Following hard on the heels of the human genome project, microbial genome versions have now begun to produce vast

amounts of information on the nucleotide sequences of specific microbes. How useful is this information and how can researchers wade through the millions of base pairs of sequence data to find genes or sequences of interest for either diagnostic or therapeutic strategies? In theory, the answer lies with the new specialty of bioinformatics, which covers genomics, proteomics and metabolomics - terms that are more recognisable to many as molecular genetics and

biochemistry

Greenwell, P.

Rughooputh, S.

Ernstorfer, R.

MPG.PuRe

Accessing Microscopic Coupling in Solids with Momentum-Resolving Ultrafast Techniques

WestminsterResearch

     University of Westminster Eprints http://eprints.wmin.ac.uk   Bioinformatics: a useful tool for the molecular microbiologist?  Pamela Greenwell Sanjiv Rughooputh School of Biosciences    This is an electronic version of an article published in The Biomedical Scientist, 48(5), pp. 494-495, May 2004.      The Eprints service at the University of Westminster aims to make the research output of the University available to a wider audience.  Copyright and Moral Rights remain with the authors and/or copyright owners. Users are permitted to download and/or print one copy for non-commercial private study or research.  Further distribution and any use of material from within this archive for profit-making enterprises or for commercial gain is strictly forbidden.        Whilst further distribution of specific materials from within this archive is forbidden, you may freely distribute the URL of the University of Westminster Eprints (http://eprints.wmin.ac.uk).   In case of abuse or copyright appearing without permission e-mail wattsn@wmin.ac.uk.  Following hard on the heels of the humangenome project, microbial genomeversions have now begun to produce vastamounts of information on the nucleotidesequences of specific microbes. Howuseful is this information and how canresearchers wade through the millions ofbase pairs of sequence data to find genesor sequences of interest for eitherdiagnostic or therapeutic strategies? Intheory, the answer lies with the newspecialty of bioinformatics, which coversgenomics, proteomics and metabolomics– terms that are more recognisable tomany as molecular genetics andbiochemistry. Many scientists have the impression thatonce the genome of an organism is sequencedthen everything about it is known. But is thisreally true? At this stage, it might be useful tolook at an organism whose genome has beensequenced completely. Take Chlamydiatrachomatis serovar D, for example.Chlamydia trachomatisThe C. trachomatis serovar D genome wassequenced in 1998 and found to be 1.045Mb in length. Full details of the genespresent, the proteins they encode and anyreferences can be obtained by logging on tothe TIGR website (www.TIGR.org) and thenclicking on the ‘Comprehensive microbialresource’ and then the ‘Visit a CMR page foran individual genome’ icons. This will revealthe names of all the organisms cloned. In thisexample, C. trachomatis serovar D would bethe destination, where everything that isknown about the genome of the organismcan be explored. So, if everything is known about C. trachomatis serovar D, why is work stillbeing undertaken on it? The truth is thateverything is known about the organism’snucleotide sequence and researchers canpredict that it codes for 877 proteins, 103 ofwhich are secreted as judged by the presenceof signal peptides and 241 havetransmembrane domains and thereforeappear to be membrane bound. However, thefunction of more than 20% of the proteinsremains a mystery.One way to identify genes is by theirhomology with other cloned genes, on theassumption that bacteria must have evolvedfrom each other and therefore will sharegenes, albeit with some changes that haveresulted from evolution. To observe suchhomology, an alignment search facility suchas BLAST (basic local alignment search tool)can be used, which permits comparison ofnucleotides or derived proteins from C. trachomatis serovar D with all other genesor proteins lodged in databases worldwide orwith subsets (eg prokaryotic genes andproteins) of that data. Bioinformatics: a useful tool for the molecularmicrobiologist?National Cancer Institute/Science Photo LibraryThe appearance of Chlamydia trachomatis infection in a cervical smear. The C. trachomatis serovar D genome was sequenced in 1998. ‘One way to identify genes is by theirhomology with other cloned genes’ARTICLEVast amounts of data are now available on individual microbial speciesthrough the application of molecular techniques such as genomics, proteomicsand metabolomics. Here, Pamela Greenwell and Sanjiv Rughooputh considerthe value of the information obtained and how it can be understood.494 THE BIOMEDICAL SCIENTIST   May 2004 495THE BIOMEDICAL SCIENTIST   May 2004The results show areas of homology asalignments and give the statistical probabilitythat the homology is significant and not dueto chance. However, work tends to be donewith the translated amino acids that arederived from the nucleotide sequencesbecause different organisms do have differentcodon preferences that can make nucleotidecomparisons difficult. Of course, as the databases contain billionsof sequences it might be expected that mostproteins could be identified by theirhomology to other known proteins. This,however, is not the case, as has been seenwith C. trachomatis serovar D. Is it anunusual microbe? No, Trichomonas vaginalishas also been studied and this has shown thatmost of its cloned genes encode proteinswith little homology to other cloned genes.Widening the focusSo, does this mean that bioinformatics is notuseful? Although it is not possible to identifyall genes by this method, some proteins canbe identified and then other useful data canbe derived from them. For example, whenproteins encoded by C. trachomatis serovar Dgenes were compared to those of Escherichiacoli and Bacillus subtilis, 195 showed betterhomology with E. coli proteins, whereas 259showed greater homology with those of B. subtilis. This implies that C. trachomatis serovar Dhas characteristics of both Gram-positive andGram-negative organisms. When C.trachomatis serovar D proteins werecompared to those of other sexuallytransmitted organisms (eg Treponemapallidum and Mycoplasma genitalium), 68 of the encoded C. trachomatis serovar Dproteins showed homology with M.genitalium proteins and 286 showedhomology with T. pallidum proteins. However, the majority of C. trachomatisserovar D proteins show no similarity withthose produced by either of the organisms.Nonetheless, the proteins that are conservedbetween these organisms may be of greatinterest as targets for therapeutics strategiesagainst more than one organismsimultaneously. Genomic comparisonOne of the most useful tools for themolecular microbiologist is whole genomecomparison, using traditional tools such asBLAST. To achieve the best visualisation,however, Artemis(www.sanger.ac.uk/Software/Artemis/) is the tool of choice, with ACT(www.sanger.ac.uk/Software/ACT/) required for whole genome comparison. ACT permits direct comparison of twogenomes and, in the case of mycobacteria,has shown that Mycobacterium leprae and M. tuberculosis originally had similargenomes, but that M. leprae went on to losegenes, which resulted in its current smallersize. However, small fragments of these lostgenes are still visible using ACT. In other organisms, comparisons ofpathogenic and non-pathogenic bacteria havehighlighted the presence of pathogenicityislands, which can be investigated aspotential therapeutics targets. Similarly, theevolution of antibiotic resistance can bestudied and areas of the microbial genomesuitable as diagnostic targets can bedetermined. In addition, nucleotide information permitsthe identification of specific areas of thegenome and tools are available (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) for designingprimers for polymerase chain reaction (PCR)-based detection.Leading questionSo, is bioinformatics useful? Yes, but itcannot tell the whole story. It is simply atool, albeit a very powerful one, to aid theunderstanding of microbial genomes. In conclusion, it is interesting to ponderthe value of the human genome project inlight of the limitations found with microbialanalysis. Clearly, the take-home messagemust be, ‘you can know everything about thenucleotides in the genome, but that does notmean that you know everything about theorganism’. nDr Pamela Greenwell and Dr SanjivRughooputh are members of the Molecularand Medical Microbiology Research Groupat the School of Biosciences, University ofWestminster, London. Comparisons ofpathogenic and non-pathogenic bacteriahave highlighted thepresence ofpathogenicity islands’