Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences

Phylogenetic analysis of 15 species of the genus Aquaspirillum based on 16S rRNA gene (rDNA) sequences indicated that the genus Aquaspirillum is phylogenetically heterogeneous and the species could be divided into four groups as follows: Aquaspirillum serpens, the type species of this genus, A. dispar and A. putridiconchylium are situated in the family Neisseriaceae; members of the second group, A. gracile, A. delicatum, A. anulus, A. giesbergeri, A. sinuosum, A. metamorphum and A. psychrophilum, are included in the family Comamonadaceae; the two members of the third group, A. arcticum and A. autotrophicum, are included in the family Oxalobacteriaceae; and members of the fourth group, A. polymorphum, A. peregrinum, and A. itersonii, are included in the alpha-subdivision of Proteobacteria. Thus, phylogenetic studies indicated that all the species excepting A. serpens, the type species, should be transferred to distinct genera.     

Relationships of Phenylobacterium immobile and purple nonsulfur bacteria on the basis of surface antigens

Abstract Indirect immunofluorescence tests with antisera against whole cells of Phenylobacterium immobile strains revealed a serological relationship to Pseudomonas vesicularis, Aquaspirillum itersonii and Rhodospirillum rubrum , three members of the purple nonsulfur bacteria (group I) and also to Gluconobacter oxydans and Azotobacter vinelandii . Antisera against whole cells of Gluconobacter oxydans and Pseudomonas vesicularis reacted positively with the Phenylobacterium immobile strains, tested. Furthermore, a serological relationship of Gluconobacter oxydans to Acetobacter aceti , and of Pseudomonas vesicularis to Pseudomonas diminuta and Aquaspirillum itersonii could be demonstrated.     

Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.     

Isolation and sequence analysis of the pmi gene encoding phosphomannose isomerase of Streptococcus mutans

Abstract The phylogenetic structure of non-sulfur purple bacteria in the Proteobacteria α group was elucidated by the comparative analysis of 16S rRNA sequences from 29 strains of phototrophs and 14 strains of related non-phototrophs. The sequences of 12 strains including 7 isolates were determined in this study. The phototrophs in the α group were found to be extremely diversified and intermingled with non-phototrophs. Rhodopseudomonas species were dispersed into 3 lines of descent and Rhodospirillum species were dispersed into 5 lines. Marine organisms were composed of 4 lineages which were independent of each other and of freshwater lineages. Rhodospirillum fulvum, Rhodospirillum molischianum and the genus Magnetospirillum were found to be monophyletic.     

The soluble c-type cytochromes from the bacterium Aquaspirillum itersonii. The complete amino acid sequence of the cytochrome c-550

A complete amino acid sequence is proposed for the cytochrome c-550 isolated from the gram-negative chemo-organotrophic bacterium Aquaspirillum itersonii. The sequence, a single polypeptide chain of 111 residues, was deduced from the sequences of peptides obtained by tryptic, thermolytic or chymotryptic digestion. The cytochrome shows a high degree of sequence homology with the cytochrome c2 from the photosynthetic bacterium Rhodospirillum rubrum, and the evolutionary implications of this are considered.     

Determination of the spiral conformation of Aquaspirillum spp. by scanning electron microscopy of elongated cells induced by cephalexin treatment

The effect of the beta-lactam antibiotic cephalexin on the spiral conformation of cells of Aquaspirillum spp. was examined by scanning electron microscopy. A. itersonii and A. peregrinum, which are known to have a left-handed spiral shape, elongated and still showed left-handed spirals in medium containing cephalexin. The spiral conformation of the elongated cells is therefore considered to represent the natural condition. The spiral conformations of A. metamorphum and A. psychrophilum grown in ordinary cultures were difficult to determine because they have short cells without a complete spiral. After cephalexin treatment, the cells of these species elongated and displayed spiral forms, right-handed in A. metamorphum and left-handed in A. psychrophilum. This elongation method may be useful for checking and determination of the spiral handedness of short spiral or curved bacteria such as vibrios.     

Phylogeny of the genus Azospirillum based on 16S rDNA sequence

Abstract The 16S rDNA of 17 strains of Azospirillum , 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum , and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens ; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.     

Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster

The genera Azospirillum, Skermanella and Rhodocista form a phylogenetic subgroup within the alfa subclass of Proteobacteria. Based on comparative 16S rRNA sequence analysis a nested set of new oligonucleotide probes was designed. It comprises probes for the whole genus cluster Azospirillum-Skermanella-Rhodocista, for the Azospirilli subcluster I including A. lipoferum, A. doebereinerae, A. largimobile, A. brasilense and A. halopraeferens, for the Azospirilli subcluster II including A. amazonense, A. irakense and the genus Skermanella, for the genus Rhodocista as well as for all Azospirilli species or species cluster. The new probes allow a fast and reliable in situ identification of bacteria belonging to the Azospirillum-Skermanella-Rhodocista-cluster at different phylogenetic levels. The specificity of the new probes was tested with 56 strains of the Azospirillum-Rhodocista-Skermanella-cluster and selected reference cells from other genera by hybridising with the complete probe set. In addition, applications of the fluorescently labelled probes for in situ identification of isolates and for the in situ localisation of A. brasilense on maize roots were demonstrated using confocal laser scanning microscopy.     

The presumptive magnetosome protein Mms16 is a poly(3-hydroxybutyrate) granule-bound protein (phasin) in Magnetospirillum gryphiswaldense

The Mms16 protein has been previously found to be associated with isolated magnetosomes from two Magnetospirillum strains. A function of this protein as a magnetosome-specific GTPase involved in the formation of intracellular magnetosome membrane vesicles was suggested. Here we present a study of the Mms16 protein from Magnetospirillum gryphiswaldense to clarify its function. Insertion-duplication mutagenesis of the mms16 gene did not affect the formation of magnetosome particles but resulted in the loss of the ability of M. gryphiswaldense cell extracts to activate poly(3-hydroxybutyrate) (PHB) depolymerization in vitro, which was coincident with loss of the most abundant 16-kDa polypeptide from preparations of PHB granule-bound proteins. The mms16 mutation could be functionally complemented by enhanced yellow fluorescent protein (EYFP) fused to ApdA, which is a PHB granule-bound protein (phasin) in Rhodospirillum rubrum sharing 55% identity to Mms16. Fusions of Mms16 and ApdA to enhanced green fluorescent protein (EGFP) or EYFP were colocalized in vivo with the PHB granules but not with the magnetosome particles after conjugative transfer to M. gryphiswaldense. Although the Mms16-EGFP fusion protein became detectable by Western analysis in all cell fractions upon cell disruption, it was predominantly associated with isolated PHB granules. Contrary to previous suggestions, our results argue against an essential role of Mms16 in magnetosome formation, and the previously observed magnetosome localization is likely an artifact due to unspecific adsorption during preparation. Instead, we conclude that Mms16 in vivo is a PHB granule-bound protein (phasin) and acts in vitro as an activator of PHB hydrolysis by R. rubrum PHB depolymerase PhaZ1. Accordingly, we suggest renaming the Mms16 protein of Magnetospirillum species to ApdA, as in R. rubrum.     

Inactivation of the flagellin gene flaA in Magnetospirillum gryphiswaldense results in nonmagnetotactic mutants lacking flagellar filaments

Magnetotactic bacteria synthesize magnetosomes, which cause them to orient and migrate along magnetic field lines. The analysis of magnetotaxis and magnetosome biomineralization at the molecular level has been hindered by the unavailability of genetic methods, namely the lack of a means to introduce directed gene-specific mutations. Here we report a method for knockout mutagenesis by homologous recombination in Magnetospirillum gryphiswaldense. Multiple flagellin genes, which are unlinked in the genome, were identified in M. gryphiswaldense. The targeted disruption of the flagellin gene flaA was shown to eliminate flagella formation, motility, and magnetotaxis. The techniques described in this paper will make it possible to take full advantage of the forthcoming genome sequences of M. gryphiswaldense and other magnetotactic bacteria.     

N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species

Cells of Geobacter metallireducens , Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N₂-dependent growth, the first anaerobically with Fe(III) as the electron acceptor, and the latter three species microaerobically in semi-solid oxygen gradient cultures. Cells of the Magnetospirillum species grown with N₂ under microaerobic conditions were magnetotactic and therefore produced magnetosomes. Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5 ± 5.9 nmol C₂H₄ produced min⁻¹ mg⁻¹ cell protein) while growing with Fe(III) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source. Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates. Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum , indicating the presence of these nitrogenase structural genes in these organisms. The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum , fix atmospheric dinitrogen.     

Development of a genetic system for <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis>

Genetic analysis of bacterial magnetosome biomineralization has been hindered by the lack of an appropriate methodology for cultivation and genetic manipulation of most magnetotactic bacteria. In this report, a genetic system for Magnetospirillum gryphiswaldense is described. The system includes a plating technique that allows the screening of magnetic vs non-magnetic colonies, and a protocol for the transfer of foreign DNA by electroporation and high-frequency conjugation. Various broad-host-range vectors of the IncQ, IncP, and pBBR1 groups were found to be capable of replication in M. gryphiswaldense. Several antibiotic resistance markers that can be expressed in M. gryphiswaldense were identified. Tn 5 transposons delivered on a suicide plasmid showed transpositional insertion into random chromosomal sites.     

Physiological versatility of the genus Rhodocista

A new purple bacterium (strain T4), capable of heterotrophic aerobic and phototrophic anaerobic growth, was isolated from waste water of a noodle factory near Hanoi, Vietnam. A comparison of 16S rDNA sequences revealed its association with the genus Rhodocista. The isolate, tentatively named "Rhodocista hanoiensis", forms cysts after growth on butyrate-containing plates at 42 degrees C. The vegetative cells form short (under aerobic conditions) or long curve-shaped rods. In contrast to other species of this genus T4 does not need any supplines (growth factors, not synthesized by the organisms). Comparative studies of T4 with Rhodocista centenaria (Rhodospirillum centenum) and Rhodocista pekingensis revealed a remarkable physiological versatility regarding nutrient spectra and survival properties of this genus.     

趋磁螺菌遗传操作体系的建立及磁小体缺失突变株的筛选

由于MagnetospirillumgryphiswaldenseMSR 1缺少简便有效的遗传操作体系和对常见抗生素的抗性 ,致使对该菌磁小体生物合成的机制等研究工作进展缓慢。为此建立了一套比较简便有效的遗传操作体系 ,其中包括 :以平板封膜培养技术获得单菌落、在选择性培养液中进行接合转移遗传因子 ,以液体培养和磁铁吸附技术筛选突变子。利用此体系 ,通过接合转座诱变技术 ,获得了 2个磁小体缺失突变株 ,为研究该菌磁小体合成的分子遗传学提供了技术支撑     

The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization

Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria.     

Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.     

Fur in Magnetospirillum gryphiswaldense influences magnetosomes formation and directly regulates the genes involved in iron and oxygen metabolism

Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe(3)O(4)). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense.     

Dynamics of magnetotactic bacteria in a rotating magnetic field

The dynamics of the motile magnetotactic bacterium Magnetospirillum gryphiswaldense in a rotating magnetic field is investigated experimentally and analyzed by a theoretical model. These elongated bacteria are propelled by single flagella at each bacterial end and contain a magnetic filament formed by a linear assembly of approximately 40 ferromagnetic nanoparticles. The movements of the bacteria in suspension are analyzed by consideration of the orientation of their magnetic dipoles in the field, the hydrodynamic resistance of the bacteria, and the propulsive force of the flagella. Several novel features found in experiments include a velocity reversal during motion in the rotating field and an interesting diffusive wandering of the trajectory curvature centers. A new method to measure the magnetic moment of an individual bacterium is proposed based on the theory developed.     

Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol

Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.