast, bacteria were not present in oocyte-associated cells or in the spermathecae of mature queens, although occasionally a small number of bacteria could be detected in the testis follicles of males. Interestingly, the number of bacteriocytes in mature queens was strongly reduced and the bacteriocytes contained only very few or no bacteria at all, although the endosymbionts were present in huge amounts in the ovaries of the same animals. During embryogenesis of the deposited egg, the bacteria were concentrated in a ring of endodermal tissue destined to become the midgut in later developmental stages. However, during larval development, bacteria could also be detected in other tissues although to a lesser extent. Only in the last-instar larvae were bacteria found exclusively in the midgut tissue within typical bacteriocytes. Tetracycline and rifampin efficiently cleansed C. floridanus workers of their symbionts and the bacteriocytes of these animals still remained empty several months after treatment had ceased. Despite the lack of their endosymbionts, these adult animals were able to survive without any obvious negative effect under normal cultivation conditions.     

"Candidatus Bacilloplasma," a novel lineage of Mollicutes associated with the hindgut wall of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda)

Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean Porcellio scaber (Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the class Mollicutes, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known Mollicutes. The lineage has been positioned as a sister group to the clade comprising the Spiroplasma group, the Mycoplasma pneumoniae group, and the Mycoplasma hominis group. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut of P. scaber and made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name "Candidatus Bacilloplasma" has been proposed for this new lineage of bacteria specifically associated with the gut surface of P. scaber.     

Development of a protocol for recognizing sulfidic sediments (potential acid sulfate soils) in freshwater wetlands

Summary   The presence of sulfidic sediments (potential acid sulfate soils) is an emerging problem in the management of inland wetlands. Using data from 81 wetlands in the Murray-Darling Basin, a simple protocol was developed to assess whether a wetland will contain sulfidic sediments at levels that could cause ecological damage. Risk factors include whether or not the wetland receives municipal waste or irrigation return water, elevated salinity in the overlying water (>1750 µS/cm) or sediment (400 µS/cm in a 1:5 soil : water extract) and high levels of sulfate in the water column (>10 mg/L). Neutral or basic sediment pH indicates that, even if the sediment does contain sulfidic sediments, there is a reduced likelihood of acidification if the sediments are oxidized.     

A highly sensitive fluorometric assay for "enkephalinase," a neutral metalloendopeptidase that releases tyrosine-glycine-glycine from enkephalins

A fluorogenic peptide, dansyl-D-Ala-Gly-Phe(pNO2)-Gly (DAGNPG), was synthesized as a selective substrate for the neutral metalloendopeptidase (EC 3.4.24.11) involved in enkephalin metabolism. This enzyme, designated "enkephalinase," cleaves the Gly-Phe(pNO2) peptide bond of DAGNPG (V = 0.65 mumol/mg protein/min and Km = 45 microM) leading to a fluorescence increase related to the disappearance of intramolecular quenching of the dansyl fluorescence by the nitrophenyl residue. This change was used for quantitative measurements of "enkephalinase" activity in different tissues and determination of inhibitory potency of various compounds. The substrate is not cleaved by aminopeptidase or dipeptidylaminopeptidase activities and the assay itself is rapid, convenient, and sensitive.