A tailored galK counterselection system for efficient markerless gene deletion and chromosomal tagging in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of the phbCAB operon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.

Magnetovibrio blakemorei gen. nov., sp. nov., a magnetotactic bacterium (Alphaproteobacteria: Rhodospirillaceae) isolated from a salt marsh.

A magnetotactic bacterium, designated strain MV-1(T), was isolated from sulfide-rich sediments in a salt marsh near Boston, MA, USA. Cells of strain MV-1(T) were Gram-negative, and vibrioid to helicoid in morphology. Cells were motile by means of a single polar flagellum. The cells appeared to display a transitional state between axial and polar magnetotaxis: cells swam in both directions, but generally had longer excursions in one direction than the other. Cells possessed a single chain of magnetosomes containing truncated hexaoctahedral crystals of magnetite, positioned along the long axis of the cell. Strain MV-1(T) was a microaerophile that was also capable of anaerobic growth on some nitrogen oxides. Salinities greater than 10 % seawater were required for growth. Strain MV-1(T) exhibited chemolithoautotrophic growth on thiosulfate and sulfide with oxygen as the terminal electron acceptor (microaerobic growth) and on thiosulfate using nitrous oxide (N2O) as the terminal electron acceptor (anaerobic growth). Chemo-organoautotrophic and methylotrophic growth was supported by formate under microaerobic conditions. Autotrophic growth occurred via the Calvin-Benson-Bassham cycle. Chemo-organoheterotrophic growth was supported by various organic acids and amino acids, under microaerobic and anaerobic conditions. Optimal growth occurred at pH 7.0 and 26-28 °C. The genome of strain MV-1(T) consisted of a single, circular chromosome, about 3.7 Mb in size, with a G+C content of 52.9-53.5 mol%.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MV-1(T) belongs to the family Rhodospirillaceae within the Alphaproteobacteria, but is not closely related to the genus Magnetospirillum. The name Magnetovibrio blakemorei gen. nov., sp. nov. is proposed for strain MV-1(T). The type strain of Magnetovibrio blakemorei is MV-1(T) ( = ATCC BAA-1436(T)  = DSM 18854(T)).

Magnetococcus marinus gen. nov., sp. nov., a marine, magnetotactic bacterium that represents a novel lineage (Magnetococcaceae fam. nov., Magnetococcales ord. nov.) at the base of the Alphaproteobacteria.

Magnetotactic bacteria are a morphologically, metabolically and phylogenetically disparate array of bacteria united by the ability to biomineralize membrane-encased, single-magnetic-domain mineral crystals (magnetosomes) that cause the cell to orientate along the Earth's geomagnetic field. The most commonly observed type of magnetotactic bacteria is the ubiquitous magnetotactic cocci, which comprise their own phylogenetic group. Strain MC-1(T), a member of this group, was isolated from water collected from the oxic-anoxic interface of the Pettaquamscutt Estuary in Rhode Island, USA, and cultivated in axenic culture. Cells of strain MC-1(T) are roughly spherical, with two sheathed bundles of flagella at a single pole (bilophotrichous). Strain MC-1(T) uses polar magnetotaxis, and has a single chain of magnetite crystals per cell. Cells grow chemolithoautotrophically with thiosulfate or sulfide as the electron donors, and chemo-organoheterotrophically on acetate. During autotrophic growth, strain MC-1(T) relies on the reductive tricarboxylic acid cycle for CO2 fixation. The DNA G+C content is 54.2 mol%. The new genus and species Magnetococcus marinus gen. nov., sp. nov. are proposed to accommodate strain MC-1(T) ( = ATCC BAA-1437(T)  = JCM 17883(T)), which is nominated as the type strain of Magnetococcus marinus. A new order (Magnetococcales ord. nov.) and family (Magnetococcaceae fam. nov.) are proposed for the reception of Magnetococcus and related magnetotactic cocci, which are provisionally included in the Alphaproteobacteria as the most basal known lineage of this class.

Magnetospira thiophila gen. nov., sp. nov., a marine magnetotactic bacterium that represents a novel lineage within the Rhodospirillaceae (Alphaproteobacteria).

A marine, magnetotactic bacterium, designated strain MMS-1(T), was isolated from mud and water from a salt marsh in Woods Hole, Massachusetts, USA, after enrichment in defined oxygen-concentration/redox-gradient medium. Strain MMS-1(T) is an obligate microaerophile capable of chemoorganoheterotrophic and chemolithoautotrophic growth. Optimal growth occurred at pH 7.0 and 24-26 °C. Chemolithoautotrophic growth occurred with thiosulfate as the electron donor and autotrophic carbon fixation was via the Calvin-Benson-Bassham cycle. The G+C content of the DNA of strain MMS-1(T) was 47.2 mol%. Cells were Gram-negative and morphologically variable, with shapes that ranged from that of a lima bean to fully helical. Cells were motile by means of a single flagellum at each end of the cell (amphitrichous). Regardless of whether grown in liquid or semi-solid cultures, strain MMS-1(T) displayed only polar magnetotaxis and possessed a single chain of magnetosomes containing elongated octahedral crystals of magnetite, positioned along the long axis of the cell. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MMS-1(T) belongs to the family Rhodospirillaceae within the Alphaproteobacteria, and is distantly related to species of the genus Magnetospirillum. Strain MMS-1(T) is therefore considered to represent a novel species of a new genus, for which the name Magnetospira thiophila gen. nov., sp. nov. is proposed. The type strain of Magnetospira thiophila is MMS-1(T) ( = ATCC BAA-1438(T) = JCM 17960(T)).

Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3.

Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that orient along magnetic fields using membrane-coated magnetic nanocrystals of magnetite (Fe(3) O(4) ) or greigite (Fe(3) S(4) ), the magnetosomes. Previous phylogenetic analysis of MTB has been limited to few cultivated species and most abundant members of natural populations, which were assigned to Proteobacteria and the Nitrospirae phyla. Here, we describe a single cell-based approach that allowed the targeted phylogenetic and ultrastructural analysis of the magnetotactic bacterium SKK-01, which was low abundant in sediments of Lake Chiemsee. Morphologically conspicuous single cells of SKK-01 were micromanipulated from magnetically collected multi-species MTB populations, which was followed by whole genome amplification and ultrastructural analysis of sorted cells. Besides intracellular sulphur inclusions, the large ovoid cells of SKK-01 harbour ∼175 bullet-shaped magnetosomes arranged in multiple chains that consist of magnetite as revealed by TEM and EDX analysis. Sequence analysis of 16 and 23S rRNA genes from amplified genomic DNA as well as fluorescence in situ hybridization assigned SKK-01 to the candidate division OP3, which so far lacks any cultivated representatives. SKK-01 represents the first morphotype that can be assigned to the OP3 group as well as the first magnetotactic member of the PVC superphylum.