cyanobacteria not the dominant N2-fixing

organism in the tropical oceans, and why areTrichodesmium spp. not able to thrive in tem-perate and polar regions, and in freshwater andbrackish environments?

Using an acetylene reduction assay, theauthors compared nitrogenase activity in theheterocystous Nodularia spumigenia strainCCY 9414 and Anabaena sp. strain CCY 9901with the nitrogenase activity in Trichodesmiumsp. strain IMS101, and showed that N

2fixation

in the dark in both heterocysts and diazocytes(the specialized N

2-fixing cells of

Trichodesmium which do not have a glycolipidlayer) is limited by O

2diffusion. In the dark,

respiratory ATP production limits N2

fixationdue to a limited influx of O

2to the heterocyst

or diazocyte. However, in the light, ATP pro-duction by the photosynthetic electron trans-port chain ensures that ATP availability is not alimiting factor.

The authors also examined the effect ofincreased temperature on the gas flux andenzyme potential in the N

2-fixing cells.

Although the concentration of dissolved O2

decreases with increased temperatures, the gasdiffusion coefficient increases — resulting in anet increase in the gas flux into the N

2-fixing

cells. However, the enzyme potential increases

As nitrogen availability is often the limitingfactor to growth and biomass production,nitrogen fixation is one of the most importantbiological processes on Earth. Now, newresearch has revealed the reasons behind thedifferences in the global distribution of hetero-cystous and non-heterocystous nitrogen-fixingcyanobacteria.

Heterocystous cyanobacteria are believedto be better adapted for N

2fixation than non-

heterocystous cyanobacteria — because theheterocysts (differentiated cells enveloped by aglycolipid layer) are thought to protect nitro-genase from inhibition by molecular oxygen.Despite this however, the dominant N

2-fixing

cyanobacteria in the tropical oceans are thenon-heterocystous Trichodesmium spp.Indeed, there are significant differences in theglobal distribution of hetercystous and non-heterocystous cyanobacteria — heterocystouscyanobacteria are the dominant species infreshwater lakes and brackish environments(such as the Baltic Sea), but Trichodesmiumspp. are dominant in the tropical oceans, yetunsuccessful in temperate or polar oceans.

Now, reporting in Nature, Staal et al. haveprovided answers to two important questionsregarding these differences in distribution;namely, why are free-living heterocystous

172 | DECEMBER 2003 | VOLUME 1 www.nature.com/reviews/micro

H I G H L I G H T S

The single flagellum of Trypanosoma brucei has been unveiled as a key player indetermining cell polarity, division, size and shape.

The flagellum, an appendage whosestructure is conserved from protists tomammals, extends from the cell body. It isassembled by adding subunits to the distaltip. However, the flagellum lacks themachinery for protein synthesis and atransport system for subunits synthesizedin the cell body is needed. A specializedsystem, known as intraflagellar transport(IFT), delivers ‘rafts’ of proteins that aresynthesised in the cell body to the flagellatip, and can also transport proteins fromthe flagellum back to the cell body. Motorproteins drive movement of rafts, whichcontain additional IFT proteins known ascargoes. Defects in IFT prevent flagellumformation in most eukaryotes tested so far.In new research published in the EMBOJournal RNAi and cell biology were

combined to probe the function of thetrypanosome flagellum.

Kohl et al. mined T. brucei genomesequences for putative IFT homologues.From several homologues identified, twogenes were selected. One gene encodes aputative cargo protein whilst the other geneencodes a putative motor protein. Bycoupling gene silencing using RNAi withmicroscopy, Kohl et al. showed that separatesilencing of either the putative motor orcargo homologue resulted in progressiveflagellum shortening. A shortenedflagellum is initially produced becauseRNAi results in a dwindling pool of IFTproteins, which in turn reduces transport ofthe proteins needed for flagellum assembly.Eventually daughter cells had no flagellum.So, IFT is functional in T. brucei and isrequired for flagellum assembly and forcontrol of flagellum length. Mutant cellslacking a flagellum lost polarity, asdemonstrated by mislocalization of

clathrin, a typical cell organzation marker.These mutants also lost their distinctiveshape and had no clear anterior or posteriorend. So, both cell polarity and internalorganization were lost when flagellaassembly was disrupted.

During induction of RNAi, mutants notonly produced a shortened flagellum but

Trypanosome flagellum — motility and more

P R OTO Z O A N PA R A S I T E S

Some like it hot

E N V I R O N M E N TA L M I C R O B I O LO G Y

Image shows a mixture of trypanosomes with full-length,shortened or no flagellum (stained green), basal body (stainedred) and DNA (stained blue) viewed by differential interferencecontrast microscopy. Courtesy of Linda Kohl and PhilippeBastin (Laboratoire de Biophysique, Muséum Nationald’Histoire Naturelle, Paris, France).

NATURE REVIEWS | MICROBIOLOGY VOLUME 1 | DECEMBER 2003 | 173

Five distinct secretion pathways are used totransport proteins across the Gram-negativebacterial double membrane. This is in additionto three transport routes across the innermembrane. Even so, Escherichia coli, the best-studied bacterium, can still spring a surprise onmicrobiologists. Reporting in Cell, Wai et al.describe a new mechanism of secretion inwhich a toxin is exported in vesicles. Vesicle orbleb formation is nothing new — but this is thefirst time vesicle-mediated secretion has beengiven clear physiological relevance.

Common laboratory strains of E. coli canproduce ClyA, a cytotoxin that is secreted intothe bacterial periplasm but acts on mammaliancells. How does the toxin access its target site?Using immunofluorescence, Wai et al. showedthat ClyA is surface-exposed. Closer examinationwith electron microscopy and atomic forcemicroscopy revealed a multitude of smallouter-membrane vesicles (OMVs) approxi-mately 50–200 nM in diameter surroundingthe bacteria. Analysing the protein content ofthese vesicles proved that they were derivedfrom the outer membrane. Strains of E. colithat do not produce ClyA still producedOMVs. Reassuringly, pathogenic E. coli andSalmonella enterica serovar Typhi strains thatproduce ClyA also localized the toxin intoOMVs, so this process isn’t restricted to labo-ratory-adapted strains. The OMVs typicallyhad ring-like pores, which were shown to beassemblies of ClyA protein.

Toxicity assays showed that ClyA present inOMVs was at least 8 times more toxic than

purified ClyA. This striking result promptedthe authors to examine ClyA multimerization,and cross-linking experiments confirmed thatperiplasmic ClyA is monomeric comparedwith multimeric ClyA present in OMVs.Further, removing the disulphide-bond oxida-tion machinery from E.coli dramaticallyincreased the action of ClyA. The authorscouldn’t detect proteins that participate indisulphide-bond formation in OMVs con-taining ClyA, raising the possibility that theredox status of ClyA contributes to oligomer-ization and toxicity. OMVs seem to contain asubset of periplasmic proteins, which meansthat E.coli could have a crude protein-sortingmechanism that selects which proteins areexported in OMVs.

OMVs are formed by a whole variety of bac-terial species, including commensal Bacteroidesspecies and pathogenic Neisseria species. Ifbacteria can sort periplasmic proteins intovesicles, the challenge will be to find out howsorting is accomplished, how it is regulatedand whether this process can be used as amodel for eukaryotic vesicle trafficking.

Susan Jones

References and linksORIGINAL RESEARCH PAPER Wai et al. Vesicle-mediatedexport and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell 115, 25–35 (2003) FURTHER READING Miller, S. I. et al. Bacterial vesicleformation as a mechanism of protein transfer to animals. Cell115, 2–3 (2003)WEB SITEBernt-Eric Uhlin’s laboratory:http://www.molbiol.umu.se/forskning/uhlin/

Bacterial secretion into vesicles

B A C T E R I A L P H Y S I O LO G Y

faster than the gas flux, so with incerasingtemperatures, nitrogenase activity becomessubstrate limited, particularly in thosecyanobacteria with an efficient diffusion barrier— heterocystous cyanobacteria. In addition,by modelling the influx of O

2to both hetero-

cysts and diazocytes, Staal et al. calculated thatO

2flux increases by approximately 25% when

the salinity is decreased from 35 (sea water) to0 (fresh water) — thereby, highlighting theneed for an efficient diffusion barrier, such asthe glycolipid envelope of the heterocyst, infreshwater conditions.

So, in freshwater conditions, the glycol-ipid envelope of the heterocyst provides aselective advantage over non-heterocystouscyanobacteria — explaining the absence ofTrichodesmium from freshwater seas. But, inpelagic environments at high temperatures, theglycolipid envelope is a disadvantage —explaining the dominance of Trichodesmiumin the tropical oceans.

Jane SaundersReferences and links

ORIGINAL RESEARCH PAPER Staal, M., Meysman, F. J. R. &Stal, L. J. Temperature excludes N2-fixing heterocystouscyanobacteria in the tropical oceans. Nature 425, 504–507(2003)WEB SITEThe Netherlands Institute of Ecology:http://www.nioo.knaw.nl/CEME/MM/index.htm

were smaller than wild-type cells. Thelength of the flagellum correlated with cellsize — the shorter the flagellum, the smaller the daughter cell. Before celldivision, a new flagellum is assembledalongside the old flagellum. By monitoringmutants during induction of RNAi theauthors deduced that the tip of the newlysynthesised flagellum defines the site ofcytokinesis. Alongside the flagellum in thecytoplasm there is a flagellar attachmentzone (FAZ) which also replicates before celldivision. Mutant trypanosomes whichcompletely lack flagella still have a FAZ but its shape is shortened and irregular.Since these cells can still divide, the FAZ isthe best candidate for a marker ofcytokinesis.

During the life cycle of T. brucei, there areseveral morphologically distinct cell-types.It’s possible that regulating IFT — andtherefore regulating flagellum length —could be used to modulate parasitedifferentiation.

Susan Jones

References and linksORIGINAL RESEARCH PAPER Kohl et al. Novel roles for the flagellum in cell morphogenesis andcytokinesis of trypanosomes. EMBO J. 22, 5336–5345 (2003)