From DNA sequence to application: possibilities and complications

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The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to
   Antonie van Leeuwenhoek   76:  3–23, 1999.© 1999  Kluwer Academic Publishers. Printed in the Netherlands.  3 From DNA sequence to application: possibilities and complications Gerard Venema 1 , ∗ , Jan Kok 1 & Douwe van Sinderen 2 1  Department of Genetics, Groningen BiomolecularSciences and BiotechnologyInstitute, University of Groningen,Kerklaan 30, 9751 NN, Haren, The Netherlands;  2  Department of Microbiology and the National Food Biotech-nology Centre, University College Cork, Ireland. ( ∗  Author for correspondence)Key words:  lactic acid bacteria, bacteriophage, inducible gene expression, regelatory DNA elements Abstract The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increaseof fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. Thisknowledge relates both to the assignments of open reading frames (ORF’s) and the function of non-coding DNAsequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed thattheir chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phaseof the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the constructionof temperature-induciblegene expression systems, gene-integration systems, and bacteriophage defence systems.The function of several LAB open reading frames and transcriptional units have been identified and char-acterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhancecheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. Moreknowledgehas also become available concerningthe functionand structure of non-codingDNA positioned at or inthevicinityofpromoters.InseveralcasesthemRNAproducedfromthisDNAcontainsatranscriptionalterminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction witha regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidencehas accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNAelements in the vicinity of promoters governing the transcription of catabolic operons.Although some biological barriers have yet to be solved, the vast body of scientific informationpresently avail-able allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraintsrather than biological hurdles impede the use of genetically modified LAB. Introduction The central molecule of a living unit is a polymerizedstring of either ribonucleotides (RNA) or deoxyribo-nucleotides (DNA). These often huge linear or cir-cular polymers serve two functions: (1) they encodeall information needed to sustain and reproduce anindividual cell and, (2) they are transmitted duringcell division with high fidelity. Alterations are keptto a minimum due to the presence of powerful re-pair mechanisms and daughter cells are essentiallyidentical to the parent cell, at least during asexualreproduction. When sexual or parasexual systems in-terfere, the genetic information can be changed moredrastically. However, in the natural context, neverbeyond certain limits set by the degree to which thegenetic exchange mechanisms operating in generalrecombination can tolerate.The natural constraints on the formation of grosslychanged DNA at high frequency in a short periodof time were definitely raised with the advent of recombinant DNA technology which permitted to in-tentionally change the genetic meaning of genomesalmost overnight in a way unlikely to occur in Nature.This development not only accelerated the progressof fundamental research to an impressive speed, butalso opened avenues for the production of cells andorganisms for the pharmaceutical and fermentationindustries. In addition, automated DNA sequencingfacilities and advanced computer programmes which  4convert nucleotide sequences to ORF’s and link to-gethernucleotidesequencesinto contigshas permittedentire microbial genomes to be sequenced as well asthat of the eukaryotic  Saccharomyces cerevisiae  and,very recently, that of the multicellular  Caenorhabditiselegans . As more genomes are being sequenced, weexpect to understand in a much more detailed waythan before, how evolution worked in shaping thegenomes in present day organisms. For the first timethe genome of a LAB has been sequenced to com-pletion. The sequence of   Lactococcus lactis  strainIL1403 is a major step toward making comparisonswith the genome sequences of other LAB, such as thatof   Lactobacillus acidophilus , which is well under wayintheframeworkofthe U.S.NationalDairyFoodsRe-search Center Programme (T.R. Klaenhammer, pers.comm.). As more LAB genomes are completed, com-paring their genetic organization and informationalcontent will undoubtedly shed light on the evolution-ary relationships among the diverse members of thisgroup.After the genomes are sequenced, determining thefunction of each and every gene contained in the gen-ome of an organism is a tremendous challenge. Thegeneral strategy to determine the function of a geneis to produce an interrupted copy by a Campbell-typeintegration and to analyze the phenotypic effect(s).However, if the gene to be interrupted is essential,this strategy does not work and should be replacedby one in which, in addition to the interrupted copy,an intact copy remains present, which can be turnedon at will. For the functional analysis of the  Bacillussubtilis  genome, the vector pMUTin2 meeting theserequirements has been developed(Vagner et al. 1998).Upon integration of this vector, transcription of thenon-interruptedcopyoftheessentialgeneiscontrolledby the Pspac promoter in an IPTG-dependent fash-ion. As far as we are aware, such a vector for LABhas not yet been constructed. However, since bothsuitable integration vectors for LAB (Leenhouts et al.1991; Maguin et al. 1992; Biswas et al. 1993; Lawet al. 1996) and tightly regulated inducible promotersare currently available, a LAB vector with proper-ties similar to pMUTin2 could be easily constructed.Nevertheless, the shear number of all the genes of agenome makes it a formidable task to uncover theirfunctions.Undoubtedly,this task will carry us throughthe next few decades, even if several groups would join forces in this enterprise for just one species of LAB. Yet, pursuing this goal is worthwhile, as it willprovide a wealth of information for both fundamentaland applications-oriented research.Although the phenotype of an organism ultimatelydepends on its genetic potential, knowledge of thefunction of all its ORF’s is only part of the completeunderstanding of the organism in its biological con-text. Ideally, one would like to be able to predict thebehaviour of that organism from its genetic composi-tion. This requires knowledge about non-coding DNAsequences, such as those to which regulatory mo-lecules attach to modulategene expression.Even then,full understanding of the organism would be incom-plete without knowledge of how proteins interact toproduce a particular phenotype. Rapid progress is be-ing made on this level, owing to the computer-assistedexploitation of the large body of informationavailableon proteins and protein domains. In those fortunatecases where the crystal structure of a protein is known,examples exist in which computer-assisted modellingof related proteins can occur and result in geneticengineering of proteins with desirable properties.Cells are continuously adapting to their surround-ings: nutrients can become limiting and, in the caseof LAB, cells are exposed to increasingly lower pHvaluesthroughtheirownmetabolicactivity.Moreover,chemical additions to industrial fermentations areroutinely made (e.g., salt during cheese making). Howthese changes affect the cell’s biochemistry, dependson the modulation of specific gene sets, which can beexamined by Northern hybridisation at the transcrip-tional level or by Western blotting at the translationallevel. These are, of course, very time consumingapproaches and in a in a majority of cases Westernanalysis is not feasible because suitable antibodies arelacking. An alternative to assess the protein-encodingpotential of the cell is to use highly standardized2D gel electrophoresis to construct a 2D protein in-dex as has been done for  B .  subtilis  (Bernhardt etal. 1997; Schmid et al. 1997; ). However, to linkparticular protein spots to the corresponding genes istime-consuming and often requires microsequencingand/or mutant production. Moreover, genes which areweakly expressed will remain hidden in this type of analysis.The recent introduction of DNA array technologyto monitor RNA expression of target genes, representsa major step forward in studying genome-wide geneexpression. These techniques have now advanced toa state that the transcriptional potential of completegenomes can be monitored on just one, or a very lim-ited number of oligonucleotide chips (for review: see  5Schena et al. 1998; Ramsay 1998, and elsewhere inthis volume), with a resolving power of only a fewmRNA molecules per cell (de Saizieu et al. 1998). Insummary, a whole spectrum of sophisticated analyt-ical tools is presently available for quickly assessinggene function, its modification in a predictable wayandtracingdifferentialgeneexpressionunderavarietyof external conditions.Due to the relatively small size of their chromo-somes, the genomic analysis of LAB bacteriophagesadvanced much more rapidly than that of their hosts.In the following text, some aspects of LAB bacterio-phage genomics will be reviewed briefly, emphasizingthose bacteriophage elements with potential interestfor the industry, This will be followed by a few se-lected examples in which the (probable) function of a lactococcal gene could be derived from sequencecomparisons with bacteria distantly related to LAB.Finally, some attention will be given to non-codingDNA, as well as to those constraints which impedeapplications of genetically modified LAB. LAB bacteriophage genomics The first LAB bacteriophage chromosome sequencedto completion was that of the lytic lactococcal bac-teriophage bIL67 (Schouler et al. 1994). Additional(complete) sequences of bacteriophage chromosomes,from both virulent and temperate phages infectingvarious LAB species, have now become available(Table 1). We are now in the position to comparetheir overall genetic organization and, by homologycomparisons, assign probable functions to a numberof their ORF’s. Figure 1 displays a schematic repres-entation of such a functional assignment for a specificgroup of small isometric-headed phages containingboth lytic and temperate representatives which infecta variety of LAB species. Comparison of the partialfunctional maps has allowed a number of general-isations. Figure 2 shows that: (1) genes involved inlysogeny are transcribed divergently from those re-quiredforthe lyticpathway; (2)theelementgoverningthe switch from the lysogenic to the lytic cycle of temperate bacteriophages is located between the lyticand lysogenic genes; and, (3) adjacent to the 3 ′ -endof the genetic switch is a cluster of genes for the ini-tiation and sustenance of bacteriophage chromosomereplication. These genes are followed by gene clustersinvolved in packaging of the chromosomes into thephage heads, and directing bacteriophagemorphogen-esis and genes required for lysis of the host. Thus,these bacteriophage chromosomes are highly modu-lar with functionally-related genes clustered togetherin a fixed order. Exchange of modules between in-terbreedingbacteriophagesby recombinationhas beeninvoked to explain the variability between and evolu-tion of bacteriophage chromosomes (Botstein 1980).However, in an extensive review on the variabilityof a number of   Streptococcus thermophilus  bacterio-phages, this evolutionary mechanism has been ques-tioned on the basis of the observation that exchangedsegments were neither functional units (modules) noreven complete genes (Brüssow et al. 1998). A numberof ORF’s of bacteriophage ØSfi21 of   S  .  thermophilus showed significant identities with ORF’s from vari-ous LAB-infecting bacteriophages and even from a  Lb .  delbrueckii plasmid.Theseobservationsfavourtheidea that  S  .  thermophilus  bacteriophages have evolvedthroughhorizontalgenetransferbetweenvariousLABas their hosts share the same ecological environment.It is interesting to note that the mechanism bywhich the chromosomes are incorporated in the bac-teriophage heads may differ profoundly, even amongclosely-related bacteriophages. For example bacterio-phage r1t uses a  cos  site and a terminase to producechromosomes with single- strand complementary ter-mini, whereas Tuc2009 employs the headful mech-anism to produce circularly permuted chromosomes.Apparently, lytic phage varieties can be derived fromtemperate bacteriophages, as illustrated by inspectionof the genome organization of bacteriophage LL-H,which, although having retained  attP  and part of theintegrase gene, appears to lack the repressor gene andthe switch governing the life cycle (Figure 1).Apart from these general deductions, the questionshould be raised as to which set of genetic informa-tion could be used for (future) applications. Obviousexamples are: (1) repressor-operator systems for theconstruction of LAB strains in which gene expressioncan be turned on or off at will; (2) genetic elementswhich could be used to provoke lysis of LAB in thecontext that lysis of LAB may facilitate cheese ripen-ing; (3) elements that could be used for the stableintegration of (foreign) genes in LAB; and, (4) ele-ments which couldbe used to combatphageinfectionsduring fermentations. An example of each of thesepossibilities will be described in the following pages.   6   Figure 1.  Schematic representation of the genomes of a number of temperate and lytic small isometric-headed bacteriophages infecting various LAB (names in the left-hand margin refer tophages; see also Table 1). Arrows and filled circles ( ) depict identified open reading frames (ORF’s). The direction of the arrows corresponds to the presumed direction of transcription.Triangles ( ) indicate the position at which an intron interrupts the coding coding sequence of an ORF. A thick black line ( ) indicates sections of phage genomes where no sequenceinformation is available. Genomic sections which were not considered because of lack of known function have been placed between two sets of perpendicular lines (–//—//–). Deduced ordetermined positions of initiation of packaging, pac or cos, are indicated by green and red filled circles, respectively. Bacteriophage attachment sites (att) are indicated by squares (    ). Otherabbreviations refer to to the presumed function of the ORF and are as follows: int, integrase; cro, small DNA-binding protein probably involved in the genetic switch; DnaA, DnaB and DnaC,components of the replisome, referring to the  E  .  coli  replisome; sp, structural protein; terS and terL, small and large subunits of the terminase, respectively; mhp, major head protein; mtp, majortail protein; minor tp, minor tail protein; portal, portal protein; SSB, single-strand DNA binding protein; topo, topoisomerase I; int-del, deleted integrase-encoding ORF.  7 Table 1.  List of LAB bacteriophages of which the genome has been partially or completely sequenced. This table only includes phages of which at least six kb of contiguous sequence has been determinedPhage cos/pac a Type Complete sequence ORF’s present LAB host Referenceavailable; base pairs on genomebIL67 cos prolate, lytic yes; 22,195 37  L. lactis  subsp  lactis  Schouler et al. 1994c2 cos prolate, lytic yes; 22,163 39  L. lactis  subsp  lactis  Lubbers et al. 1995sk1 cos small isometric, lytic yes; 28,451 54  L. lactis  subsp.  cremoris  Chandry et al. 1997bIL41 cos small isometric, lytic no NA  L. lactis  subsp.  lactis  Parreira et al. 1996BK5-T cos small isometric, temperate no NA  L. lactis  subsp.  cremoris  Boyce et al. 1995r1t cos small isometric, temperate yes; 33,350 50  L. lactis  subsp  cremoris  van Sinderen et al. 1996Tuc2009 pac small isometric, temperate yes; 38,347 56  L. lactis  subsp  cremoris  van Sinderen et al. 1 TP901-1 pac small isometric, temperate no NA  L. lactis  subsp  cremoris  Christiansen et al. 1996Johnsen et al. 1996Madsen et al. 1998LL-H pac small isometric, lytic yes; 34,659 52  Lb. delbrueckii  subsp.  lactis  Mikkonen et al. 1996øg1e (pac) small isometric, temperate yes; 42,259 62  Lactobacillus  species Kodaira et al. 1997øO1205 pac small isometric, temperate yes; 43,075 57  S. thermophilus  Stanley et al. 1997ø7201 cos small isometric, lytic yes; 35,466 44  S. thermophilus  van Sinderen et al. 2 TP-J34 pac small isometric, temperate no NA  S. thermophilus  Neve et al. 1998øSfi11 pac small isometric, lytic no NA  S. thermophilus  Lucchini et al. 1998øSfi19 cos small isometric, lytic no NA  S. thermophilus  Lucchini et al. 1998Desiere et al. 1998øSfi21 cos small isometric, temperate no NA  S.thermophilus  Desiere et al. 1998Bruttin et al. 1997Desiere et al. 1997 a cos/pac: phages using cos or pac type of DNA packaging, respectively; NA: not applicable. Figure 2.  Modular genomic organization found in a large group of small isometric-headed bacteriophages (see Figure 1 and Table 1). Arrowspositioned at the top of the figure and coloured in green and red indicate the transcription direction during the lysogenic and lytic life cycle,respectively (note that in lytic phages the DNA region transcribed from lysogenic phages is not, or only partially present). The presumedfunction of specific DNA modules is indicated in the middle part of the figure and regions with no known function are indicated by questionmarks. Vertical arrows pointing towards the bottom part of the figure point at additional information on functionality or composition of thespecific DNA module.
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