The Green Sulphur Bacteria

There was a time when we really didn't know what to make of bacterial systematics. We knew that there were a lot of different species out there (not, it turns out, any near as many as there actually are, but still...) but prior to the molecular revolution of the last few decades we lacked the facilities to tell how many of them were related to each other. Nevertheless, there are some bacterial groupings that are distinctive enough to have been recognised even before the advent of regular genetic sequencing. One such group is the green sulphur bacteria.

Culture of Chlorobium phaeobacteroides, from here.

Two things must you know of green sulphur bacteria. One, they are (commonly) green. Two, they are associated with sulphur. Like the more familiar blue-green algae, green sulphur bacteria are photosynthetic, using light energy collected by coloured pigments to assimilate carbon dioxide. In some species the photosynthetic pigments are bacteriochlorophyll c or d, giving the cells a grass green coloration. In others, the pigment is bacteriochlorophyll e, and the cells are a chocolate brown. In contrast to blue-green algae, green sulphur bacteria are anaerobic: instead of using water as an electron donor to produce oxygen, they oxidise sulphide or sulphur to produce sulphur or sulphate (a single species, C. ferrooxidans, uses ferrous iron instead of sulphur). As a result, they are found growing in habitats that light reaches but oxygen doesn't. Many species are found in thermally stratified lakes or brackish lagoons with little mixing between upper and lower water layers, and form a distinct planktonic layer at the optimum intersection between light and sulphide gradients. They are also common in sulphur-rich hot springs. The cell's bacteriochlorophylls are concentrated into structures referred to as chlorosomes attached to the cytoplasmic membrane, maximising their ability to gather light at the low intensities. A number of species contain gas vacuoles to improve buoyancy. Most green sulphur bacteria are non-motile, though one species Chloroherpeton thalassium has long, filamentous cells with gliding motility. Molecular phylogenetic analyses have placed this species as the sister taxon to all other described green sulphur bacteria.

Scanning electron micrograph of 'Chlorochromatium aggregatum', showing the green sulphur bacteria wrapped around a central (concealed) non-photosynthetic partner. Copyright American Society for Microbiology.

An interesting characteristic of many green sulphur bacteria is their propensity for forming close symbiotic relationships with other, non-photosynthetic bacteria. These associations (referred to as consortia) are so closely integrated that many were described as formal bacterial species before their composite nature was realised, and are commonly still referred to by their old 'species' names for convenience (especially as none of the bacteria involved can yet be cultured independently). In the majority of consortia, referred to as 'Chlorochromatium' and 'Pelochromatium', the non-motile green sulphur bacteria form a layer around the surface of a larger flagellated, non-photosynthetic bacterium. The motile bacterium is able to swim towards sulphide concentrations that are used for energy by the green sulphur bacteria. The oxidised sulphur or sulphate produced by the sulphur bacteria is then believed to be used by the non-photosynthetic partner for its own metabolic purposes. A slightly different type of consortium, referred to as "Chloroplana vacuolata", grows as non-motile films made up of alternating rows of the green sulphur bacteria and their colourless partners, with the one converting sulphides to sulphur or sulphates and the other converting them back again.

Short and long individuals of the non-green, not-always-sulphur bacterium Ignavibacterium album, from here.

In 2010, a group of researchers described Ignavibacterium album, currently the closest known non-photosynthetic relative of the green sulphur bacteria, from a sulphide-rich hot spring in Japan (Liu, Frigaard et al. 2012). Unlike the green sulphur bacteria, Ignavibacterium is only a facultative anaerobe, being also capable of growing in the presence of oxygen. It uses a number of electron donors including sulphide (though not elemental sulphur) and also has limited abilities to fix carbon dioxide. However, it cannot use carbon dioxide as its only carbon source in the way that the green sulphur bacteria can; as it lacks the ability to synthesise some vital amino acids, it still depends on being able to obtain those compounds from external sources. When first described, Ignavibacterium was believed to be non-motile; however, further study of its genome has identified complete versions of the genes used in flagella production. It is not unknown for motile bacteria to lose their flagella in the process of being cultured, and its seems likely that this happened to the original Ignavibacterium isolate.

A further link between Ignavibacterium and the green sulphur bacteria is provided by an organism that currently goes by the label 'Candidatus Thermochlorobacter aerophilum' (Liu, Klatt et al. 2012). As indicated by the term 'Candidatus', Thermochlorobacter has not been cultured in the laboratory. Instead, it is one of an ever-increasing number of bacterial taxa that have been identified from genetic samples extracted directly from the environment, in this case from hot springs in Yellowstone National Park. Even though these organisms have, in a sense, never been directly 'seen', we can still infer a great deal from their genomic data about what their characters are likely to be. We know that Thermochlorobacter is photosynthetic like the green sulphur bacteria, able to produce chlorosomes containing bacteriochlorophyll (probabably bacteriochlorophyll d) to obtain energy from sunlight. However, unlike the green sulphur bacteria, Thermochlorobacter lacks the ability to meet all its carbon needs by fixing carbon dioxide; like Ignavibacterium, it depends on external sources of nutrients. It is also aerobic rather than anaerobic, and lacks the ability to oxidise sulphides or sulphur. It does resemble the green sulphur bacteria in lacking the ability to produce flagella. Interestingly, however, it retains some genes that are associated in Ignavibacterium with movement towards nutrient sources; as these genes are also present in Chloroherpeton, I find myself wondering if Thermochlorobacter may be capable of gliding motility in the way that Chloroherpeton is.

REFERENCES

Garrity, G. M., & J. G. Holt. 2001. Phylum BXI. Chlorobi phy. nov. In: Boone, D. R., & R. W. Castenholz (eds) Bergey's Manual of Systematic Bacteriology 2nd ed. vol. 1. The Archaea and the Deeply Branching and Phototrophic Bacteria pp. 601–623. Springer.

Liu, Z., N.-U. Frigaard, K. Vogl, T. Iino, M. Ohkuma, J. Overmann & D. A. Bryant. 2012. Complete genome of Ignavibacterium album, a metabolically versatile, flagellated, facultative anaerobe from the phylum Chlorobi. Frontiers in Microbiology 3: 185. doi: 10.3389/fmicb.2012.00185.

Liu, Z., C. G. Klatt, M. Ludwig, D. B. Rusch, S. I. Jensen, M. Kühl, D. M. Ward & D. A. Bryant. 2012. ‘Candidatus Thermochlorobacter aerophilum:’ an aerobic chlorophotoheterotrophic member of the phylum Chlorobi defined by metagenomics and metatranscriptomics. ISME Journal 6: 1869–1882. doi:10.1038/ismej.2012.24.

Alpha Bacteria

Two budding individuals of Caulobacter crescentus, from the US Dept of Energy.

From about the 1980s onwards, the increasing application of molecular data (particularly the sequences of ribosomal RNA genes) to bacterial phylogeny meant that what had previously been an intractable mass of diversity began to emerge into some sort of order. One of the major new groups of bacteria to be recognised in this way was the Proteobacteria, a hyperdiverse array that includes many of those bacteria of direct significance to ourselves. Within this bacterial supergroup, the phylogeneticists also resolved five major subgroups that, in the absence of any more obvious markers, they labelled alphabetically: the alpha, beta, gamma, delta and epsilon Proteobacteria. Eventually these convenient labels would become formalised, and it is with the group known as the Alphaproteobacteria that I am concerned today.

Like the other proteobacterial lineages, the Alphaproteobacteria are diverse in features and habits. To the best of my knowledge, no uniting characteristic has yet been identified for members of this group other than their shared ribosomal heritage. Many of the Alphaproteobacteria are associated with anoxic habitats. Many are at least facultatively photosynthetic, obtaining energy from sunlight by means of bacteriochlorophyll a and/or carotenoids; these factors give such bacteria a purple coloration. Other Alphaproteobacteria are intracellular parasites of eukaryotes, including a number that are of medical significance to humans. Earlier posts on this site have covered particular subgroups of the Alphaproteobacteria: the nitrogen fixers and plant pathogens of the Rhizobiales, and the diverse order Rhodospirillales. Another group of Proteobacteria, the Epsilonproteobacteria, was the subject of another post.

Culture of vinegar bacteria Acetobacter aceti, copyright Эрг.

A recent study of Alphaproteobacteria ribosomal phylogeny by Ferla et al. (2013) recognised three major lineages within the group which they dubbed the Magnetococcidae, Rickettsidae and Caulobacteridae. The Caulobacteridae include the greater number of the named free-living Alphaproteobacteria: both of the orders covered in earlier posts, for instance, belong to this lineage. Detailed coverage of the various members of Caulobacteridae would fill a book, so I'll just mention some highlights. The earlier post on Rhodospirillales mentioned the family Acetobacteraceae, but one important detail I neglected to mention was that many members of this family obtain their energy by oxidising ethanol to acetic acid: these are the bacteria responsible for producing ethanol. Also potentially belonging to the Rhodospirillales is Sporospirillum, a candidate genus of enormous bacteria that have been found in the intestines of tadpoles. Individuals of Sporospirillum reach up to one-tenth of a millimetre in length, potentially large enough to be observed with a standard dissecting microscope, though they are only up to 5 µm in width. Because Sporospirillum have never been cultured or studied from a molecular perspective, their relationships remain uncertain: they may alternatively belong to the Spirillaceae, a family of the Betaproteobacteria (Brenner et al. 2005).

Also belonging to the Caulobacteridae are the Caulobacterales. As recognised by Ferla et al. (2013), this order contains two families, the Caulobacteraceae and Hyphomonadaceae. Many (but not all) of the members of these families have a distinctive life cycle, in which a previously motile individual loses its flagellum and grows an elongate stalk. This now-immotile individual then produces a motile offspring by budding at one end. The manner of budding differs between the two families: in the Caulobacteraceae, the stalk functions as an attachment to the substrate and the offspring buds from the unattached end of the cell, but in the Hyphomonadaceae the stalk is not an attachment organ and the offspring buds from the end of the stalk. Similar modes of growth and budding are found in other families of the Caulobacteridae, such as the Hyphomicrobiaceae in the Rhizobiales.

TEM view of Magnetococcus marinus, showing the line of magnetic particles (magnetosomes).

The other subclasses of the Alphaproteobacteria are smaller than the Caulobacteridae in terms of numbers of named species, but this may reflect our low appreciation of bacterial diversity more than environmental reality. The Magnetococcidae are represented by only a single named species, Magnetococcus marinus. This is an aquatic chemolithoautotroph, obtaining energy from sulphur compounds. Cells of Magnetococcus contain a row of magnetic particles that the bacterium uses to orient itself. Though only one species of magnetococcid has been named to date, environmental DNA samples indicate that many more await description (Bazylinski et al. 2013).

The ciliate Paramecium, infected with Holospora (in the swollen nucleus in the lower part of the photo). Photo from here, by this network's own Psi Wavefunction (wherever she may be...)

The named members of the Rickettsidae are mostly placed in the order Rickettsiales, an assemblage of intracellular parasites of eukaryotes. This order contains two families, the Rickettsiaceae and Anaplasmataceae; a third family, the Holosporaceae, that contains intracellular endosymbionts of large protozoans such as Paramecium and Acanthamoeba, was found by Ferla et al. (2013) to be potentially closer to the Caulobacteridae than the Rickettsidae. Members of the Rickettsiales of significance to humans include those causing such diseases as typhus or spotted fever. The Anaplasmataceae also includes the genus Wolbachia which has come under the spotlight in recent years for the significance that its effects on reproductive compatibility may have for the evolution of insects.

The only free-living bacterium associated with the Rickettsidae to date is the marine Pelagibacter ubique but, again, environmental DNA samples suggest that this is merely a representative of a larger undescribed group, commonly referred to as the 'SAR11' clade. Indeed, Pelagibacter and its relatives may be the most numerous organisms on the entire planet, making up about a third of the planktonic cells in the surface layers of the world's oceans (Morris et al. 2002). Even by bacterial standards, Pelagibacter cells are small, and it has one of the smallest known genomes for any free-living organisms.

Stained sample of Pelagibacter ubique, copyright Thomas Lankiewicz & Matthew Cottrell.

There is one final important subgroup of the alphaproteobacterial lineage that I haven't mentioned yet: us. At some point in the distant past, a member of the Alphaproteobacteria developed a close and personal relationship with another micro-organism, either a member of the Archaea or a close relative thereof. Phylogenetic studies indicate that this early alphaproteobacterium was probably a close relative of the Rickettsiales. Over time, this relationship became ever closer, until the one became inseparable from the other. Together, these two microbes were to give rise to the eukaryotes, with the alphaproteobacteria becoming transformed into the mitochondria of a eukaryote cell. From the perspective of descent, then, we are all Alphaproteobacteria.

REFERENCES

Bazylinski, D. A., T. J. Williams, C. T. Lefèvre, R. J. Berg, C. L. Zhang, S. S. Bowser, A. J. Dean & T. J. Beveridge. 2013. 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. International Journal of Systematic and Evolutionary Microbiology 63: 801–808.

Brenner, D. J., N. R. Krieg & J. T. Staley. 2005. Bergey's Manual of Systematic Bacteriology 2nd ed. vol. 2 pt C. The Alpha-, Beta-, Delta-, and Epsilonproteobacteria. Springer.

Ferla, M. P., J. C. Thrash, S. J. Giovannoni & W. M. Patrick. 2013. New rRNA gene-based phylogenies of the Alphaproteobacteria provide perspective on major groups, mitochondrial ancestry and phylogenetic instability. PLoS One 8 (12): e83383. doi:10.1371/journal.pone.0083383.

Morris, R. M., M. S. Rappé, S. A. Connon, K. L. Vergin, W. A. Siebold, C. A. Carlson & S. J. Giovannoni. 2002. SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420: 806–810.

Friend and/or Foe: Separating Rhizobium and Agrobacterium

Roots of cowpea Vigna unguiculata with nodules containing Rhizobium, copyright Dave Whitinger.

For as far back as we have records to know, farmers have recognised the value of crop rotation: varying the crops grown on a particular patch of land in order to avoid exhausting the soil of nutrients. It also did not take these ancient farmers long to realise the value of one of these rotated crops being a legume, the group of plants including peas, beans, lentils and the like. What these pioneers of agriculture did not know was that the rejuvenating effect that legumes seemed to have on the soil was due to bacteria living in their roots, the organisms that we now know as Rhizobium.

The value of Rhizobium to agriculture comes from its ability to fix nitrogen. Nitrogen compounds are essential for all living organisms (proteins, for instance, contain nitrogen). But while nitrogen is also the most abundant element in our planet's atmosphere, most of it exists in a form that cannot be used directly by most organisms. Nitrogen-fixing bacteria are the exception, able to extract the nitrogen directly from the atmosphere and 'fix' it into more tractable compounds. Rhizobium is not the only genus of bacteria able to fix nitrogen, but it is certainly one of the most predominant. One of the limitations of nitrogen fixation is that it generally involves enzymes that do not work well in the presence of oxygen. Rhizobium cells induce the growth of nodules on legume roots, within which they are sheltered from that polluting gas. Not all Rhizobium live in legumes, however: they may also be found in large numbers free in the soil, with concentrations of tens of millions of cells per gram of soil having been recorded (Kuykendall et al. 2005).

Crown gall caused by Agrobacterium tumefaciens, copyright Christoph Müller.

Rhizobium has been regarded as closely related to another bacterial genus called Agrobacterium, whose significance for agriculture has been seen somewhat less favourably. As classically distinguished, Agrobacterium species do not fix nitrogen like Rhizobium, but they do resemble Rhizobium in causing growths on plant roots. These might be tumours, as in Agrobacterium tumefaciens (which can also cause galls to form elsewhere on the plant), or an overabundance of small rootlets, as in A. rhizogenes. While not necessarily fatal to the host plant, these deformities do often stunt growth, causing a loss in yield. A third species, A. radiobacter, has been recognised for non-pathogenic Agrobacterium.

However, as microbiologists gained a better understanding of the underlying genetics of Rhizobium and Agrobacterium, the picture became more complicated. The ability of Rhizobium to fix nitrogen, and of Agrobacterium to cause root deformities, is due to particular sets of genes in each. These genes are not contained in the main chromosome of each bacterium, but are held in little 'mini-chromosomes' called plasmids. And plasmids can be readily transferred from one bacterial cell to another. Through the transfer of the right plasmids, an Agrobacterium might gain the ability to fix nitrogen, or a Rhizobium might start inducing tumours. Phylogenetic analyses of the genera also indicated that some 'Rhizobium' were more closely related to 'Agrobacterium', and vice versa. As a result, at least one group of researchers has proposed uniting the two genera into one, Rhizobium. But others have been loathe to abandon a name as long-used in both the microbiological and agricultural literature as Agrobacterium (Farrand et al. 2003). There are recognisably distinct phylogenetic lines within the family Rhizobiaceae that includes the two genera, and even differences in plasmids are not entirely uninformative: not all strains can utilise all plasmids.

Cells of Rhizobium trifolii on root hair of clover, copyright Frank Dazzo.

There are also some significant genomic differences involved. Some of you may have learnt in biology class that the normal arrangement for bacterial cells is to have the bulk of the genome contained in a single circular chromosome, possibly with a scattering of small plasmids. The difference between the two is that the cell can function without the plasmids, but not without the chromosome. Rhizobium leguminosarum, the type species of Rhizobium, keeps to this arrangement, as do most other Rhizobium (though the plasmid containing the nitrogen-fixation genes is a bit of a whopper by usual standards). However, Agrobacterium tumefaciens*, the type species of its genus, is more unusual in having not one but two chromosomes: some of its vital genes have been transferred to what was originally a large plasmid (Slater et al. 2009). What is more, this second chromosome is not formed as a circle like other bacterial chromosomes, but is linear like the chromosomes of eukaryotes. Another 'Agrobacterium' species, A. vitis, has the second chromosome like A. tumefaciens but it remains circular. The type strain of A. rhizogenes, on the other hand, has only a single circular chromosome, and appears to be closer to Rhizobium.

*The type strains of 'Agrobacterium tumefaciens' and 'A. radiobacter' are close enough that the two names should be synonymised into a single species. However, there seems to be an on-going dispute over which of the two names should be used for the combined taxon. I'm using A. tumefaciens for convenience, but I wouldn't be able to judge which of the sides is correct.

Phylogenetic analysis also supports the recognition of a further genus of Rhizobiaceae, Ensifer, sitting outside the clade including Rhizobium and Agrobacterium. The type species of Ensifer, E. adhaerens, is a soil-dwelling bacterium that can live as a predator of other bacteria. It attaches to them end-wise (when multiple E. adhaerens attach to a single target cell, they may form a palisade) and causes them to burst open. Ensifer adhaerens is not an obligate predator—when suitable prey is not available, it can survive on free nutrients in the soil—and when analysed it turns out to be related to a clade of nitrogen-fixing bacteria previously recognised as the genus 'Sinorhizobium'. Indeed, one species of this genus turned out to be simply a non-parasitic form of E. adhaerens (Young 2003).

REFERENCES

Farrand, S. K., P. B. van Berkum & P. Oger. 2003. Agrobacterium is a definable genus of the family Rhizobiaceae. International Journal of Systematic and Evolutionary Microbiology 53 (5): 1681-1687.

Kuykendall, L. D., J. M. Young, E. Martínez-Romero & H. Sawada. 2005. Genus I. Rhizobium Frank 1889, 337^AL. In: Garrity, G., D. J. Brenner, N. R. Krieg & J. T. Staley (eds) Bergey's Manual of Systematic Bacteriology vol. 2. The Proteobacteria, Part C. Springer.

Slater, S. C., B. S. Goldman, B. Goodner, J. C. Setubal, S. K. Farrand, E. W. Nester, T. J. Burr, L. Banta, A. W. Dickerman, I. Paulsen, L. Otten, G. Suen, R. Welch, N. F. Almeida, F. Arnold, O. T. Burton, Z. Du, A. Ewing, E. Godsy, S. Heisel, K. L. Houmiel, J. Jhaveri, J. Lu, N. M. Miller, S. Norton, Q. Chen, W. Phoolcharoen, V. Ohlin, D. Ondrusek, N. Pride, S. L. Stricklin, J. Sun, C. Wheeler, L. Wilson, H. Zhu & D. W. Wood. 2009. Genome sequences of three Agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria. Journal of Bacteriology 191 (8): 2501-2511.

Young, J. M. 2003. The genus name Ensifer Casida 1982 takes priority over Sinorhizobium Chen et al. 1988, and Sinorhizobium morelense Wang et al. 2002 is a later synonym of Ensifer adhaerens Casida 1982. Is the combination ‘Sinorhizobium adhaerens’ (Casida 1982) Willems et al. 2003 legitimate? Request for an Opinion. International Journal of Systematic and Evolutionary Microbiology 53 (6): 2107-2110.