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Appl Environ Microbiol. 2010 May; 76(10): 3220–3227.
Published online 2010 April 2. doi:  10.1128/AEM.00408-10
PMCID: PMC2869151

Nonmagnetotactic Multicellular Prokaryotes from Low-Saline, Nonmarine Aquatic Environments and Their Unusual Negative Phototactic Behavior[down-pointing small open triangle]

Abstract

Magnetotactic multicellular prokaryotes (MMPs) are unique magnetotactic bacteria of the Deltaproteobacteria class and the first found to biomineralize the magnetic mineral greigite (Fe3S4). Thus far they have been reported only from marine habitats. We questioned whether MMPs exist in low-saline, nonmarine environments. MMPs were observed in samples from shallow springs in the Great Boiling Springs geothermal field and Pyramid Lake, both located in northwestern Nevada. The temperature at all sites was ambient, and salinities ranged from 5 to 11 ppt. These MMPs were not magnetotactic and did not contain magnetosomes (called nMMPs here). nMMPs ranged from 7 to 11 μm in diameter, were composed of about 40 to 60 Gram-negative cells, and were motile by numerous flagella that covered each cell on one side, characteristics similar to those of MMPs. 16S rRNA gene sequences of nMMPs show that they form a separate phylogenetic branch within the MMP group in the Deltaproteobacteria class, probably representing a single species. nMMPs exhibited a negative phototactic behavior to white light and to wavelengths of ≤480 nm (blue). We devised a “light racetrack” to exploit this behavior, which was used to photoconcentrate nMMPs for specific purposes (e.g., DNA extraction) even though their numbers were low in the sample. Our results show that the unique morphology of the MMP is not restricted to marine and magnetotactic prokaryotes. Discovery of nonmagnetotactic forms of the MMP might support the hypothesis that acquisition of the magnetosome genes involves horizontal gene transfer. To our knowledge, this is the first report of phototaxis in bacteria of the Deltaproteobacteria class.

One of the most interesting and unusual examples of prokaryotic morphology is that of the organism known as the magnetotactic multicellular prokaryote (MMP; also known as the magnetotactic multicellular aggregate [MMA] [16, 37] and the magnetotactic multicellular organism [MMO] [30]). The acronym MMP originally stood for many-celled magnetotactic prokaryote (45, 46, 53, 54), but in more recent reports, because of a number of recent findings suggesting that individual cells interact and/or communicate with each other, many investigators use MMP for multicellular magnetotactic prokaryote (for an example, see reference 59).

The MMP is a large prokaryotic microorganism that ranges from about 3 to 12 μm in diameter (5, 45, 46). It is best described as an aggregation of about 10 to 40 (5, 45, 46) Gram-negative, genetically similar cells (53) that swim only as an intact unit and not as individual cells (5, 45, 46). Cells that separate from the intact unit die quickly according to viability studies (1). The surface of the cell exposed to the surrounding environment is covered with numerous flagella (44, 46, 52). Most described MMPs are spherical (2, 5, 30, 33, 45, 46, 59), although some are ovoid in morphology (36), and all appear to possess a central, acellular compartment (30, 33). The MMP divides as aggregates without an individual cell stage (32, 33). In all known instances, MMPs have been found to be magnetotactic, and iron sulfide minerals, magnetic greigite (Fe3S4) and others, in magnetotactic bacteria were first discovered in this microorganism (17, 39, 43, 44), although magnetite, Fe3O4, has also been identified in some types (31, 38).

MMPs appear to be cosmopolitan in distribution and have been observed to be present on most continents in numerous saline aquatic environments, ranging from brackish (lowest salinity reported, ~12 ppt) (5) to hypersaline (highest salinity reported, ~60 ppt) (2, 30, 32, 40). In all cases, the salinity is due to the input of seawater, and many consider these organisms obligately marine (53). More specifically, MMPs have been found in stratified sediments of salt marshes (5, 12, 44, 53) and coastal lagoons (15-17, 40), the water column of meromictic salt water lagoons (44, 54), and some oceanic sediments (e.g., Santa Barbara Basin [D. A. Bazylinski, unpublished data]) and coastal tidal salt flat sediments (59). In general, these organisms, like other greigite-producing magnetotactic bacteria, are found in their highest numbers in the anoxic zone of these environments where sulfide is noticeably present (5, 53, 54).

Phylogenetically, MMPs are members of the Deltaproteobacteria, where they form a single cluster within the sulfate-reducing bacteria (2, 12, 53). Simmons and Edwards (53) sequenced 16S rRNA genes from a natural population of MMPs from the Little Sippewissett salt marsh (Falmouth, MA) and found that MMPs from this population were phylogenetically made up of five lineages separated by at least 5% sequence divergence. Because of their apparently unique morphology, the MMP was considered by many to consist of a single species, but this phylogenetic diversity shows that the MMP probably represents a separate genus in the Deltaproteobacteria that includes several species. All cells in each aggregate expressed identical small-subunit rRNAs, suggesting that the aggregates are composed of a single MMP phylotype (53). Based on the information presented above, the biogeographical distribution and phylogenetic diversity of the MMP appears to be great, although not completely known.

While examining a number of mud and water samples taken from low-saline, nonmarine aquatic environments for greigite-producing magnetotactic bacteria, we observed MMP-like organisms. Surprisingly, none of these organisms were magnetotactic (we refer to them here as nMMPs), a finding not previously reported. This is also the first description of the MMP-like morphological form from a nonmarine habitat. We also noted that these nMMPs demonstrated an unusual type of negative phototaxis that could be exploited to purify enough of them for phylogenetic analysis and electron microscopy. In this paper, we describe these features in detail.

MATERIALS AND METHODS

Sampling sites.

In this study, water and sediment samples were taken from Pyramid Lake and several shallow temperate pools in the Great Boiling Springs (GBS) geothermal field in Gerlach, Nevada. Pyramid Lake, one of the largest lakes in the United States, is an endorheic salt lake that covers approximately 490 km2 in area. It is located in the Great Basin in the northwestern part of Nevada. GBS is a series of hot springs that range from ambient temperatures to ~96°C (4, 11). The geology, chemistry, and microbial ecology of the springs have been described in some detail (4, 11). Pyramid Lake was connected with Gerlach between 18,000 and 7,000 BC, during the last ice age, by prehistoric Lake Lahontan, which also extended into northeastern California and southern Oregon (7, 29). The temperate pools sampled at GBS are designated GL1, GL2, and GL3, and the water temperature in these pools was ambient. GL2 and GL3 are pools about 3 m in diameter formed by old hot springs that no longer have a connection with geothermally heated water, while GL1 is a shallow marsh close to an artificial pond used for the introduction of fish to control the breeding of mosquitoes. For a comparison, we also collected samples containing magnetic MMPs from the Salton Sea, which is a saline, endorheic rift lake located on the San Andreas Fault in southern California. Samples from the Salton Sea were taken from the southeast shore where the salinity was 52 ppt. Salinities of the samples were determined with a hand-held Palm Abbe PA203 digital refractometer (MISCO Refractometer, Cleveland, OH).

One-liter glass bottles were filled to about 0.2 to 0.3 of their volume with sediment, and the remainder of the bottles were filled to their capacity with water that overlaid the sediment in the pool. Air bubbles were excluded. Once in the laboratory, samples were stored in the dark at room temperature (~25°C). We observed nMMPs in these samples over a period of several months. MMPs were never found in any of these samples at any time, although single-celled magnetotactic bacteria were always present.

Light and electron microscopy.

The presence and behavior of microorganisms was observed using light microscopy with a Zeiss (Carl Zeiss MicroImaging, Inc., Thornwood, NY) AxioImager M1 light microscope equipped with fluorescence, phase-contrast, and differential interference contrast capabilities. The hanging drop technique (49) was used routinely in the examination of samples and was also employed in phototactic behavioral experiments as described below. In experiments where numbers of nMMPs required accurate counting, those that accumulated at the “dark” edge of the drop (the farthest edge of the drop from the light beam) were quickly exposed to fluorescent light in the microscope light using a Lumar 49 filter set (the same filter set used for cells stained with 4′,6-diamidino-2-phenylindole [DAPI]; emission BP445/50, excitation G 365; Carl Zeiss MicroImaging, Inc., Thornwood, NY) which permanently immobilized them without disaggregation and facilitated an accurate nMMP count. Because of their large size and conspicuous morphology, nMMPs were easily distinguishable from single bacterial cells or sediment particles. The presence of magnetosomes was determined using electron microscopy with a Tecnai (FEI Company, Hillsboro, OR) model G2 F30 Super-Twin transmission electron microscope.

To determine which visible wavelengths of light caused the negative phototactic response in the nMMPs, a hanging drop of 5 μl was prepared and the microscope was focused on one side of the drop. Colored filters were tested with wavelengths of 435 (violet), 480 (blue), 530 (green), and 640 (orange-red) nm (Carl Zeiss MicroImaging, Inc., Thornwood, NY). After 5 min of exposure of light of a specific wavelength at one edge of the drop, nMMPs that swam to and accumulated at the opposite edge of the drop were counted as described above.

Determination of 16S rRNA gene sequences and phylogenies of nMMPs.

16S rRNA genes of photoconcentrated nMMPs were amplified using bacterium-specific primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGHTACCTTGTTACGACTT-3′) (35). PCR products were cloned into pGEM-T Easy Vector (Promega Corporation, Madison, WI) and sequenced (Functional Biosciences, Inc., Madison, WI).

Alignment of 16S rRNA genes was performed using the ClustalW multiple alignment accessory application in the BioEdit sequence alignment editor (24). Phylogenetic trees were constructed using Mega version 4 (57), applying the neighbor-joining method (47). Bootstrap values were calculated with 1,000 replicates.

Nucleotide sequence accession numbers.

16S rRNA gene sequences of nMMPs from GBS pools GL1 and GL3 were deposited under GenBank accession numbers GU732821 to GU732823 and GU732824 and GU732825, respectively. Those sequences from nMMPs from Pyramid Lake are under GenBank accession numbers GU732826 and GU732827. The 16S rRNA gene sequence of the MMP from the Salton Sea is listed under accession number GU784824.

RESULTS

Description of sampling sites and samples.

In all the locations sampled, sediments were black and reducing; the odor of hydrogen sulfide was readily apparent when sediments were disturbed. Photosynthetic sulfide-oxidizing bacteria were observed macroscopically as patches of pink and microscopically in some of the samples that morphologically resembled Chromatium species.

The salinities of our samples were low compared to those of seawater (~35 ppt) and other sites where MMPs have been found (Pyramid Lake, 6 ppt; GL1, 5 ppt; GL2, 8 ppt; and GL3, 11 ppt). There is no marine input to the sampling sites. Despite these low salinities, these values are still higher than those accepted to be freshwater (<0.5 ppt), and thus the water at the sites must be considered brackish.

Microscopic observations and ultrastructure.

One of the most sensitive methods of detecting the presence of magnetotactic bacteria in natural samples is through the use of the hanging drop technique (49). In addition, the numbers of north- and south-seeking magnetotactic cells can be readily determined. While we were examining samples taken from Pyramid Lake and pools and hot springs from the GBS thermal field in Nevada using the hanging drop technique, we observed relatively high numbers of single-celled, north-seeking magnetotactic bacteria as expected. When we moved to the opposite side of the drop to look for south-seeking cells, we noted organisms that were identical in size, morphology, and style of motility to the MMP (see Video S1 in the supplemental material). Most of the described MMPs range from 3 to 12 μm in diameter and consist of about 10 to 40 individual Gram-negative cells (5, 45, 46). The size range of the MMP-like aggregates from Pyramid Lake and GL1 to GL3 was 7 to 11 μm, and the average was 7.5 ± 1.0 μm (n = 24) (Fig. (Fig.1).1). Disruption of the MMPs into their component cells is known to occur when they are left under the microscope for periods ranging from 15 min to 1 h or when they are placed in distilled water (45, 46). This allows for accurate counting of individual cells without the use of electron microscopy. The nMMPs from Pyramid Lake and the GBS pools did not disaggregate as easily as normal MMPs even when left in wet mounts or hanging drops for very long periods of time. Thus, we were unable to obtain a large number of cell counts for individual nMMPs. In addition, when nMMPs did disrupt, individual cells remained tightly associated with each other (Fig. (Fig.1A).1A). For the nMMPs whose cells we could count, there were between 40 and 60 cells per nMMP (Fig. (Fig.1A).1A). It seems that the overall structure of the nMMP aggregates is more robust than that previously observed for MMPs, possibly due to the connection between cells or the material holding them together being stronger than in MMPs examined thus far. Like other MMPs, an internal acellular compartment (30, 33) appeared to be present in these nMMPs if the light microscope was focused appropriately (Fig. (Fig.1C).1C). Transmission electron microscopy confirmed the multicellular nature of the nMMPs as well as the tight association of the cells (Fig. 2A and B) and the presence of flagella (Fig. (Fig.2B2B).

FIG. 1.
Differential interference contrast (DIC) light micrographs of the nonmagnetotactic multicellular prokaryotes (nMMPs). (A) Time course microscopy showing disaggregation of the nMMP. (B) Several nMMPs demonstrating variation in size of the organism. (C) ...
FIG. 2.
Transmission electron microscope (TEM) images of nMMPs and an MMP. (A) TEM image of an unstained nMMP showing the absence of magnetosomes. (B) TEM image of a negatively stained nMMP showing the numerous flagella (at dark arrows) similar to that of the ...

The nMMPs did not accumulate at the edge of the drop as would typical MMPs and other magnetotactic bacteria in a magnetic field, nor did they exhibit the unusual “ping-pong” behavior (22, 45, 46) or “escape” motility (33), motility in a magnetic field described as spontaneous linear reversals of hundreds of micrometers with decreasing speed, followed by forward swimming with increasing velocity (22). Generally this occurs when MMPs swim and reach the edge of the hanging drop in the magnetic field. Instead, the nMMPs showed no magnetotactic behavior and migrated away from the light to the other side of the drop after several minutes (see Video S2 in the supplemental material). When the light beam was placed on them again at the north side of the drop, they swam toward the south side. This negative phototactic response to white light was observed for nMMPs collected from all sampling sites.

Using the hanging drop technique and colored filters, specific wavelengths of light were tested for the ability to induce the negative phototactic response in nMMPs. Exposure of a drop of water containing nMMPs to wavelengths of 435 and 480 nm for 5 min resulted in nMMP migration to the extreme edge of the other side of the drop (the “dark” side of the drop) (Fig. (Fig.3).3). Exposure to wavelengths of 530 and 640 nm under similar conditions showed little if any effect (Fig. (Fig.33).

FIG. 3.
Histogram showing the total number of nMMPs that swam to and accumulated at the dark far edge of a 5-μl drop exposed at the opposite end to a specific wavelength of light for 5 min. Data are represented as the means and standard deviations from ...

Photoconcentration of nMMPs.

The negative phototactic response in the nMMPs appeared robust enough to exploit for their concentration as well as to decrease or eliminate the number of contaminating bacteria. Because the nMMPs were only a very small component of the population of bacteria in the samples, this would prove important for DNA extraction for 16S rRNA gene cloning and sequencing and also for electron microscopic examination. To test this possibility, we modified the capillary racetrack of Wolfe et al. (60) that was originally used for the concentration and/or physical isolation of magnetotactic bacteria to be used as inocula for cultivation (50), for DNA extraction (18, 56), and for electron microscopic examination (36). We used the same general setup and apparatus of the capillary racetrack as follows (Fig. (Fig.4):4): a sterile Pasteur pipette sealed at the thin end and containing a cotton plug was filled with filter-sterilized water from the sample up to the cotton filter. Typically, 1 ml of mud and water from the sample was used to fill the open end of the pipette above the cotton plug. In the original racetrack, magnets were placed at the ends of and parallel to the racetrack to create a magnetic field to separate north- or south-seeking magnetotactic bacteria (60). In this case, magnets were not used and a strong light source, a Fiber-L-Lite high-intensity illuminator series 180 (Dolan-Jenner Industries, Boxborough, MA), set at the maximum intensity, was placed at the open end of the racetrack (Fig. (Fig.4).4). The sealed end of the capillary was covered with black tape to negate any effects of extraneous light in the room. The idea was that the motile nMMPs exposed to the strong light would be driven through the cotton plug to the dark sealed end of the pipette, where they would accumulate. Contaminating cells would be significantly delayed in passage into the sterile water by the cotton plug. All light racetracks were operated for 30 min. Purified nMMPs were retrieved by breaking off the sealed end of the pipette and quantified as described above where appropriate or used for DNA extraction for phylogenetic analysis.

FIG. 4.
Light “racetrack” modified from the magnetic capillary racetrack of Wolfe et al. (60) that was originally used to concentrate magnetotactic bacteria. Here the light racetrack was used to photoconcentrate nMMPs. The Pasteur pipette is filled ...

The system was tested to see if we could quantitatively prove that light could be used to concentrate the nMMPs. In this experiment, all racetracks were cut so the capillary end of the pipette was 8 cm in length (the distance from the cotton plug at the junction to the sealed end). The samples tested were those from pool GL1 from GBS and Pyramid Lake. After the open end containing 1 ml of the sample was exposed to the light for 30 min, the dark sealed end of the capillary was broken off approximately 2 cm from the end, and the water containing nMMPs was removed and brought up to a final volume of 50 μl from which the hanging drops (3 per racetrack) were made and nMMPs counted. Results from these experiments which clearly indicate that intense white light can be effectively used to photoconcentrate nMMPs are shown in Fig. Fig.5.5. The relatively large standard deviations in the counts of nMMPs in the racetracks in Fig. Fig.55 reflect the variability in numbers of nMMPs in the original samples.

FIG. 5.
Effect of light on nMMPs in capillary light racetracks (shown in Fig. Fig.4).4). Histogram of numbers of nMMPs refers to nMMPs from samples from Great Boiling Springs pool GL1 (GL1) and Pyramid Lake (PL) that swam to and accumulated at the end ...

Testing for magnetotaxis and the presence of magnetosomes.

Because magnetosomes in the MMP are often not as organized as well as they are in other magnetotactic bacteria, the lack of magnetotaxis in nMMPs could be due to (i) the presence of disorganized magnetosomes and/or magnetosome chains that could result in the lack of a north and south end of the organism (i.e., a magnetic dipole), (ii) too few magnetosomes to elicit a magnetotactic response, or (iii) a lack of magnetosomes altogether. Examination of nMMPs from all sites with electron microscopy showed the latter to be the case (Fig. (Fig.2).2). We were unable to confirm the presence of a single magnetosome in any nMMP.

Phylogeny of nMMPs.

Three of the seven 16S rRNA genes cloned and sequenced from photoconcentrated racetracks of nMMPs from site GL1, two of the eight sequenced for site GL3, and two of the eight sequenced for Pyramid Lake were closely related (≥99.3% sequence similarity) to sequences of the MMP group in the Deltaproteobacteria class (Fig. (Fig.6).6). Because the sequence divergence between the different nMMPs clones was very small, we considered that it may be the result of errors in sequencing. The minimum difference in 16S rRNA gene sequence between nMMPs was 3/1,527 bases and the maximum was 11/1,527 bases. The maximum possible error we obtained with our sequencing facility was 5 bases on an entire 16S rRNA gene sequence of an axenic culture. Thus, it is possible that errors in sequencing might contribute to the observed sequence divergence in the nMMPs in our study. However, even taking this possibility into consideration, the sequences from the nMMPs clearly form a new phylogenetic branch within the MMP group (Fig. (Fig.6)6) and show that the nMMPs sequenced in our study probably represent a single species within the group.

FIG. 6.
Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of the nMMPs in the Deltaproteobacteria class. GL1 and GL3 refer to two different pools from the Great Boiling Springs site, and PL refers to Pyramid Lake where the nMMPs ...

DISCUSSION

The uncultured microorganism known as the MMP has intrigued microbiologists for a number of years because of the implications related to its obligately multicellular nature and the fact that it was the first magnetotactic bacterium discovered that biomineralized greigite (39) from nonmagnetic iron sulfide precursors (43, 44) and that could incorporate a metal other than iron (copper) in its magnetosomes (6). More specifically, the MMP's multicellularity suggests to many that some kind of intercellular communication must occur in this organism for it to function—for example, the apparent coordination of the numerous flagella for directed motility—and to divide.

The first important question to be answered is whether nMMPs simply share the same apparently unusual morphology and ultrastructure of MMPs or are phylogenetically related to them; i.e., are they actually an unrecognized form of MMP? Phylogenetic analyses of the nMMPs clearly demonstrate that they are members of the Deltaproteobacteria as part of a large group of sulfate-reducing bacteria and that they are closely related to MMPs. However, the nMMPs appear to form a separate, coherent phylogenetic branch within the MMP group. Considering the degree of 16S rRNA gene sequence similarity (≥99.3%) between the nMMPs and the sequence difference between these and the MMPs (≤95.8%), nMMPs appear to represent a new form of MMP that consists of a single species that is distinct from the several species represented by the MMPs (53).

MMP-like organisms had previously only been found in or associated with marine environments with salinities that are relatively high even compared to hypersaline environments (2, 30, 32, 40). For this reason, many consider the MMP to be an exclusively marine organism (53). Our results show that MMP-like organisms are not confined to marine environments, but because the aquatic habitats that we studied are still saline (i.e., brackish), that does not eliminate the possibility that MMP-like organisms require elevated levels of certain salts compared to freshwater bacteria. Different types of MMPs appear to have distinct salinity ranges for growth and survival that would presumably reflect the extent of any salt requirement. For example, viability studies involving the MMP “Candidatus Magnetoglobus multicellularis,” from the hypersaline Araruama lagoon in Brazil (2), showed that they died when exposed to salinities of >55 and <40 ppt in microcosms (40). Interestingly, however, viable organisms were found in the lagoon even when the salinity was 60 ppt, although in lower numbers when the salinity was intermediate between 40 and 55 ppt (average salinity of the lagoon, 40 ppt). Nonetheless, the known salinities in which MMP-like organisms exist have now been extended to as low as 5 ppt. Pure cultures will be required to determine whether specific salts at specific concentrations are required for growth and survival of the MMP-like organisms.

The nMMPs did not exhibit magnetotaxis, nor did they biomineralize magnetosomes of any type. Environmental conditions are also known to affect magnetosome production, and it is possible that chemical and redox conditions present at the sampling sites are not conducive to intracellular greigite biomineralization in magnetosomes. This notion is unlikely because other greigite-producing magnetotactic bacteria, as well as magnetite producers, were also present in the same samples. The greigite producers in these samples, large rod-shaped cells with a double chain of magnetosomes, were similar to those often found together with MMPs from marine sites (5, 6; see also Video S2 in the supplemental material).

Some magnetotactic bacteria are known to frequently lose the phenotypic trait of magnetotaxis and the ability to biomineralize magnetosomes in culture (14, 20, 34, 58). It has been shown that the genes encoding magnetosome (membrane) proteins are localized on a 130-kb magnetosome genomic island (MAI) in Magnetospirillum gryphiswaldense (48, 58). Similar magnetosome islands appear to be present in the genomes of other Magnetospirillum strains (20, 27, 28), as well as in the marine vibrio strain MV-1 (28). The MAIs in these organisms are flanked and interrupted in some cases by several types of mobile genetic elements, including direct repeats, insertion sequences, integrases, transposases, proximal transfer RNAs, and areas of atypical G+C content, which are responsible for the mobilization of the island (28). Because of these mobile elements, the MAI, like other genomic islands, is likely capable of horizontal gene transfer (13, 23). In addition, genomic islands not only have a strong tendency to be deleted from genomes with high frequency, they also can undergo duplications, amplifications, and rearrangements (13, 23, 25). Horizontal gene transfer of the MAI may explain why the trait of magnetotaxis is widespread, occurring in many diverse, unrelated bacteria, and why the trait is easily lost in various strains of magnetotactic bacteria. Strong evidence from tetranucleotide usage patterns in Magnetospirillum species and strain MV-1 showing that the MAIs in these organisms are transferred by horizontal gene transfer was recently described (28). Another suggestive piece of evidence for horizontal gene transfer of the MAI is the discovery of Magnetospirillum species that are not magnetotactic and do not biomineralize magnetosomes (10, 21, 51). Amann et al. (3) point out that the 98% 16S rRNA gene similarity between nonmagnetotactic Aquaspirillum polymorphum and M. gryphiswaldense is significantly greater than that between M. magnetotacticum and M. gryphiswaldense, and they pose the question of whether A. polymorphum represents a Magnetospirillum strain that recently lost the genes required for magnetotaxis, or whether M. gryphiswaldense recently acquired these genes. A similar situation seems to exist here between the MMPs and nMMPs. The nMMPs clearly represent a different species of the MMPs that may have lost the genes for greigite biomineralization or has not (yet?) acquired them. If this is true, then the genes for greigite biomineralization might be organized similarly (i.e., as an MAI) to those for magnetite production in the magnetospirilla.

One of the most interesting behaviors we observed in the nMMPs was their strong negative phototactic reaction to white light. This response was robust enough to exploit for the photoconcentration of nMMPs for DNA extraction for phylogenetic analysis and for the preparation of grids for electron microscopy. When nMMPs were tested for this negative phototactic response to specific wavelengths of visible light, only wavelengths of ≤480 nm (blue to violet) elicited the behavior. Longer wavelengths (≥530 nm; green to red) did not. There is phylogenetic, genetic, and ecological evidence that strongly suggests that all forms of MMPs are anaerobic sulfate-reducing bacteria (2, 5, 12, 53, 59). The most obvious explanation for this negative phototactic response is that light would drive nMMPs, and perhaps MMPs, in the environment toward and into the anaerobic zone of aquatic habitats where they are found in their highest numbers (54). It is significant that the shorter wavelengths of visible light that caused the response, ≤480 nm (blue to violet), are those that generally penetrate the water column the deepest (9). The negative phototactic response of the nMMPs is clearly efficient, and it is interesting that in this case, it might function similarly to magnetotaxis. That is, if light causes the nMMPs in nature to swim more or less vertically, then, like magnetotaxis (19), it would help to reduce a three-dimensional search problem to a one-dimensional search problem for an organism that must locate and maintain a position where specific chemical and redox conditions are optimal in a vertical chemical and redox gradient common in aquatic habitats. In this way, negative phototaxis in this case might increase the efficiency of chemotaxis as does polar magneto-aerotaxis (19). Although phototaxis as a trait is distributed widely among prokaryotes (26), as far as we know, this is the first report of a form of phototaxis in the Deltaproteobacteria class.

Other prokaryotes show a similar response to blue light for apparently different reasons. For example, the halophilic purple bacterium Ectothiorhodospira (Halorhodospira) halophila exhibits a negative phototactic response to blue light, presumably to avoid the damaging effects of UV light (8, 55). The photoactive yellow protein PYP (41, 42) is thought to be the photoreceptor responsible for this negative photoresponse, and it appears that many prokaryotes synthesize similar proteins or other blue light receptors (8). Cells of Escherichia coli display tumbling behavior upon short exposure (1 s) to intense blue light (8). After tumbling, cells run and soon become immotile and die, presumably from the formation of reactive oxygen species (8).

Despite the fact that no MMP-like organisms have yet been isolated and cultivated, a great deal of information regarding these unusual prokaryotes has been obtained through the use of culture-independent means and ecological studies. Results presented in this paper significantly advance and extend what we know about these microorganisms. Evidence in this paper clearly shows that the MMP group contains forms that are not magnetotactic, the nMMPs, which represent a new subgroup of the MMP group, probably a single species. Our results also show that MMP-like organisms are not restricted to marine environments, although, because the habitats we describe are still brackish by definition, elevated levels of certain salts may be required for these organisms to exist. Nonetheless, the lower limit of salinity for these types of organisms to exist is now ~5 ppt, which now gives cause to look for them in freshwater environments. The discovery of the nMMPs also raises many important questions. MMPs also do not evidently need to be magnetotactic and biomineralize magnetosomes, which leads to the question of the function of these organelles in the MMP. It seems possible that the nMMP's negative phototactic response might replace magnetotaxis in increasing the efficiency of chemotaxis in these organisms. It would be interesting now to know whether MMPs show this same phototactic response. It seems apparent that MMP-like microorganisms will continue to intrigue microbiologists in the future.

Supplementary Material

[Supplemental material]

Acknowledgments

We dedicate this paper to the memory of the late Karl Canter, a physicist whose research focus took on a major microbiological component when he was introduced to the MMP.

We thank David and Sandie Jamieson for access to the GBS field site and J. Dodsworth for help collecting samples.

This work was supported by U.S. National Science Foundation grant EAR-0920718 to D.A.B. U.L. and F.A. acknowledge partial financial support from the National Council for Scientific and Technological Development (CNPq) of Brazil.

Footnotes

[down-pointing small open triangle]Published ahead of print on 2 April 2010.

Supplemental material for this article may be found at http://aem.asm.org/.

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