Episode 84: Bioinformatics in the noughties with Mark Pallen

Mark Pallen on microbial bioinformatics in the noughties

Professor Mark Pallen joins Lee Katz, Andrew Page and Nabil-Fareed Alikhan to revisit microbial bioinformatics around the turn of the millennium. He describes sequencing Tropheryma whipplei, using PSI-BLAST to discover protein families across newly available genomes, and finding unexpected complexity in secretion systems. These experiences challenged assumptions about model organisms and showed the value of combining comparative genomics, evolutionary thinking and laboratory experiments.

Listen to Episode 84: Bioinformatics in the noughties with Mark Pallen on SoundCloud

In this episode

First genomes and the race to sequence Tropheryma whipplei

Pallen recalls the excitement of seeing complete microbial genomes for the first time. Even for E. coli, studied for roughly a century, he says half its genes had remained undiscovered until genome sequencing. Alongside work on Campylobacter, he became involved in sequencing Tropheryma whipplei, the unusual, slow-growing organism responsible for Whipple’s disease.

The project brought him together with David Relman, whom he regarded as a scientific hero. Relman had established the organism’s distinct identity using a 16S sequence. A French group had managed to grow it in association with human cells, but a proposed collaboration did not proceed. Pallen recalls that the team working with Relman needed 18 months to grow sufficient biomass for sequencing—longer than the sequencing itself. The UK-led project finished ahead of a competing French effort.

PSI-BLAST reveals more than one sortase story

After moving to Belfast in late 1999, Pallen approached Tim Foster in Dublin about cross-border research funding. Foster wanted to identify the full set of Staphylococcus aureus proteins targeted to the cell surface by sortase. These substrates carried an LPXTG motif. Pallen used PSI-BLAST, whose iterative searches build a model from homologues and use it to find more distant matches. He remembers the repeated discovery of unexpected matches as intensely addictive.

The work identified roughly half a dozen or more new sortase substrates in S. aureus, followed by laboratory characterisation. Extending the searches across dozens of other genomes overturned the simple picture: substrate genes often clustered with sortase genes, and some organisms contained multiple sortases and substrate clusters. In some actinobacteria, sortases also helped assemble Gram-positive fimbriae rather than simply attach individual proteins to the surface. Pallen connects this experience to Alfred North Whitehead’s advice to seek simplicity while distrusting it.

ESAT-6, WXG100 and unanswered questions about function

A discussion about a tuberculosis T-cell test prompted another comparative search. Pallen asked what ESAT-6 did for the bacterium: being recognised as an antigen described its interaction with the host, not necessarily its biological function. Searches involving the protein family and an associated ATPase revealed a much wider distribution than the original Mycobacterium tuberculosis setting suggested.

The WXG100 proteins and type VII secretion systems appeared in contexts including S. aureus, with related proteins also found in some Gram-negative bacteria. Pallen stresses that these unexpected distributions raised questions that remained unresolved. When Lee asks about reverse vaccinology, Pallen places that approach alongside the opportunities created by early genomes, but notes that ESAT-6 was discovered before the genome was sequenced. Despite its use in the tuberculosis ELISpot test, its function was still not entirely clear twenty years later.

E. coli K-12 is another strain, not a perfect model

Work on E. coli exposed the limits of treating K-12 as an ideal representative of bacterial biology. Pallen describes ETT2, a type III secretion gene cluster that was widespread among the E. coli strains examined. In every case his group inspected, frameshifts and deletions suggested that it could not function as a type III secretion system.

K-12 also contained a small remnant of a second flagellar gene cluster: two incomplete genes lacking start codons and promoters. When the group sequenced enteroaggregative E. coli, they found a complete cluster, Flag-2, at that position; only a small number of strains retain this ancestral state. Pallen compares these remnants with Darwin’s vestigial organs. Extensive gene degeneration was already familiar from Mycobacterium leprae, but finding such remnants in a revered model organism initially felt surprising. His retrospective lesson is straightforward: K-12 is simply another E. coli strain.

Fresh isolates and an ecological–evolutionary perspective

Lee asks whether a more representative strain would have been preferable to K-12. Pallen explains that early sequencing projects often prioritised type strains and familiar laboratory strains. Long periods of subculture complicated their interpretation; he mentions Salmonella Typhimurium LT2 and notes irradiation in K-12’s ancestry. Gordon Dougan advocated sequencing fresh, minimally passaged isolates alongside laboratory strains to obtain a less laboratory-shaped view of bacterial genomes.

Pallen and Brendan Wren developed the broader argument in a Nature review, emphasising an ecological–evolutionary, or eco-evo, perspective. A genome needs to be interpreted through both its evolutionary ancestry and its ecological setting. Features associated with virulence also occurred in commensal and environmental organisms. Pallen points out that bacteria interact with eukaryotes throughout soils and oceans, particularly unicellular eukaryotes. Against that background, amoebae and invertebrates were not merely artificial substitutes for studying interactions with human hosts.

From predicted effectors to laboratory confirmation

Scott Beatson joined Pallen’s group after securing a Dorothy Hodgkin fellowship. Although Beatson had worked on Pseudomonas aeruginosa, Pallen persuaded him to investigate type III secretion in E. coli. The opportunity was to look beyond the assumption that the system’s effectors were all confined to one secretion-associated gene cluster.

Homology searches instead identified dozens of new candidate effectors elsewhere in the chromosome. Working with collaborators at Imperial and in Japan, the team showed experimentally that many of these proteins were secreted by the system. The work culminated in a PNAS paper. Pallen describes this as especially rewarding because the project connected bioinformatics predictions with laboratory confirmation, rather than stopping at a list of sequence similarities.

Highlights

00:00:47 — Nabil introduces Mark Pallen and the return to bioinformatics around the turn of the millennium.
00:01:17 — Pallen recalls the first microbial genomes and the Tropheryma whipplei sequencing project.
00:15:28 — Lee asks whether E. coli K-12 is representative enough to serve as a model.
00:15:47 — Pallen explains the limitations of long-cultured laboratory strains and the case for fresh isolates.
00:20:17 — Lee asks whether the ESAT-6 discussion connects with reverse vaccinology.
00:20:38 — Pallen discusses reverse vaccinology, the tuberculosis T-cell test and uncertainty about ESAT-6 function.

In their own words

one of the great things about a career in academic life is you do get to meet your heroes and sometimes you can get to work with your heroes as time goes on

— Mark Pallen, 00:01:17

But for me, that was one of the most rewarding things where we actually tied the bioinformatics predictions into laboratory confirmation in a very coherent way.

— Mark Pallen, 00:15:47

Who is talking

Mark Pallen (guest, University of East Anglia; Quadram Institute)
Lee Katz (host)
Andrew Page (host)
Nabil-Fareed Alikhan (host)

Also mentioned: David Relman, Tim Foster, Alfred North Whitehead, Gordon Dougan, Brendan Wren, Scott Beatson.

Tools and resources mentioned

PSI-BLAST, BLAST, ELISpot, reverse vaccinology.

Questions this episode answers

How did Mark Pallen use PSI-BLAST to study sortases?

He used iterative homology searches to identify sortases and their substrates, initially in Staphylococcus aureus and then across other newly available genomes. The broader searches revealed multiple sortases, clustered substrate genes and roles in assembling Gram-positive fimbriae.

Why does the episode question E. coli K-12 as a representative model?

Pallen describes vestigial flagellar genes in K-12 and explains how long laboratory histories can complicate interpretation of model strains. He argues for comparing laboratory strains with fresh, minimally passaged isolates rather than treating K-12 as an idealised E. coli genome.

Was the biological function of ESAT-6 understood?

Pallen says its function was still not entirely clear at the time of the episode. Its recognition by T cells made it useful in a tuberculosis test, but did not explain everything it did for the bacterium or the roles of related proteins in other organisms.

How were the newly predicted E. coli type III secretion effectors tested?

Pallen and Scott Beatson used homology searches to identify dozens of new candidates. Collaborators then demonstrated secretion of many candidates experimentally, connecting the computational predictions with laboratory evidence in work published in PNAS.

This page was written from a machine transcript of the episode and checked against it. Listen to the episode for the whole conversation.

Show notes

Mark Pallen explains how exciting it was to be in microbial bioinformatics around the turn of the millennium, as we gained genomes for the first time from model organisms and fearsome pathogens.

He recounts working with his hero David Relman on the genome sequencing of the strange slow-growing organism called Tropheryma whipplei in competition with a French team. Mark moved to Belfast in late 1999 collaborating with another Englishman working on the island of Ireland, Tim Foster in Dublin. Pallen describes the addictive exhilaration of using PSI-BLAST to find new sortases and sortase substrates across a range of new genomes—for him this was the bioinformatics equivalent of crack cocaine. He quotes the philosopher Alfred North Whitehead in saying that the goal of every scientist is to seek simplicity but distrust it. What Mark found was that in most organisms sortases were behaving quite differently from the rather simple scenario seen in Staphylococcus aureus. He made similar observations on the WXG100 proteins and type VII secretion, which he found in many new contexts quite different from the original context of ESAT-6 as an antigen in Mycobacterium tuberculosis. Mark makes clear that we still don’t really fully understand the role of ESAT-6 twenty years on. The focus of Pallen’s work then shifted to E. coli, where he described vestigial gene clusters for non-functional type III secretion systems in this model organism. He came to realize that E. coli K-12 was not handed to microbiologist by God as a model organism but was just another strain of E. coli and nothing special. Many of the earliest genomes to be sequenced came from worn-out lab strains. To counter this problem, Gordon Dougan at the Wellcome Trust Sanger Institute moved the focus to genome-sequencing freshly isolated minimally passaged isolates. With Brendan Wren, Pallen wrote a review article for nature, emphasizing the importance of adopting an eco-evo perspective when trying to interpret bacterial genomes. Around that time, Scott Beatson joined Pallen’s group. Mark managed to persuade Scott to work on type III secretion in E. coli rather than Pseudomonas aeruginosa. The result was the discovery of dozens of new type III secretion effectors, tying together bioinformatics and lab work to culminate in a PNAS paper.

Selective bibliography

Konings, F, Perkins, MD, Kuhn, JH, Pallen, MJ, Alm, EJ, Archer, BN, Barakat, A, Bedford, T, Bhiman, JN, Caly, L, Carter, LL, Cullinane, A, de Oliveira, T, Druce, J, El Masry, I, Evans, R, Gao, GF, Gorbalenya, AE, Hamblion, E, Herring, BL, Hodcroft, E, Holmes, EC, Kakkar, M, Khare, S, Koopmans, MPG, Korber, B, Leite, J, MacCannell, D, Marklewitz, M, Maurer-Stroh, S, Rico, JAM, Munster, VJ, Neher, R, Munnink, BO, Pavlin, BI, Peiris, M, Poon, L, Pybus, O, Rambaut, A, Resende, P, Subissi, L, Thiel, V, Tong, S, van der Werf, S, von Gottberg, A, Ziebuhr, J, Van Kerkhove, MD (2021) SARS-CoV-2 Variants of Interest and Concern naming scheme conducive for global discourse Nature Microbiology 6 (7) 821 823
Pallen, MJ, Alikhan, NF (2021) Naming the Unnamed: Over 45,000 Candidatus Names for Unnamed Archaea and Bacteria in the Genome Taxonomy Database Preprints https://doi.org/10.20944/preprints2021110557v1
Pallen MJ (2021) Bacterial nomenclature in the era of genomics New Microbes New Infect 44 100942 https://doi.org/10.1016/jnmni2021100942
Pallen, MJ, Telatin, A, Oren, A (2021) The Next Million Names for Archaea and Bacteria Trends in Microbiology 29 (4) 289 298 https://doi.org/10.1016/jtim202010009
Gilroy, R, Ravi, A, Getino, M, Pursley, I, Horton, DL, Alikhan, N-F, Baker, D, Gharbi, K, Hall, N, Watson, M, Adriaenssens, EM, Foster-Nyarko, E, Jarju, S, Secka, A, Antonio, M, Oren, A, Chaudhuri, RR, Ragione, RL, Hildebrand, F, Pallen, MJ (2021) Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture PeerJ 9 e10941 https://doi.org/10.7717/peerj10941
Ravi, A, Halstead, FD, Bamford, A, Casey, A, Thomson, NM, Van Schaik, W, Snelson, C, Goulden, R, Foster-Nyarko, E, Savva, GM, Whitehouse, T, Pallen, MJ, Oppenheim, BA (2019) Loss of microbial diversity and pathogen domination of the gut microbiota in critically ill patients Microbial Genomics 5 (9) 293 https://doi.org/10.1099/mgen0000293
Connor, TR, Loman, NJ, Thompson, S, Smith, A, Southgate, J, Poplawski, R, Bull, MJ, Richardson, E, Ismail, M, Thompson, SE, Kitchen, C, Guest, M, Bakke, M, Sheppard, SK, Pallen, MJ (2016) CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online resource for the medical microbiology community Microbial genomics 2 (9) e000086 https://doi.org/10.1099/mgen0000086
Loman NJ, Pallen MJ (2015) Twenty years of bacterial genome sequencing. Nat Rev Microbiol 13:787–794. https://doi.org/10.1038/nrmicro3565
Smith O, Momber G, Bates R, Garwood P, Fitch S, Pallen M*, Gaffney V, Allaby RG (2015) Sedimentary DNA from a submerged site reveals wheat in the British Isles 8000 years ago. Science 347:998–1001. https://doi.org/10.1126/science.1261278
Kay GL, Sergeant MJ, Zhou Z, Chan JZ, Millard A, Quick J, Szikossy I, Pap I, Spigelman M, Loman NJ, Achtman M, Donoghue HD, Pallen MJ (2015) Eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in Europe. Nat
Halachev MR, Chan JZ, Constantinidou CI, Cumley N, Bradley C, Smith-Banks M, Oppenheim B, Pallen MJ (2014) Genomic epidemiology of a protracted hospital outbreak caused by multidrug-resistant Acinetobacter baumannii in Birmingham, England. Genome Med 6:70. https://doi.org/10.1186/s13073-014-0070-x
Sergeant MJ, Constantinidou C, Cogan TA, Bedford MR, Penn CW, Pallen MJ (2014) Extensive microbial and functional diversity within the chicken cecal microbiome. PLoS One 9:e91941. https://doi.org/10.1371/journal.pone.0091941
Pallen MJ (2014) Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections. Parasitology 141:1856–1862. https://doi.org/10.1017/S0031182014000134 Citations: 103
Doughty EL, Sergeant MJ, Adetifa I, Antonio M, Pallen MJ (2014) Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer. PeerJ 2:e585. https://doi.org/10.7717/peerj.585
Chan JZ, Sergeant MJ, Lee OY, Minnikin DE, Besra GS, Pap I, Spigelman M, Donoghue HD, Pallen MJ (2013) Metagenomic analysis of tuberculosis in a mummy. N Engl J Med 369(3):289–290. https://doi.org/10.1056/NEJMc1302295
Robinson ER, Walker TM, Pallen MJ (2013) Genomics and outbreak investigation: from sequence to consequence. Genome Med 5:36. https://doi.org/10.1186/gm440
Loman NJ, Constantinidou C, Christner M, Rohde H, Chan JZ, Quick J, Weir JC, Quince C, Smith GP, Betley JR, Aepfelbacher M, Pallen MJ (2013) A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4. JAMA 309:1502–1510. https://doi.org/10.1001/jama.2013.3231
Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, Pallen MJ (2012) Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol 30:434–439. https://doi.org/10.1038/nbt.2198
Loman NJ, Constantinidou C, Chan JZ, Halachev M, Sergeant M, Penn CW, Robinson ER, Pallen MJ (2012) High-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity. Nat Rev Microbiol 10:599–606. https://doi.org/10.1038/nrmicro2850
Chan JZ, Pallen MJ, Oppenheim B, Constantinidou C (2012) Genome sequencing in clinical microbiology. Nat Biotechnol 30:1068–1071. https://doi.org/10.1038/nbt.2410
Chan JZ, Halachev MR, Loman NJ, Constantinidou C, Pallen MJ (2012) Defining bacterial species in the genomic era: insights from the genus Acinetobacter. BMC Microbiol 12:302. https://doi.org/10.1186/1471-2180-12-302
Mol Microbiol 79:468–483. https://doi.org/10.1111/j.1365-2958.2010.07460.x
Rohde H, Qin J, Cui Y, Li D, Loman NJ, Hentschke M, Chen W, Pu F, Peng Y, Li J, Xi F, Li S, Li Y, Zhang Z, Yang X, Zhao M, Wang P, Guan Y, Cen Z, Zhao X, Christner M, Kobbe R, Loos S, Oh J, Yang L, Danchin A, Gao GF, Song Y, Li Y, Yang H, Wang J, Xu J, Pallen MJ*, Wang J, Aepfelbacher M, Yang R (2011) Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N Engl J Med 365:718–724. https://doi.org/10.1056/NEJMoa1107643
Lewis T, Loman NJ, Bingle L, Jumaa P, Weinstock GM, Mortiboy D, Pallen MJ (2010) High-throughput whole-genome sequencing to dissect the epidemiology of Acinetobacter baumannii isolates from a hospital outbreak. J Hosp Infect 75:37–41. https://doi.org/10.1016/j.jhin.2010.01.012
Pallen MJ (2010) High-throughput sequencing as a tool in the discovery, diagnosis and epidemiology of nosocomial pathogens. Journal of Hospital Infection 76:S1. https://doi.org/10.1016/S0195-6701(10)00376-2
Pallen MJ, Loman NJ, Penn CW (2010) High-throughput sequencing and clinical microbiology: progress, opportunities and challenges. Curr Opin Microbiol 13:625–631. https://doi.org/10.1016/j.mib.2010.08.003
Pallen MJ, Nelson KE, Preston GM (2007) Bacterial Pathogenomics (ASM Press, Washington, DC, [Edited Book]
Pallen MJ (2007) Introduction to Pathogenomics in Bacterial Pathogenomics, eds Pallen
Tobe T, Beatson SA, Taniguchi H, Abe H, Bailey CM, Fivian A, Younis R, Matthews S, Marches O, Frankel G, Hayashi T, Pallen MJ (2006) An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination. Proc Natl Acad Sci U S A 103:14941–14946. https://doi.org/10.1073/pnas.0604891103
Pallen MJ, Beatson SA, Bailey CM (2005) Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective. FEMS Microbiol Rev 29:201–229. https://doi.org/10.1016/j.femsre.2005.01.001
Pallen MJ, Beatson SA, Bailey CM (2005) Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion. BMC Microbiol 5:9. https://doi.org/10.1186/1471-2180-5-9
Pallen MJ, Penn CW, Chaudhuri RR (2005) Bacterial flagellar diversity in the post-genomic era. Trends in Microbiology 13:143–149. https://doi.org/10.1016/j.tim.2005.02.008
Ideses D, Gophna U, Paitan Y, Chaudhuri RR, Pallen MJ, Ron EZ (2005) A degenerate type III secretion system from septicemic Escherichia coli contributes to pathogenesis. J Bacteriol 187:8164–8171. https://doi.org/10.1128/JB.187.23.8164-8171.2005
Ren CP, Chaudhuri RR, Fivian A, Bailey CM, Antonio M, Barnes WM, Pallen MJ (2004) The ETT2 gene cluster, encoding a second type III secretion system from Escherichia coli, is present in the majority of strains but has undergone widespread mutational attrition. J Bacteriol 186:3547–3560. https://doi.org/10.1128/JB.186.11.3547-3560.2004
Zhang L, Chaudhuri RR, Constantinidou C, Hobman JL, Patel MD, Jones AC, Sarti D, Roe AJ, Vlisidou I, Shaw RK, Falciani F, Stevens MP, Gally DL, Knutton S, Frankel G, Penn CW, Pallen MJ (2004) Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157:H7. Infect Immun 72:7282–7293. https://doi.org/10.1128/IAI.72.12.7282-7293.2004
Chaudhuri RR, Khan AM, Pallen MJ (2004) coliBASE: an online database for Escherichia coli, Shigella and Salmonella comparative genomics. Nucleic Acids Res 32:D296–9. https://doi.org/10.1093/nar/gkh031
Cerdeno-Tarraga AM, Efstratiou A, Dover LG, Holden MT, Pallen M, Bentley SD, Besra GS, Churcher C, James KD, De Zoysa A, Chillingworth T, Cronin A, Dowd L, Feltwell T, Hamlin N, Holroyd S, Jagels K, Moule S, Quail MA, Rabbinowitsch E, Rutherford KM, Thomson NR, Unwin L, Whitehead S, Barrell BG, Parkhill J (2003) The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129. Nucleic Acids Res 31:6516–6523. https://doi.org/10.1093/nar/gkg874
Pallen MJ, Francis MS, Futterer K (2003) Tetratricopeptide-like repeats in type-III-secretion chaperones and regulators. FEMS Microbiol Lett 223:53–60. https://doi.org/10.1016/S0378-1097(03)00344-6
Bentley SD, Maiwald M, Murphy LD, Pallen MJ, Yeats CA, Dover LG, Norbertczak HT, Besra GS, Quail MA, Harris DE, von Herbay A, Goble A, Rutter S, Squares R, Squares S, Barrell BG, Parkhill J, Relman DA (2003) Sequencing and analysis of the genome of the Whipple’s disease bacterium Tropheryma whipplei. Lancet 361:637–644. https://doi.org/10.1016/S0140-6736(03)12597-4
Roche FM, Massey R, Peacock SJ, Day NP, Visai L, Speziale P, Lam A, Pallen M, Foster TJ (2003) Characterization of novel LPXTG-containing proteins of Staphylococcus aureus identified from genome sequences. Microbiology 149:643–654. https://doi.org/10.1099/mic.0.25996-0
Pallen MJ (2002) The ESAT-6/WXG100 superfamily – and a new Gram-positive secretion system? Trends in Microbiology 10:209–212. https://doi.org/10.1016/S0966-842X(02)02345-4
Spiers A, Lamb HK, Cocklin S, Wheeler KA, Budworth J, Dodds AL, Pallen MJ, Maskell DJ, Charles IG, Hawkins AR (2002) PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide. J Biol Chem 277:39443–39449. https://doi.org/10.1074/jbc.M202790200
Pallen M (2002) From sequence to consequence: In silico hypothesis generation and testing. Methods in Microbiology 33:27–48. https://doi.org/10.1016/S0580-9517(02)33003-4
Pallen M, Chaudhuri R, Khan A (2002) Bacterial FHA domains: neglected players in the phospho-threonine signalling game? Trends in Microbiology 10:556–563. https://doi.org/10.1016/S0966-842X(02)02476-9
Pallen MJ, Lam AC, Loman N (2001) Tricorn-like proteases in bacteria. Trends in Microbiology 9:518–521. https://doi.org/10.1016/S0966-842X(01)02199-0
Pallen MJ, Lam AC, Antonio M, Dunbar K (2001) An embarrassment of sortases - a richness of substrates? Trends in Microbiology 9:97–102. https://doi.org/10.1016/S0966-842X(01)01956-4
Pallen MJ, Lam AC, Loman NJ, McBride A (2001) An abundance of bacterial ADP-ribosyltransferases–implications for the origin of exotoxins and their human homologues. Trends in Microbiology 9:302–7; discussion 308. https://doi.org/10.1016/S0966-842X(01)02074-1
Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, Chillingworth T, Davies RM, Feltwell T, Holroyd S, Jagels K, Karlyshev AV, Moule S, Pallen MJ, Penn CW, Quail MA, Rajandream MA, Rutherford KM, van Vliet AH, Whitehead S, Barrell BG (2000) The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665–668. https://doi.org/10.1038/35001088
Ponting CP, Pallen MJ (1999) beta-propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection. FEMS Microbiol Lett 179:447–451.
Ponting CP, Pallen MJ (1999) A beta-propeller domain within TolB. Mol Microbiol 31:739–740. https://doi.org/10.1046/j.1365-2958.1999.01168.x
Pallen MJ, Ponting CP (1997) PDZ domains in bacterial proteins. Mol Microbiol 26(2):411–413. https://doi.org/10.1046/j.1365-2958.1997.5591911.x
Pallen MJ (1996) Medicine and the Internet: dreams, nightmares and reality. Br J Hosp Med 56:506–509.
Pallen M, Kiley R (1996) Medical Information on the Internet. British Medical Journal 313:1411. https://doi.org/10.1136/bmj.313.7069.1411a
Mullany P, Pallen M, Wilks M, Stephen JR, Tabaqchali S (1996) A group II intron in a conjugative transposon from the gram-positive bacterium, Clostridium difficile. Gene 174:145–150. https://doi.org/10.1016/0378-1119(96)00511-2
Pallen MJ (1995) Medicine and the internet: dreams, nightmares and reality. British journal of hospital medicine 56:506–509.
Pallen M (1995) Guide to the Internet: Introducing the Internet. British Medical Journal 311:1422–1424. https://doi.org/10.1136/bmj.311.7017.1422
Pallen M (1995) Guide to the Internet: Electronic mail. British Medical Journal 311:1487–1490. https://doi.org/10.1136/bmj.311.7018.1487
Pallen M (1995) Guide to the Internet. The world wide web. British Medical Journal 311:1552–1556. https://doi.org/10.1136/bmj.311.7019.1552
Pallen M (1995) Guide to the Internet. Logging in, fetching files, reading news. British Medical Journal 311:1626–1630. https://doi.org/10.1136/bmj.311.7020.1626
Pallen MJ, Hay AJ, Puckey LH, Efstratiou A (1994) Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxin. J Clin Pathol 47:353–356. https://doi.org/10.1136/jcp.47.4.353
Mullany P, Clayton CL, Pallen MJ, Slone R, al-Saleh A, Tabaqchali S (1994) Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome. FEMS Microbiol Lett 124:61–67. https://doi.org/10.1111/j.1574-6968.1994.tb07262.x
Clayton CL, Pallen MJ, Kleanthous H, Wren BW, Tabaqchali S (1990) Nucleotide sequence of two genes from Helicobacter pylori encoding for urease subunits. Nucleic Acids Res 18:362. https://doi.org/10.1093/nar/18.2.362