10 | JANUARY 2004 | VOLUME 2 www.nature.com/reviews/micro

Humpty Dumpty had a famous problem — hesat on a wall, had a great fall and…couldn’t beput back together again. Genome biologistshave had a similar problem until now — theycould break genomes up and sequence them butnot synthesize them de novo, or at least not inany efficient way. A group led by Craig Venterhas now devised a method for assembling thecomplete genome of a bacteriophage from apool of chemically synthesized oligonucleotides.Although still in need of a correcting mecha-nism, the method might in future be used tomake synthetic chromosomes.

Driven by the desire to improve on previousattempts to assemble active viral genomes, Smithet al. chose the φX174 bacteriophage — the firstliving organism to have its 5,386 bp genome

sequenced — to test their three-step approach tosynthetic genome assembly. The first stepinvolves oligonucleotide synthesis, based on bothstrands of the phage genome. In the second step,the oligonucleotides are phosphorylated, mixedand ligated to give, on average, 700-bp double-stranded fragments. Two rounds of polymerasecycle assembly are used in the third step toassemble these fragments into full-lengthgenome molecules, which are PCR-amplified,linearized and tested for size by electrophoresis.

As the ultimate test of successful in vitro syn-thesis is that of molecular function, recircular-ized synthetic phages were electroporated intoEscherichia coli to test for their infectivity.Careful comparison of infection efficiencybetween the wild-type and the synthetic phagesindicated that some 9–10 inactivating muta-tions were present per synthetic genome,although at least one synthetic isolate was 100%identical to the original φX174 sequence. Smithet al. suggest that these point mutations proba-bly result either from replication errors or arealready present in the oligonucleotides.

Given the mutation rate, the authors pointout that without an ability to select for the wild-type sequence, synthetic forms will carry, on

average, a point mutation per 2 kb. Undeterredby this, they propose that, in combination withDNA sequencing and site-directed repair, theirmethod can be used to assemble larger genomes.

Synthetic genomics could solve the problemsof dealing with degraded biological material orextinct species. Genome sequences could alsobe modified during synthesis to provide analternative to genetic engineering. Althoughthis application of synthetic genomics might bearound the corner for microbial genomes,much larger, metazoan genomes might remainin the ‘Humpty Dumpty’ class for a while yet.

Magdalena SkipperEditor, Nature Reviews Genetics

References and linksORIGINAL RESEARCH PAPER Smith, H. O. et al. Generating asynthetic genome by whole genome assembly: phiX174bacteriophage from synthetic oligonucleotides. Proc. Natl Acad.Sci. USA (2003) doi: 10.1073/pnas.2237126100

Phagocytes invaded by microorganismsfight back with the innate immune systemand produce potent broad-spectrumantimicrobial molecules that can inhibit thegrowth of intracellular pathogens. Theseinclude reactive nitrogen species (RNS),such as nitric oxide (NO) and S-nitroso-glutathione (GSNO). Unsurprisingly,pathogenic microorganisms have evolvedstrategies to overcome innate host defences— but until now antimicrobial defenceagainst reactive nitrogen species has notbeen linked to microbial virulence.Reporting in the latest issue of CurrentBiology, Joseph Heitman and colleagues atDuke University have established thatfungal enzymes that counteract NO do havea role in virulence.

Cryptococcus neoformans is a fungalpathogen that can cause life-threateninginfections of the central nervous system inimmunocompromised patients. Followingpulmonary infection, patrolling alveolarmacrophages are a first line of defenceagainst cryptococcosis. NO — generated by

NO synthase, the product of the iNOS locus— exerts a fungistatic effect against C. neoformans in vitro and it is known that C. neoformans detoxifies NO activity inmacrophages. Enzymes that are active againstNO include flavohaemoglobin denitrosylase— found in yeasts and bacteria — whichconverts NO to nitrate, and GSNO reductase,which is conserved from bacteria to man andreduces GSNO to ammonia and glutathionedisulphide. The C. neoformans flavohaemo-globin denitrosylase (FHB1) and GSNOreductase (GNO1) genes were identified bymining the TIGR and Stanford genomicsequences for C. neoformans with genesequences from Saccharomyces cerevisiae.

By analysing single and double genedisruption mutants, de Jesús-Berríos et al.showed that C. neoformans converts GSNOto NO (by the action of Gno1), which issubsequently metabolized by flavohaemo-globin denitrosylase (by the action of Fhb1).Using a mouse model for C. neoformans,which mimics human disease progression,they established that flavohaemoglobin

denitrosylase activity contributes tovirulence. By itself, GSNO reductase doesnot; however, mutants that lacked bothenzymes were more attenuated for virulencethan either single mutant, so GSNO reduc-tase can contribute to counteract nitrosativechallenge and promote disease progression.

The use of a mutant mouse that is unableto produce NO because it lacks the genesencoding the inducible form of NOsynthase, restored the virulence of thefungal mutant lacking Fhb1. This validatesthe link between host defence (NO produc-tion) and microbial counterattack (flavo-haemoglobin) with consequences forvirulence. Combining mutations inflavohaemoglobin and superoxide dismutase(which defends against oxidative attack byphagocytes) attenuated virulence further.Plants and animals both produce NO todefend against pathogens, and it seems thatthe flavohaemoglobin family might havebeen conserved because these enzymes havea general role in microorganism retorts tothe defences mounted by their hosts.

Susan JonesReferences and links

ORIGINAL RESEARCH PAPER de Jesús-Berríos, M. et al.Enzymes that counteract nitrosative stress promote virulence.Curr. Biol. 13, 1963–1968 (2003) WEB SITEJoseph Heitman’s laboratory:http://mgm.duke.edu/microbial/mycology/heitmn/

Fungal defence? NO stress!

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