Strategies for isolation of in vivo expressed genes from bacteria

Strategies for isolation of in vivo expressed genes from bacteria

Martin Hand¢eld 1, Roger C. Levesque *Molecular Microbiology and Protein Engineering, Health and Life Sciences Research Center, Charles-Eugeéne Marchand Bld.,

and Faculty of Medicine, Laval University, Quebec, Que. G1K 7P4, Canada

Received 2 February 1998; received in revised form 1 July 1998; accepted 12 October 1998

Abstract

The discovery and characterization of genes specifically induced in vivo upon infection and/or at a specific stage of theinfection will be the next phase in studying bacterial virulence at the molecular level. Genes isolated are most likely to encodevirulence-associated factors or products essential for survival, bacterial cell division and multiplication in situ. Identification ofthese genes is expected to provide new means to prevent infection, new targets for antimicrobial therapy, as well as new insightsinto the infection process. Analysis of genes and their sequences initially discovered as in vivo induced may now be revealed byfunctional and comparative genomics. The new field of virulence genomics and their clustering as pathogenicity islands makesfeasible their in-depth analysis. Application of new technologies such as in vivo expression technologies, signature-taggedmutagenesis, differential fluorescence induction, differential display using polymerase chain reaction coupled to bacterialgenomics is expected to provide a strong basis for studying in vivo induced genes, and a better understanding of bacterialpathogenicity in vivo. This review presents technologies for characterization of genes expressed in vivo. z 1999 Federation ofEuropean Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: In vivo induced; Gene expression; Gene fusion; Transposon; Genomics; Proteome; Di¡erential display; Subtractive hybrid-

ization; Di¡erential hybridization; Oligonucleotide array; Signature-tagged mutagenesis ; In vivo expression technology; Arbitrarily primed

polymerase chain reaction

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702. Gene fusions and transposons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713. Two-dimensional gel electrophoresis and bacterial proteomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724. Subtractive and di¡erential hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765. In vivo expression technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786. Signature-tagged mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827. Di¡erential £uorescence induction (DFI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 858. Di¡erential display using arbitrarily primed PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

0168-6445 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 3 3 - 3

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* Corresponding author. Tel. : +1 (418) 656-3070; Fax: +1 (418) 656-7176; E-mail: [email protected]

1 Present address: University of Florida College of Dentistry, Department of Oral Biology, P.O. Box 100424, Gainesville, FL 32610-0424, USA.

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9. The cDNA representational di¡erence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8610. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

1. Introduction

Virulence factors that de¢ne bacterial pathogenic-ity were initially discovered following Koch's postu-lates and by mimicking the environmental conditionspresent in infections. In some cases bacterial geneticspermitted the analysis of genes and protein productsinduced under these de¢ned conditions. Many viru-lence-associated proteins including cholera and diph-theria toxins were discovered and characterized invitro using bacterial mutants and biochemical anal-ysis of toxins. Expression of these virulence factorswas found to be dependent upon de¢ned conditions,i.e. iron and nutrient starvation, oxygen tension, pHand stress [1]. Studies on bacterial pathogenicity us-ing tissue culture as host de¢ned some host-pathogeninteractions at the cellular level ; Listeria, Shigella,and Salmonella are good examples that have beenreviewed recently and are not presented here [2].However, in vitro systems initially described didnot always allow the reconstruction of exact interac-tions between bacteria and the host [3].

Infection processes have been demonstrated to becoordinately regulated or stimulated by host factorsencountered in vivo, and these were found to bemultifactorial and dynamic. An understanding andthe de¢nition of virulence evolved as we becameaware that the pathogenic potential of microorgan-isms could not be explained by the sole contributionof one or a few so-called virulence determinants, i.e.toxins, adhesins, invasins, etc. This concept of viru-lence was continuously re-de¢ned by innovative ap-proaches designed to identify and characterize genesessential for pathogenicity; several genes were foundto be environmentally regulated in many pathogenicbacteria.

Bacteria such as Escherichia coli and Salmonellaenterica serovar typhimurium were used as model or-ganisms to study pathogenicity. It is not surprisingthen to observe that most novel molecular biology orbacterial genetic methods emerged from studies withthese two microorganisms as models. Historically,

there was a close relationship between the engineer-ing of new technologies and the evolution of ourunderstanding of bacterial pathogenicity and viru-lence at the level of the gene. In turn, bacterial ge-netics provided new insights from which to explorethe biology and physiology of these bacteria. Today,these ideas, concepts and technologies of bacterialgenetics and molecular biology have expanded forstudies in vivo and in situ.

Bacteria used in conjunction with animal modelsof infections have always been a concern in studyingand in de¢ning pathogenicity. Early work tended touse animal models, but this practice was greatly re-duced with increasing concern for ethical, technical,and economic reasons. Although they represent acomplex system in which many variables cannot becontrolled, animal models still represent one of thebest approaches for studying in vivo induced (ivi)genes, genes de¢ned by the process of being ex-pressed solely in vivo. In many instances, culturedcell lines provide a simpler, more easily controlledmodel for investigating the host-bacterium interac-tions in bacterial pathogenicity, even though cul-tured cell lines remain an arti¢cial system with di¡er-ences when compared to `normal' cells such as tissue-speci¢c cell surface molecules acting as receptors forbacterial adhesins, low a¤nity receptors, or naturalpolarity of mucosal cells. Nevertheless, data obtainedfor intracellular pathogens are remarkable. Such lim-itations have been addressed with research on organculture, and with the use of cells in primary culture[3,4].

In the past 5 years, new technologies have emergedto study gene regulation of microorganisms in situ,¢lling gaps in our understanding of bacterial patho-genicity occurring in vivo. These new methods willprovide the basis for understanding the possible met-abolic shifts of bacteria during an infection and sur-vival in the host. This is expected to open new ave-nues for the development of new antimicrobialagents, vaccines, as well as new prophylactics andtherapeutic strategies.

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M. Hand¢eld, R.C. Levesque / FEMS Microbiology Reviews 23 (1999) 69^9170

This review focuses essentially on recent technicaldevelopments for identi¢cation and for studying bac-terial genes essential for survival in vivo and theirregulation. Technologies presented here focus onthe study of human bacterial pathogens. However,the analysis of ivi genes and their regulation is alsoof interest and applicable in other areas of micro-biology such as plant-pathogen interactions, bacte-ria-matrix interactions in bio¢lms, and bioremedia-tion, etc. Technologies presented here can be easilymodi¢ed for use in any of these systems and an over-view of the latest strategies, as well as advantagesand disadvantages, is given. General guidelines areprovided for selecting a technology and studyinggene regulation in vivo for any bacterium. Wepresent a description of the strategies and importantparameters to consider, the limitations of these meth-ods and how such technology has contributed to thestudy and understanding of bacterial pathogenicity.Signi¢cant results will be presented to illustrate thesemethods and facilitate their applications to other bi-ological systems.

2. Gene fusions and transposons

Genetic fusions have from the earliest times ofmolecular genetics provided an important means ofanalyzing basic biological systems. The basic princi-ple of the genetic fusion approach was to put anassayable gene product under the control of anothergene of interest, and thus have a means to monitorits expression. In the early 1960s, an approach usingthe well-characterized E. coli lac operon was used forobtaining fusions and provided many advantages ingenetic selection that could be easily monitored usingchromogenic substrates. Beginning in 1976, a seriesof publications described ¢rst in vivo and then invitro methods for using the lac operon fused to awide variety of genes. Methods for the constructionof lac fusions could not be adapted directly to micro-organisms other than E. coli or closely related organ-isms because the lac transcription/translation signalswere not universally recognized by all bacteria; how-ever, the concept initially thought to be generallyapplicable has proven true in many cases [5]. Today,a wide range of reporter genes are available for mostorganisms. These include the chloramphenicol ace-

tyltransferase (CAT) and luciferase (Lux), which al-low selection and quanti¢cation of di¡erent gene ex-pression levels, and the green £uorescent protein(GFP) which allows real-time measurement of geneactivity in vivo.

To generalize the gene fusion approach, genetictechniques were devised which would allow the fu-sion of the lac operon to any gene. Casadaban con-ceptualized and engineered the two bacteriophagesMu and V p1(209) as tools for the ¢rst general meth-ods for constructing lac fusions to any target gene.The discovery of transposable genetic elements in theearly 1970s provided the means to move the lac geneto any position on the chromosome. It became ap-parent that mobile elements demonstrated more ver-satility since transposons were found to exist in var-ious life forms [5,6].

Transposons carrying an antibiotic resistance genewere ¢rst described in 1974 based on the observationthat plasmids that acquired penicillin resistance al-ways showed the same increase in molecular mass[7]. New methods in bacterial genetics were devel-oped using transposons conferring drug resistanceto isolate mutants and for construction of gene andoperon fusions [8].

A plethora of natural and engineered transposonsbecame available, varying in marker selection, spe-ci¢city of insertion, size, and polarity, but commonlybased upon the well-known Tn3, Tn5, Tn10 andTn916 derivatives. For example, a collection ofwidely used Tn5-derived minitransposons has beenconstructed that substantially simpli¢es the genera-tion of insertion mutants, in vivo fusions with re-porter genes, and the introduction of foreign DNAfragments into the chromosome of a variety ofGram-negative bacteria [9,10]. In addition to a vari-ety of antibiotic resistance genes as markers, severalderivatives also contain lacZ, phoA, luxAB or XylEgenes devoid of their native promoters located nextto the terminal repeats in an orientation that a¡ordsthe generation of gene-operon fusions. The transpo-sons are located on a R6K-based suicide deliveryplasmid that provides the IS50r transposase tnpgene in cis but external to the mobile element andwhose conjugal transfer to recipients is mediated byRP4 mobilization functions in the donor.

The initial use of Mu and transposons permittedthe rapid construction of a large number of gene

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M. Hand¢eld, R.C. Levesque / FEMS Microbiology Reviews 23 (1999) 69^91 71

fusions to promoters of genes identi¢ed solely by thecriterion of regulation, with no knowledge a priori oftheir phenotype, chromosome position, or biologicalfunction [8]. The Mu-lac fusions were used to studygenetic expression of ara and mal promoters in E.coli, genes from operons responding to environmen-tal signals. The Mu-lac system was used to character-ize E. coli genes of the SOS system induced by DNAdamage in the cell but not identi¢ed because of thephenotype, genes derepressed in conditions of phos-phate starvation or genes expressed in stress condi-tions [11^13]. Genes regulated under starvation con-ditions were identi¢ed with this technique in S.typhimurium [14]. Genes discovered by these genefusion methods led to the concept of coordinatedgene regulation in response to signals from the envi-ronment and presumably from the host [1].

Gene fusions to alkaline phosphatase were engi-neered to speci¢cally study the regulation of cell en-velope proteins in response to environmental signals.One of the most popular and widely used has beenTnphoA which can be inserted randomly in the bac-terial chromosome and when inserted in the properorientation and proper open reading frame, will fusealkaline phosphatase to the amino-terminus of theprotein. Cytoplasmic alkaline phosphatase wasfound to be unstable and requires export from thecytoplasm for folding and enzymatic activity [15].The promoting signal can correspond to those foundin periplasmic, outer membrane or cytoplasmicmembrane proteins. In contrast to the Mudlac sys-tem, which will generate Lac� fusions whenever it isinserted in the proper orientation into an expressedgene, the TnphoA system will generate PhoA� fu-sions only when it is inserted into a gene encodingan extracytoplasmic protein. The use of this ap-proach to the study of osmotically regulated genesin E. coli identi¢ed two genes already known to beinducible by osmolarity and eight other genes, des-ignated osm. Isolation of orally attenuated S. typhi-murium and Vibrio cholerae following TnphoA muta-genesis identi¢ed membrane associated virulencefactors following oral infection [16]. Several advan-tages to the use of TnphoA allowed screening for ane¡ect of a given regulatory parameter at any step ofgene expression, including transcription, mRNAprocessing or stability, and translational or post-translational modi¢cations [17].

The major restriction to all transposon-based ap-proaches is that their use is limited to characterizingnon-essential genes since insertional inactivation ofan essential gene would give a lethal phenotype.Transposons creating transcriptional fusions over-come this problem. Many transposons were shownto give strongly polar mutations in operons. Whatwas initially perceived as a limitation is now beingused to probe speci¢cally for isolation of mutantsand genes expressed in de¢ned conditions. Compre-hensive screens and selections are needed to convertsequence data into meaningful biological informa-tion.

Genomic footprinting uses a retroviral integrase togenerate a comprehensive library of mutants, each ofwhich bears a single insertion of a de¢ned oligonu-cleotide at a random position in the gene of interest[18]. This mutant library is selected for gene functionen masse. DNA samples are isolated from the libraryboth before and after the selection, and the muta-tions represented in each sample are then analyzed.The analysis is designed so that a mutation at aparticular location gives rise to an electrophoreticband of discrete mobility. For the whole library,this results in a ladder of bands, each band repre-senting a speci¢c mutation. Mutants in which theinserted sequence disrupts a feature that is requiredfor the selected function, ipso facto, fail the selection.The corresponding bands are therefore absent fromthe ladder of bands obtained from the library afterselection, giving rise to a footprint representing fea-tures of the gene that are essential for the selectedfunction. A simple system for performing transposonmutagenesis on naturally transformable organismsalong with a technique to rapidly identify essentialor conditionally essential DNA segments [19], calledGAMBIT, for genomic analysis and mapping by invitro transposition was applied to Haemophilus in£u-enzae and Streptococcus pneumoniae, in whichknown essential genes and several open readingframes (ORF) of unknown function were detectedas essential.

3. Two-dimensional gel electrophoresis and bacterialproteomes

Two-dimensional gel electrophoresis (2DGE) of

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total proteins was the ¢rst attempt to address geneexpression from total cell content. Due to its highresolution, reproducibility, and sensitivity, this ap-proach remains a powerful method for the compara-tive analysis and detection of proteins from bacterialcells grown in di¡erent conditions. In 2DGE, pro-teins are separated according to isoelectric point byelectrofocussing in the ¢rst dimension and by molec-ular mass with sodium dodecyl sulfate electrophore-sis in the second dimension; variations are also pos-sible [20]. Since these two parameters are unrelated,it is possible to obtain an almost uniform distribu-tion of proteins spots across a gel and in two dimen-sions. This technique resolved 1100 di¡erent proteinsfrom E. coli. A protein which constitutes as little as1034^1035% of total proteins can be detected andquanti¢ed by autoradiography. For example, a pro-tein (MW 40 000 kDa) present at a concentration ofone molecule per cell in E. coli would constitute2U1035% of total proteins.

Proteins with changes in charge caused by a singlemutation could be identi¢ed. This was initially dem-onstrated by a single missense mutation in gene 32 ofbacteriophage T4 infecting E. coli. Slight di¡erencesin growth conditions of an E. coli mutant in cyclicadenosine 3P :5P-monophosphate binding proteingrown in the presence of adenosine 5P-monophos-phate or cyclic guanosine 3P :5P-monophosphate re-sulted in several quantitative di¡erences visualizedon two-dimensional electrophoretograms. These ex-periments opened new avenues for sensitive, qualita-tive and quantitative analysis of gene induction inresponse to environmental stimuli at a high resolu-tion [20]. The recent application of electron sprayionization (ESI) and matrix-assisted laser desorp-tion/ionization (MALDI) as tools for protein analy-sis coupled to gel electrophoresis has become a keyto proteome approaches.

The 2DGE system has been used to compare pro-teins from S. typhimurium grown in vitro comparedto growth in macrophages and demonstrated that anumber of proteins were induced in vivo, amongwhich GroEL and DnaK, known as immunodomi-nant antigens in several bacterial pathogens [21].

An alternative scheme was developed for E. coli inwhich detection, cloning, and mapping of a respond-ing gene or coregulated gene in vivo was achieved(Fig. 1). This technique employed hybridization to

measure mRNA levels expressed from various re-gions of the chromosome by using an overlappingset of clones in V vectors. Comparisons betweenmRNA levels transcribed as control and in de¢nedexperimental conditions allowed detection of inducedor repressed genes in speci¢c conditions. The tech-nique was applied to the study of E. coli K-12 strainW3110 grown during L-galactosidase induction withisopropyl-L-D-thiogalactopyranoside (IPTG), duringthe entrance of cells into stationary phase, and dur-ing anaerobic growth conditions. Growth under nu-trient starvation, heat shock, or osmolarity varia-tions were used to mimic conditions outside theanimal gut. Gene expression from bacteria grownin gnotobiotic animals was compared to the geneexpression levels in vitro under anaerobic conditions[21]. Mutations of genes which gave pleiotropic ef-fects were studied in vitro to uncover the molecularmechanisms behind the adaptive responses in E. coli.Examples included rpoH, himA, topA, and crp, en-coding the heat shock-speci¢c transcriptional initia-tion factor c32, the K subunit of the integration hostfactor (IHF), DNA topoisomerase I, and the cyclicAMP (cAMP) receptor protein. Most mapped genesknown to be regulated in de¢ned conditions weresuccessfully detected. Many chromosomal regionscontaining undescribed genes were discovered andresponded to various stimuli [22].

One limitation of this technique was that the aver-age insert size of a V clone in the library ranged from9 to 21 kb; DNA inserts may contain over a dozengenes. Individual genes of a given V clone may havedi¡erent activity and respond di¡erently resulting ina reduced signal. This limitation was addressed byconstructing a library containing smaller inserts andsuch a library could ¢t on a single 80U80 plaque[22]. Instability of prokaryotic mRNA in the initialsteps of the construction of a representative cDNAlibrary can be limiting. Obviously, applicability ofsuch genomic-based techniques now requires less ef-fort in the construction of a representative gene li-brary, known to be the major limiting step. Recently,the resolution and sensitivity of protein detectionduring expression were improved by coupling geldata and their visualization to informatics, to devel-opments in automation, microscale sequencing andmass spectrometry. These combined methods cangive a picture of total protein expression of the ge-

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nome, and in situ in living cells in de¢ned conditions[23].

Knowledge of the proteome provides informationon gene translation patterns, relative concentrationof gene products, and the extent of posttranslationalmodi¢cations; this information is very di¤cult orimpossible to predict accurately from the nucleicacid sequence alone [24,25]. Proteome data frommodel organisms are expected to shed light on thefunction of genes that are revealed by genomics butwith no known functions [26].

Although this method is technically demanding, itsgeneral applicability is evident. Proteome studieshave been partly completed for model organismssuch as E. coli, Bacillus subtilis and Saccharomycescerevisiae. [27]. 2DGE and Edman sequencing werecombined to sequence Coomassie-stained 2DGEspots representing the abundant proteins of wild-

type E. coli K-12 strains. More than 90% of theabundant proteins in the E. coli proteome lie in asmall isoelectric point and molecular mass windowof 4^7 and 10^100 kDa, respectively [28]. These pro-teome methods applied to E. coli have given theEcoCyc encyclopedia of genes and metabolism withapplications to related organisms for visualizing thelayout of genes, an individual biochemical reactionor a complete biochemical pathway [29]. The data-base allows complex computations related to bacte-rial metabolism including studies on the design, evo-lution and simulation of novel biochemicalpathways. Metabolic databases are a new type ofbioinformatics resource with a wide variety of poten-tial uses including the study of bacterial metabolismexpressed in vivo. [30]. The complete sequence of theE. coli genome is available as well as the sequences ofvarious loci encoding virulence factors [31]. For ex-

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Fig. 1. Global transcriptional response for isolation of environmentally coregulated genes (adapted from [22]).


ample, the 35-kb locus encoding enterocyte e¡ace-ment from enteropathogenic E. coli has a G+C con-tent of only 38.4% compared to 50.8% for E. coli ;analysis of its 41 predicted ORFs in vitro and com-parison to in vivo expression coupled to the pro-teome of E. coli would be the next obvious step inde¢ning its role in virulence [32].

Analysis of the S. typhimurium proteome will al-low rapid delineation of virulence factors when com-pared to E. coli, detected as novel genes/proteinsamongst those in Salmonella which have no closehomologs in the former. To test the feasibility ofdetermining the proteome of Salmonella, the identi-ties of 53 randomly sequenced cell envelope proteinshave been determined by N-terminal sequencing ofspots separated by 2DGE [33]. It was found thatapproximately 20% of these proteins had no matchesin databases including E. coli. Results suggest thatproteome analysis provides a useful approach for thestudy of Salmonella virulence.

The genome of Haemophilus in£uenzae was the¢rst to be fully sequenced for a self-replicating or-ganism [34]. Proteome studies have now been com-pleted for H. in£uenzae, particularly for speci¢c phe-notypes where from 303 spots obtained by massspectrometry and 2DGE, 263 were identi¢ed asunique proteins [35]. In addition, a two-dimensionalmap of basic proteins was constructed [36]. Based onvariety and abundance, it was found that H. in£uen-zae proteins involved in energy metabolism and mac-romolecular synthesis are the dominant classes. Un-expectedly, tryptophanase was identi¢ed as a highlyabundant protein in strain NTCC8143 whose se-quence is not present in the genome of the Rd strain.Comparative studies of the proteins from othermembers of the Haemophilus genus, H. parain£uen-zae, H. haemolyticus and H. parahaemolyticus iden-ti¢ed 21^37% of the H. in£uenzae proteins whichcomigrated with proteins in the other isolates fromthe Haemophilus genus [37]. This compared with 62%and 64% comigration of proteins when H. in£uenzaestrain Hi-64443 was compared with the Eagan andRd strains. The capacity of 2DGE to investigateglobal interaction of gene expression was applied tothe comparative analysis of superoxide dismutase(SOD) in H. in£uenzae Eagan and a knockout mu-tant. In addition to SOD, quantitative changes inexpression of two other proteins in the SOD mutant

were also detected by comparison with the parentalisolate.

The proteome methods can also be applied to or-ganisms poorly studied at the molecular level. Pro-teome analysis of Spiroplasma melliferum with a ge-nome size of 1460 bp encoding 800^1000 proteinsgave a reference map of 506 silver-stained and repli-cated protein spots [38,39]. For the ¢rst time, pro-teins with close relationships to those previously de-termined from other species were identi¢ed acrossthe species barrier. The levels of expression of S.melliferum gene products were determined with re-spect to total optical intensity associated with thevisible proteins expressed in exponentially growingcells. Gene products from major families such asglycolysis, translation, transcription, cellular proc-esses, energy metabolism and protein synthesis wereidenti¢ed, as well as novel proteins not present in thesequenced genomes of the closely related Mycoplas-ma genitalium and Mycoplasma pneumoniae. A sim-ilar method was used to study the proteome of Lis-teria monocytogenes obtained in di¡erent stressconditions [40]. The stress imposed with pH 4, pH10, 0.015% SDS, 0.03% sodium deoxycholate and 4%ethanol gave a proteome analysis where more than50% of the proteins normally synthesized by Listeriacells were repressed, and where each stress factorinduced or repressed a set of novel proteins.

Proteomic contigs of Mycobacterium tuberculosisH37Rv and Mycobacterium bovis BCG identi¢ed atotal of 772 and 638 proteins, respectively [41]. Curi-ously, a bimodal distribution was observed for pro-teins separated from M. bovis BCG across both mo-lecular masses and isoelectric values. Somedi¡erences in protein expression were observed be-tween these two organisms, contrary to what mayhave been expected considering the high degree ofsimilarity between homologous genes. Further anal-ysis of both proteomes will allow more accurate di-agnosis between vaccination and active tuberculosis,epidemiological studies and patient management.The interaction between the intramacrophage patho-gen Mycobacterium avium and the macrophage ana-lyzed by 2DGE using particle containing phago-somes and metabolic labeling of macrophagesrevealed only minor di¡erences in protein pro¢lesbetween M. avium and IgG bead phagosomes, de-spite the marked di¡erences in the fusigenicity of

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the respective vacuoles [42]. The majority of poly-peptides in the bacilli were common to both growthconditions. Despite these similarities, intracellular M.avium expresses several unique proteins, most nota-bly one abundant 51-kDa protein. In addition, thebacterium manifests a restricted set of proteins ex-pressed while in stasis shortly after infection. Signi¢-cant advances in genetic studies of M. tuberculosisincluding methods for inactivating genes, expressionwith reporter genes, gene transfer, gene fusion andgenome sequencing have prepared a scenario forproteome analysis as well as for applications of invivo expression technology (IVET), signature-taggedmutagenesis (STM) and other techniques describedhere [43].

Proteomics was used for identi¢cation of prokary-otic developmental stages of Streptomyces coelicolorby statistical analysis of 2DGE products using clus-ter, principal component and correlation analysesclassifying gel patterns into four distinct groups,each re£ecting a stage-speci¢c pattern of gene expres-sion [44]. By focusing studies on phase-arrestedgrowth as a key regulatory transition leading to sec-ondary metabolism and a phase of renewed growth,21 and 18 proteins were identi¢ed whose synthesiswas switched on or o¡ during the transitional phase.

One of the challenges to proteome and bacterialgenomics is the analysis of low molecular mass pro-teins. The prediction of low molecular mass proteins(small), arbitrarily de¢ned as sequences 150 aminoacids in length, has been studied using the EcoGeneproject which identi¢es new small genes in other or-ganisms and conserved motifs [45]. In most casesthese proteins are unknown and it remains to bedetermined if they have a function and/or a role inbacterial survival in vivo.

4. Subtractive and di¡erential hybridization

Di¡erential subtractive hybridization systems havebeen developed to identify transcriptionally inducedgenes in cells that do not necessarily have a well-de¢ned genetic background. These techniques wereinitially developed in eukaryotic systems for theanalysis of di¡erences between closely related spe-cies, genes expressed di¡erentially in di¡erent tissues,or di¡erential expression in relation to external stim-

uli [24^26]. Recently, these systems have been ap-plied to prokaryotes. Although based on mRNA ex-pression levels, these schemes di¡er considerablyfrom well-known and widely used Northern blot,primer extension and S1 mapping techniques. Inthese traditional mRNA analysis methods, a speci¢cprobe is required to assess expression of a known orsingle gene, while schemes for di¡erential expressionrely essentially on random isolation of unknown anddi¡erentially expressed genes. The characterizationof genes that are di¡erentially expressed has beendi¤cult in prokaryotes mostly because of mRNAinstability and because of the technical challenge inthe isolation of large amounts of high qualitymRNA used for the construction of a cDNA library.Initially, libraries were screened with two speci¢cprobes, referred to as di¡erential hybridization. Analternative, referred to as subtractive hybridization,was done using cDNA libraries screened with sub-tracted probes prepared from labeled cDNA corre-sponding to mRNA isolated from treated cells, andsubtracted with mRNA isolated from untreated cells[26,27,46].

An interesting application of di¡erential hybridiza-tion in prokaryotes was analysis of gene expressionof M. avium following phagocytosis by humanmacrophages [49]. These experiments determinedwhether speci¢c genes were induced to express pro-teins that facilitated adaptation and survival in thephagosome. As presented in Fig. 2, the scheme reliedon the preparation of cDNA from M. avium grownin parallel in human-derived macrophages and insynthetic broth culture. Total RNA was isolatedfrom M. avium grown in macrophages and in broth.Mycobacterial mRNA was converted to cDNA byreverse transcription. Biotin-labeled cDNA preparedfrom M. avium grown in broth was used to subtracthousekeeping genes from the cDNA of the macro-phage-derived M. avium using streptavidin-coatedparamagnetic beads. After each round of subtrac-tion, a sample of the unsubtracted cDNA was am-pli¢ed, labeled, and hybridized to a cosmid library ofM. avium. After three rounds of subtraction, theampli¢ed DNA hybridized to approximately 1% ofthe cosmid clones under stringent conditions.Although the majority of the genes that are inducedin phagocytized M. avium cells are expressed in thebroth-grown bacilli, one DNA fragment that was

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identi¢ed encoded an mRNA that is highly speci¢cfor M. avium in phagosomes. The gene(s) encoded bythis sequence did not appear to be present in E. coliK-12 or M. tuberculosis. Nucleotide sequence analy-sis indicated codon usage typical for mycobacterialcoding sequences; an open reading frame was iden-ti¢ed as encoding a putative protein of 27 kDa witha leader peptide having a deduced protein with nosigni¢cant homolog in databases.

Di¡erential genome analysis of bacteria by ge-nomic subtractive hybridization and pulsed ¢eld gelelectrophoresis was applied to Pseudomonas aerugi-nosa choosing two clone C isolates, one from thelung of a cystic ¢brosis (CF) patient and the otherfrom a river habitat [50]. The subtractive methodresulted in the production of a library of speci¢csequence tags present in only one strain, while theconstruction of macrorestriction maps of the bacte-

rial chromosome yielded data about the overall ge-nome organization and the arrangement and dis-tance of gene loci. Comparison of the physical andgenetic maps and determination of the map positionsof the strain-speci¢c DNA sequences reveals grosschromosomal modi¢cations, insertions, deletionsand transpositional events. The 35 clones obtainedfrom the subtractive hybridization procedure usingthe two clone C variants of P. aeruginosa containedinserts speci¢c for the CF isolate; the non-randomdistribution of the strain-speci¢c clones by macrores-triction mapping and hybridization agreed with thegenomic mapping data of the two strains. Furtherstudies should con¢rm if these regions were acquiredby lateral transfer and determine their potential aspathogenicity islands.

A panoramic view of bacterial transcription cannow be obtained by using high density oligonucleo-

FEMSRE 634 8-2-99

Fig. 2. Application of subtractive and di¡erential hybridization to identify phagocytosis-induced mRNAs of M. avium (adapted from[49]).


tide probe arrays to monitor gene expression in bac-teria, a technique initially developed for S. cerevisiae[51,52]. Bacterial transcript imaging was designed ona chip containing probes for 106 H. in£uenzae and100 S. pneumoniae genes and demonstrated the ap-plicability of oligo arrays to global gene expressionmonitoring of a bacterial genome [53]. The apparentlack of polyadenylated transcripts excludes enrich-ment of mRNA and total chemically biotinylatedRNA was used as a hybridization probe. In additionto allowing simultaneous quanti¢cation of the tran-script level, the sensitivity was found to be in therange of one to ¢ve transcripts per cell and the quan-titative chip results were in agreement with conven-tional Northern blot analysis of selected genes. Dif-ferential gene expression was studied duringdevelopment of competence in S. pneumoniae in thepresence or absence of a competence-stimulatingpeptide (CSP). Most of the 100 genes present onthe chip were expressed at similar levels with lowerthan two-fold changes in the presence or absence ofCSP. However, cinA, recA and lytA genes, presenton the same operon, were induced at levels of 30-fold, 18-fold and 10-fold, respectively, in the compe-tent strain. RNA samples were also compared forexponentially growing cells to early stationary phasecells. The genes capX and cpsADEF encoding en-zymes for polysaccharide capsule biosynthesis, accCinvolved in long chain fatty acid biosynthesis andftsA involved in cell division were transcribed at lev-els three- to eight-fold lower in stationary phase thanin exponential phase. The genes nanB (neuramini-dase), clpl (protease), groEL (heat shock) and riba(GTP cyclohydrolase II) were induced in stationaryphase. With the increasing number of fully se-quenced bacterial genomes, highly parallel methodsfor monitoring gene expression should provide se-quence information for understanding bacterial re-sponse in an infection and studying virulence induc-tion in vivo.

The advantage of subtractive and di¡erential hy-bridization compared to IVET and STM mutants isthat it considerably reduces the number of genes tobe analyzed and does not depend on the constructionof transcriptional fusions of promoter sequences, orthe use of a well-characterized mutant host strain todetect expression of transcriptional fusions. In addi-tion, this technique does not require a clean genetic

background and the re-introduction of the mutantgene back into the chromosome of the host strainas IVET does [55]. Furthermore, the use of subtrac-tive libraries considerably reduces the number ofconstitutively expressed housekeeping genes and re-petitive sequences. However, induced genes having avery low constitutive expression level, a transientburst of expression, or expression of genes with ho-mology to but distinct from constitutively expressedgenes may not be well represented in a subtractivelibrary. Isolation of su¤cient amounts of mRNAeven with optimal protocols is still a limiting step.This system cannot be generalized to tissue culturesystems for studying in vivo interactions becausemany species give low concentrations of bacteria invivo. Furthermore, upregulated genes showing a lowexpression level in vitro or transiently expressedgenes could be missed by this method. However,this technique may be universally applicable to bac-teria having minimal genetic characterization [49].

An alternative scheme has been reported usingsubtraction with magnetic beads and PCR. In thisscheme, PCR was used as an ampli¢cation step priorto cloning of the cDNA recovered from the subtrac-tion. This additional step facilitated handling ofsmall amounts of cDNA and subsequent cloning ofthe sequences of interest. However, it is believed thatPCR preferentially ampli¢es short cDNA sequences,and sequences not capable of forming secondarystructures; this could limit the representativity of alibrary. Because of the importance of studying genesin situ, the advantages of using PCR outweigh thedisadvantages [54].

5. In vivo expression technology

The IVET system allowed for the ¢rst time thestudy of the bacterial response to the host environ-ment in situ using a gene expression scheme withinthe animal host itself for selection of genes that arespeci¢cally expressed during the infection. As shownin Fig. 2, the prototype pIVET1 and pIVET2 plas-mids were built with a promoterless operon fusion ofthe lacZY genes fused to the purA or thyA genesdownstream of a unique BglII cloning site (Fig. 3).The synthetic operon fusion was constructed in asuicide delivery plasmid. Cloning chromosomal

FEMSRE 634 8-2-99


DNA in the BglII site resulted in the construction ofa pool of transcriptional fusions driven by promoterspresent in the cloned DNA. The pool of fusions wastransferred in a vpurA or vthyA auxotroph and se-lected for integration into the chromosome by ho-mologous recombination. In the conditions prevail-ing in vivo, the wild-type auxotroph or recombinantstrains having no functional promoters are not via-ble. The fusions containing a promoter either con-stitutive or induced in vivo allowed transcription andbacterial survival. In vivo complemented fusionstrains were tested in vitro for their levels of L-gal-actosidase activity. Clones that contained fusions togenes speci¢cally induced in the animal modelshowed little lacZ expression in synthetic laboratory

media, although these clones had su¤cient transcrip-tion levels to survive within the mouse [55].

The conception of the IVET system met three im-portant criteria. The fusions were present in singlecopy in the chromosome, avoiding possible compli-cations arising from the use of plasmids or transpo-sons. The integration of fusions by homologous re-combination in the host chromosome generated aduplication of the cloned DNA in which the nativepromoter drove the synthetic fusion, and contained afunctional copy of the wild-type gene. This duplica-tion also assured that no polar e¡ects were present.The use of lacZY as gene reporters was demon-strated to be e¤cient in monitoring the transcrip-tional activity of the fusion strains in vitro in agar

FEMSRE 634 8-2-99

Fig. 3. IVET system developed to positively select for genes induced speci¢cally in the host (adapted from [55]). Abbreviations: bla, AmR

gene; lacZY, promoterless E. coli lac operon genes; mob, mobilization functions; oriR6K, origin of replication of the R6K plasmid; pir,gene encoding Pi; purA, promoterless adenine biosynthesis gene; XP, cloned chromosomal DNA fragment; X�, chromosomal homologueof the cloned DNA fragment.


plates with a chromogenic substrate and in vivo us-ing £uorometry with the chromophore FDG [56^59].

The original application of IVET in a murine ty-phoid fever model, and for P. aeruginosa in a mousemodel of septicemia, in a rat model of chronic lunginfection, and in direct contact with mucins isolatedfrom CF patients identi¢ed ivi genes encoding vari-ous known virulence factors, genes involved in inter-mediate metabolic pathways, and in housekeepingfunctions. Approximately 25^50% of genes identi¢edas ivi had no known functions and no signi¢canthomology to sequences available in the databases[55,57,59^61].

The IVET technology has also been used for thestudy of Brucella suis [62], and Mycobacterium tuber-culosis [63]. In these cases, the attP and int genesfrom V were part of the IVET plasmid vector forstable gene integration in the bacterial chromosome;the leuC, leuD, or leuCD gene products were used asa selection. These IVETs gave insight into the possi-ble changes in metabolism, gene regulation, and cellsurface properties that may enhance bacterial growthand infectivity in host tissues.

IVET has been expanded with a selection based onantibiotic resistance in the host rather than allevia-tion of nutritional de¢ciencies. In pIVET8, the catgene encoding chloramphenicol acetyltransferaseused as a selection marker is expressed as fused tolacZY. This scheme was tested in cell culture [64] butcould present problems in systemic distribution ofchloramphenicol and present limitations for selectionof chloramphenicol resistance in certain animal mod-els. Study of the virulence of S. typhimurium in aBALB/c model of septicemia, and in cultured macro-phages for induction indicated that the isolated ivigenes encoded regulatory functions. In addition, thefunction of many metabolic genes may not representtheir sole contribution to virulence. Thus, the hostecology was e¡ectively probed for biochemical func-tions via recovered ivi genes. Finally, nutrient limi-tation was thought to play a dual signaling role inpathogenesis in inducing metabolic functions thatcomplement host nutritional de¢ciencies and in in-ducing virulence functions required for immediatesurvival and the dissemination of bacterial pathogensto other sites in the host [65].

Comparable CAT-based IVET approaches havebeen reported for identi¢cation of novel virulence

genes from Yersinia enterolitica using a mouse mod-el ; plasmids encoding CAT instead of integratingvectors have also been used [66,67]. Since gene acti-vation and expression is not an all or none phenom-enon, the quantitative nature of CAT-based IVETscan distinguish between various levels of expressionin the infective cycle. CAT-based IVETs avoidedproblems (e.g. read-through) linked to the high e¤-ciency of resolution with resolvase. Invasion ofHenle cells by S. typhi identi¢ed promoter-contain-ing DNA sequences that activated bacterial gene ex-pression inside eukaryotic cells. One promoter-con-taining region exhibited sequence homology to c54-dependent promoter, whereas another appeared tobe dependent on the stationary phase RNA polymer-ase subunit cs. S. typhi containing the S1 subunitgene of the pertussis toxin cloned under the controlof these promoters selectively expressed the S1 sub-unit in di¡erent phagocytic and non-phagocytic celllines. These results e¤ciently and selectively ex-pressed proteins as intracellular antigens for vac-cines. Since the expression of heterologous antigensis known in some cases to compromise bacterialphysiology and competitiveness, it could be very ad-vantageous in terms of the viability and e¡ectivityof a live vaccine if the heterologous genes are notexpressed until the carrier strain attaches to andinvades the host. Promising vaccine applicationsappear to be the e¤cient expression of the recombi-nant protein in key antigen-presenting cells such asin macrophages and in dendritic cells [67].

Another antibiotic resistance-based IVET system(pPGIVET) is based on an operon fusion of the pro-moterless tetA, conferring tetracycline resistance, andgalK encoding galactokinase. Although the design ofthis system allows its use as an ivi promoter, it wasoriginally used to demonstrate speci¢c in vivo tran-scription of genes coding for adherence in the oralpathogen Porphyromonas gingivalis [68].

The prototype IVET systems required the avail-ability of a bacterial auxotroph; this nutritional de-¢ciency needed to be supplemented in vitro and com-plemented in vivo by expression from the ivipromoter fused to the gene used as selection at lowcopy number. The pIVET1 and pIVET2 vectorshave relied on the use of purA (purine metabolism)and thyA (thymine biosynthesis). Another system hasbeen described [60] in which the products of purEK

FEMSRE 634 8-2-99


(purine metabolism) were used as selection in an ad-enine auxotroph genetic background. The particular-ity of this last system was that it relied solely oncolony size rather than expresssion from a reportergene to measure in vitro activity of gene fusions. Therationale here was that a lower activity would resultin smaller colony size on minimal medium contain-ing limited amounts of adenine. Furthermore, athree-way translational stop codon was inserted infront of the promoterless purEK selection gene toensure the generation of transcriptional fusions in-stead of translational fusions that might producenon-functional fusion proteins. Application of thisstrategy to a neutropenic mouse infection modelidenti¢ed ivi genes inducible by respiratory mucusisolated from CF patients [60,61].

An alternative IVET relied on the use of tnpRoperon fusions encoding a site-speci¢c resolvase asthe selection gene and lacZY as reporter genes. Inthis system, the resolvase excision could be screenedfor and designed for identi¢cation of genes havingconstitutive or transient bursts of expression in vivo[69,70]. The basis for this IVET is the pre-selectionof strains carrying tnpR operon fusions which arenot expressed in vitro, followed by screening for asubset of these strains that subsequently express re-solvase in the host, recognized as in vivo recombi-nants that had deleted a resolvase-speci¢c reporter.A modi¢cation was constructed by generating genefusions to a tnpR allele in which the ribosome bind-ing site would be mutated to reduce subsequent lev-els of resolvase expression [71]. Recently, new report-er systems including sensitivity to sucrose and GFPexpression have been used with this system, thusbroadening the set of ivi genes isolated with higherbasal levels of transcription [69].

Plasmids were recently described (pIVET-GFPand pIVPRO) which relied on aspartate-L-semi-alde-hyde dehydrogenase (asd) as a selection gene. ASD isan enzyme essential for bacterial cell wall biosynthe-sis in Gram-negative bacteria, and one of its end-products, diaminopimelic acid, is absent from mam-malian cells, unlike adenine or thymine which maybe present (albeit at low concentrations), comple-ment the auxotrophs and give false positive results.Unless DAP is supplemented in vitro, or ASD com-plemented in vivo, ASD3 strains undergo cell lysis.The use of GFP as an alternative reporter system

(pIVET-GFP) will facilitate construction of fusionlibraries because of the smaller size of plasmids:pIVET1 10.3 kb, pIVET-GFP 5.5 kb. GFP can beused with automated £uorescence-activated cell sort-ing technology for recovery and analysis of ivi geneseither upregulated or strictly induced [58].

Overall, one minor drawback of the IVET systemwas that the duplication generated by integration offusions by homologous recombination may create anunstable genetic system that went through excisionor duplication at a frequency of 1035^1036 per cellper division, giving rise to loss of the cointegrate orto an ampli¢cation artifact. Because of the antibioticselective pressure in vitro, loss of cointegrates wasnot observed. However, the ampli¢cation e¡ect wasobserved by the presence of colonies with enhancedLac� phenotype originating from a single clone. TheIVET selection inherently selects for promoters thatare constitutively and highly expressed in vivo. Thegenes that are transiently expressed during infectioncould be missed. This limitation was overcome withthe pIVET5 vector (tnpR-lacZY) for ivi genes havinglow levels or temporal burst of transcription [69].Another limitation of the IVET system was the iso-lation of ivi genes dependent upon transcriptionalregulation. Genes activated or regulated posttran-scriptionally would not be isolated by such an ap-proach. Importantly, e¤cient transfer of genetic ma-terial is required. Transformation, conjugation, andelectroporation are known to be limited in severalspecies of bacteria. The IVET has to be used withcare in heterologous bacterial systems because tran-scription and translation signals of recombinant fu-sion may not be recognized.

Five years after the ¢rst description of the originalIVET system by Mahan et al. [55], it has alreadybeen used and proven applicable to a wide varietyof microorganisms, including Gram-positives, Gram-negatives, and some fastidious bacteria includingMycobacteria. Analysis of the data generated byIVETs showed that di¡erential patterns of acquiredvirulence genes could distinguish di¡erent Salmonellastrains and serotypes [72] Using IVETs, acquired se-quences were identi¢ed from S. typhimurium thatwere distinct within and between Salmonella sero-vars. Analysis of over 100 ivi genes had previouslyshown that approximately 25% of these had no se-quence homologs in databases. Sequence analysis of

FEMSRE 634 8-2-99


at least 10 of these `unknown' genes showed thatthey could be mapped into six di¡erent regions ofthe chromosome; several of these genes had an atyp-ical base composition. Removal of a portion of theseregions in the chromosome conferred a virulence de-fect in strains tested in animal models with a com-petitive index assay. These data will undoubtedlycontribute to a better understanding of the diseasemanifestation, host range, as well as new avenuesand hypotheses on the evolution of species, and onthe acquisition by horizontal transfer of virulencedeterminants associated with mobile genetic elementssuch as phages, insertion sequences, and pathogenic-ity islands that contribute to the ¢tness of a speci¢cpathogen within its host [72].

IVET was applied to Staphylococcus aureus usinga promoter trap that relies on genetic recombinationof the site-speci¢c resolvase of tnpR from gammadelta (Tn1000) as a reporter gene expression [73].A collection of 45 genes were found induced duringinfection in a murine renal abscess model. Of these,only six had been known previously; 11 others havehomology to known non-staphylococcal genes. Theknown staphylococcal genes included agrA, a keylocus regulating numerous virulence products; anda glycerol ester hydrolase which may enhance bacte-rial survival in abscesses. Many of the ivi genes iden-ti¢ed were not classical virulence factors but ratherare involved in adaptation to the in vivo environ-ment; knowledge of these biochemical pathways isclearly the next phase in IVET systems.

6. Signature-tagged mutagenesis

Several di¡erent approaches have been used toexploit transposon mutagenesis for the isolation ofbacterial virulence genes. Comprehensive screeningof bacterial genomes for virulence genes has notbeen possible because of the inability to identify mu-tants with attenuated virulence within pools of mu-tagenized bacteria and the impracticability of sepa-rately assessing the virulence of each of the severalthousand mutants necessary to screen a bacterial ge-nome. These limitations were circumvented by devel-oping a transposon mutagenesis system termed sig-nature-tagged mutagenesis in which each transposonmutant was tagged with a di¡erent DNA sequence,

or tag [74]. This tag allowed the identi¢cation ofbacteria recovered from hosts infected with a mixedpopulation of mutants as well as the selection ofmutants with attenuated virulence. The ability to re-spond to various stimuli and to regulate gene expres-sion to counteract disadvantageous conditions suchas host defense or nutritional deprivation is impor-tant for virulence; as such STM represents a wholegenome scan for habitat-speci¢c genes [75]. As pre-sented in Fig. 4, STM was used to identify virulencegenes from S. typhimurium in a murine model oftyphoid fever. The tag is prepared as pools and in-cluded variable central regions prepared as oligosand £anked by arms of invariant sequences. Thecentral region sequences were designed with su¤cientvariability to ensure that the same sequence shouldoccur only once in 2U1017 molecules. The complexmixture of double stranded DNA tags was generatedby oligonucleotide synthesis and PCR. Each tagcomprises a di¡erent sequence of 40 bp ([NK]20 ;N = A, C, G, or T; K = G or T). The arms weredesigned so that the ampli¢cation of the tags inPCR reactions ampli¢ed with speci¢c primers wouldproduce probes with 10 times more label in the cen-tral region than in each arm. The double strandedtags were ligated into the mini-Tn5 Km2 transposonand transferred from E. coli to S. typhimurium byconjugation. A library of 1510 exconjugants resultingfrom transposition events was stored in microtiterdishes. Twelve pools of inoculum, each comprising96 di¡erent sequence-tagged insertion mutants, werescreened for attenuated virulence by intraperitonealinjection in BALB/c mice. Bacteria were recoveredby plating spleen homogenates on synthetic medium,and DNA extracted from pools of infection. Thetags present in these DNA samples were ampli¢edand labeled by PCR, and colony blots were probedand compared with the hybridization patterns ob-tained with the use of tags ampli¢ed from the inoc-ulum as a probe. Twenty-eight mutants with attenu-ated virulence were identi¢ed by use of tags thatwere present in the inoculum in vitro but not inbacteria recovered from infected mice in vivo. Thir-teen of the mutations were in previously identi¢ed S.typhimurium virulence genes, six were in homologs ofknown genes of S. typhimurium and other bacteria,and nine were in sequences without similarity in da-tabases [74]. STM and mapping of the insertion

FEMSRE 634 8-2-99


FEMSRE 634 8-2-99

Fig. 4. STM used for simultaneous identi¢cation of S. typhimurium virulence genes based on transposon mutagenesis and negative selec-tion (adapted from [74,75]). Abbreviations and symbols: P, primers ; Kp, KpnI ; H, HindIII; I and O, ends of mini-Tn5 ; Km, kanamycinresistance gene; *, labelled tag.


points in the S. typhimurium chromosome identi¢edthe 40-kb SPI2 pathogenicity island encoding thecomponents of a new type III secretion system. Fur-ther analysis con¢rmed that the virulence of SPI2mutants was e¡ectively attenuated in two animalmodels [76].

STM has been used to study virulence-associatedgenes from Proteus mirabilis using a mouse model ofurinary infection, from V. cholerae in a sucklingmouse model of infection, from S. aureus in threeanimal models and from P. aeruginosa in a ratchronic lung infection model [77^80]. STM of S.aureus with Tn917 was used to screen 1248 mutantsin pools of 96 clones in a murine model of bacter-emia and resulted in the provisional identi¢cation of50 mutants; their subsequent individual analysis con-¢rmed attenuated virulence [79]. DNA sequenceanalysis of regions £anking insertion endpoints re-vealed that approximately 25 of these genes haveno known function; the remainder are involved innutrient biosynthesis and cell surface metabolism.It was concluded that many components of the S.aureus cell surface are critical for the survival andreplication of this pathogen in the blood. Comparingthe pro¢les of attenuated mutants obtained with dif-ferent models of infection such as endocarditis, os-teomyelitis, soft tissue abscesses, pneumonia and ar-thritis with those of the bacteremia model shouldidentify genes required for speci¢c S. aureus infec-tions as well as those for more than one type ofinfection. In another S. aureus STM analysis, poolsof S. aureus Tn917 mutants were screened in mouseabscess, bacteremia and wound infection models forgrowth attenuation after in vivo passages [81]. Whencompared to wild-type, one of the mutants identi¢eddisplayed a 10-fold attenuation following screeningin all three animal models. Sequence analysis showed99% identity to the high a¤nity proline permease(putP) gene characterized in another strain of S. au-reus. Transduction of the putP mutation into anotherS. aureus strain displayed attenuated virulence invivo. These results suggested that proline scavengingby bacteria is important for in vivo growth and pro-liferation and that analogs of proline may serve aspotential antistaphylococcal therapeutic agents.

STM was used to conduct a screen of randominsertion mutations that a¡ect colonization in thesuckling mouse model for cholera [78]. Of approxi-

mately 1100 mutants screened, ¢ve had insertions inone of the 15 TCP biogenesis genes; insertions werealso found in lipopolysaccharide, biotin, purine bio-synthetic, homologs of phosphate transfer genes andinsertions in novel genes; all of these caused coloni-zation defects in V. cholerae. Some of the mutantstrains were initially identi¢ed as attenuated wheninfected as one of a pool of 48 but were non-infectedas one of two competing strains, suggesting that V.cholerae strains would show in vivo attenuation ifinoculated in lower numbers. Thus, STM will leadto a better understanding of the processes by whichV. cholerae establishes a successful infection in thehost.

Screening of 480 miniTn5-Gm mutants of P. aeru-ginosa in pools of 48 identi¢ed mutants attenuatedfor virulence in a murine groin abscess model [82].The majority of mutants were con¢rmed for viru-lence attenuation when tested individually in thegroin model, while DNA sequencing identi¢ed inser-tions in popB and pscK, both encoding part of thetype III secretion system. An alternative method ofSTM was applied to P. aeruginosa strain PAO1 usingminiTn5-Km, but based upon a simpler library con-struction using 12 unique tags with clone pools andPCR screening rather than hybridization [80]. Invivo screening of clones was done in a rat chroniclung infection model, while attenuation of virulencewas con¢rmed by intraperitoneal injection in amouse model and calculation of the infectivity indexof attenuated mutants. This double screening strat-egy identi¢ed 14 clones, while the infectivity indexidenti¢ed two mutants with 10-fold attenuation. Se-quencing of insertion endpoints identi¢ed severalORFs with no homologs in databases and a homo-log to ftsX.

There are two factors in STM that restrict thecomplexity of pools of di¡erent mutants for use asinocula in infection studies. As the complexity of thepool increased, so did the probability that some vir-ulent mutants would not be present in su¤cientnumbers in vivo to produce enough labeled probefor hybridization analysis. In the hybridization stepof STM, the quantity of labeled tag for each trans-poson was inversely proportional to the complexityof the tag pool, so that there was a limit to the poolsize above which hybridization signals became tooweak to be detected by autoradiography. These lim-

FEMSRE 634 8-2-99


itations have been solved by ¢rst restricting the tagcomplexity to 12, 24, 48 and possibly 96 unique oli-gonucleotide sequences that do not cross-hybridize.Pooling of clones from each tag library is only lim-ited by redundancy in insertions. Hybridization istime-consuming and demanding but has now largelybeen replaced by PCR [83]. In theory, STM was de-signed to ¢nd a general applicability to other animaland plant pathogens. The recent modi¢cations andrapid evolution of STM technology now make gen-eral applicability feasible.

7. Di¡erential £uorescence induction (DFI)

To explore the genetic basis of intracellular surviv-al of S. typhimurium, a genetic selection was designedto allow identi¢cation of genes that are di¡erentiallyexpressed within murine macrophages [84]. DFI uti-lized a £uorescence-enhanced GFP and a £uores-cence activated cell sorter (FACS) to separate bacte-ria or infected cells on the basis of GFP £orescence[85]. In the original application, the technique wasused to screen a S. typhimurium library for pro-moters that are upregulated at pH 4.5. Macro-phage-like cell lines were also infected with thesame library of S. typhimurium bearing randomgene fusions. Cells that became £uorescent due totheir association with a gfp-expressing S. typhimu-rium were collected by FACS. These bacteria wererecovered, grown in the absence of cells and sampledby FACS. Bacteria that were no longer £uorescent inthe extracellular environment were sorted and usedfor a second round of macrophage infection. S. ty-phimurium present within these £uorescent macro-phages contained gfp fusions that were upregulatedin the host's cell intracellular environment. A partialscreen identi¢ed 18 macrophage-inducible pro-moters. Approximately half the genes isolatedshowed homology to genes previously described. Asubset of these genes had been reported either to beupregulated intracellularly or to be important for invivo survival [86,87].

One of the advantages of DFI is automation forinitial screening compared to manual screening. An-other advantage is that DFI allows the study of up-regulation as opposed to on-o¡ types of gene induc-tion.

8. Di¡erential display using arbitrarily primed PCR

Di¡erential display approaches can be used toquantitate environmental stimuli on bacterial geneexpression [88]. Arbitrarily primed (AP) PCR canidentify di¡erent gene expression levels betweenwell-de¢ned conditions [89,90]. RNA arbitrarilyprimed PCR (RAP-PCR) provided a complex phe-notype re£ecting changes in the abundance of hun-dreds of mRNAs simultaneously and under variousconditions. The analogy with 2DGE for proteins isquite obvious [91^94]. As shown in Fig. 5, RAP-PCR was based on initial synthesis of the ¢rst strandDNA from an arbitrary primer at those sites in theRNA that best matched the primer. Second strandsynthesis was achieved by arbitrary priming wherethe primer found the highest matches. Poorermatches at one end of the ampli¢ed sequence couldbe compensated for by very good matches at theother end. These two steps resulted in a collectionof DNA molecules that were £anked at their 3P and5P ends by the exact sequence (and complement) ofthe arbitrary primer. These in turn served as tem-plates for high-stringency PCR ampli¢cation.Although the intensities of di¡erent bands withinthe same ¢ngerprint vary, the intensity of a bandbetween ¢ngerprints appears to be proportional tothe concentration of its corresponding template ormRNA [94]. A complete pattern of all mRNAs ex-pressed in a particular cell is possible using a reason-able number of primer pairs [61]. Di¡erential displayusing AP-PCR generally consists of six steps: (1)isolation of mRNA; (2) reverse transcription in frac-tions using a de¢ned set of primers; (3) ampli¢cationof cDNA species from each fraction using a set ofarbitrary primers; (4) electrophoretic separation ofthe resulting fragments; (5) reampli¢cation of frag-ments that are di¡erent between two conditions,cloning and sequencing; (6) con¢rmation of di¡er-ential expression by an independent RNA analysistechnique.

Disadvantages of di¡erential display include la-bor-intensive and time-consuming performance, andthe requirement for labeling multiple probes corre-sponding to the di¡erentially displayed cloned can-didates [95]. Di¡erential display presents several ad-vantages compared to the alternative methods ofsubtractive hybridization, di¡erential screening, or

FEMSRE 634 8-2-99


2DGE: it allows simultaneous display of all di¡er-ences in mRNAs, it detects upregulation and down-regulation at the same time, it allows comparison ofmore than two situations, and it is technically faster[89].

9. The cDNA representational di¡erence analysis

This method combines an in-solution di¡erentialhybridization approach with PCR ampli¢cationsteps to isolate sequences unique to one out of twosamples. The original technique was applied to com-paring genome diversity between Neisseria meningi-tidis and Neisseria gonorrhoeae. Other methods de-pending on the use of arbitrary primer systems were

successfully applied in isolating adherence inducedgenes from E. coli, stress induced sequences fromS. typhimurium and host induced transcripts of Le-gionella pneumophila [88].

The e¡ect of mRNA abundance on the e¤ciencyof sampling by RNA ¢ngerprinting was a problemtypical to most AP-PRC methods. Nested RAP-PCRwas designed to normalize the ¢ngerprint with re-spect to mRNA abundance. The strategy was verysimilar to standard nested PCR methods, except thatthe internal sequences ampli¢ed were not known apriori. In this method, the ¢rst RAP-PCR ¢ngerprintwas further ampli¢ed at high stringency using a sec-ond nested primer having one, two, or three addi-tional arbitrarily chosen nucleotides at the 3P end ofthe ¢rst primer sequence. Since there were more mol-

FEMSRE 634 8-2-99

Fig. 5. AP-PCR for detection and identi¢cation of di¡erentially expressed genes [47,48] (adapted from [92]). A: Ampli¢cation of di¡eren-tially expressed genes using arbitrary primers from mRNA template isolated from bacteria grown in di¡erent conditions. B: Electrophore-sis of PCR-ampli¢ed products allowing detection and identi¢cation of induced/repressed genes.


ecules of low abundance in the background, mostampli¢ed products obtained by the high-stringencynesting step should have originated from the low-abundance, high-complexity class [95,96].

So far, di¡erential display based on AP-PCR hasbeen adapted for the identi¢cation of genes regulatedby antibiotics in P. aeruginosa found in sputum ofCF patients [97]. AP-PCR was used for the study ofgene expression in Enterococcus faecalis grown underaerobic and anaerobic conditions [98], and is cur-rently used to study virulence associated genesfrom Streptococcus mutans (D. Cvitkovitch, Univer-sity of Florida, personal communication), and for ivigenes from two oral pathogens Actinobacillus actino-mycetemcomitans and Porphyromonas gingivalis (A.Progulske-Fox, and J.D. Hillman, University ofFlorida, personal communication).

Obviously, di¡erential display is not restricted towell-characterized bacteria and genetic systems fromGram-negative and Gram-positive bacteria. AP-PCRis limited by the lower stability of prokaryoticmRNA, and by the relative concentration of targetmRNA. AP-PCR currently allows rapid and directpuri¢cation of di¡erentially expressed genes usingGel Scan with the automated gel sequencing appara-tus for the rapid separation of PCR products.

10. Concluding remarks

The basic techniques to develop new systems forthe study of in vivo expressed genes in bacteria wereactually in place as early as 1985 (Table 1). Thefundamental work with phages Mu, V, and transpo-sons combined with gene fusion technologies pro-vided the bacterial genetics that would yield generalmethods for constructing transcriptional gene fu-sions to any target gene as reviewed here withIVET, DFI or STM. Bacterial genetics producedthe methods to study genetics of regulatory systems,especially with genes that gave no visible phenotypes.2DGE of proteins coupled to informatics for analy-sis of separated proteins and automation of themethod can now compare proteins simultaneouslyin response to a single or a combination of speci¢cstimuli in vitro or in vivo. 2DGE has now advancedinto proteomes particularly in the ¢eld of bacterialproteomics. Subtractive hybridization coupled toPCR allows identi¢cation, cloning, and characteriza-tion of induced and/or repressed proteins in situ, andstudies of genes selected in vitro compared to invivo.

It is apparent that with IVET, STM, AP-PCR andDFI, we have entered a new era for studying genes

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Table 1Tools and strategies for isolation of environmentally regulated bacterial genes

Year Tool or strategy Relevant characteristics Investigator

1907 Cultured cell lines In vitro model for host-parasite interactions Harrison1962 Gene fusions Reporter gene under the translational control

of a gene of interestChampe

1963 Transposable elements Random insertion, polar e¡ect, gene markers, gene fusions Taylor1975 Two-dimensional electrophoresis Protein induction pattern of inducible/repressible genes O'Farrell1979 Mu-lac fusions Translocation and fusion in one step and in vivo Casadaban1979 Di¡erential and subtractive hybridization Low limit of detection. For systems of unde¢ned

genetic characteristicsSt-John

1985 Tnlac and TnphoA Intracellular localization of gene expression Manoil1988 Z-regulated suicide vectors oriR6K (Z-mediated), mob�, tra�, selectable marker Miller1992 Arbitrarily primed polymerase chain reaction

(AP-PCR)Di¡erential display using PCR Liang

1993 In vivo expression technology (IVET) Natural selection within the host Mahan1993 Global transcription response Genomic response to stress. Detection, cloning,

and mapping in one stepChuang

1995 tnpA-based IVET Transient burst of gene expression detectable Camilli1995 Multicycling system using Gm Distinguish di¡erent expression levels Staendner1996 Signature-tagged mutagenesis Identi¢cation of mutants with attenuated virulence Hensel1997 Di¡erential £uorescence induction (DFI) FACS separation of regulated genes Valdivia1997 asd and gfp-based IVETs Higher stringency, automated Hand¢eld


expressed or mostly induced in vivo, some of whichare crucial in host-bacteria interactions. Bacterialpathogenicity can now be studied in situ at the ge-nomics and proteomics levels and in real time. Thesenew methods will undoubtedly lead to unexpectedand stimulating insights which in turn promise newapproaches to ¢ght bacterial infections. Recent ad-vances in bacterial genomics, particularly with theadvent of available sequences of more than 50 bac-terial genomes, technologies for large DNA fragmentmanipulations, and miniaturization and automationof techniques support this view.

The frequent recovery of so called housekeepinggenes, when probing for virulence genes, and forgenes presumably essential for survival in vivo, couldmotivate the general acceptance of a more globalde¢nition of a virulence factor. Survival and persis-tence of the infective bacterium within its host maybe the most important virulence determinant whenthe production of a speci¢c set of classical virulencefactors is not obvious, as is the case for variouspathogens, and speci¢cally for certain opportunisticmicroorganisms.

One redundant problem ¢nally arises from the factthat these new schemes still rely on animal models,with the limitations that such models may impose.Only a few exceptions exist where in vivo gene ex-pression and regulation can readily be probed di-rectly in human infections. There is growing interestin modifying certain approaches described so far toallow in vivo gene expression probing directlyfrom human subjects. Such an approach is currentlyunder investigation for the periopathogens P. gingi-valis and A. actinomycetemcomitans (M. Hand¢eld,A. Progulske-Fox, and J.D. Hillman, unpublisheddata).

Acknowledgments

Work in R.C. Levesque's laboratory is funded bythe Medical Research Council of Canada, by theCanadian Cystic Fibrosis Foundation, and by theCanadian Bacterial Diseases Network via the Cana-dian Centers of Excellence. R.C.L. is a Scholar ofExceptional Merit from Le Fonds de la Rechercheen Santeè du Queèbec. M.H. obtained a studentshipfrom the Canadian Cystic Fibrosis Foundation.

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