Microbial genetics

Outline of the topic

Genetic Information in Microbes Genome Organization Mutation and Selection Exchange of Genetic Information Recombinant DNA and Gene loning Regulation of Gene Expression

INTRODUCTION
Genetic Information In Microbes
!he genetic material of bacteria and plasmids is DNA" #acterial $iruses %bacteriophages or phages& ha$e DNA or RNA as genetic material" !he t'o essential functions of genetic material are replication and expression" Genetic material must replicate accuratel( so that progen( inherit all of the specific genetic determinants %the genot(pe& of the parental organism" Expression of specific genetic material under a particular set of gro'th conditions determines the obser$able characteristics %phenot(pe& of the organism" #acteria ha$e fe' structural or de$elopmental features that can be obser$ed easil() but the( ha$e a $ast arra( of biochemical capabilities and patterns of susceptibilit( to antimicrobial agents or bacteriophages" !hese latter characteristics are often selected as the inherited traits to be anal(zed in studies of bacterial genetics"

Nucleic Acid Structure

Nucleic acids are large pol(mers consisting of repeating nucleotide units %*ig" +,-&" Each nucleotide contains one phosphate group) one pentose or deox(pentose sugar) and one purine or p(rimidine base" In DNA the sugar is D,.,deox(ribose/ in RNA the sugar is D,ribose" In DNA the purine bases are adenine %A& and guanine %G&) and the p(rimidine bases are th(mine %!& and c(tosine % &" In RNA) uracil %0& replaces th(mine" hemicall( modified purine and p(rimidine bases are found in some bacteria and bacteriophages" !he repeating structure of pol(nucleotides in$ol$es alternating sugar and phosphate residues) 'ith phosphodiester bonds lin1ing the 23,h(drox(l group of one nucleotide sugar to the +3,h(drox(l group of the ad4acent nucleotide sugar" !hese as(mmetric phosphodiester lin1ages define the polarit( of the pol(nucleotide chain" A purine or p(rimidine base is lin1ed at the -3,carbon atom of each sugar residue and pro4ects from the repeating sugar,phosphate bac1bone" Double,stranded DNA is helical) and the t'o strands in the helix are antiparallel" !he double helix is stabilized b( h(drogen bonds bet'een purine and p(rimidine bases on the opposite strands" At each position) A on one strand pairs b( t'o h(drogen bonds 'ith ! on the opposite strand) or G pairs b( three h(drogen bonds 'ith " !he t'o strands of double,helical DNA are) therefore) -

complementar(" #ecause of complementarit() double,stranded DNA contains e5uimolar amounts of purines %A 6 G& and p(rimidines %! 6 &) 'ith A e5ual to ! and G e5ual to ) but the mole fraction of G 6 in DNA $aries 'idel( among different bacteria" Information in nucleic acids is encoded b( the ordered se5uence of nucleotides along the pol(nucleotide chain) and in double,stranded DNA the se5uence of each strand determines 'hat the se5uence of the complementar( strand must be" !he extent of se5uence homolog( bet'een DNAs from different microorganisms is the most stringent criterion for determining ho' closel( the( are related"

Fig 1 Double !elical structure of DNA !he diagram sho's the structure of DNA represented as a helical ladder" !he bac1bone of each pol(nucleotide strand %represented as a ribbon& consists of alternating phosphate and deox(ribose residues lin1ed b( phosphodiester bonds) and the strands ha$e opposite polarities %arro's&" !he purine or p(rimidine base of each nucleotide on one strand pro4ects to'ard the complementar( base of the corresponding nucleotide from the other strand and is lin1ed to it b( h(drogen bonds" !he double helix has a diameter of . nm" Each full turn of the double helix contains -7 nucleotide pairs and is 2"8 nm in length"

DNA Re"lication
During replication of the bacterial genome) each strand in double,helical DNA ser$es as a template for s(nthesis of a ne' complementar( strand" Each daughter double,stranded DNA molecule thus contains one old pol(nucleotide strand and one ne'l( s(nthesized strand" !his t(pe of DNA replication is called semiconser$ati$e" Replication of chromosomal DNA in bacteria starts at a specific chromosomal site called the origin and proceeds bidirectionall( until the process is completed %*ig" -&" 9hen bacteria di$ide b( binar( fission after completing DNA replication) the replicated chromosomes are partitioned into each of the .

daughter cells" !he origin regions specificall( and transientl( associate 'ith the cell membrane after DNA replication has been intitiated) leading to a model 'hereb( membrane attachment directs separation of daughter chromosomes %the replicon model&" !hese characteristics of DNA replication during bacterial gro'th fulfill the re5uirements of the genetic material to be reproduced accuratel( and to be inherited b( each daughter cell at the time of cell di$ision"

Gene #$"ression
Genetic information encoded in DNA is expressed b( s(nthesis of specific RNAs and proteins) and information flo's from DNA to RNA to protein" !he DNA,directed s(nthesis of RNA is called transcription" #ecause the strands of double,helical DNA are antiparallel and complementar() onl( one of the t'o DNA strands can ser$e as template for s(nthesis of a specific mRNA molecule" Messenger RNAs %mRNAs& transmit information from DNA) and each mRNA in bacteria functions as the template for s(nthesis of one or more specific proteins" !he process b( 'hich the nucleotide se5uence of an mRNA molecule determines the primar( amino acid se5uence of a protein is called translation" Ribosomes) complexes of ribosomal RNAs %rRNAs& and se$eral ribosomal proteins) translate each mRNA into the corresponding pol(peptide se5uence 'ith the aid of transfer RNAs %tRNAs&) amino,ac(l tRNA s(nthesases) initiation factors and elongation factors" All of these components of the apparatus for protein s(nthesis function in the production of man( different proteins" A gene is a DNA se5uence that encodes a protein) rRNA) or tRNA molecule %gene product&" !he genetic code determines ho' the nucleotides in mRNA specif( the amino acids in a pol(peptide" #ecause there are onl( 8 different nucleotides in mRNA %containing 0) A) and G&) single nucleotides do not contain enough information to specif( uni5uel( all .7 of the amino acids" In dinucleotides -: %8 x 8& arrangements of the four nucleotides are possible) and in trinucleotides :8 %8 x 8 x 8& arrangements are possible" !hus) a minimum of three nucleotides is re5uired to pro$ide at least one uni5ue se5uence corresponding to each of the .7 amino acids" !he ;uni$ersal; genetic code emplo(ed b( most organisms %!able -& is a triplet code in 'hich :- of the :8 possible trinucleotides %codons& encode specific amino acids) and an( of the three remaining codons %0AG) 0AA or 0GA& results in termination of translation" !he chain,terminating codons are also called nonsense codons because the( do not specif( an( amino acids" !he genetic code is described as degenerate) because se$eral codons ma( be used for a single amino acid) and as nono$erlapping) because ad4acent codons do not share an( common nucleotides" Exceptions to the ;uni$ersal; code include the use of 0GA as a tr(ptophan codon in some species of M(coplasma and in mitochondrial DNA) and a fe' additional codon differences in mitochondrial DNAs from (easts) Drosophila) and mammals" !ranslation of mRNA is usuall( initiated at an A0G codon for methionine) and ad4acent codons are translated se5uentiall( as the mRNA is read in the +3 to 23 direction" !he corresponding pol(peptide chain is assembled beginning at its amino terminus and proceeding to'ard its carbox( terminus" !he se5uence of amino acids in the pol(peptide is) therefore) colinear 'ith the se5uence of nucleotides in the mRNA and the corresponding gene" Expression of genetic determinants in bacteria in$ol$es the unidirectional flo' of information from DNA to RNA to protein" In bacteriophages) either DNA or RNA can ser$e as genetic 2

material" During infection of bacteria b( RNA bacteriophages) RNA molecules ser$e as templates for RNA replication and as mRNAs" Studies 'ith the retro$irus group of animal $iruses re$eal that DNA molecules can be s(nthesized from RNA templates b( enz(mes designated as RNA,dependent DNA pol(merases %re$erse transcriptases&" !his re$ersal of the usual direction for flo' of genetic information) from RNA to DNA instead of from DNA to RNA) is an important mechanism for enabling information from retro$iruses to be encoded in DNA and to become incorporated into the genomes of animal cells"

Genome Organi%ation
DNA molecules that replicate as discrete genetic units in bacteria are called replicons" In some Escherichia coli strains) the chromosome is the onl( replicon present in the cell" Other bacterial strains ha$e additional replicons) such as plasmids and bacteriophages"

C!romosomal DNA
#acterial genomes $ar( in size from about 7"8 x -7< to =": x -7< daltons %Da&) some of the smallest being obligate parasites %M(coplasma& and the largest belonging to bacteria capable of complex differentiation such as M(xococcus" !he amount of DNA in the genome determines the maximum amount of information that it can encode" Most bacteria ha$e a haploid genome) a single chromosome consisting of a circular) double stranded DNA molecule" >o'e$er linear chromosomes ha$e been found in Gram,positi$e #orrelia and Streptom(ces spp") and one linear and one circular chromosome is present in the Gram, negati$e bacterium Agrobacterium tumefaciens" !he single chromosome of the common 8

intestinal bacterium E coli is 2 x -7< Da %8)+77 1ilobase pairs ?1bp@& in size) accounting for about . to 2 percent of the dr( 'eight of the cell" !he E coli genome is onl( about 7"-A as large as the human genome) but it is sufficient to code for se$eral thousand pol(peptides of a$erage size %87 1Da or 2:7 amino acids&" !he chromosome of E coli has a contour length of approximatel( -"2+ mm) se$eral hundred times longer than the bacterial cell) but the DNA is supercoiled and tightl( pac1aged in the bacterial nucleoid" !he time re5uired for replication of the entire chromosome is about 87 minutes) 'hich is approximatel( t'ice the shortest di$ision time for this bacterium" DNA replication must be initiated as often as the cells di$ide) so in rapidl( gro'ing bacteria a ne' round of chromosomal replication begins before an earlier round is completed" At rapid gro'th rates there ma( be four chromosomes replicating to form eight at the time of cell di$ision) 'hich is coupled 'ith completion of a round of chromosomal replication" !hus) the chromosome in rapidl( gro'ing bacteria is replicating at more than one point" !he replication of chromosomal DNA in bacteria is complex and in$ol$es man( different proteins"

&lasmids
Blasmids are replicons that are maintained as discrete) extrachromosomal genetic elements in bacteria" !he( are usuall( much smaller than the bacterial chromosome) $ar(ing from less than + to more than se$eral hundred 1bp) though plasmids as large as . Mbp occur in some bacteria" Blasmids usuall( encode traits that are not essential for bacterial $iabilit() and replicate independentl( of the chromosome" Most plasmids are supercoiled) circular) double, stranded DNA molecules) but linear plasmids ha$e also been demonstrated in #orrelia and Streptom(ces" losel( related or identical plasmids demonstrate incompatibilit(/ the( cannot be stabl( maintained in the same bacterial host" lassification of plasmids is based on incompatibilit( or on use of specific DNA probes in h(bridization tests to identif( nucleotide se5uences that are characteristic of specific plasmid replicons" Some h(brid plasmids contain more than one replicon" on4ugati$e plasmids code for functions that promote transfer of the plasmid from the donor bacterium to other recipient bacteria) but noncon4ugati$e plasmids do not" on4ugati$e plasmids that also promote transfer of the bacterial chromosome from the donor bacterium to other recipient bacteria are called fertilit( plasmids) and are discussed belo'" !he a$erage number of molecules of a gi$en plasmid per bacterial chromosome is called its cop( number" Carge plasmids %D87 1ilobase pairs& are often con4ugati$e) ha$e small cop( numbers %- to se$eral per chromosome&) code for all functions re5uired for their replication) and partition themsel$es among daughter cells during cell di$ision in a manner similar to the bacterial chromosome" Blasmids smaller than E"+ 1ilobase pairs usuall( are noncon4ugati$e) ha$e high cop( numbers %t(picall( -7,.7 per chromosome&) rel( on their bacterial host to pro$ide some functions re5uired for replication) and are distributed randoml( bet'een daughter cells at di$ision" Man( plasmids control medicall( important properties of pathogenic bacteria) including resistance to one or se$eral antibiotics) production of toxins) and s(nthesis of cell surface structures re5uired for adherence or colonization" Blasmids that determine resistance to antibiotics are often called R plasmids %or R factors&" Representati$e toxins encoded b( plasmids include heat,labile and heat,stable enterotoxins of E coli) exfoliati$e toxin of Staph(lococcus aureus) and tetanus toxin of lostridium tetani" Some plasmids are cr(ptic and ha$e no recognizable effects on the bacterial cells that harbor them" omparing plasmid profiles is a useful method for assessing possible relatedness of indi$idual clinical isolates of +

a particular bacterial species for epidemiological studies" !he role of plasmids in the e$olution of resistance to antibiotics is discussed belo'"

'acterio"!ages
#acteriophages %bacterial $iruses) phages& are infectious agents that replicate as obligate intracellular parasites in bacteria" Extracellular phage particles are metabolicall( inert and consist principall( of proteins plus nucleic acid %DNA or RNA) but not both&" !he proteins of the phage particle form a protecti$e shell %capsid& surrounding the tightl( pac1aged nucleic acid genome" Bhage genomes $ar( in size from approximatel( . to .77 1ilobases per strand of nucleic acid and consist of double,stranded DNA) single,stranded DNA) or RNA" Bhage genomes) li1e plasmids) encode functions re5uired for replication in bacteria) but unli1e plasmids the( also encode capsid proteins and nonstructural proteins re5uired for phage assembl(" Se$eral morphologicall( distinct t(pes of phage ha$e been described) including pol(hedral) filamentous) and complex" omplex phages ha$e pol(hedral heads to 'hich tails and sometimes other appendages %tail plates) tail fibers) etc"& are attached" A single c(cle of phage gro'th is sho'n in *ig" ." Infection is initiated b( adsorption of phage to specific receptors on the surface of susceptible host bacteria" !he capsids remain at the cell surface) and the DNA or RNA genomes enter the target cells %penetration&" #ecause infecti$it( of genomic DNA or RNA is much less than that of mature $irus) there is a time immediatel( after infection called the eclipse period during 'hich intracellular infectious phage cannot be detected" !he infecting phage RNA or DNA is replicated to produce man( ne' copies of the phage genome) and phage,specific proteins are produced" *or most phages assembl( of progen( occurs in the c(toplasm) and release of the progen( occurs b( cell l(sis" In contrast) filamentous phages are formed at the cell en$elope and released 'ithout 1illing the host cells" !he eclipse period ends 'hen intracellular infectious progen( appear" !he latent period is the inter$al from infection until extracellular progen( appear) and the rise period is the inter$al from the end of the latent period until all phage are extracellular" !he a$erage number of phage particles produced b( each infected cell) called the burst size) is characteristic for each $irus and often ranges bet'een +7 and se$eral hundred" *or discussions of structure) multiplication) and classification of animal $iruses)

Figure ()

One*ste" gro+t! of bacterio"!age. A culture of susceptible bacteria is synchronously infected with bacteriophage added at time 0 at low multiplicity of infection. Unabsorbed phage is inactivated shortly thereafter by addition of anti-phage antiserum, and the culture is then diluted to prevent further activity of the antiserum. Samples are taken at intervals for phage assays. Total phage intracellular plus e!tracellular" is determined by testing the sample after treating it to disrupt infected bacteria, and e!tracellular phage is determined by testing supernatant after removal of bacteria by centrifugation or ultrafiltration. #hage titers are as the ratio of phage per infected bacterial cell. Bhages are classified into t'o ma4or groupsF $irulent and temperate" Gro'th of $irulent phages in susceptible bacteria destro(s the host cells" Infection of susceptible bacteria b( temperate phages can ha$e either of t'o outcomesF l(tic gro'th or l(sogen(" C(tic gro'th of temperate and $irulent bacteriophages is similar) leading to production of phage progen( and death of the host bacteria" C(sogen( is a specific t(pe of latent $iral infection in 'hich the phage genome replicates as a prophage in the bacterial cell" In most l(sogenic bacteria the genes re5uired for l(tic phage de$elopment are not expressed) and production of infectious phage does not occur" *urthermore) the l(sogenic cells are immune to superinfection b( the $irus 'hich the( harbor as a prophage" !he ph(sical state of the prophage is not identical for all temperate $iruses" *or example) the prophage of bacteriophage l in E coli is integrated into the bacterial chromosome at a specific site and replicates as part of the bacterial chromosome) 'hereas the prophage of bacteriophage B- in E coli replicates as an extrachromosomal plasmid" C(tic phage gro'th occurs spontaneousl( in a small fraction of l(sogenic cells) and a fe' extracellular phages are present in cultures of l(sogenic bacteria" *or some l(sogenic bacteria) s(nchronous induction of l(tic phage de$elopment occurs in the entire population of l(sogenic bacteria 'hen the( are treated 'ith agents that damage DNA) such as ultra$iolet light or mitom(cin " !he loss of prophage from a l(sogenic bacterium) con$erting it to the E

nonl(sogenic state and restoring susceptibilit( to infection b( the phage that 'as originall( present as prophage) is called curing" Some temperate phages contain genes for bacterial characteristics that are unrelated to l(tic phage de$elopment or the l(sogenic state) and expression of such genes is called phage con$ersion %or l(sogenic con$ersion&" Examples of phage con$ersion that are important for microbial $irulence include production of diphtheria toxin b( or(nebacterium diphtheriae) er(throgenic toxin b( Streptococcus p(ogenes %group A b,hemol(tic streptococci&) botulinum toxin b( lostridium botulinum) and Shiga,li1e toxins b( E coli" In each of these examples the gene 'hich encodes the bacterial toxin is present in a temperate phage genome" !he specificit( of O antigens in Salmonella can also be controlled b( phage con$ersion" Bhage t(ping is the testing of strains of a particular bacterial species for susceptibilit( to specific bacteriophages" !he patterns of susceptibilit( to the set of t(ping phages pro$ide information about the possible relatedness of indi$idual clinical isolates" Such information is particularl( useful for epidemiological in$estigations"

Mutation and Selection

Mutations are heritable changes in the genome" Spontaneous mutations in indi$idual bacteria are rare" Some mutations cause changes in phenot(pic characteristics/ the occurrence of such mutations can be inferred from the effects the( produce" In microbial genetics specific reference organisms are designated as 'ild,t(pe strains) and descendants that ha$e mutations in their genomes are called mutants" !hus) mutants are characterized b( the inherited differences bet'een them and their ancestral 'ild,t(pe strains" Gariant forms of a specific genetic determinant are called alleles" Genot(pic s(mbols are lo'er case) italicized abbre$iations that specif( indi$idual genes) 'ith a %6& superscript indicating the 'ild t(pe allele" Bhenot(pic s(mbols are capitalized and not italicized) to distinguish them from genot(pic s(mbols" *or example) the genot(pic s(mbol for the abilit( to produce b, galactosidase) re5uired to ferment lactose) is lacH6) and mutants that cannot produce b, galactosidase are lacH" !he lactose,fermenting phenot(pe is designated Cac6) and inabilit( to ferment lactose is Cac,"

Detection of Mutant &!enot,"es

Selecti$e and differential media are helpful for isolating bacterial mutants" Some selecti$e media permit particular mutants to gro') but do not allo' the 'ild,t(pe strains to gro'" Rare mutants can be isolated b( using such selecti$e media" Differential media permit 'ild,t(pe and mutant bacteria to gro' and form colonies that differ in appearance" Detection of rare mutants on differential media is limited b( the total number of colonies that can be obser$ed" onsider a 'ild,t(pe strain of E coli that is susceptible to the antibiotic streptom(cin %phenot(pe Strs& and can utilize lactose as the sole source of carbon %phenot(pe Cac6&" Spontaneousl( occurring Strr mutants are rare and are usuall( found at fre5uencies of less than one per -7< bacteria in cultures of 'ild,t(pe E coli" Ne$ertheless) Strr mutants can be isolated easil( b( using selecti$e media containing streptom(cin) because the 'ild,t(pe Strs bacteria are 1illed" Isolation of lactose,negati$e %phenot(pe Cac,& mutants of E coli poses a different problem" On minimal media 'ith lactose as the sole source of carbon) Cac6 'ild, t(pe strains 'ill gro') but Cac, mutants cannot gro'" On differential media such as Mac on1e(,lactose agar or eosin,meth(lene blue,lactose agar) Cac6 'ild,t(pe and Cac, mutant strains of E coli can be distinguished b( their color) but spontaneous Cac, mutants are =

too rare to be isolated easil(" Selecti$e media for Cac, mutants of E coli can be made b( incorporating chemical analogs of lactose that are con$erted into toxic metabolites b( Cac6 bacteria but not b( Cac, mutants" !he Cac, mutants can then gro' on such media) but the Cac6 'ild,t(pe bacteria are 1illed" Mutations that inacti$ate essential genes in haploid organisms are usuall( lethal) but such potentiall( lethal mutations can often be studied if their expression is controlled b( manipulation of experimental conditions" *or example) a mutation that increases the thermolabilit( of an essential gene product ma( pre$ent bacterial gro'th at 8.I ) although the mutant bacterium can still gro' at .+I " on$ersel() cold,sensiti$e mutants express the mutant phenot(pe at lo' temperature) but not at high temperature" !emperature,sensiti$e and cold,sensiti$e mutations are examples of conditional mutations) as are suppressible mutations described later in this chapter" A conditional lethal phenot(pe indicates that the mutant gene is essential for $iabilit("

S"ontaneous and Induced Mutations

!he mutation rate in bacteria is determined b( the accurac( of DNA replication) the occurrence of damage to DNA) and the effecti$eness of mechanisms for repair of damaged DNA" *or a particular bacterial strain under defined gro'th conditions) the mutation rate for an( specific gene is constant and is expressed as the probabilit( of mutation per cell di$ision" In a population of bacteria gro'n from a small inoculum) the proportion of mutants usuall( increases progressi$el( as the size of the bacterial population increases" Mutations in bacteria can occur spontaneousl( and independentl( of the experimental methods used to detect them" !his principle 'as first demonstrated b( the fluctuation test %*ig" 2&" !he numbers of phage,resistant mutants of E coli in replicate cultures gro'n from small inocula 'ere measured and compared 'ith those in multiple samples ta1en from a single culture" If mutations to phage resistance occurred onl( after exposure to phage) the $ariabilit( in numbers of mutants bet'een cultures should be similar under both sets of conditions" In contrast) if phage,resistant mutants occurred spontaneousl( before exposure of the bacteria to phage) the numbers of mutants should be more $ariable in the independentl( gro'n cultures) because differences in the size of the bacterial population 'hen the first mutant appeared 'ould contribute to the obser$ed $ariabilit(" !he data indicated that the mutations to phage resistance in E coli occurred spontaneousl( 'ith constant probabilit( per cell di$ision"

<

FIGURE 3. The fluctuation test. $ifferences in numbers of colonies of phage-resistant mutants in replicate samples from single subculture were small and reflected only e!pected fluctuations due to sampling errors. %n contrast, numbers of phage-resistant colonies in samples from individual subcultures were more variable and reflected both sampling errors and the independent origins of mutants in individual subcultures. Si&es of clonal populations of mutants in each culture reflected numbers of generations of growth between times that mutations occurred and time of sampling.

Replica plating confirmed that mutations in bacteria can occur spontaneousl() 'ithout exposure of bacteria to selecti$e agents %*ig" 8&" *or replica plating) a flat) sterile) $el$eteen surface is used to pic1 up an inoculum from the surface of an agar master plate and transfer samples to other agar plates" In this manner) samples of the bacterial population from the master plate are transferred to the replica plates 'ithout distorting their spatial arrangement" If the replica plates contain selecti$e medium and the master plates do not) the positions of selected mutant colonies on the replica plates can be noted) and bacteria that 'ere not exposed to the selecti$e conditions can be isolated from the same positions on the master plate" Mutants of E coli resistant to bacteriophage !- or to streptom(cin ha$e been isolated in this 'a() 'ithout exposing the 'ild,t(pe bacteria to the bacteriophage or the antibiotic"

-7

FIGUR# - Detecting "ree$isting bacterial mutants b, re"lica "lating Master plate 'as hea$il( inoculated 'ith sample from pure cultures of phage,susceptible bacterium" After incubation) bacteria from master plate 'ere transferred b( replica plating to duplicate agar plates impregnated 'ith bacteriophage" Bhage,susceptible bacteria 'ere 1illed b( the bacteriophage" olonies of phage,resistant bacteria appeared at identical positions on duplicate plates) indicating that phage,resistant bacteria had been transferred to each replica plate from the corresponding locations on master plate" #acterial inocula selected from appropriate locations on master plate contained a higher proportion of phage,resistant mutants than original bacterial culture" #( repeating these procedures se$eral times) it 'as possible to isolate pure cultures of phage,resistant bacterial mutants that had ne$er been exposed to bacteriophage" #oth en$ironmental and genetic factors affect mutation rates" Exposure of bacteria to mutagenic agents causes mutation rates to increase) sometimes b( se$eral orders of magnitude" Man( chemical and ph(sical agents) including J,ra(s and ultra$iolet light) ha$e mutagenic acti$it(" hemicals that are carcinogenic for animals are often mutagenic for bacteria) or can be con$erted b( animal tissues to metabolites that are mutagenic for bacteria" Standardized tests for mutagenicit( in bacteria are used as screening procedures to identif( en$ironmental agents that ma( be carcinogenic in humans" Mutator genes in bacteria cause an increase in spontaneous mutation rates for a 'ide $ariet( of other genes" Expression of these genes) induced b( DNA damage %see SOS response later&) enables the repair of DNA lesions that 'ould other'ise be lethal) but b( an error,prone mechanism that increases the rate of mutation" !he o$erall mutation ratethe probabilit( that a mutation 'ill occur some'here in the bacterial genome per cell di$isionis relati$el( constant for a $ariet( of organisms 'ith genomes of different sizes and appears to be a significant factor in determining the fitness of --

a bacterial strain for sur$i$al in nature" Most mutations are deleterious) and the ris1 of ad$erse mutations for indi$idual bacteria must be balanced against the positi$e $alue of mutabilit( as a mechanism for adaptation of bacterial populations to changing en$ironmental conditions" Molecular 'asis of Mutations Mutations are classified on the basis of structural changes that occur in DNA %!able .&" Some mutations are localized 'ithin short segments of DNA %for example) nucleotide substitutions) microdeletions) and microinsertions&" Other mutations in$ol$e large regions of DNA and include deletions) insertions) or rearrangements of segments of DNA"

9hen a nucleotide substitution occurs in a region of DNA that codes for a pol(peptide) one of the three nucleotides 'ithin a single codon of a corresponding mRNA molecule 'ill be changed" Silent mutations cause no change in pol(peptide structure or function) because one codon in mRNA is changed to another for the same amino acid" Other substitutions cause one amino acid to be replaced b( another at the specific position 'ithin the pol(peptide corresponding to the altered codon" Mutations that result in replacement of one amino acid for another 'ithin a pol(peptide chain are called missense mutations" !he effects of amino acid replacements on the function of a pol(peptide gene product $ar( and depend on the location and the identit( of the amino acid replacement" Mutant pol(peptides containing amino acid replacements usuall( share antigenic determinants 'ith the 'ild,t(pe pol(peptide and often ha$e some residual biologic acti$it(" Mutations that result in replacement of an amino acid codon 'ith a termination codon are called nonsense mutations" !his results in production of an amino,terminal fragment of the normal pol(peptide 'hen the mutant mRNA is translated" Nonsense mutations often result in complete loss of acti$it( of the gene product" #ecause of the triplet nature of the genetic code) the conse5uences of mutations caused b( insertions or deletions of small numbers of nucleotides %microinsertions) microdeletions& depend on both the number and se5uence of nucleotides in$ol$ed" Deletion or addition of multiples of three nucleotide pairs does not affect the reading frame) but causes deletion or addition of appropriate numbers of amino acids at one site 'ithin the pol(peptide" If a ne' -.

chain,terminating codon is introduced) premature chain termination occurs 'ithin the pol(peptide" In contrast) addition or deletion of other numbers of nucleotide pairs alters the reading frame for the entire segment of mRNA from the mutation to the distal end of the gene" !herefore) frameshift mutations are li1el( to cause drastic changes in the structure and acti$it( of pol(peptide gene products) and the( are often classified as nonsense mutations" Com"lementation Tests !o determine if mutations are located in the same gene or different genes) complementation tests are performed 'ith partiall( diploid bacterial strains %*ig" +,:&" !'o copies of the region of the bacterial chromosome harboring a mutation are present in the same bacterium) 'ith each cop( containing a different mutation %mutations are in the trans arrangement&" A 'ild, t(pe phenot(pe indicates that the mutations are in different genes" !his phenomenon is called complementation" If a mutant phenot(pe is obser$ed) a control experiment should be performed 'ith the mutations in the cis arrangement to exclude the possibilit( that the 'ild, t(pe alleles cannot be expressed normall( in a partiall( diploid bacterial strain" omplementation tests 'ere originall( called ;cis,trans; tests) and the term cistron is sometimes used as a s(non(m for gene" omplementation tests can be performed and interpreted e$en if the specific biochemical functions of the gene products are un1no'n"

FIGUR# . Com"lementation is a met!od to test for functional gene "roducts !'o mutants 'ith similar phenot(pes %inabilit( to con$ert substrate J to product H& 'ere isolated" Mutations in these strains are designated a and b) respecti$el() and the 'ild t(pe alleles are a6 and b6" Bartiall( diploid heteroz(gous strains 'ere tested to determine if mutations a and b 'ere in the same structural gene %cistron& and inacti$ated the same gene products" A&) If a and b are in the same structural gene %e"g") encoding the enz(me that con$erts J to K&) neither the a6b nor the ab6 allele codes for an acti$e enz(me) substrate J cannot be utilized) the mutant phenot(pe is expressed) and no complementation occurs" #&) If a and b are in different cistrons %e"g") encoding the enz(mes that con$ert J to K and K to H&) the a6 and b6 alleles encode acti$e enz(mes) substrate J is con$erted to product H) the 'ild t(pe phenot(pe is expressed) and complementation occurs" -2

As an example) consider using a complementation test to characterize t'o independentl( deri$ed Cac, mutants of E coli" !he biochemical path'a( for utilization of lactose re5uires L, galactoside permease %genot(pic s(mbol lacK& to transport lactose into the bacterial cell and b,galactosidase %genot(pic s(mbol lacH& to con$ert lactose into D,glucose and D,galactose" Mutants that lac1 b,galactoside permease or b,galactosidase cannot utilize lactose for gro'th" If the mutations in both Cac, mutants inacti$ated the same protein %e"g") b,galactoside& then a partial diploid strain containing the lacH genes from both mutants in the trans arrangement 'ould be unable to utilize lactose" In contrast) if the genot(pes of the t'o mutants 'ere lacH6 lacK and lacH lacK6) the partiall( diploid bacterium 'ould produce acti$e b,galactosidase from the lacH6 determinant and acti$e b,galactoside permease from the lacK6 determinant" omplementation 'ould occur) and the partiall( diploid strain 'ould utilize lactose"

Re/ersion and Su""ression

Mutations that con$ert the phenot(pe from 'ild,t(pe to mutant are called for'ard mutations) and mutations that change the phenot(pe from mutant bac1 to 'ild,t(pe are called re$erse mutations %re$ersions&" #acterial strains that contain re$erse mutations are called re$ertants" Anal(sis of mutations that cause phenot(pic re$ersion (ields useful information" Re$erse mutations that restore the exact nucleotide se5uence of the 'ild,t(pe DNA are true re$ersions" !rue re$ertants are identical to 'ild,t(pe strains both genot(picall( and phenot(picall(" Re$erse mutations that do not restore the exact nucleotide se5uence of the 'ild,t(pe DNA are called suppressor mutations %suppressors&" Some re$ertants that harbor suppressor mutations are phenot(picall( indistinguishable from 'ild,t(pe strains" Other re$ertants) called pseudore$ertants) can be distinguished phenot(picall( from 'ild,t(pe strains) for example) b( subtle differences in the characteristics of an enz(matic acti$it( that has been regained %such as specific acti$it() substrate specificit() 1inetic constants) or susceptibilit( to thermal or chemical inacti$ation&" Recognition of pseudore$ertant phenot(pes suggests the presence of suppressor mutations" Suppressor mutations can be intragenic or extragenic" Intragenic suppressors are located in the same gene as the for'ard mutations that the( suppress" !he possible locations and nature of intragenic suppressors are determined b( the original for'ard mutation and b( the relationships bet'een the primar( structure of the gene product and its biologic acti$it(" Extragenic suppressors are located in different genes from mutations 'hose effects the( suppress" !he abilit( of extragenic suppressors to suppress a $ariet( of independent mutations can be tested" Some extragenic suppressors are specific for particular genes) some are specific for particular codons) and some ha$e other specificit( patterns" Extragenic suppressors that re$erse the phenot(pic effects of chain,terminating codons ha$e been 'ell characterized and found to alter the structure of specific tRNAs" A particular suppressor tRNA can permit a specific chain,terminating codon to be translated) resulting in incorporation of a specific amino acid into the nascent pol(peptide at the position corresponding to the chain, terminating codon" In a bacterium that has a chain,terminating mutation and an appropriate extragenic suppressor) translation of the mRNA containing the mutant codon can therefore result in formation of a full,length pol(peptide" !he biologic acti$it( of the full,length pol(peptide formed as a conse5uence of suppression depends both on the amount of protein made and on the functional conse5uences of the specific amino acid replacement determined b( the suppressor tRNA" -8

#$c!ange of Genetic Information

!he biologic significance of sexualit( in microorganisms is to increase the probabilit( that rare) independent mutations 'ill occur together in a single microbe and be sub4ected to natural selection" Genetic interactions bet'een microbes enable their genomes to e$ol$e much more rapidl( than b( mutation alone" Representati$e phenomena of medical importance that in$ol$e exchanges of genetic information or genomic rearrangements include the rapid emergence and dissemination of antibiotic resistance plasmids) flagellar phase $ariation in Salmonella) and antigenic $ariation of surface antigens in Neisseria and #orrelia" Sexual processes in bacteria in$ol$e transfer of genetic information from a donor to a recipient and result either in substitution of donor alleles for recipient alleles or addition of donor genetic elements to the recipient genome" !ransformation) transduction) and con4ugation are sexual processes that use different mechanisms to introduce donor DNA into recipient bacteria %*ig" :&" #ecause donor DNA cannot persist in the recipient bacterium unless it is part of a replicon) recombination bet'een donor and recipient genomes is often re5uired to produce stable) h(brid progen(" Recombination is most li1el( to occur 'hen the donor and recipient bacteria are from the same or closel( related species"

FIGUR# 0 #$c!ange of genetic information in bacteria !ransformation) transduction) and con4ugation differ in means for introducing DNA from donor cell into recipient cell" A& In transformation) fragments of DNA released from donor bacteria are ta1en up b( competent recipient bacteria" #& In transduction) abnormal bacteriophage particles containing DNA from donor bacteria in4ect their DNA into recipient bacteria" & on4ugation occurs b( formation of c(toplasmic connections bet'een donor and recipient bacteria) 'ith direct transfer of -+

ne'l( s(nthesized donor DNA into the recipient cells" In all three cases) recombination bet'een donor and recipient DNA molecules is re5uired for formation of stable recombinant genomes" #acterial genome is represented diagrammaticall( as a circular element in bacterial cells" Donor and recipient DNA are indicated b( fine lines and hea$( lines) respecti$el(" In each recombinant genome) the a6 allele from donor strain has replaced the a allele from recipient strain) and the b6 allele is deri$ed from recipient strain" *or a recombinant to be detected) its phenot(pe must be different from both parental phenot(pes" Gro'th or cell di$ision ma( be re5uired before the recombinant phenot(pe is expressed" Dela( in expression of a recombinant phenot(pe until a haploid recombinant genome has segregated is called segregation lag) and dela( until s(nthesis of products encoded b( donor genes has occurred is called phenot(pic lag"

Transformation
In transformation) pieces of DNA released from donor bacteria are ta1en up directl( from the extracellular en$ironment b( recipient bacteria" Recombination occurs bet'een single molecules of transforming DNA and the chromosomes of recipient bacteria" !o be acti$e in transformation) DNA molecules must be at least +77 nucleotides in length) and transforming acti$it( is destro(ed rapidl( b( treating DNA 'ith deox(ribonuclease" Molecules of transforming DNA correspond to $er( small fragments of the bacterial chromosome" otransformation of genes is unli1el() therefore) unless the( are so closel( lin1ed that the( can be encoded on a single DNA fragment" !ransformation 'as disco$ered in Streptococcus pneumoniae and occurs in other bacterial genera including >aemophilus) Neisseria) #acillus) and Staph(lococcus" !he abilit( of bacteria to ta1e up extracellular DNA and to become transformed) called competence) $aries 'ith the ph(siologic state of the bacteria" Man( bacteria that are not usuall( competent can be made to ta1e up DNA b( laborator( manipulations) such as calcium shoc1 or exposure to a high,$oltage electrical pulse %electroporation&" In some bacteria %including >aemophilus and Neisseria& DNA upta1e depends on the presence of specific oligonucleotide se5uences in the transforming DNA) but in others %including Streptococcus pneumoniae& DNA upta1e is not se5uence,specific" ompetent bacteria ma( also ta1e up intact bacteriophage DNA %transfection& or plasmid DNA) 'hich can then replicate as extrachromosomal genetic elements in the recipient bacteria" In contrast) a piece of chromosomal DNA from a donor bacterium usuall( cannot replicate in the recipient bacterium unless it becomes part of a replicon b( recombination" >istoricall() characterization of ;transforming principle; from S pneumoniae pro$ided the first direct e$idence DNA is genetic material"

Transduction
In transduction) bacteriophages function as $ectors to introduce DNA from donor bacteria into recipient bacteria b( infection" *or some phages) called generalized transducing phages) a small fraction of the $irions produced during l(tic gro'th are aberrant and contain a random fragment of the bacterial genome instead of phage DNA" Each indi$idual transducing phage carries a different set of closel( lin1ed genes) representing a small segment of the bacterial genome" !ransduction mediated b( populations of such phages is called generalized transduction) because each part of the bacterial genome has approximatel( the same probabilit( of being transferred from donor to recipient bacteria" 9hen a generalized transducing phage infects a recipient cell) expression of the transferred donor genes occurs" Aborti$e transduction refers to the transient expression of one or more donor genes 'ithout -:

formation of recombinant progen() 'hereas complete transduction is characterized b( production of stable recombinants that inherit donor genes and retain the abilit( to express them" In aborti$e transduction the donor DNA fragment does not replicate) and among the progen( of the original transductant onl( one bacterium contains the donor DNA fragment" In all other progen( the donor gene products become progressi$el( diluted after each generation of bacterial gro'th until the donor phenot(pe can no longer be expressed" On selecti$e medium upon 'hich onl( bacteria 'ith the donor phenot(pe can gro') aborti$e transductants produce minute colonies that can be distinguished easil( from colonies of stable transductants" !he fre5uenc( of aborti$e transduction is t(picall( one to t'o orders of magnitude greater than the fre5uenc( of generalized transduction) indicating that most cells infected b( generalized transducing phages do not produce recombinant progen(" Specialized transduction differs from generalized transduction in se$eral 'a(s" It is mediated onl( b( specific temperate phages) and onl( a fe' specific donor genes can be transferred to recipient bacteria" Specialized transducing phages are formed onl( 'hen l(sogenic donor bacteria enter the l(tic c(cle and release phage progen(" !he specialized transducing phages are rare recombinants 'hich lac1 part of the normal phage genome and contain part of the bacterial chromosome located ad4acent to the prophage attachment site" Man( specialized transducing phages are defecti$e and cannot complete the l(tic c(cle of phage gro'th in infected cells unless helper phages are present to pro$ide missing phage functions" Specialized transduction results from l(sogenization of the recipient bacterium b( the specialized transducing phage and expression of the donor genes" Bhage con$ersion and specialized transduction ha$e man( similarities) but the origin of the con$erting genes in temperate con$erting phages is un1no'n"

Con1ugation
In con4ugation) direct contact bet'een the donor and recipient bacteria leads to establishment of a c(toplasmic bridge bet'een them and transfer of part or all of the donor genome to the recipient" Donor abilit( is determined b( specific con4ugati$e plasmids called fertilit( plasmids or sex plasmids" !he * plasmid %also called * factor& of E coli is the protot(pe for fertilit( plasmids in Gram, negati$e bacteria" Strains of E coli 'ith an extrachromosomal * plasmid are called *6 and function as donors) 'hereas strains that lac1 the * plasmid are *, and beha$e as recipients" !he con4ugati$e functions of the * plasmid are specified b( a cluster of at least .+ transfer %tra& genes 'hich determine expression of * pili) s(nthesis and transfer of DNA during mating) interference 'ith the abilit( of *6 bacteria to ser$e as recipients) and other functions" Each *6 bacterium has - to 2 * pili that bind to a specific outer membrane protein %the ompA gene product& on recipient bacteria to initiate mating" An intercellular c(toplasmic bridge is formed) and one strand of the * plasmid DNA is transferred from donor to recipient) beginning at a uni5ue origin and progressing in the +3 to 23 direction" !he transferred strand is con$erted to circular double,stranded * plasmid DNA in the recipient bacterium) and a ne' strand is s(nthesized in the donor to replace the transferred strand" #oth of the excon4ugant bacteria are *6) and the * plasmid can therefore spread b( infection among geneticall( compatible populations of bacteria" In addition to the role of the * pili in con4ugation) the( also function as receptors for donor,specific %male,specific& phages" !he * plasmid in E coli can exist as an extrachromosomal genetic element or be integrated into the bacterial chromosome %*ig" E&" #ecause the * plasmid and the bacterial chromosome -E

are both circular DNA molecules) reciprocal recombination bet'een them produces a larger DNA circle consisting of * plasmid DNA inserted linearl( into the chromosome" E coli contains multiple copies of se$eral different genetic elements called insertion se5uences %see section on transposons for more detail&) at $arious locations in its chromosome and in the * plasmid" >omologous recombination bet'een insertion se5uences in the chromosome and the * plasmid leads to preferential integration of the * plasmid at chromosomal sites 'here insertion se5uences are located" !he chromosomal sites 'here insertion se5uences are found $ar() ho'e$er) among strains of E coli"

FIGUR# 2 Role of F "lasmid in determining donor and reci"ient states of E coli. !he * plasmid is representati$e of specific con4ugati$e plasmids that control donor abilit( in E coli" *, strains lac1 the * plasmid and are genetic recipients" *6 strains harbor the * plasmid as a c(toplasmic element) express * pili) and are genetic donors" !he * plasmid can become integrated into bacterial chromosome at $arious locations to produce >fr %high,fre5uenc( recombination& donor strains" Abnormal excision of * plasmid can result in formation of *3 plasmids that contain segments of bacterial chromosome and the corresponding bacterial genes" !he arro'head in * plasmid defines origin for transfer of DNA during con4ugation" * plasmid and chromosomal DNA are indicated b( hea$( and fine lines) respecti$el(" *or additional data concerning the genomes of >fr and *3 strains) see *ig" E" An E coli strain 'ith an integrated * plasmid retains its abilit( to function as a donor in con4ugal matings" #ecause donor strains 'ith integrated * factors can transfer chromosomal genes to recipients 'ith high efficienc() the( are called >fr %>igh fre5uenc( recombination& strains" !ransfer of single,stranded DNA from an >fr donor to a recipient begins from the origin 'ithin the * plasmid and proceeds as described abo$e) except that the transferred DNA is the h(brid replicon consisting of * plasmid integrated into the bacterial chromosome" !ransfer of this entire replicon) including the bacterial chromosome) re5uires approximatel( -77 minutes" !he identit( of the first chromosomal gene to be transferred and the polarit( of -=

chromosomal transfer are determined b( the site of integration of the * plasmid and its orientation 'ith respect to the bacterial chromosome" #ecause the mating bacteria usuall( separate spontaneousl( before the entire chromosome is transferred) con4ugation t(picall( transfers onl( a fragment of the donor chromosome into the recipient" !he probabilit( that a donor gene 'ill enter the recipient bacterium during con4ugation decreases) therefore) as its distance from the * origin %and therefore the time of its transfer& increases" Mating cells can also be bro1en apart experimentall( b( sub4ecting them to strong shearing forces in a mechanical blender/ this is called interrupted mating" *ormation of recombinant progen( re5uires recombination bet'een the transferred donor DNA and the genome of the recipient bacterium" Anal(sis of progen( from matings that are interrupted after different inter$als demonstrates 'hich chromosomal genes are transferred first b( particular donor strains) the se5uential times of entr( for genes that are transferred subse5uentl() and the progressi$el( lo'er probabilit( that genes transferred later 'ill appear in recombinant progen(" !he circularit( of the genetic map of E coli 'as originall( deduced from the o$erlapping) circularl( permuted groups of lin1ed genes that 'ere transferred earl( b( indi$idual donor strains in 'hich the * factor 'as integrated at different chromosomal locations" In matings bet'een *6 and *, bacteria) onl( the * plasmid is transferred 'ith high efficienc( to recipients" hromosomal genes are transferred 'ith $er( lo' efficienc() and it is the spontaneous >fr mutants in *6 populations that mediate transfer of donor chromosomal genes" In matings bet'een >fr and *, strains) the segment of the * plasmid containing the tra region is transferred last) after the entire bacterial chromosome has been transferred" Most recombinants from matings bet'een >fr and *, cells fail to inherit the entire set of * plasmid genes and are phenot(picall( *," In matings bet'een *6 and *, strains) the * plasmid spreads rapidl( throughout the bacterial population) and most recombinants are *6" Integrated * plasmids in >fr strains can sometimes be excised from the bacterial chromosome" If excision precisel( re$erses the integration process) *6 cells are produced" On rare occasions) ho'e$er) excision occurs b( recombinations in$ol$ing insertion se5uences or other genes on the bacterial chromosome that are located at some distance from the original integration site" In such cases segments of the bacterial chromosome can become incorporated into h(brid * plasmids that are called *3 plasmids %see *ig" +,<&" #( similar processes) segments of the bacterial chromosome can sometimes become incorporated into R plasmids to produce h(brid R3 plasmids" on4ugati$e R3 plasmids can function as fertilit( plasmids because the( can integrate into the bacterial chromosome b( homologous recombination and mediate transfer of chromosomal genes during matings 'ith recipient bacteria" *3 plasmids) R3 plasmids) specialized transducing phages) and recombinant plasmids or phages constructed b( gene cloning %described belo'& are h(brid replicons that can include segments of the bacterial chromosome" !herefore) an( of these genetic elements can be used to construct the partiall( diploid bacterial strains that are re5uired for complementation tests and other purposes" on4ugation also occurs in Gram,positi$e bacteria" Gram,positi$e donor bacteria produce adhesins that cause them to aggregate 'ith recipient cells) but sex pili are not in$ol$ed" In some Streptococcus species) recipient bacteria produce extracellular sex pheromones that cause the donor phenot(pe to be expressed b( bacteria that harbor an appropriate con4ugati$e plasmid) and the con4ugati$e plasmid pre$ents the donor cells from producing the corresponding pheromone"

Recombination
-<

Recombination in$ol$es brea1age and 4oining of parental DNA molecules to form h(brid) recombinant molecules" Se$eral distinct 1inds of recombination ha$e been identified that depend on different features of the participating genomes and re5uire the acti$ities of different gene products" Specific enz(mes that act on DNA %for example) exonucleases) endonucleases) pol(merases) ligases& participate in recombination" Detailed discussion of the biochemical e$ents in recombination is be(ond the scope of this chapter" Generalized recombination in$ol$es donor and recipient DNA molecules that ha$e homologous nucleotide se5uences" Reciprocal exchanges can occur bet'een an( homologous donor and recipient sites" In E coli) the product of the recA gene is essential for generalized recombination) but other gene products also participate" Site,specific recombination in$ol$es reciprocal exchanges onl( bet'een specific sites in donor and recipient DNA molecules" !he recA gene product is not re5uired for site,specific recombination" Integration of the temperate bacteriophage l into the chromosome of E coli is a 'ell,studied example of site,specific recombination %*ig" +,-7&" !he specific attachment %att& sites on the E coli chromosome and l phage DNA ha$e a common core se5uence of -+ nucleotides) 'ithin 'hich reciprocal recombination occurs) flan1ed b( ad4acent se5uences that are not homologous in the phage and bacterial genomes" In phage l the product of the int gene %integrase& is re5uired for the site,specific integration e$ent in l(sogenization/ the products of the int and xis %excisionase& genes are both needed for the complementar( site, specific excision e$ent that occurs during induction of l(tic phage de$elopment in l(sogenic cells" Illegitimate recombination is the term used to describe nonhomologous) aberrant recombination e$ents such as those in$ol$ed in formation of specialized transducing phages" !he mechanisms of illegitimate recombination are un1no'n"

.7

FIGUR# 3 Integration and e$cision of bacterio"!age l are e$am"les of site*s"ecific recombination l DNA is sho'n b( thin lines and chromosomal DNA b( thic1 lines" Attachment %att& sites are closed boxes for the bacterial chromosome and open boxes for the l chromosome" !he gal and bio operons) 'hich determine utilization of galactose and bios(nthesis of biotin) are located ad4acent to the bacterial attachment site" In an infected E coli the l DNA becomes circular b( 4oining ends m and m3) and site,specific recombination bet'een phage and bacterial att sites results in insertion of the l genome into the bacterial chromosome" !he arrangement of the prophage DNA %m and m3 located internall(& is) therefore) a circular permutation of l $irion DNA %m and m3 located terminall(&"

Trans"osons
!ransposons are segments of DNA that can mo$e from one site in a DNA molecule to other target sites in the same or a different DNA molecule" !he process is called transposition and occurs b( a mechanism that is independent of generalized recombination" !ransposons are important genetic elements because the( cause mutations) mediate genomic rearrangements) function as portable regions of genetic homolog() and ac5uire ne' genes and contribute to their dissemination 'ithin bacterial populations" Insertion of a transposon often interrupts the linear se5uence of a gene and inacti$ates it" !ransposons ha$e a ma4or role in causing deletions) duplications) and in$ersions of DNA segments as 'ell as fusions bet'een replicons" !ransposons are not self,replicating genetic elements) ho'e$er) and the( must integrate into other replicons to be maintained stabl( in bacterial genomes" Most transposons share a number of common features" Each transposon encodes the functions necessar( for its transposition) including a transposase enz(me that interacts 'ith specific se5uences at the ends of the transposon" During transposition a short se5uence of target DNA is duplicated) and the transposon is inserted bet'een the directl( repeated target se5uences" !he length of this short duplication $aries) but is characteristic for each transposon" !he duplication is presumed to in$ol$e as(mmetric clea$age of DNA at the target site) follo'ed b( s(nthesis of ne' complementar( strands corresponding to the region bet'een the clea$age sites" Some transposons insert into almost an( target se5uence) 'hereas others ha$e relati$el( stringent target specificit(" !'o t(pes of transposition are recognized" Excision of the transposon from a donor site follo'ed b( its insertion into a target site is called nonreplicati$e transposition" If the transposon at a donor site is replicated and a cop( is inserted into the target site) ho'e$er) the process is called replicati$e transposition" !he process of replicati$e transposition can in$ol$e formation of a cointegrate) a single circular DNA molecule consisting of t'o replicons 4oined 'ith copies of the transposon in an alternating se5uence" Resolution of the cointegrate into its component replicons is often accomplished b( a transposon,encoded resol$ase that catal(zes site,specific recombination bet'een the transposons" Generalized recombination bet'een homologous transposons can also lead to the formation or resolution of cointegrates" !ransposition differs from site, specific recombination b( duplicating a segment of the target se5uence and b( using a $ariet( of different target se5uences for a single donor se5uence" Most transposons in bacteria can be separated into three ma4or classes %*ig" +,--&" Insertion se5uences and related composite transposons comprise the first class" Insertion se5uences are simplest in structure and encode onl( the functions needed for transposition" !he 1no'n insertion se5uences $ar( in length from approximatel( E=7 to -+77 nucleotide pairs) ha$e short %-+,.+ base pair& in$erted repeats at their ends) and are not closel( related to each other" !he DNA bet'een the in$erted terminal repeats contains one %or rarel( t'o& transposase .-

genes and does not encode a resol$ase" omplex transposons $ar( in length from about .)777 to more than 87)777 nucleotide pairs and contain insertion se5uences %or closel( related se5uences& at each end) usuall( as in$erted repeats" !he entire complex element can transpose as a unit" !he DNA bet'een the terminal insertion se5uences of complex transposons encodes multiple functions that are not essential for transposition" In medicall( important bacteria) genes that determine production of adherence antigens) toxins) or other $irulence factors) or specif( resistance to one or more antibiotics) are often located in complex transposons" 9ell,1no'n examples of complex transposons are !n+ and !n-7) 'hich determine resistance to 1anam(cin and tetrac(cline) respecti$el(" !he complex transposons probabl( e$ol$e b( transposition of homologous insertion se5uences to nearb( sites 'ithin a DNA molecule"

FIGUR# 4 Features of re"resentati/e trans"osons 5!ea/, lines6 integrated into t!e bacterial c!romosome 5fine lines6 !ransposons are important genetic elements because the( cause mutations) mediate genomic rearrangements) function as portable regions of genetic homolog() and ac5uire ne' genes and contribute to their dissemination 'ithin bacterial populations" -A& IS- insertion se5uence %E=: base pairs& has transposase gene flan1ed b( in$erted terminal repeats %hatched bars 'ith arro's abo$e them&" !he IS- element is flan1ed b( copies of target site %open arro's& 'ith same orientation" -#& omposite transposon !n+ %+=-: base pairs& consists of 1anam(cin resistance determinant flan1ed b( in$erted copies of IS+7 insertion element" .&) !ransposon !nA %8<+E base pairs& contains ampicillin resistance determinant) transposase and resol$ase genes bet'een terminal in$erted repeat se5uences %hatched bars 'ith arro's abo$e them&) flan1ed b( direct repeats of target site %open arro's&" 2& Bhage Mu %2E 1ilobase pairs& encodes transposase that catal(zes recombination bet'een the ends of Mu DNA and target DNA" Direct repeats of the target site %open arro's& flan1 the integrated Mu genome" Mu $irion DNA is longer than Mu prophage ..

and contains chromosomal se5uences at both ends) reflecting the process b( 'hich prophage Mu is excised and pac1aged" !he second class of transposons consists of the highl( homologous !nA famil(" !hese transposons ha$e longer %2+ to 87 base pair& terminal in$erted repeats than the complex transposons described abo$e) but the( lac1 terminal insertion se5uences" All members of the famil( encode both transposase and resol$ase functions" 9ell 1no'n examples from the !nA transposon famil( include the ampicillin resistance transposon !n2 and !n-777 %the gamma, delta transposon& found in the * plasmid" !he !nA famil( has an important place in the histor( of medical microbiolog(" !he de$elopment of high,le$el resistance to ampicillin in >aemophilus influenzae and Neisseria gonorrhoeae during the -<E7s) 'hich se$erel( limited the usefulness of ampicillin for treatment of gonorrhea and >aemophilus infections in areas 'here such strains became pre$alent) 'as caused b( dissemination of ampicillin resistance determinants from !nA transposons in plasmids of the Enterobacteriaceae to plasmids in >aemophilus and Neisseria" !he third class of transposons consists of bacteriophage Mu and related temperate phages" !he entire phage genome functions as a transposon) and replication of the phage DNA during $egetati$e gro'th occurs b( replicati$e transposition" Brophage integration can occur at man( different sites in the bacterial chromosome and often causes mutations" *or that reason Mu and related phages are sometimes called mutator phages" A fourth class of transposons) disco$ered in Gram,positi$e bacteria and represented b( !n<-E) consists of con4ugati$e transposons that are completel( different from the transposons described abo$e" !he con4ugati$e transposon does not generate a duplication of the target se5uence into 'hich it inserts) and in Gram,positi$e bacteria the host strain carr(ing the transposon can act as a con4ugal donor" Recipient bacteria need not be closel( related to the donor bacterium" !he transposon is excised from the chromosome of the donor and transmitted b( con4ugation to the recipient) 'here it integrates randoml( into the chromosome" !n<-E encodes tetrac(cline resistance) but other larger con4ugati$e transposons ma( encode additional antibiotic resistances" on4ugati$e transposons appear to be a ma4or cause of the spread of antibiotic resistance in Gram,positi$e bacteria" Some roles of transposons in bacterial e$olution are illustrated b( considering enteric Gram, negati$e bacteria and the structure of their plasmids" #acteria collected during the pre, antibiotic era contained man( plasmids) but the( usuall( lac1ed resistance determinants" Man( of the R plasmids from current clinical isolates belong to the same incompatibilit( groups as plasmids found pre$iousl() but the( also determine resistance to multiple antibiotics" !he close relationships bet'een their replicons pro$ide strong e$idence that man( current R plasmids e$ol$ed from the older plasmids b( ac5uisition of resistance determinants" Some of the multiple antibiotic resistant plasmids ha$e indi$idual transposons 'ith se$eral resistance determinants) others ha$e multiple resistance transposons located at separate sites) and still others contain complex h(brid resistance transposons formed b( integration of one transposon into another" !he step'ise ac5uisition of resistance determinants can lead) in some cases) to the formation of composite transposons that encode multiple resistance determinants" !herapeutic use of antibiotics and their incorporation into animal feeds pro$ide selecti$e ad$antages for bacteria 'ith R plasmids) 'hereas con4ugation) transformation and transfection pro$ide means for dissemination of R plasmids 'ithin and bet'een bacterial species" After a plasmid carr(ing a transposon is introduced into a ne' bacterial host) the transposon and its determinants can 4ump into the chromosome or indigenous plasmids of the .2

ne' host" !herefore) stabilit( of the mobilizing plasmid in a ne' bacterial host is not essential for persistence of genetic determinants located on a transposon"

Recombination DNA and Gene Cloning

Man( methods are a$ailable to ma1e h(brid DNA molecules in $itro %recombinant DNA& and to characterize them" Such methods include isolating specific genes in h(brid replicons) determining their nucleotide se5uences) and creating mutations at designated locations %site, directed mutagenesis&" A clone is a population of organisms or molecules deri$ed b( asexual reproduction from a single ancestor" Gene cloning is the process of incorporating foreign genes into h(brid DNA replicons" loned genes can be expressed in appropriate host cells) and the phenot(pes that the( determine can be anal(zed" Some 1e( concepts underl(ing representati$e methods are summarized here" !he first step in gene cloning is to ma1e fragments of the donor DNA b( mechanical or enz(matic methods" ertain restriction endonucleases) designated as class II) are particularl( useful for preparing defined fragments of DNA molecules" !he( clea$e both strands of double,stranded DNA molecules at specific) palindromic se5uences %restriction sites& that usuall( $ar( from four to eight nucleotides in length) and the resulting DNA fragments are called restriction fragments" Some restriction endonucleases clea$e at coincident sites to create blunt,ended DNA fragments) and others cut at staggered positions to create DNA fragments 'ith short) self,complementar() single,stranded +3 or 23 ends %see !able 2&" !he random probabilit( that n ad4acent nucleotides in a DNA strand 'ill correspond to a specific restriction site is approximatel( -M8n" Sites for enz(mes that recognize uni5ue 8) :) or = nucleotide targets are li1el( to occur about once in e$er( .+:) 87<:) or :+)+2: nucleotides) respecti$el(" #( choosing appropriate restriction enz(mes) specific DNA molecules) including bacterial chromosomes) plasmids) and phage genomes) can be digested into sets of restriction fragments that ha$e appropriate sizes for specific applications"

.8

A restriction map identifies the positions of target sites for specific restriction endonucleases in a DNA molecule" Restriction maps are a$ailable for man( cloned DNA fragments) plasmids and phage genomes) as 'ell as for the entire chromosome of E coli and se$eral other bacteria" !he second step in gene cloning is to create h(brid replicons consisting of donor DNA fragments and a cloning $ector %*ig" +,-.&" loning $ectors are small plasmid or phage replicons that ha$e one or more restriction sites into 'hich foreign DNA can be inserted" >(brid replicons are produced b( using DNA ligase to 4oin the restricted $ector DNA 'ith donor DNA fragments that ha$e compatible ends) or) alternati$el() s(nthetic oligonucleotides are used as lin1ers to create compatibilit( bet'een donor and $ector DNA molecules 'ith different ends" Cigating a $ector to a heterogeneous set of DNA fragments from a donor genome is called shotgun cloning) and the collection of recombinant DNA molecules that contains the $arious fragments is called a genomic librar(" If a specific DNA fragment is a$ailable) it can be incorporated into a recombinant replicon b( direct cloning into an appropriate $ector chosen from the 'ide $ariet( of $ectors a$ailable" Blasmid and phage $ectors are used mainl( to clone small inserts usuall( less than -7 1bp" Examples of more special purpose $ectors include cosmids) 'hich are plasmid $ectors that can be pac1aged into phage capsids %lambda cosmids accept inserts up to 27,87 1bp&) and phagemids) 'hich are plasmid,phage h(brid replicons that can exist either as plasmids or as single,stranded DNA phages under different experimental conditions" Bhage B- cosmids can accept inserts up to -77 1bp) and still larger DNA molecules can be cloned in (east artificial chromosomes %KA s& 'hich can stabl( maintain inserts up to and exceeding - Mbp in size" Other specialized $ectors detect promoters) transcription termination signals) or other regulator(

.+

elements 'ithin foreign DNA inserts or) con$ersel() pro$ide promoters from 'hich transcription of cloned genes can be initiated"

FIGUR# 17 Diagrammatic re"resentation of gene cloning e$"eriment Blasmid cloning $ector

p#R2.. is 8"2: 1ilobase pairs in size) has genes for resistance to ampicillin %ampr& and to tetrac(cline %tetr&) and has onl( one >indIII restriction site that is located 'ithin the tetr locus" >indIII is used to treat samples of DNA from plasmid p#R2.. and from a donor organism 'ith a gene) designated a6) to be cloned" !he donor can be a pro1ar(otic or a eu1ar(otic organism" If >indIII restriction sites are located ad4acent to a6 in donor DNA) but do not occur 'ithin a6) a restriction fragment carr(ing intact a6 mar1er can be generated from donor DNA" >(brid plasmids can be formed b( random association and ligation of the >indIII,treated donor and $ector DNA fragments" Although p#R2.. is tetr) h(brid plasmids 'ill be tets because the donor DNA fragments are inserted at the >indIII restriction site 'ithin the tetr locus" After transformation of amps,recipient bacteria that also lac1 a6) transcon4ugants 'ith h(brid plasmids can be selected b( their ampr tets phenot(pes" Strains in 'hich the a6 gene is present can then be identified b( expresion of a6 or b( testing for the pol(nucleotide se5uence corresponding to a6" !he p#R2.. plasmid contains other uniu5e restriction sites that can also be used for cloning %e"g") BstI in ampr and #am>I in tetr&" Man( other cloning $ectors and restriction endonucleases ha$e also been used for gene cloning experiments"

!he final steps in gene cloning are to introduce h(brid replicons into appropriate recipient cells and test them for expression of donor genes of interest" Bro1ar(otic cells %including bacteria& or eu1ar(otic cells %including (east) animal or plant cells& can be used as recipients) but the( differ 'ith respect to their permissi$eness for specific replicons) the transcriptional signals that the( recognize) and the post,translational modifications of protein structure that the( can accomplish" Recombinant DNA molecules produced in $itro can be introduced directl( into recipient cells b( transformation or transfection" In addition) clones in cosmid or .:

phage $ectors can be pac1aged into phage coats and introduced into susceptible recipient cells b( transduction" #( using specialized $ectors %shuttle $ectors& that can replicate in multiple cell t(pes) genes from an( organism can be cloned and manipulated in a con$enient bacterial s(stem and subse5uentl( reintroduced into cells of the original organism for anal(sis in their natural en$ironment" Man( methods are a$ailable to identif( bacteria that contain recombinant DNA molecules" Most cloning $ectors ha$e genes for traits that can be positi$el( selected) such as resistance to antibiotics" *urthermore) it is often possible to introduce foreign DNA into the cloning $ector at a site that inacti$ates a nonessential) but easil( recognizable) $ector function" If both of these conditions are fulfilled) bacteria that contain recombinant molecules can be selected and distinguished easil( from bacteria that contain onl( the $ector" #acteria in a genomic librar( that contain a particular cloned gene can be identified b( using biochemical or immunologic methods to test for the desired gene product" Alternati$el() the cloned gene of interest can be detected directl( b( using nucleic acid h(bridization methods) pro$ided that a specific DNA or RNA probe is a$ailable" #ecause insertion of foreign DNA into a cloning $ector at an appropriate site does not inacti$ate its abilit( to replicate in appropriate recipient cells) h(brid replicons of interest can be amplified b( replication) and the recombinant DNA molecules or their gene products can be purified and studied" !he abilit( to purif( specific DNA molecules made it feasible to de$elop enz(matic and chemical methods for determining their nucleotide se5uences) and current methods for introducing mutations at defined sites in cloned genes are based on 1no'ing their restriction maps or nucleotide se5uences" Recombinant DNA methods ma1e it feasible to clone specific DNA fragments from an( source into $ectors that can be studied in 'ell,characterized bacteria) in eu1ar(otic cells) or in $itro" Applications of DNA cloning are expanding rapidl( in all fields of biolog( and medicine" In medical genetics such applications range from the prenatal diagnosis of inherited human diseases to the characterization of oncogenes and their roles in carcinogenesis" Bharmaceutical applications include large,scale production from cloned human genes of biologic products 'ith therapeutic $alue) such as pol(peptide hormones) interleu1ins) and enz(mes" Applications in public health and laborator( medicine include de$elopment of $accines to pre$ent specific infections and probes to diagnose specific infections b( nucleic acid h(bridization or pol(merase chain reaction %B R&" !he latter process uses oligonucleotide primers and DNA pol(merase to amplif( specific target DNA se5uences during multiple c(cles of s(nthesis in $itro) ma1ing it possible to detect rare target DNA se5uences in clinical specimens 'ith great sensiti$it("

Regulation of Gene #$"ression

!he phenot(pic properties of bacteria are determined b( their genot(pes and gro'th conditions" *or bacteria in pure culture) changes in gro'th conditions often result in predictable ph(siological adaptations in all members of the population" !(picall() essential gene products are made in amounts that permit fastest gro'th in the gi$en en$ironment) and products re5uired under special circumstances are made onl( 'hen the( are needed" Bh(siological adaptations are often associated 'ith changes in metabolic acti$ities" !he flo' of metabolites through particular biochemical path'a(s can be controlled both b( regulating the s(nthesis of specific enz(mes and b( altering the acti$ities of existing enz(mes" .E

Mechanisms that regulate expression of genes b( affecting s(nthesis of specific gene products are discussed here" Specific regulation in$ol$es a gene or group of genes in$ol$ed in a particular metabolic process" Induction and repression enable bacteria to regulate production of specific gene products in response to appropriate signals" Generall( catabolic enz(mes are induced 'hen the substrate for the path'a( is present in the gro'th medium) and bios(nthetic enz(mes are repressed b( the product of the path'a(" Enz(mes that participate in a single biochemical path'a( often occup( ad4acent positions on the bacterial chromosome and are coordinatel( induced or repressed" !he( form an operon) a group of contiguous genes that is transcribed as a single unit and translated to produce the corresponding gene products" Organization into an operon is an important strateg( for coordinatel( regulating the expression of genes in bacteria" Operons that can be induced or repressed are controlled b( binding of specific regulator( proteins to particular nucleotide se5uences that function as regulator( sites 'ithin the operon" omparison of the amino acid se5uences of man( of these different regulator( proteins sho'ed that the( could be grouped together into families of regulators %e"g" the l(sR famil( of proteins& that ma( ha$e e$ol$ed from common ancestoral genes" Members of the l(sR famil( include regulators of such di$erse phenomena as l(sine) c(steine and methionine metabolism in E coli and iron repression in G cholerae" Global regulation simultaneousl( alters expression of a group of genes and operons) collecti$el( called a regulon) that are controlled b( the same regulator( signal" Global regulation determines responses of bacteria to basic nutrients such as carbon) nitrogen or phosphate) reactions to stresses such as DNA damage or heat shoc1) and s(nthesis b( pathogens of specific $irulence factors during gro'th in their host animals" !he amount of a specific protein in a bacterial cell can $ar( from none to man( thousands of molecules" !his 'ide range is often determined b( the combined action of se$eral regulator( mechanisms that affect expression of the corresponding structural gene" Regulation is achie$ed b( determining ho' often a gene is transcribed into functional mRNA) ho' efficientl( the mRNA is translated into protein) ho' rapidl( the mRNA is degraded) ho' rapidl( the protein product turns o$er) and 'hether the acti$it( of the protein product can be altered b( allosteric effects or co$alent modifications"

mRNAs as Transcri"tional Units

Gene expression begins 'ith DNA,dependent RNA pol(merase %RNA pol(merase& catal(zing the transcription of specific mRNA from one strand of a DNA template" #inding of RNA pol(merase to DNA occurs at specific sites called promoters) and transcription begins ad4acent to the promoter" Strong promoters can interact efficientl( 'ith RNA pol(merase and initiate transcription at a high rate/ 'ea1 promoters initiate transcription at slo' rates" In either case) mRNA is s(nthesized from its +3 end to'ard its 23 end at an approximatel( constant rate until the RNA pol(merase recognizes another specific site called a terminator" RNA pol(merase then dissociates from the template) and transcription of the mRNA is completed" Indi$idual mRNA molecules ma( code for one or more pol(peptides" !ranscription of an operon produces a pol(cistronic mRNA that codes for se$eral pol(peptides" !ranslation of pol(cistronic mRNAs leads to coordinate s(nthesis of the encoded pol(peptides) but each .=

pol(peptide is s(nthesized as a separate molecule" A specific ribosome binding site is located 4ust upstream from the start of each coding se5uence on the mRNA molecule" Messenger RNAs in bacteria are degraded rapidl( 'ith an a$erage half life of se$eral minutes) in contrast to tRNAs and rRNAs 'hich are much more stable" Although mRNAs represent about half of the ne'l( s(nthesized RNA) the( represent onl( a small fraction of the total RNA" !he short half,life of mRNAs has important conse5uences for gene expression" If the s(nthesis of a specific mRNA is pre$ented) production of the corresponding pol(peptides declines rapidl(" ontrol of gene expression occurs b( regulating one or more of the steps in the path'a( from the DNA template to the acti$e gene product" Simultaneous regulation at se$eral le$els permits greater control o$er gene expression than 'ould be possible 'ith a single regulator( mechanism" !he most common 'a( to regulate gene expression in bacteria is to control the production of specific mRNAs" Since the rate of elongation of an RNA molecule is approximatel( constant) the ma4or factors that control mRNA s(nthesis are the rate of initiation and the probabilit( that a full length transcript 'ill be produced"

Regulation of Transcri"tion Initiation

Some mRNAs in bacteria are s(nthesized at constant rates) resulting in constituti$e production of the encoded pol(peptides" !he amounts of specific mRNAs and pol(peptides produced from different constituti$e genes $ar( greatl() ho'e$er) and often reflect differences in strength of the promoters for those genes" !ranscription of man( operons is regulated in response to changing en$ironmental conditions" !he promoters determine the maximum rate of transcription initiation for such operons) but regulator( proteins participate in controlling transcription" Nucleotide se5uences in operons to 'hich specific regulator( proteins bind are called regulator( sites or operators" Operators and promoters are located close together 'ithin operons and ma( ha$e o$erlapping DNA se5uences" !he binding of regulator( proteins to operators can either increase %positi$e regulation& or decrease %negati$e regulation& the fre5uenc( of transcription initiation" Broteins that function as negati$e regulators are usuall( called repressors" #ecause regulator( proteins can diffuse through the c(toplasm) the structural genes for regulator( proteins do not ha$e to be lin1ed to the target operons" !he abilit( to sense the presence or absence of specific compounds and change the rates of s(nthesis of appropriate gene products are central to the control of gene expression" Regulator( proteins offer one solution to this problem of stimulus,response coupling" Man( regulator( proteins are bifunctional and bind not onl( to appropriate operators but also to specific effectors) 'hich are small molecules such as particular sugars) amino acids) and other metabolites" *urthermore) regulator( proteins are allosteric) meaning that the( can exist in different conformations 'hich exhibit different binding affinities for their cognate operators and effectors" A sufficient concentration of effector fa$ors formation of the regulator( protein,effector complex) 'hich has either high or lo' affinit( for the operator in an( specific case" In negati$el( regulated s(stems the effector functions as a corepressor if the regulator( protein,effector complex is the acti$e repressor) and the effector functions as an inducer and causes derepression if the free regulator( protein is the acti$e repressor" on$ersel() in positi$el( regulated s(stems) the effector stimulates expression of the operon .<

if the regulator( protein,effector complex is the positi$e regulator) and the effector inhibits expression of the operon if the free regulator( protein is the positi$e regulator" !he lactose %lac& operon of E coli is an example of an inducible) negati$el( regulated operon %*ig" +,-2&" !he lacI gene codes for a repressor that binds to the lac operator and pre$ents transcription from the lac promoter" !he structural gene for this repressor is separate from the lac operon) and the repressor is s(nthesized constituti$el( at a lo' rate" 9hen inducer binds to the lac repressor) the complex cannot bind to the operator and cannot pre$ent binding of the RNA pol(merase to the promoter" If other conditions are fa$orable) the lac operon is expressed) resulting in s(nthesis of b,galactosidase) b,galactoside permease and b,galactoside transacet(lase" !he lac operon can be induced b( lactose or b( structurall( related compounds such as isoprop(l,b,D,thiogalactoside %IB!G&" IB!G is called a gratuitous inducer because it induces the lac operon) but is not a substrate for b,galactosidase" Negati$e regulation also occurs in man( bios(nthetic operons in E coli" In such operons a product of the bios(nthetic path'a( functions as the effector for the negati$e regulator( s(stem"

FIGUR# 11 Regulation of lac o"eron in # coli Structural genes lacH) lacK) and lacA code for b, galactosidase) b,galactoside permease) and b,galactoside transacet(lase) respecti$el(" !he ph(siologic role of lacA is un1no'n" !he lac repressor is product of lacI gene in separate regulator( operon" !ranscription of mRNA encoding lacH) lacK) and lacA is negati$el( regulated" and binding of lac repressor to operator lacO pre$ents initiation of transcription at promoter lacB" Inducer binds to lac repressor and inacti$ates it" atabolite acti$ator protein % AB& forms a complex 'ith c(clic AMB) and binding of the complex to a site immediatel( ad4acent to the lac promoter stimulates transcription of the lac operon b( RNA pol(merase" An expanded diagram of the lac operator,promoter region sho's the binding sites for AB) RNA pol(merase) and lac repressor"

27

!he arabinose %ara& operon in E coli is both positi$el( and negati$el( regulated" In the presence of arabinose the regulator( protein stimulates transcription of the ara operon" In the absence of arabinose) ho'e$er) the regulator( protein represses the ara operon" Operons are often controlled b( more than one mechanism" 9hen E coli is gro'n in a medium containing glucose and an alternati$e carbon source such as lactose or arabinose) induction of the lac or ara operon and utilization of the lactose or arabinose are dela(ed until the glucose has been consumed" !his phenomenon is called diauxic gro'th" !he failure to induce the lac or ara operon in the presence of glucose is an example of catabolite repression" !he lac and ara operons are positi$el( regulated b( c(clic,23)+3,adenosine monophosphate %cAMB& and the catabolite gene acti$ator % AB& protein %the product of the crp gene&" !he cAMB, AB complex interacts 'ith AB binding sites in the regulator( regions of some operons) including the lac and ara operons) and stimulates transcription from the corresponding promoters" !he le$el of intracellular cAMB in E coli is high during gro'th in the absence of glucose) and lo' during gro'th in the presence of glucose" atabolite repression is due) therefore) to lac1 of acti$ation of cAMB,dependent operons 'hen the bacteria are gro'n in the presence of glucose or certain other rapidl( metabolizable carbon sources"

Regulation of Transcri"tion Termination

Attenuation is a mechanism for regulating operons b( terminating transcription of mRNA prematurel(" Attenuation is common in bios(nthetic operons) including the trp) histidine %his&) threonine %thr&) isoleucine,$aline %il$&) and phen(lalanine %phe& operons" !he trp operon in E coli is controlled both b( repression and attenuation" In the presence of excess tr(ptophan) initiation of transcription from the trp promoter is repressed" In addition) ho'e$er) those transcripts that are initiated from the trp promoter are usuall( terminated before an( of the structural genes of the trp operon are transcribed" !he concentration of intracellular tr(ptophan re5uired to maintain repression exceeds that needed for attenuation" Such dual control enables the cell to fine tune the expression of the trp operon in response to decreasing concentrations of tr(ptophan" !he secondar( structure of mRNA has an important role in the mechanism of attenuation" All mRNAs ha$e a leader se5uence bet'een the transcriptional start site and the beginning of the coding se5uence for the first structural gene" *or amino acid bios(nthetic operons that are sub4ect to attenuation) the mRNA leader se5uence has t'o distincti$e features" It encodes a short peptide containing the amino,acid produced b( the regulated path'a() and it can form alternati$e) mutuall( incompatible) double,stranded RNA structures that participate in regulator( e$ents" *or example) the peptide encoded b( the trp mRNA leader se5uence contains t'o ad4acent tr(ptophan residues) and the peptide encoded b( the his mRNA leader se5uence has a series of se$en consecuti$e histidine residues" *ig" +,-8 sho's the trp operon and illustrates alternati$e secondar( structures in the leader se5uence of trp mRNA" !here are three possible secondar( structures for this region) called the pause site %segments -6.&) the anti,terminator %segments .62&) and the attenuator %segments 268&" Segment - of the pause site o$erlaps 'ith the coding region for the trpC peptide" 9hich secondar( structures are formed depends on efficienc( of translation of the trpC peptide" 9hen segments - and . are transcribed) the( immediatel( anneal and cause the RNA pol(merase to pause temporaril(" Subse5uent initiation of translation of the trpC peptide disrupts the pause site and allo's RNA pol(merase to continue transcription" If tr(ptophan is present) transcription of segments 2 and 8 and formation of the attenuator structure occurs 'hile the ribosome is bloc1ing 2-

segment .) causing the RNA pol(merase to terminate transcription" If tr(ptophan is deficient) ho'e$er) tr(ptophan(l,tRNA is also deficient) and the ribosome stalls at the tr(ptophan codons in segment -" !his allo's segment . to anneal 'ith ne'l( s(nthesized segment 2 to form the antiterminator) thereb( ma1ing segment 2 una$ailable to anneal 'ith segment 8" *ormation of the attenuator is therefore pre$ented) and the RNA pol(merase transcribes the entire trp operon" In this manner depletion of tr(ptophan %actuall( the suppl( of tr(ptophan(l, tRNA& is coupled to regulation of transcription of the bios(nthetic operon for tr(ptophan"

FIGUR# 1( Regulation of tr" o"eron in # coli !he organization of the trp operon is sho'n at the top of the figure" !he fi$e structural genes trpE) trpD) trp ) trp#) and trpA encode enz(mes that catal(ze terminal se5uence of reactions in tr(ptophan formation" !ranscription initiation is controlled at the promoter,operator %p,o& locus) and signals 'ithin the -:. nucleotide trp mRNA leader se5uence control termination of transcription b( attenuation" !he leader se5uence of trp mRNA is expanded to sho' locations of the trpC coding se5uence) the complementar( segments -) .) 2) and 8) and their possible alternati$e secondar( structures 'hich function as pause site) anti,terminator) or attenuator %see text&"

In E coli transcription and translation are functionall( coupled" Nonsense mutations that cause premature termination of translation often cause decreased transcription of more distal genes in the same operon" !his phenomenon is called polarit(" Ribosomes usuall( initiate translation of a gro'ing mRNA molecule prior to completion of transcription) and such translation mas1s sites that 'ould other'ise cause the RNA pol(merase to terminate transcription" Bremature termination of translation b( a nonsense codon dissociates the ribosomes from the mRNA and enables RNA pol(merase to interact 'ith the unmas1ed transcription termination sites" In some biological s(stems) including phage lambda) antitermination is used as a positi$e regulator( mechanism to control gene expression" Immediatel( after infection of E coli b( 2.

lambda) RNA pol(merase binds to t'o promoters in lambda DNA and initiates di$ergent primar( transcripts 'hich terminate at specific sites on the lambda genome" A protein encoded b( one of the primar( transcripts interacts 'ith RNA pol(merase and enables it to continue transcription through the primar( termination sites) thereb( expressing a second set of lambda genes" One of the products encoded b( a secondar( transcript bloc1s termination of another mRNA and acti$ates expression of a third set of genes" Antitermination has a 1e( role) therefore) in controlling the cascade of gene expression during l(tic gro'th of phage lambda" Antitermination is also in$ol$ed in the regulation of E coli rRNA operons"

Regulation of Translation
!he ribosome binding site on mRNA is complementar( to a se5uence at the 23 end of -:S rRNA" Interaction bet'een these se5uences facilitates formation of the initiation complex for protein s(nthesis" #oth the extent of homolog( 'ith -:S rRNA and the spacing of the ribosome binding site from the initiation codon affect the efficienc( of translation initiation" odon usage in mRNA also influences translation efficienc(" Messenger RNAs for proteins that are re5uired in large amounts tend to use codons that are translated b( the most abundant species of tRNA) and the con$erse is also true" !ranslational control is important for regulation of s(nthesis of ribosomal proteins" Broduction of ribosomes in$ol$es a high metabolic cost for bacteria) and at high gro'th rates ribosomes can constitute nearl( one,half of the cell 'eight" Most ribosomal proteins and rRNAs are found assembled into ribosomes) and the pool of free ribosomal subunits is $er( small" !he genes for ribosomal proteins are organized into se$eral operons" ertain of the free ribosomal proteins directl( inhibit the translation of the pol(cistronic mRNAs that encode them) thereb( ensuring that s(nthesis of ribosomal proteins is balanced 'ith the re5uirement for their utilization"

Regulons and signal transducing "roteins

A regulon is a group of genes or operons controlled b( a common regulator" !here are se$eral ad$antages to placing different operons under control b( the same regulator" It enables the sensing of a single stimulus to be coupled to expression of a large number of genes that ma( be needed for an appropriate response) and it eliminates the re5uirement for the coordinatel( regulated genes to be lin1ed on the bacterial chromosome" !he stimulus to 'hich the regulon responds can be an intracellular component or an en$ironmental signal" Indi$idual operons ma( also be sub4ect to regulation b( se$eral different mechanisms and expressed under conditions that differ from those affecting the 'hole regulon" More than 87 different regulons ha$e been identified in E coli" Specific examples of regulons that respond to intracellular components include the cAMB, AB regulon described pre$iousl( and the regulons controlled b( the stringent response and the SOS response" 9hen ribosomes encounter uncharged tRNA molecules during protein s(nthesis) the stringent response is acti$ated and results in prompt cessation of rRNA s(nthesis" A no$el nucleotide called guanosine,23,diphosphate,+3,diphosphate %ppGpp& accumulates during amino,acid star$ation" !he ppGpp produced b( idling ribosomes appears to be a mediator of the stringent response) but the precise mechanism causing inhibition of rRNA s(nthesis is un1no'n" !he SOS response is associated 'ith damage to DNA and in$ol$es induction of more than .7 genes in$ol$ed in se$eral DNA repair path'a(s" !he product of the recA gene detects 22

inhibition of DNA s(nthesis and initiates e$ents leading to proteol(tic clea$age and inacti$ation of the repressor for the SOS path'a() encoded b( the lexA gene" Some regulons are induced b( specific en$ironmental stimuli) such as nutrient limitation or osmotic stress" Often operons from more than one regulon ma( be induced) and the term stimulon has been used to describe the set of genes so induced" !(picall() bacteria sense such en$ironmental conditions b( t'o component s(stems" !he first component is a membrane, spanning protein 'ith extracellular and intracellular domains" Its extracellular domain detects the en$ironmental stimulus) and its c(toplasmic domain transmits the signal" !he second component is a bifunctional c(toplasmic protein" It has a recei$er domain that interacts 'ith the transmitter module of the first component) as 'ell as an effector domain that controls expression of the corresponding regulon" !he transmitter and recei$er modules of the t'o component regulator( s(stems from a 'ide $ariet( of regulons are geneticall( related and share amino,acid homolog(" !he signal,detecting and effector domains of the proteins from different regulons $ar() ho'e$er) and determine the signal that is detected and the operons that are acti$ated or repressed in response to that signal" Global regulation has an important role in the ph(siolog( of pathogenic bacteria" *or example) Gibrio cholerae and #ordetella pertussis express man( of their $irulence determinants under the control of signal transducing s(stems that are related to the t'o component s(stems described abo$e" !he expression of proteins needed for the in$asi$e phenot(pe is controlled b( temperature in Shigella" Kersinia enterocolitica senses both the en$ironmental temperature and the concentration of calcium ions and couples these signals to the expression of genes and cellular location of the gene products that are appropriate for an intracellular or extracellular en$ironment" In host tissues the concentration of free iron is extremel( lo') and most pathogenic bacteria ha$e high affinit( iron transport s(stems that are induced under lo',iron conditions" !he s(nthesis of diphtheria toxin b( diphtheriae) Shiga toxin b( Shigella d(senteriae) exotoxin A b( Bseudomonas aeruginosa) and other specific proteins in man( pathogenic bacteria is induced under conditions of iron,limited gro'th" !hese examples illustrate ho' en$ironmental factors can regulate the expression of $irulence genes in pathogenic bacteria"

R#F#R#NC#S
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Miller GC) Oaper N#) Bortno( DA et al" %eds&F Molecular genetics of bacterial pathogenesis" American Societ( for Microbiolog() 9ashington D ) -<<8 Sa(lers AA) 9hitt DDF #acterial pathogenesisF a molecular approach" American Societ( for Microbiolog() 9ashington D ) -<<8 Singer M) #erg B" Genes and genomesF a changing perspecti$e" 0ni$ersit( Science #oo1s) Mill Galle() A) -<<-

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