Culture‐independent characterization of a novel magnetotactic member affiliated to the Beta class of the Proteobacteria phylum from an acidic lagoon

TLDR: This is the first report of an MTB phylogenetically affiliated with Betaproteobacteria class from an acidic freshwater lagoon in Brazil and some of the retrieved gene sequences belonged to the genus Herbaspirillum within the BetAProteob bacteria class of the Proteobacteria phylum.

Abstract: Magnetotactic bacteria (MTB) comprise a group of motile microorganisms common in most mesothermal aquatic habitats with pH values around neutrality. However, during the last two decades, a number of MTB from extreme environments have been characterized including: cultured alkaliphilic strains belonging to the Deltaproteobacteria class of the Proteobacteria phylum; uncultured moderately thermophilic strains belonging to the Nitrospirae phylum; cultured and uncultured moderately halophilic or strongly halotolerant bacteria affiliated with the Deltaproteobacteria and Gammaproteobacteria classes and an uncultured psychrophilic species belonging to the Alphaproteobacteria class. Here, we used culture-independent techniques to characterize MTB from an acidic freshwater lagoon in Brazil (pH ∼ 4.4). MTB morphotypes found in this acidic lagoon included cocci, rods, spirilla and vibrioid cells. Magnetite (Fe3 O4 ) was the only mineral identified in magnetosomes of these MTB while magnetite magnetosome crystal morphologies within the different MTB cells included cuboctahedral (present in spirilla), elongated prismatic (present in cocci and vibrios) and bullet-shaped (present in rod-shaped cells). Intracellular pH measurements using fluorescent dyes showed that the cytoplasmic pH was close to neutral in most MTB cells and acidic in some intracellular granules. Based on 16S rRNA gene phylogenetic analyses, some of the retrieved gene sequences belonged to the genus Herbaspirillum within the Betaproteobacteria class of the Proteobacteria phylum. Fluorescent in situ hybridization using a Herbaspirillum-specific probe hybridized with vibrioid MTB in magnetically-enriched samples. Transmission electron microscopy of the Herbaspirillum-like MTB revealed the presence of many intracellular granules and a single chain of elongated prismatic magnetite magnetosomes. Diverse populations of MTB have not seemed to have been described in detail in an acid environment. In addition, this is the first report of an MTB phylogenetically affiliated with Betaproteobacteria class.

Figures

Fig. 3. Phylogenetic analysis and corresponding fluorescent in situ hybridization (FISH) of the MTB collected from the Comprida Lagoon belonging to the Betaproteobacteria class. (A) Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences showing the position of the strain CLV-1 among other MTB and Herbaspirillum species. Bootstrap values were calculated for 1,000 replicates. GenBank accession numbers are given in parentheses. (B) DIC microscopy image of magnetic-enriched MTB, showing two cells; one containing a chain of magnetosomes and two intracellular granules (arrow) and another cell (arrowhead). (C) Fluorescence microscopy image of the same region shown in (B) stained with 4',6-diamidino2-phenylindole (DAPI). Note that all cells stain with DAPI (arrow and arrowhead). (D) Fluorescence microscopy image of the same cells hybridized with the Bacteria-specific probes. Note the fluorescent signal on both cells (arrow and arrowhead). (E) Fluorescence microscopy image of the same cells hybridized with the Herbaspirillum-specific probe. The vibrioid-shaped cell with two granules hybridized to this specific probe (arrow) but not the other cell.

Fig. 4. Characterization of magnetosomes in strain CLV-1. (A) TEM image of a cell of the vibrioid MTB (strain CLV-1) Comprida Lagoon, showing the presence of an intracellular single chain of elongated prismatic magnetosomes as well a number of granule-like inclusions. (B) TEM image of an ultrathin section of a cell of strain CLV-1 showing that magnetosomes are enveloped by an electron-dense membrane (arrows). (C) High resolution TEM (HRTEM) image of a single magnetosome from strain CLV-1. Inset: fast Fourier transform (FFT) image corresponding to the [1,-1,0] zone axis of magnetite (Fe3O4). (D) High-angle annular dark-field (HAADF) image of a cell of strain CLV-1 showing a chain of magnetosomes with

Fig. 1. Association of specific MTB morphotypes with sediment depth in the Comprida Lagoon. (A) Oxygen concentration [O2] profile correlated with MTB distribution in sediment layers and water pH. (B) Differential interference contrast (DIC) microscopy image of MTB morphotypes present in magnetically-enriched samples from the Comprida Lagoon. Note the presence of vibrioid cells with cytoplasmic inclusions (black arrowheads), and cocci (white arrows), spirilla (white arrowheads) and a rod (inset, black arrow).

Fig. 2. Intracellular pH analysis of MTB using the fluorescent acid-sensitive dye pHrodo Green. (A) DIC microscopy images showing three MTB morphotypes from Comprida Lagoon: spirillar (helical), coccoid and vibrioid cells; (B) only vibrioid cells; (C) only rod-shaped cells. The inset in each image shows the fluorescent cell reaction of designated MTB corresponding to cells indicated by white squares in (A), (B) and (C), respectively, using fluorescence

Fig. 5. Characterization of magnetosomes by electron microscopy. (A) Scanning TEM (STEM) image a magnetosome chain in a cell of CLV-1. Inset: Higher magnification of the region indicated by the white square in (A). Note the defects around the magnetosome magnetite crystal. (B) Three-dimensional reconstruction based on STEM tomography showing the relative spatial distribution of magnetosomes organized as a single chain within the cell.

Full text

Culture-independent characterization of a novel
magnetotactic member affiliated to the Beta
class of the Proteobacteria phylum from an acidic
lagoon
Fernanda Abreu, Pedro Leão, Gabriele Vargas, Jefferson Cypriano, Viviane
Figueiredo, Alex Enrich Prast, Dennis A. Bazylinski and Ulysses Lins
The self-archived postprint version of this journal article is available at Linköping
University Institutional Repository (DiVA):
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-150986
N.B.: When citing this work, cite the original publication.
Abreu, F., Leão, P., Vargas, G., Cypriano, J., Figueiredo, V., Enrich Prast, A., Bazylinski, D. A., Lins, U.,
(2018), Culture-independent characterization of a novel magnetotactic member affiliated to the Beta
class of the Proteobacteria phylum from an acidic lagoon, Environmental Microbiology, 20(7), 2615-
2624. https://doi.org/10.1111/1462-2920.14286
Original publication available at:
https://doi.org/10.1111/1462-2920.14286
Copyright: Wiley (12 months)
http://eu.wiley.com/WileyCDA/

1
Culture-independent characterization of a novel, uncultivated magnetotactic member of
the Betaproteobacteria class of the Proteobacteria phylum from an acidic lagoon
Fernanda Abreu
1§
, Pedro Leão
1
, Gabriele Vargas
1
, Jefferson Cypriano
1
, Viviane Figueiredo
2
,
Alex Enrich-Prast
2,3
, Dennis A. Bazylinski
4
and Ulysses Lins
1
1
Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-902,
Rio de Janeiro, RJ, Brazil.
2
Instituto de Biologia, Departamento de Botânica, Universidade Federal do Rio de Janeiro,
21941-902, Rio de Janeiro, RJ, Brazil.
3
Department of Environmental Change, Linköping University, 58183, Linköping, Sweden.
4
School of Life Sciences, University of Nevada at Las Vegas, Las Vegas, Nevada 89154-4004,
USA.
§
Corresponding author: Fernanda Abreu. Avenida Carlos Chagas Filho, 373; Centro de
Ciências da Saúde; Instituto de Microbiologia; UFRJ; 21941-902; Rio de Janeiro, RJ, Brazil.
E-mail: fernandaaabreu@micro.ufrj.br; Tel: +55-21-2560-8344 ext. 170; Fax: +55-21-2560-
8028
Running head: Magnetotactic bacteria from Betaproteobacteria class
Conflict of Interest Statement: The authors declare no conflict of interest.

2
Originality Significance Statement
Magnetotactic bacteria are widely distributed in aquatic environments and are
phylogenetically diverse with representatives in several different phyla. These bacteria are
common members of microbial communities in most mesothermal environments with pH
values around neutrality. However, uncultured alkaliphilic, moderately thermophilic, halophilic
or strongly halotolerant, and psychrophilic magnetotactic bacteria have also been described in
extreme environments across the globe. Here we report the presence of magnetotactic bacteria
in sediments of a permanently acidic freshwater lagoon, which includes the first description of
a magnetotactic bacterium belonging to the Betaproteobacteria class of the Proteobacteria
phylum.

3
Summary
Magnetotactic bacteria (MTB) comprise a group of motile microorganisms common in
most mesothermal aquatic habitats with pH values around neutrality. However, during the last
two decades, a number of MTB from extreme environments have been characterized including:
cultured alkaliphilic strains belonging to the Deltaproteobacteria class of the Proteobacteria
phylum; uncultured moderately thermophilic strains belonging to the Nitrospirae phylum;
cultured and uncultured moderately halophilic or strongly halotolerant bacteria affiliated with
the Delta- and Gammaproteobacteria classes and an uncultured psychrophilic species
belonging to the Alphaproteobacteria class. Here we used culture-independent techniques to
characterize MTB from an acidic freshwater lagoon in Brazil (pH ~4.4). MTB morphotypes
found in this acidic lagoon included cocci, rods, spirilla and vibrioid cells. Magnetite (Fe
3
O
4
)
was the only mineral identified in magnetosomes of these MTB while magnetite magnetosome
crystal morphologies within the different MTB cells included cuboctahedral (present in
spirilla), elongated prismatic (present in cocci and vibrios) and bullet-shaped (present in rod-
shaped cells). Intracellular pH measurements using fluorescent dyes showed that the
cytoplasmic pH was close to neutral in most MTB cells and acidic in some intracellular
granules. Based on 16S rRNA gene phylogenetic analyses, some of the retrieved gene
sequences belonged to the genus Herbaspirillum within the Betaproteobacteria class of the
Proteobacteria phylum. Fluorescent in situ hybridization using a Herbaspirillum-specific probe
hybridized with vibrioid MTB in magnetically-enriched samples. Transmission electron
microscopy of the Herbaspirillum-like MTB revealed the presence of many intracellular
granules and a single chain of elongated prismatic magnetite magnetosomes. Diverse
populations of MTB have not seemed to have been described in detail in an acid environment.
In addition, this is the first report of an MTB phylogenetically affiliated with
Betaproteobacteria class.

4
Introduction
Magnetotactic bacteria (MTB) biomineralize intracellular, membrane-bounded
magnetite (Fe
3
O
4
) or greigite (Fe
3
S
4
) magnetic nanocrystals called magnetosomes (Bazylinski
and Frankel, 2004). Magnetosomes are prokaryotic organelles used as part of a navigation
system to more efficiently locate and maintain an optimal position in chemically-stratified
aquatic environments for survival and growth (Cornejo et al., 2014).
MTB are ubiquitous in aquatic environments, being more commonly observed in
sediments, but have also been described in wet soils (Fassbinder et al., 1990) and in the water
columns of chemically-stratified habitats (Spring and Bazylinski, 2006). MTB were first
reported in freshwater, brackish or marine environments with pH values close to neutrality and
temperatures close to ambient (Bazylinski and Frankel, 2004). Mesophilic MTB include
cultured species belonging to the Alpha-, Delta- and Gammaproteobacteria classes within the
Proteobacteria phylum and several uncultured bacteria phylogenetically affiliated with the
Proteobacteria and Nitrospirae phyla, and candidate phylum “Omnitrophica” (formerly OP3)
(Lefèvre and Bazylinski, 2013). MTB signature genes in metagenomic sequences obtained from
Sakinaw Lake (Canada), a lake where the pH varies from 5.7 to 6.5 and the temperature ranges
from 8 to 19
o
C (Perry, 1990), strongly indicate the possibility of a novel greigite-producing
MTB belonging to the candidate phylum “Latescibacteria” (formerly WS3) (Lin and Pan,
2015) although an MTB of this type was not physically observed from the lake.
In the last two decades, a number of extremophilic MTB have been described and
include halophilic, alkaliphilic, psychrophilic and thermophilic representatives (Bazylinski and
Lefèvre, 2013). Some examples of these extremophiles include Candidatus Magnetoglobus
multicellularis, an uncultured, slightly halophilic magnetotactic multicellular prokaryote
(MMP) that inhabits a hypersaline lagoon in Brazil (Abreu et al., 2007). Strain SS-5, a rod-
shaped, as yet unnamed MTB of the Gammaproteobacteria, capable of producing elongated

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Culture-independent characterization of a novel magnetotactic member affiliated to the beta class of the proteobacteria phylum from an acidic lagoon" ?

N. B.: When citing this work, cite the original publication. 

Q2. What are the future works mentioned in the paper "Culture-independent characterization of a novel magnetotactic member affiliated to the beta class of the proteobacteria phylum from an acidic lagoon" ?

Future metagenomic studies of MTB from Comprida Lagoon should provide important information regarding magnetosome biomineralization in acidic environments. 

Q3. What are the common types of bacteria in the world?

uncultured alkaliphilic, moderately thermophilic, halophilic or strongly halotolerant, and psychrophilic magnetotactic bacteria have also been described in extreme environments across the globe. 

Q4. What is the morphology of the different MTB cells?

Magnetite (Fe3O4) was the only mineral identified in magnetosomes of these MTB while magnetite magnetosome crystal morphologies within the different MTB cells included cuboctahedral (present in spirilla), elongated prismatic (present in cocci and vibrios) and bullet-shaped (present in rodshaped cells). 

Q5. What is the pH of the cytoplasm?

Intracellular pH measurements using fluorescent dyes showed that the cytoplasmic pH was close to neutral in most MTB cells and acidic in some intracellular granules. 

Q6. What is the way to describe MTB?

Fluorescent in situ hybridization using a Herbaspirillum-specific probe hybridized with vibrioid MTB in magnetically-enriched samples. 

Q7. What is the morphology of the MTB?

Transmission electron microscopy of the Herbaspirillum-like MTB revealed the presence of many intracellular granules and a single chain of elongated prismatic magnetite magnetosomes. 

Q8. What is the name of the bacterium?

Based on 16S rRNA gene phylogenetic analyses, some of the retrieved gene sequences belonged to the genus Herbaspirillum within the Betaproteobacteria class of the Proteobacteria phylum. 

Q9. What are the characteristics of MTB from extreme environments?

during the last two decades, a number of MTB from extreme environments have been characterized including: cultured alkaliphilic strains belonging to the Deltaproteobacteria class of the Proteobacteria phylum; uncultured moderately thermophilic strains belonging to the Nitrospirae phylum; cultured and uncultured moderately halophilic or strongly halotolerant bacteria affiliated with the Delta- and Gammaproteobacteria classes and an uncultured psychrophilic species belonging to the Alphaproteobacteria class. 

Q10. What is the name of the bacteria?

Here the authors report the presence of magnetotactic bacteria in sediments of a permanently acidic freshwater lagoon, which includes the first description of a magnetotactic bacterium belonging to the Betaproteobacteria class of the Proteobacteria phylum.