Bacterial Intracellular Sulfur Globules: Structure and

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Transcript of Bacterial Intracellular Sulfur Globules: Structure and

MMB351335.inddBacterial Intracellular Sulfur Globules: Structure and Function
James S. Maki
Introduction
Sulfur is an important bioelement in the global eco- system. On the cellular level, sulfur is required for impor- tant amino acids, cysteine and methionine and is found in the vitamins thiamine pyrophosphate, coenzyme A and biotin, as well as in sulfolipids like α-lipoic acid [Gottschalk, 1986]. Inorganic sulfur compounds have many oxidation states [Dahl et al., 2008a; Steudel, 1989] and numerous transformations of sulfur in the environ- ment are mediated by special groups of prokaryotes, members of both Bacteria and Archaea. In its reduced forms, sulfur compounds are important electron donors and energy sources for those microbes that can utilize them. Chemolithotrophic prokaryotes use the energy for both respiration and fixation of CO 2 into organic carbon. Alternatively, the anaerobic photolithoautotrophic bac- teria use the energy primarily for CO 2 fixation [Dahl et al., 2008a; Ghosh and Dam, 2009]. In the process of oxi- dizing reduced sulfur compounds like sulfides and thio- sulfate into sulfate, a number of bacteria transiently form sulfur globules both extracellularly and intracellularly [Dahl and Prange, 2006]. In this review, the focus will be on the bacteria that form intracellular sulfur globules, intracellular meaning being found inside the cell wall, and what is currently known about globule structure, formation and degeneration.
Key Words
Abstract
Bacteria that oxidize reduced sulfur compounds like H 2 S of- ten transiently store sulfur in protein membrane-bounded intracellular sulfur globules; intracellular in this case mean- ing found inside the cell wall. The cultured bacteria that form these globules are primarily phylogenetically classi- fied in the Proteobacteria and are chemotrophic or photo- autotrophic. The current model organism is the purple sul- fur bacterium Allochromatium vinosum . Research on this bacterium has provided the groundwork for understanding the protein membranes and the sulfur contents of globules. In addition, it has demonstrated the importance of different genes (e.g. sulfur oxidizing, sox ) in their formation and in the final oxidation of sulfur in the globules to sulfate (e.g. dis- similatory sulfite reductase, dsr ). Pursuing the characteris- tics of other intracellular sulfur globule-forming bacteria through genomics, transcriptomics and proteomics will eventually lead to a complete picture of their formation and breakdown. There will be commonality to some of the ge- netic, physiological and morphological characteristics in- volved in intracellular sulfur globules of different bacteria, but there will likely be some surprises as well.
Copyright © 2013 S. Karger AG, Basel
Published online: August 5, 2013
Dr. James S. Maki Marquette University Department of Biological Sciences PO Box 1881, Milwaukee, WI 53201-1881 (USA) E-Mail james.maki   @   marquette.edu
© 2013 S. Karger AG, Basel 1464–1801/13/0235–0270$38.00/0
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The early microbiological study of intracellular sulfur globules, their structure and contents involves the re- search of some of the most well-known microbiologists in the history of the discipline [e.g. van Niel, 1931; Wino- gradsky, 1889]. Those interested in the early research on sulfur bacteria are referred to discussions by Waksman [1922], Waksman and Joffe [1922], Shively [2006], Dahl and Prange [2006], Trüper [2008], and papers referenced therein. The focus of this review will be on more recent information.
Phylogenetic Distribution of Genera with
Intracellular Sulfur Globules
The ability to form intracellular sulfur globules is pres- ent in a wide variety of cultured genera largely found in the α-, β-, γ- and ε-Proteobacteria ( fig. 1 ). Magnetotactic bacteria vary in their ability to form intracellular sulfur globules [Bazylinski et al., 2004; Bazylinski and Williams, 2006; Keim et al., 2005]. Those that do are primarily in the α-Proteobacteria ( fig. 1 ) and have been demonstrated to form globules using sulfide and/or thiosulfate [Bazy- linski and Williams 2006; Williams et al., 2006]. These genera include Magnetococcus , which branches out early from the other α-Proteobacteria [Bazylinski and Wil- liams, 2006] and the newly described genera Magnetovi- brio [Bazylinski et al., 2004, 2012] in which sulfur globules are formed when cells are grown on sulfide, and Magne-
tospira [Williams et al., 2012] in which they form when cells are grown on thiosulfate. However, the ability of magnetotactic bacteria to form globules is not restricted to the α-Proteobacteria. Recently, a magnetotactic bacte- rium that forms intracellular sulfur globules was discov- ered that phylogenetically belongs in the γ-Proteobacteria [Lefevre et al., 2012].
Members of two genera, Thermothrix and Macromo- nas , in the β-Proteobacteria have been observed to form globules. While one species of the genus Thermothrix ,
Thiorhodococcus Allochromatium
Thioalkalivibrio Achromatium
Thiothrix Beggiatoa
Thiomargarita Thioploca
Macromonas Thermothrix
Magnetococcus Aquifex
Fig. 1. Phylogenetic tree based on nearest neighbor analysis of 16S rRNA genes of the different genera, all in the Proteobacteria, in which intracellular sulfur globules have been reported. The tree is rooted with the genus Aquifex .
Fig. 2. Thiothrix spp. found on an aquatic macrophyte in the flume of a freshwater hydrothermal vent in Yellowstone Lake, Wyo., USA. Arrows show intracellular sulfur globules. Details about the habitat can be found in Konkol et al. [2010]. Scale bar = 1.0 μm [unpubl. micrograph, J.S. Maki].
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T. thiopara , deposits sulfur globules extracellularly [Cald- well et al., 1976], the other, T. azorensis , forms globules intracellularly when there is incomplete oxidation of thiosulfate and the pH is >7.0 [Odintsova et al., 1996]. In the genus Macromonas , the species M. bipunctata , forms sulfur globules in the periplasm when cells are grown in the presence of sulfides but not thiosulfate [Dubinina et al., 2005].
By far the majority of genera in which cultured bacte- ria produce intracellular sulfur globules are found in the γ-Proteobacteria ( fig. 1 ). This includes bacteria in genera like Thiothrix (e.g., fig. 2) [Bland and Staley, 1978; Konkol et al., 2010; Larkin, 1980], Beggiatoa [Strohl et al., 1981], Thiomargarita [Schulz et al., 1999] and Thioploca [Jør- gensen and Gallardo, 1999; Maier and Murray, 1965], some of the largest microorganisms found in nature. It also includes the purple sulfur bacteria in the family Chromatiaceae, but not the Ectothiorhodospiraceae which form extracellular sulfur globules [Frigaard and Dahl, 2009]. Endosymbiotic bacteria [Cavanaugh et al., 1981] found in invertebrates associated with hydrother- mal vents and sulfide gradients have also been indicated to be members of the γ-Proteobacteria [Distel, 1998; Stewart and Cava naugh, 2006].
Additional genera to those presented in figure 1 have been shown to produce internal sulfur globules. Primary among these is the genus Thiovulum . Members of this ge- nus have resisted attempts to get them in pure culture, but 16S rRNA gene analysis places them in the ε-Pro teo- bacteria and a genome analysis has been completed [Mar- shall et al., 2012]. Although no complete 16S rRNA se- quence has been reported for the genus Thiobacterium , a recent publication [Grünke et al., 2010] using fluorescence in situ hybridization indicated that this genus also belongs in the γ-Proteobacteria. However, not all sulfur globule- producing bacteria are in the Proteobacteria. Two genera, Thermonanerobacter and Thermoanaerobacterium in the Firmicutes have also been reported to produce internal sulfur globules [Lee et al., 2007]. A large spirillum, Titano- spirillum velox , that had internal sulfur globules has also been described [Guerrero et al., 1999]; however, the name has not been validated, no culture has been submitted to a collection and no 16S rRNA gene sequence has been sub- mitted to the Ribosomal Database Project or GenBank.
Although most of the described bacteria that construct intracellular sulfur globules are in the Proteobacteria, the fact that the vast majority of microbes in the world have yet to be isolated leads to the speculation that this ability could be more phylogenetically widespread than is cur- rently apparent.
Globule Protein Membranes
In light micrographs sulfur globules appear as translu- cent or clear areas ( fig. 2 ), while in electron micrographs sulfur globules appear as clear empty areas within thin sec- tions of cells ( fig. 3 a). Electron microscopy can be a bit of an art form and revealing cellular fine structures is some- times difficult depending on the processes of fixation, de- hydration and embedding. However, it has been reported from a variety of bacteria that intracellular sulfur globules are bounded by a protein membrane, e.g. Thiothrix [Bland and Staley, 1978], purple sulfur bacteria [Nicolson and Schmidt, 1971; Remsen, 1978, 1982; Remsen and Trüper, 1973], Beggiatoa [Strohl et al., 1981] and Thiovulum [Wir- sen and Jannasch, 1978]. This protein membrane can be observed on globules using freeze-etching, when it is in the correct plane ( fig. 3 b), and in thin sections using transmis- sion electron microscopy ( fig. 3 c). The proteins encasing the sulfur globules from most of these bacteria have yet to be characterized.
The vast majority of what is known about the protein membranes of sulfur globules comes from investigations involving purple sulfur bacteria in the family Chromatia- ceae. Initially, using centrifugation to isolate globules, ex- traction with chloroform to remove sulfur and retain the protein membrane, Schmidt et al. [1971] reported the pres- ence of a 13.5-kDa protein from the globule membrane from Allochromatium vinosum [formerly Chromatium vinosum , see Imhoff et al., 1998] . Similarly, the presence of a single 18.5-kDa protein from the sulfur globule in Iso- chromatium buderi [formerly C. buderi , Imhoff et al., 1998] was discovered [Gonye, Schroeder and Remsen, unpubl. data; reported by Remsen, 1978]. Outside of the Chroma- tiaceae, a 13-kDa, and more particularly, a 15-kDa protein were evident in sulfur inclusions from Beggiatoa when cells were grown in a medium with sulfide [Schmidt et al., 1986].
In the case of A. vinosum , subsequent analysis and sep- aration of the proteins using HPLC revealed that the 13.5- kDa protein was actually composed of two slightly small- er homologous proteins roughly 10.6 kDa in size (called SgpA and SgpB) [Brune, 1995]. In addition, a third pro- tein, 8.5 kDa (SgpC), was also found associated with the globules [Brune, 1995]. These three proteins make up the membrane of the sulfur globule ( fig.  4 ). Two proteins were also reported from the sulfur globules of Thiocapsa roseopersicina , the larger one approximately 10.6 kDa and the smaller one 8.7 kDa, and both homologous to the large and small proteins of A. vinosum , respectively [Brune, 1995]. These proteins were found to be similar to structural proteins in protein databases [Brune, 1995].
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The presence of two proteins in the original 13.5-kDa protein in A. vinosum raises the interesting question as to whether the 18.5-kDa protein band reported in I. buderi [Remsen, 1978] and the 15-kDa protein in Beggiatoa are also composed of more than one protein.
The genes for the sulfur globule proteins (sgp) from A. vinosum were cloned and sequenced and, interestingly, were found to not be in the same operon [Pattaragulwanit et al., 1998], which was confirmed when the genome was sequenced [Weissgerber et al., 2011]. When the gene for SgpA was inactivated, globules still formed indicating that only one of the two homologous proteins, SgpA or SgpB, was necessary. The data collected also indicated an extra- cytoplasmic location of the Sgp’s and implied the proteins were exported into the periplasm [Pattaragulwanit et al., 1998]. It was later determined that the genes for these three proteins were expressed constitutively under both photolithotrophic and photoorganotrophic conditions [Prange et al., 2004]. Inactivation of sgpA or sgpB did not show any differences compared to wild-type, while inac- tivation of sgpC resulted in the formation of smaller sulfur
globules [Prange et al., 2004]. SgpC appeared to have a role in the expansion of the globule and when the protein is not available the cell compensates by forming more, smaller globules [Prange et al., 2004]. When a double mutant of sgpB and sgpC was created, the mutant was unable to grow on sulfide and no globules were formed, which indicated that the protein membrane is required for the intracellular globule [Prange et al., 2004].
Doing a BLASTp search of the amino acid sequence of SgpA against all microbial genomes revealed that it has similar sequences to a number of hypothetical proteins and sgp from other γ-Proteobacteria ( fig. 5 ). Also includ- ed were a putative sgp (accession No. ZP 08816665.1) from an endosymbiont of the vestimentiferan tubeworm Tevnia jerichonana , one of the pioneer species found at new deep-sea hydrothermal vents [Mullineaux et al., 2000]. In addition to those sequences in figure 5 , the SgpA sequence also matched to a hypothetical protein in Brachybacterium squillarum (ZP 09850192.1) isolated from salt-fermented seafood [Park et al., 2011], a hypo- thetical protein (YP 003768744.1) from the rifamycin-
a b
c
Fig. 3. Structure of I. buderi (formerly C. buderi ) showing sulfur globules. a Section of I. buderi showing sulfur globules (S). b Freeze-etching of I. buderi showing membrane-bound sulfur globules. c Sec- tion of I. buderi showing membrane-bound sulfur globules. Arrows indicate mem- brane. Scale bars = 1.0 μm [unpubl. micro- graphs provided by C.C. Remsen].
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producing actinomycete Amycolatopsis mediterranei , an integral membrane protein (ZP 07979829.1) from Strep- tomyces spp. and a hypothetical protein (ZP 21109042.1) from Streptomyces viridochromogenes .
A similar search with SgpB also revealed matches to a wide variety of hypothetical proteins and sgp from other microbes also found in the γ-Proteobacteria ( fig.  6 ). It also matched a hypothetical protein (ZP 02532721) found in an endosymbiont from the giant tubeworm Riftia pa- chyptila ( fig. 6 ) called Candidatus Endoriftia persephone [Robidart et al., 2008]. The SgpC amino acid sequence also matched to hypothetical proteins and sgp in other γ-Proteobacteria ( fig. 7 ).
Globule Sulfur Content
Even though both light and electron microscopy show globules associated with bacteria that oxidize reduced sulfur compounds, and that when the source of reduced sulfur disappears the globules reduce in size [Winograd- sky, 1889], the question remained as to whether the glob- ules were actually completely filled with sulfur [Steudel, 1989]. The process of dehydration during electron mi-
croscopy removes the sulfur from the globule [Remsen and Trüper, 1973]. Elemental sulfur (S 0 ) forms polymer- ic chains and rings, with cyclooctasulfur (S 8 ) being ther- modynamically the most stable form [Steudel, 1989].
X-ray diffraction was used to analyze sulfur from sul- fur globules in Thiovulum [LaRiviere, 1963] and photo- synthetic sulfur bacteria [Hageage et al., 1970; Trüper and Hathaway, 1967]. Freshly isolated, wet sulfur globules had diffraction patterns similar to that of liquid sulfur while after drying they converted into the cyclooctasulfur form [Hageage et al., 1970]. Analyses of dense inclusion bodies in air-dried filaments of Beggiatoa alba with ener- gy-dispersive X-ray microanalysis showed that the inclu- sions were composed of sulfur [Lawry et al., 1981] but could not designate the form of the sulfur, whether poly- meric chain or cyclooctasulfur. Energy-dispersive X-ray has also been used to identify sulfur-containing globules in magnetotactic bacteria [Keim et al., 2005].
One technique that has been effective in analysis of sulfur globules is a form of X-ray spectroscopy called X- ray absorption near-edge structure (XANES) spectrosco- py. For a description of XANES, the reader is referred to the review by Prange et al. [2008] and references therein. When purple sulfur bacteria were grown photoautotro- phically on sulfide the XANES spectra showed the sulfur in the globules with a structure of long sulfur chains (R- S n -R) with unidentified organic residues at one or both ends [Prange et al., 1999]. No sulfur rings were detected. When globules in Beggiatoa and Thioploca were com- pared to purple sulfur bacteria, the sulfur in the former were found to be dominated by cyclooctasulfur, while the purple sulfur bacteria contained sulfur chains [Prange et al., 2002]. However, using the XANES methodology and examining A. vinosum globules, George et al. [2008] re- ported the presence of cyclooctasulfur (S 8 ), not chains. These two research groups used different detection strat- egies which ultimately provided dissimilar results and conclusions. Further discussions on the controversy can be found in George et al. [2008] and Prange et al. [2008]. Other methods like Raman spectroscopy have also shown the sulfur globules in Thioploca and Beggiatoa to contain cyclooctasulfur (S 8 ) [Pasteris et al., 2001].
Storage/Formation
How does the bacterium go about forming the globule and storing sulfur? For A. vinosum , it is known that the proteins making up the membrane are essential for this to occur [Prange et al., 2004]. Members of the Chroma-
SgpB
SgpB
SgpBSgpB
SgpB
SgpB
SgpC
SgpC
SgpC
SgpC
SgpC
SgpC
SgpA
SgpA
SgpA
SgpA
SgpA
SgpA
S0
Fig. 4. Schematic diagram of sulfur globule membrane proteins identified from A. vinosum . This photosynthetic purple sulfur bac- terium forms transient globules in the periplasm and requires the membrane proteins. SgpA and SgpB are interchangeable and only one of these two proteins is necessary. SgpC is required for expan- sion of the globule. See text for details.
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tiaceae, C. okenii and A. warmingii , were observed using cinematography to form globules at any location within the cell [Hermann and Truper, unpubl. data; reported by Pattaragulwanit et al., 1998; Trüper, 2008]. The model or- ganism A. vinosum can form globules from sulfur (S 0 ), sulfides and thiosulfate ( fig. 8 ).
When using sulfur (S 0 ) to form globules, it was shown that A. vinosum required direct cell contact with the sul- fur and strongly preferred only polymeric sulfur (sulfur chain), not cyclooctasulfur [Franz et al., 2007]. Similarly, it was shown that when T. roseopersicina and two species of Halorhodospira also formed sulfur globules from ele- mental sulfur, the former intracellularly and the latter extracellularly, they also preferred uptake of chain-like polymers [Franz et al., 2009b]. Although cell contact with the sulfur is essential the mobilization of the sulfur does not appear to involve excretion of soluble sulfur-contain- ing substances [Franz et al., 2009a].
When sulfides were the source of sulfur for the sulfur globules in A. vinosum two primary enzymes were pos- sibly involved in the oxidation: a periplasmic flavocyto- chrome c, and a membrane-bound sulfide:quinone oxi- doreductase [Dahl and Prange, 2006; Frigaard and Dahl, 2009]. By constructing a flavocytochrome c-deficient mutant, it was shown that there was no effect on sulfide oxidation compared to the wild-type cells and pointed to an enhanced role for the sulfide:quinone oxidoreductase [Reinartz et al., 1998]. However, under certain growth conditions as yet unidentified, the flavocytochrome c may play a role [see discussion in Frigaard and Dahl, 2009].
The sulfur-oxidizing enzyme system (Sox) has been indicated to be important in the oxidation of thiosulfate to sulfate [Friedrich et al., 2005; Ghosh and Dam, 2009]. Bacteria that form intracellular sulfur globules do not possess all the genes necessary [Friedrich et al., 2005;
Fig. 5. Amino acid sequence of the SgpA from A. vinosum and the results of BLAST search of microbial genomes for similar protein sequences. Underlined amino ac- ids correspond to the sequence determined by Brune [1995] from the purified sgp. GenBank accession numbers are in paren- theses at the end of each sequence. hp = Hy- pothetical protein; psgp = putative sgp.
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Hensen et al., 2006]. In A. vinosum , two independent loci were discovered which contained five sox genes, soxBXA and soxYZ , but lacked soxCD [Hensen et al., 2006]. Sul- fane sulfur binds to SoxY and the sulfur dehydrogenase encoded by soxCD is important for further oxidation.
Without soxCD , A. vinosum transfers the sulfane to the sulfur globule in the periplasm [Hensen et al., 2006]. In the absence of reduced sulfur compounds the sox genes are expressed at a low constitutive level [Grimm et al., 2011].
Fig. 6. Amino acid sequence of the SgpB from…