Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequences
Jeffrey S.McKinney 1,2,*,Haifeng Zhang 1,Tomoko Kubori 3,Jorge E.Gala
?n 3and Sidney Altman 1
1
Department of Molecular,Cellular and Developmental Biology,Yale University,New Haven,CT 06520,USA,2Department of Pediatrics,Division of Infectious Diseases,Yale University School of Medicine,New Haven,CT 06520,USA and 3Section of Microbial Pathogenesis,Yale University School of Medicine,New Haven,CT 06536,USA
Received October 15,2003;Revised and Accepted December 16,2003
ABSTRACT
The type III secretion system involved in Salmonella enterica serovar Typhimurium invasion of host cells has been disrupted using inducibly expressed oli-gonucleotide external guide sequences (EGSs)complementary to invB or invC mRNA.These EGSs direct single site cleavage in these mRNAs by endo-genous RNase P,and their expression in Salmonella results in invC mRNA and InvC protein depletion,decreased type III secretion and interference with host cell https://www.doczj.com/doc/205405468.html,parison of these effects with those from studies of Salmonella invB and invC mutants suggests that invB EGSs have polar effects on invC mRNA.INTRODUCTION
External guide sequence (EGS)oligonucleotides target com-plementary mRNA for speci?c cleavage catalyzed by RNase P (1).EGS oligonucleotides require an accessible single-stranded region on their target mRNA to base-pair with and create the stem structure recognized as a cleavage substrate by RNase P (1).Using EGSs complementary to essential genes,Escherichia coli viability can be decreased in a manner which is EGS oligonucleotide sequence speci?c,dose dependent and dependent on time elapsed after EGS expression (2).Here,EGS studies are extended to Salmonella ,using EGSs complementary to two Salmonella pathogenicity island SPI-1genes (3),invB and invC ,neither of which are essential for bacterial viability (4).The invB and invC DNA sequences occur directly adjacent to each other in the multigene pathogenicity island SPI-1of Salmonella ,with the last nucleotide in the ?nal codon of invB also serving as the ?rst nucleotide in the ?rst codon of invC (4).Prior studies of Salmonella invB and invC mutants have shown that invC is required for host cell invasion and that the gene encodes a protein with ATPase activity (4).The ATPase encoded by invC is postulated to provide energy to power the type III secretion system involved in host cell invasion (4)and
pathogenesis (5)by Salmonella .In contrast,invB does not appear necessary for invasion.InvB is a type III secretion chaperone speci?c for SipA,a translocated Salmonella protein which facilitates actin rearrangements within infected eukar-yotic cells (6).Mutations in invB do not alter the secretion of other type III secreted proteins (6)and do not disrupt invasion (4).Using a tightly regulated inducible EGS expression system in Salmonella (7),we show that EGSs complementary to either invB or invC mRNA can disrupt type III secretion and Salmonella invasion assayed in vitro .MATERIALS AND METHODS Plasmids and bacterial strains
The EGSs listed below were cloned as previously described into high copy number EGS expression plasmids,derived from pUC19(2)or into low copy number plasmids derived from pWKS30(8).These plasmids were transformed into the Salmonella enterica serovar Typhimurium strain SB300A#1(7).SB300A#1has a T7RNA polymerase gene integrated with an adjacent araC-P(BAD)control element into the bacterial chromosome of parent strain SB300.SB300A#1allows tightly controlled arabinose-inducible T7promoter-driven transcription of our EGSs in Salmonella (7).The invA -de?cient Salmonella strain SB136(4),which is disrupted for type III secretion,was used as a control.An invC deletion
mutant Salmonella (J.E.Gala
?n and Y.Akeda)was used as a negative control strain for studies of InvC intracellular protein level and of type III secretion.Salmonella was grown in 0.3M NaCl Luria±Bertani (LB)medium.Liquid culture incubation conditions and EGS induction with arabinose at 0.2%?nal concentration are as previously described (7).Following addition of arabinose for EGS induction,Salmonella liquid cultures were grown to late log phase prior to northern blot analysis,assay of Salmonella type III secretion or quanti?ca-tion of bacterial entry,as detailed below.Design of external guide sequences
EGS oligonucleotides were designed to be complementary to single-stranded regions of invB and invC mRNA,followed by
*To whom correspondence should be addressed.Tel:+13142862912;Fax:+13142862895;Email:McKinney_J@https://www.doczj.com/doc/205405468.html,
Present address:Jeffrey S.McKinney,Departments of Pediatrics and Molecular Microbiology,Washington University,Saint Louis,MO 63110,USA
848±854Nucleic Acids Research,2004,Vol.32,No.2DOI:10.1093/nar/gkh219
Nucleic Acids Research,Vol.32No.2?Oxford University Press 2004;all rights reserved
Published online February 3, 2004
an additional3￠-ACCA EGS terminal sequence.This strategy allows formation of a duplex EGS±mRNA molecule recognized as a substrate by endogenous RNase P with resultant cleavage of target mRNA(9).The individual EGS oligonucleotide sequences were named according to their predicted site of target mRNA cleavage.For example, invB108EGS(5￠-AAUGCAAAUAAAUCCacca-3￠)is complementary to invB mRNA nucleotides108±122 (5￠-GGAUUUAUUUGCAUU-3￠)and will result in RNase P cleavage of invB mRNA at nucleotide number108. The other invB or invC EGS sequences were:invC98 EGS(5￠-GGCGUGAUUUCACAAacca-3￠),invC269EGS (5￠-ACCGCGCCUAAUACCacca-3￠)and invC293EGS (5￠-ACGAUUUUCCCUGUCacca-3￠).Two previously char-acterized EGSs which are not complementary to invB or invC were also used:synthC5EGS21and synthC5EGS45(2).The EGSs synthC5EGS21and synthC5EGS45are complemen-tary to,and can guide the RNase P cleavage of,mRNA used for the recombinant synthesis of the C5protein subunit of the RNase P holoenzyme of E.coli,but are not complementary (containing at least?ve unpaired nucleotides per EGS)to the mRNA encoding C5in Salmonella.Herein,the EGSs synthC5 EGS21and synthC5EGS45are referred to as synthC5EGS1 and2,respectively.
Partial RNase T1digest mapping of invB and invC mRNA
Single-stranded regions of invB and invC mRNA were identi?ed using RNase T1digestion(1).Two mRNAs were digested:(i)a joint in vitro transcript containing invC mRNA 3￠to invB mRNA,transcribed from the plasmid pSB553(4) DNA after digestion with BamHI;and(ii)an invC mRNA in vitro transcript alone,expressed from plasmid pIC001(a pSB553derivative,with invB coding sequence removed via KpnI and BspEI excision)DNA after digestion with EcoRI. In vitro RNase P assays
Assays of mRNA cleavage in vitro by RNase P were performed as previously described(10),using the EGS sequences and the invB and invC mRNA targets detailed above.RNase P M1RNA was folded in a buffer containing 10mM magnesium,using a heat block to?rst heat the sample at65°C for5min and then slowly cool the sample to room temperature.For conditions of substrate excess,reagent concentrations were:11fmol labeled substrate(1100c.p.m.), 1,5and10pmol EGS,and1pmol of enzymatically active recombinant E.coli RNase P M1RNA.For conditions of limited substrate,10fmol of labeled substrate RNA (1000c.p.m.)and50,100and500fmol of EGS were used. Samples were electrophoresed in5%polyacrylamide±7M urea gels.
Northern blots
Northern blots were performed on total RNA extracts of Salmonella,using previously published techniques(11).Each lane of a2.5%agarose gel was loaded with4m g of total RNA. The UV transillumination pattern of rRNA bands after separation of each sample on an agarose gel revealed similarity in rRNA band patterns in terms of both gross quantity and quality.Northern blot probes were5￠-end-labeled DNA oligonucleotide probes.They included probes comple-mentary to invC mRNA,5S rRNA and each of the four invB and invC EGS oligonucleotides listed above.In each case, 8pmol of oligonucleotide labeled with4pmol(30m Ci)of [g-32P]ATP was used per40ml of rapid hybridization buffer (Amersham).Signal was detected using a phosphoimager (Fuji)and quantitated using image analysis software(Fuji ImageGauge).Quantitative results are reported as the ratio, expressed as a percentage,of northern blot invC mRNA band signal intensity for the matched culture specimens of a given Salmonella strain with EGS induction versus without EGS induction.
Assay of Salmonella type III secretion
Salmonella culture supernatant proteins were prepared and analyzed as previously described(12).Western blots were probed with polyclonal antibodies against SipB and SipC and chemiluminescent signals produced using ECL Plus western blotting detection reagents from Amersham Biosciences. Signal was detected using autoradiograph?lm(Kodak)and quantitated using image analysis software(Fuji ImageGauge). Quantitative results are reported as the ratio,expressed as a percentage,of western blot band signal intensity for the matched culture specimens of a given Salmonella strain with EGS induction versus without EGS induction.
Matched culture specimens were employed as internal controls for western blots.Speci?cally,a given Salmonella transformant was grown to early log phase in a single liquid culture,then split into paired cultures which were grown simultaneously either with or without arabinose induction of EGS expression.Equal volumes of paired cultures were harvested for protein preparation at the same point of their late log phase growth,as assessed by optical density.Equal volumes of these protein preparations were loaded per well for western blot analysis.
Assay of intracellular InvC protein level
Salmonella cultures in liquid media were pelleted and resuspended in one-twentieth volume of phosphate-buffered saline(PBS)±Tris(77mM Tris±HCl,pH8.0).Samples were denatured by boiling with SDS±PAGE loading buffer and separated on a9%polyacrylamide±SDS gel.Western blots were probed with a polyclonal antibody against InvC(J.E. Gala?n and Y.Akeda),and chemiluminescent signals produced using ECL Plus western blotting detection reagents from Amersham Biosciences.Signals were detected using auto-radiograph?lm(Kodak).Quantitation of results,using matched culture specimens,was performed as described for type III secretion assays,above.
Quanti?cation of bacterial entry
Entry of different Salmonella strains into Henle-407cells in a gentamicin protection assay of bacterial entry into host tissue culture cell monolayers was performed and quanti?ed as previously described(13).
RESULTS
Design of EGSs for invB and invC mRNA in vitro
To design the EGSs reported here,mRNA transcripts of invB and invC made in vitro were mapped using partial RNase T1 Nucleic Acids Research,2004,Vol.32,No.2849
nuclease digestion to suggest EGS-accessible single-stranded mRNA regions.The ?rst nucleotide of the start codon of each gene is labeled as nucleotide 1;single-stranded guanine residues of invB mRNA were identi?ed via partial RNase T1nuclease digestion at invB nucleotides 108and 217.For invC mRNA,single-stranded guanines were identi?ed at invC nucleotides 98,237,269and 293.Given uncertainty about whether invC mRNA exists in cells independently from invB mRNA or as a joint transcript with invB ,both possibilities were examined in RNase T1digestion in vitro .RNase T1digestions were performed on two in vitro transcripts:invC mRNA alone,as well as a tandem transcript of invC mRNA immediately 3￠to invB mRNA.Single-stranded regions of invC identi?ed in the joint invB ±invC in vitro transcript were notably also found for the invC in vitro transcript alone (data not shown).EGS oligonucleotides were designed to be complementary to the RNase T1-accessible mRNA sequences invB 108±122,invC 98±112,invC 269±283and invC 293±307,and were named for their predicted nucleotide cleavage sites by RNase P:invB 108,invC 98,invC 269and invC 293,respectively.
RNase P-speci?c cleavage of mRNA in vitro
RNase P hydrolyzes the phosphodiester bond (in the target mRNAs)that precedes the ?rst base pair in the 5￠end of the
target mRNA±EGS complex,akin to the site-speci?c cleavage reaction the enzyme catalyzes in the 5￠processing of precursor tRNA (9).The reaction ingredients for RNase P assays in vitro were E.coli RNase P,internally radiolabeled invB and invC mRNA target transcribed in vitro ,and EGSs complementary to portions of invB or invC mRNA.All four EGSs guide RNase P to cleave the mRNA at the predicted sites of EGS mRNA hybridization,yielding appropriately sized 5￠and 3￠cleavage products (Fig.1).RNase P cleavage of mRNA increases with increasing EGS dose,with the EGSs invB 108and invC 293most ef?cient at guiding mRNA cleavage in vitro in conditions of limited substrate (data not shown).Speci?c mRNA targeting in Salmonella
Induction of expression of invB or invC EGSs in Salmonella is followed by a decrease in invC mRNA compared with identical Salmonella transformants lacking EGS induction.Northern blots of equal microgram amounts of total RNA isolated from various Salmonella liquid cultures are shown in Figure 2,where northern blot signals for invC mRNA in matched Salmonella cultures decreased between 27and 50%following relevant EGS induction.The invB or invC EGS expression effects appear speci?c,in that there is no similar change detected in the level of constitutive 5S rRNA after EGS induction (Fig.2).Arabinose was used to induce Salmonella EGS expression.It did not have as pronounced an effect on invC mRNA in the absence of EGS expression plasmids,either in the case of Salmonella which was not transformed with an EGS expression plasmid,or in the case of Salmonella for which the EGS expression plasmid was presumably lost after prolonged culture (Fig.2).
Plasmid
Figure 1.RNase P±EGS cleavage of mRNA in vitro .Target substrates for cleavage include a joint transcript including both invB and invC mRNA (lane 1),and a transcript of invC mRNA alone (lane 6).The former was incubated with increasing amounts of the invB EGS 108(lanes 2±4);the lat-ter as a target for invC EGSs invC 98(lanes 7±9),invC 269(lanes 10±12)and invC 293(lanes 13±15).Lanes labeled N (lanes 5and 16)lack any EGS,but do have active RNase P with the invB and invC joint transcript (lane 5)or the invC transcript (lane 16)and show no non-speci?c target cleavage in the absence of EGS.Cleavage products were separated by size using electrophoresis in a 5%polyacrylamide±7M urea gel.Cleavage prod-uct sizes are consistent with RNase P cleavage occurring at the predicted site at the 5￠end of the mRNA region to which each EGS hybridizes.Predicted sizes of reaction products following RNase P enzymatic cleavage of in vitro transcripts are listed on the left and right of the image (e.g.invB and invC joint transcript mRNA cleavage products of 585and 123nucleo-tides for EGS invB 108;invC transcript mRNA cleavage products of 485and 198nucleotides for EGS invC 98,
etc.).
Figure 2.Northern blots in Salmonella with inducible expression of EGS molecules.RNA was isolated from Salmonella ,electrophoresed in a 2.5%agarose gel,and probed for invC mRNA or for constitutively produced non-targeted 5S rRNA.The partial invC in vitro transcript expressed from plas-mid pIC001DNA after digestion with EcoRI (as described for T1digest mapping)serves as a size marker (683nt).The invA mutant is SB136(also used for invasion assays).SB300is the parent Salmonella strain,from which SB300A#1was constructed.Other lanes are paired by SB300A#1Salmonella transformant type,either with (+)or without (±)the addition of arabinose for the induced expression of the EGS molecules listed.Longer term induction (++)was suspected to be accompanied by the loss of EGS expression plasmid invC 293,and an accompanying lack of effect on target mRNA.Note the decrease in invC mRNA after the induction of EGS expression,whereas non-targeted constitutive 5S rRNA levels are independ-ent of EGS expression.
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maintenance in transformants was assessed by parallel quan-titative plating on LB or LB ampicillin plates as previously reported (2,11).Plasmid loss was considered to have occurred when colony counts on LB plates were greater than on LB ampicillin plates by at least an order of magnitude.As previously demonstrated for other EGS transcription in this Salmonella system (7),northern blot probes complementary to EGS oligonucleotides detected EGS expression only after arabinose induction (data not shown).
The effect of expression of EGSs on type III secretion by Salmonella
A standard functional assay of InvC-dependent type III secretion was employed,in which proteins secreted by the type III secretion system were measured in cell culture supernatants (12).These secreted proteins,Sip
B and SipC,are also encoded in the Salmonella pathogenicity island I gene complex and are reviewed elsewhere (14,15).
A panel of Salmonella transformants,containing EGSs in various inducible expression plasmid vectors,was used for InvC-dependent type III secretion assays.The concurrent expression of two EGSs using any one of four high copy number plasmids derived from pUC19(2)consistently decreased Sip
B secretion by b 65%from that detected for the same Salmonella transformant grown in parallel under non-EGS-inducing conditions,based on the relative signal intensities of SipB western blot bands for a given Salmonella strain with versus without EGS induction (Fig.3).Using the same method of quantitative analysis,induction of the same set of EGS pairs in Salmonella transformed with a low copy number plasmid derived from pWKS30showed a 20±30%decrease in secretion (data not shown),as did any of the four EGSs when expressed alone from pUC19-derived plasmids (data not shown).These ?ndings are consistent with prior studies of EGS dose±response features in E.coli comparing EGS expression plasmids encoding single versus multiple EGSs (2)and comparing EGS expression plasmids with strong versus weak promoters (10).
To assess sequence speci?city,the negative control EGSs,synthC5EGS 2and 1,against the synthetic C5component of E.coli RNase P,described above and in McKinney et al .(2),was also used.Induction of this negative control EGS was accompanied by a <10%decrease in SipB secretion as compared with non-induced parallel cultures (Fig.3).In addition,the loss of an invB or invC EGS expression plasmid from a bacterial strain was accompanied by a loss of previously observed inhibitory effects on secretion (data not shown).
The effect of EGSs on intracellular InvC protein in Salmonella
A polyclonal antibody (J.E.Gala
?n and Y.Akeda)raised against recombinant InvC protein was used for western blot analysis of cellular InvC protein level as an assessment of EGS effects (Fig.4,top).A band consistent with the 47kDa InvC protein is found in the Salmonella strain SB300A#1with no EGS.An SB300strain (constructed by Y.Akeda)in which InvC expression has been disrupted via an invC deletion mutation serves as a negative control for InvC expression.Intracellular expression of InvC was also assessed in SB300A#1transformed with the EGS expression plasmid for the concurrent expression of either the EGSs invB 108/invC 98or the negative control EGSs synthC5EGS 2and 1(also shown in Fig.3).After the induction of EGS expression,the relevant protein band is not detected for SB300A#1with the invB /invC EGSs.No effect on the detection of this protein band is seen after the induction of the negative control EGS.Figure 4is representative of repeated assays in that the decrease in putative ~47kDa InvC signal cannot be explained by a general decrease in the intensity of other bands
detected
Figure 3.Western blots of cell culture supernatants from Salmonella with (+)or without (±)the induced expression of EGS molecules listed.Western blot detects the proteins SipB and SipC,secreted into cell culture superna-tant by the type III secretion system and subsequently electrophoresed in a 9%polyacrylamide±SDS gel.The transformants shown contain a pUC19-derived high-copy number plasmid (2)from which two EGSs are concur-rently expressed.InvBB denotes inducible expression of invB 108and invB 108EGSs in tandem,InvBC denotes invB 108and invC 98EGS expression,InvCB denotes invC 293and invB 108EGS expression,and invCC denotes invC 293and invC 98EGS expression.The control concurrently expresses two EGSs targeting the mRNA used for synthetic C5protein over-expres-sion in E.coli .Migration patterns of SipB and SipC bands relative to protein molecular weight 66and 45kDa markers are as shown in Figure
4.
Figure 4.Western blots for Salmonella intracellular InvC (top)and for SipB and SipC secreted into Salmonella cell culture supernatants (bottom,as for Fig.3).SB300A#1without an EGS expression plasmid (no EGS)and a SB300invC deletion mutant which does not express InvC (D invC )(pro-duced by J.E.Gala
?n and Y.Akeda)are shown to the left of protein mol-ecular weight markers.At right:SB300A#1transformed with the high copy number plasmids for arabinose-inducible expression of either invB 108and invC 98EGSs (InvB/C EGS)or the control EGSs (Control EGS)described for Figure 3.EGS induction status by arabinose addition is shown as (+)or (±).Arrowheads denote the predicted locations of InvC,SipB and SipC after electrophoresis in 9%polyacrylamide±SDS gels.
Nucleic Acids Research,2004,Vol.32,No.2851
by the polyclonal antibody.Indeed,for the two cases of EGS induction shown here,invB/invC EGS induction is accom-panied by decreased~47kDa InvC signal in the context of strong signals for other bands,whereas negative control EGS induction is accompanied by strong~47kDa InvC signal in the context of relatively weak signals for other bands.
Cell supernatants from the same experiment were used for type III secretion assays(Fig.4,bottom)as above.In each case,detection of the band representing intracellular InvC protein correlates with type III secretion.Type III secretion is decreased when intracellular InvC protein levels are de-creased,either by the static invC deletion mutation,or by the dynamic disruption of InvC expression following the induc-tion of the invB/invC EGSs.While the presumptive InvC protein band disappears in both situations,the decrease in type III secretion is more pronounced following the static deletion of the invC gene than following the induction of the invB/invC EGS.This functional assay of InvC cellular activity suggests that our invB/invC EGS system inhibits InvC-dependent type III secretion less completely than does the invC deletion-mediated ablation of InvC expression.Wild-type levels of secretion are observed for SB300A#1with no EGS plasmid, without induction of invB/invC EGSs,or with negative control EGS expression.
EGS impact on Salmonella invasion into host cells Salmonella strains containing EGSs in various inducible expression plasmid vectors were also tested for their ability to invade Henle-407cells in tissue culture.In each case,a given Salmonella strain was grown in parallel liquid cultures,either with arabinose added to induce EGS induction or without the addition of arabinose.For invasion,these Salmonella strains were?rst incubated with Henle cells for45min in Hanks buffered salt solution.This was followed by a2h treatment with gentamicin to kill extracellular bacteria,and subsequent washes with tissue culture buffer.Invasion was quanti?ed as the percentage of bacteria inoculated into the tissue culture wells which were recovered from lysed Henle cells.
As shown in Table1,a control Salmonella strain,SB136, with a null mutation in invA and a previously documented functional defect in type III secretion and host cell invasion (4),achieves<1%invasion into Henle cells,independent of arabinose addition.Salmonella SB300A#1transformants which express an EGS complementary to mRNA encoding synthetic C5(also shown as a negative control for type III secretion assays,above)have a b20%rate of invasion,with a small decrease in invasion observed after arabinose addition. Salmonella SB300A#1transformants which inducibly express EGSs against invB or invC from a high copy number pUC19-derived plasmid exhibit an8±12%rate of invasion without arabinose addition,which decrease to2.8±3.5%after arabi-nose induction of EGS expression.The same EGS constructs, expressed from low copy number pWKS30-derived plasmids, did not affect invasion rates after EGS induction.
DISCUSSION
The pathogenicity island genes invC and invB of Salmonella provide intriguing targets for gene product disruption.In the case of invC,mutagenesis studies clearly show that the ATPase encoded by invC is required for type III secretion in assays in vitro and is important for pathogenicity in animal models(4,5).Following appropriate EGS expression,we observe a decrease in invC mRNA,InvC intracellular protein, InvC-powered type III secretion and type III secretion-dependent host cell invasion.
The inhibition of type III secretion and of Salmonella invasion using EGSs to disrupt invC mRNA is less complete than that resulting from invC deletion mutagenesis.This suggests that a certain critical level of mRNA disruption is able to partially inhibit type III secretion and host cell invasion.The level of mRNA disruption required for phenotypic changes probably varies for different target mRNAs,depending on factors such as the ratio of EGS to target mRNA(2,11),the relative ef?ciency of various EGSs and the functional reserve capacity a cell has for a given target mRNA and the protein that mRNA encodes.The EGSs reported here show greater phenotypic effects when expressed from high copy number,rather than low copy number, plasmids.This is consistent with prior EGS dose±response observations in bacteria.Phenotypic effects in E.coli are greater following EGS expression driven by a strong promoter as compared with a weak promoter(11),and the concomitant expression of different EGSs in E.coli results in phenotypic effects exhibiting additive synergy(2).The application of EGS technologies to regulate gene expression in bacteria in in vivo models of infection may bene?t from EGS expression plasmids which can be stably maintained in bacteria within an animal.We produced the low copy number plasmids reported here in an initial effort toward this end.The fact that EGSs expressed from our low copy number plasmid system did partially inhibit type III secretion but showed no apparent effect on host cell invasion suggests a possible threshold effect,in which inhibition of type III secretion must reach a critical threshold to result in inhibition of cell invasion.
In contrast to the static effects on gene product disruption produced by mutagenesis techniques,our techniques of gene
Table1.Invasion of Henle-407cells by Salmonella strains Salmonella strain%Invasion%Invasion
No arabinose Plus arabinose
SB136(invA-)0.08T0.040.1T0.06 SB300A#127T4.7
SB300A#1transformants
pUC synthC5EGS12620
pUC synthC5EGS2and129T2.1
pUC InvB/B EGS10T1.5 2.9T0.2 pUC InvB/C EGS9.6T1.2 2.9T0.5 pUC InvC/B EGS8.8T1.0 2.8T0.3 pUC InvC/C EGS12 3.5 pWKS InvB/B EGS2519
pWKS InvB/C EGS2323
pWKS InvC/B EGS1818
pWKS InvC/C EGS1313
Invasiveness of different Salmonella strains into Henle-407cells,in a standard gentamicin protection assay.The numbers of internalized bacteria are shown as a percentage of bacteria input.Means T SDs are shown for triplicate well assays,with no standard deviations shown for single-well assays.Salmonella strains include SB300A#1,SB300A#1transformants expressing various EGSs described in the text,and the invA-de?cient SB136.
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product disruption involve a more dynamic process of EGS-guided,RNase P-mediated,mRNA cleavage commencing after EGS induction.As observed in past studies of EGSs complementary in sequence to essential genes (2)or to antimicrobial resistance genes (11)in E.coli ,a lag occurs between initiation of EGS expression and the observed molecular and phenotypic effects.This lag is consistent with the time required to accrue suf?cient amounts of mRNA cleavage for observable phenotypic effects.
Reconciling the relative magnitude of effects we observe after EGS expression upon invC gene product expression and functions (Fig.5)requires consideration of the kinetics of several steps of cellular physiology and our measurement methods.For example,the assays we employed showed a smaller relative effect of EGSs on type III secretion than on invasion.Our invasion assays detect host cell invasion over a short time period commencing at a time when differences between conditions of EGS or no EGS are relatively great.In contrast,our secretion assays detect the accumulated SipB or SipC secreted into supernatants throughout the period of EGS induction and include a lag time when EGS effects are minimal.Accordingly,the relative difference between condi-tions of EGS expression and no EGS expression which we detect for secretion might be predicted to be less than the relative difference we detect for invasion.
Effects following expression of invB EGSs were somewhat surprising given the results of prior invB studies.InvB is a type III secretion chaperone speci?c for SipA (6).Mutations of invB do not affect the type III secretion of SipB or SipC (6)or the invasion of host cells (4).Yet,an EGS complementary in nucleotide sequence to invB and which cleaves invB mRNA in vitro ,does inhibit type III secretion of SipB and SipC and
host cell invasion.Furthermore,this EGS against invB decreases invC mRNA levels in Salmonella .
The coding regions of the invB and invC genes overlap,such that the 3￠nucleotide of the ?nal codon of invB serves as the 5￠nucleotide of the ?rst codon of invC .We postulate that the effects of invB EGSs are consistent with the existence of a joint invB ±invC transcript in Salmonella (Fig.5),which becomes destabilized or functionally disrupted after cleavage of its invB portion.If this is true,the fact that an EGS targeting invB mRNA also has effects on invC suggests that,in polycistronic mRNA transcripts,EGS targeting the 5￠portions of mRNAs may also have effects on mRNAs encoded by genes 3￠to the EGS target site.
Ultimately,pathogenicity island gene product disruption may lead to novel anti-pathogenicity agents and strategies.Since these genes are not essential to bacterial viability,interfering with their functions could conceivably decrease morbidity without the relatively broad-spectrum effects and selective pressure for resistance seen with many current antimicrobial agents.ACKNOWLEDGEMENTS
We thank Yukihiro Akeda for antibody against InvC and for the invC Salmonella mutant.This research was conducted while J.M.was a P?zer postdoctoral fellow.The work was supported by an NIH research training program (NIH T32AI07210-17),a Yale Children's Health Center Scholar Award and a Washington University Digestive Diseases Research Core Center Grant (DDRCC P30DK52574)to J.M.,and NIH grants AI030492to J.E.G.and GM19422to S.A.REFERENCES
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Figure 5.Summary of invB and invC EGSs and their effects.A schematic of the invB and invC mRNA transcripts is shown,with the predicted sites of RNase P-mediated cleavage identi?ed for each invB or invC EGS (invB 108EGS,invC 98EGS,invC 269EGS and invC 293EGS).The last nucleotide in the ?nal codon of invB also serves as the ?rst nucleotide in the ?rst codon of invC .Effects of these EGSs on Salmonella invC mRNA,type III secretion,InvC protein and host cell invasion are summarized,and relevant ?gures cited.Further details are provided in the text.
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比较级与最高级 1.as...as 与(not) as(so)...as as...as...句型中，as的词性 第一个as是副词，用在形容词和副词的原级前，常译为“同样地”。第二个as是连词，连接与前面句子结构相同的一个句子(相同部分常省略)，可译为“同..... He is as tall as his brother is (tall) . (后面的as 为连词) 只有在否定句中，第一个as才可换为so 改错: He is so tall as his brother.（X） 2.在比较状语从句中，主句和从句的句式结构一般是相同的 与as...as 句式中第二个as一样，than 也是连词。as和than这两个连词后面的从句的结构与前面的句子大部分情况下结构是相同的，相同部分可以省略。 He picked more apples than she did. 完整的表达为: He picked more apples than she picked apples. 后而的picked apples和前面相同，用did 替代。 He walked as slowly as she did.完整表达为: He walked as slowly as she walked slowly. she后面walked slowly与前面相同，用did替代。
3.谓语的替代 在as和than 引导的比较状语从句中，由于句式同前面 主句相同，为避免重复，常把主句中出现而从句中又出现的动词用do的适当形式来代替。 John speaks German as fluently as Mary does. 4.前后的比较对象应一致 不管后面连词是than 还是as,前后的比较对象应一致。The weather of Beijing is colder than Guangzhou. x than前面比较对象是“天气”，than 后面比较对象是“广州”，不能相比较。应改为: The weather of Bejing is colder than that of Guangzhou. 再如: His handwriting is as good as me. 应改为: His handwriting is as good as mine. 5.可以修饰比较级的词 常用来修饰比较级的词或短语有: Much,even,far,a little,a lot,a bit,by far,rather,any,still,a great deal等。 by far的用法: 用于强调，意为“...得多”“最最...”“显然”等，可修饰形容词或副词的比较级和最高级，通常置于其后，但是若比较级或最高级前有冠词，则可置于其前或其后。
The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法） 举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法： 在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because
人教版（新目标）初中英语形容词与副词的比较级与最高级 （一）规则变化： 1．绝大多数的单音节和少数双音节词，加词尾-er ，-est tall—taller—tallest 2．以不发音的e结尾的单音节词和少数以-le结尾的双音节词只加-r，-st nice—nicer—nicest , able—abler—ablest 3．以一个辅音字母结尾的重读闭音节词或少数双音节词，双写结尾的辅音字母，再加-er，-est big—bigger—biggest 4．以辅音字母加y结尾的双音节词，改y为i再加-er，-est easy—easier—easiest 5．少数以-er，-ow结尾的双音节词末尾加-er，-est clever—cleverer—cleverest, narrow—narrower—narrowest 6．其他双音节词和多音节词，在前面加more，most来构成比较级和最高级 easily—more easily—most easily （二）不规则变化 常见的有： good / well—better—best ; bad (ly)/ ill—worse—worst ; old—older/elder—oldest/eldest many / much—more—most ; little—less—least ; far—farther/further—farthest/furthest
用法： 1．原级比较：as + adj./adv. +as（否定为not so/as + adj./adv. +as）当as… as中间有名字时，采用as + adj. + a + n.或as + many / much + n. This is as good an example as the other is . I can carry as much paper as you can. 表示倍数的词或其他程度副词做修饰语时放在as的前面 This room is twice as big as that one. 倍数+as+adj.+as = 倍数+the +n.+of Your room is twice as larger as mine. = Your room is twice the size of mine. 2．比较级+ than 比较级前可加程度状语much, still, even, far, a lot, a little, three years. five times,20%等 He is three years older than I (am). 表示“（两个中）较……的那个”时，比较级前常加the（后面有名字时前面才能加冠词） He is the taller of the two brothers. / He is taller than his two brothers. Which is larger, Canada or Australia? / Which is the larger country, Canada or Australia? 可用比较级形式表示最高级概念，关键是要用或或否定词等把一事物（或人）与其他同类事物（或人）相分离 He is taller than any other boy / anybody else.
大多数形容词有三种形式,原级,比较级和最高级, 以表示形容词说明的性质在程度上的不同。 形容词的原级: 形容词的原级形式就是词典中出现的形容词的原形。例如: poor tall great glad bad 形容词的比较级和最高级: 形容词的比较级和最高级形式是在形容词的原级形式的基础上变化的。分为规则变化和不规则变化。 规则变化如下: 1) 单音节形容词的比较级和最高级形式是在词尾加 -er 和 -est 构成。 great (原级) (比较级) (最高级) 2) 以 -e 结尾的单音节形容词的比较级和最高级是在词尾加 -r 和 -st 构成。wide (原级) (比较级) (最高级) 3)少数以-y, -er, -ow, -ble结尾的双音节形容词的比较级和最高级是在词尾加 -er 和 -est 构成。 clever(原级) (比较级) (最高级) 4) 以 -y 结尾,但 -y 前是辅音字母的形容词的比较级和最高级是把 -y 去掉,加上 -ier 和-est 构成. happy (原形) (比较级) (最高级) 5) 以一个辅音字母结尾其前面的元音字母发短元音的形容词的比较级和最高级是双写该辅音字母然后再加 -er和-est。 big (原级) (比较级) (最高级) 6) 双音节和多音节形容词的比较级和最高级需用more 和 most 加在形容词前面来构成。 beautiful (原级) (比较级) (比较级) difficult (原级) (最高级) (最高级) 常用的不规则变化的形容词的比较级和最高级: 原级------比较级------最高级 good------better------best many------more------most much------more------most bad------worse------worst far------farther, further------farthest, furthest 形容词前如加 less 和 least 则表示"较不"和"最不 形容词比较级的用法: 形容词的比较级用于两个人或事物的比较,其结构形式如下: 主语+谓语(系动词)+ 形容词比较级+than+ 对比成分。也就是, 含有形容词比较级的主句+than+从句。注意从句常常省去意义上和主句相同的部分, 而只剩下对比的成分。
The way的用法及其含义（二） 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中
英语比较级和最高级的用法归纳 在学习英语过程中，会遇到很多的语法问题，比如比较级和最高级的用法，对于 这些语法你能够掌握吗？下面是小编整理的英语比较级和最高级的用法，欢迎阅读！ 英语比较级和最高级的用法 一、形容词、副词的比较级和最高级的构成规则 1.一般单音节词和少数以-er，-ow结尾的双音节词，比较级在后面加-er，最高级 在后面加-est; (1)单音节词 如：small→smaller→smallest short→shorter→shortest tall→taller→tallest great→greater→greatest (2)双音节词 如：clever→cleverer→cleverest narrow→narrower→narrowest 2.以不发音e结尾的单音节词，比较在原级后加-r，最高级在原级后加-st; 如：large→larger→largest nice→nicer→nicest able→abler→ablest 3.在重读闭音节(即：辅音+元音+辅音)中，先双写末尾的辅音字母，比较级加-er，最高级加-est; 如：big→bigger→biggest hot→hotter→hottest fat→fatter→fattest 4.以“辅音字母+y”结尾的双音节词，把y改为i，比较级加-er，最高级加-est; 如：easy→easier→easiest heavy→heavier→heaviest busy→busier→busiest happy→happier→happiest 5.其他双音节词和多音节词，比较级在前面加more，最高级在前面加most; 如：bea utiful→more beautiful→most beautiful different→more different→most different easily→more easily→most easily 注意：(1)形容词最高级前通常必须用定冠词 the，副词最高级前可不用。 例句： The Sahara is the biggest desert in the world. (2) 形容词most前面没有the，不表示最高级的含义，只表示"非常"。 It is a most important problem. =It is a very important problem.
定冠词the的用法： 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...
More than的用法 A. “More than+名词”表示“不仅仅是” 1)Modern science is more than a large amount of information. 2)Jason is more than a lecturer; he is a writer, too. 3) We need more than material wealth to build our country.建设我们国家，不仅仅需要物质财富. B. “More than+数词”含“以上”或“不止”之意，如： 4)I have known David for more than 20 years. 5)Let's carry out the test with more than the sample copy. 6) More than one person has made this suggestion. 不止一人提过这个建议. C. “More than+形容词”等于“很”或“非常”的意思，如： 7)In doing scientific experiments, one must be more than careful with the instruments. 8)I assure you I am more than glad to help you. D. more than + (that)从句，其基本意义是“超过（=over）”，但可译成“简直不”“远非”.难以，完全不能（其后通常连用情态动词can） 9) That is more than I can understand . 那非我所能懂的. 10) That is more than I can tell. 那事我实在不明白。 11) The heat there was more than he could stand. 那儿的炎热程度是他所不能忍受的 此外，“more than”也在一些惯用语中出现，如： more...than 的用法 1. 比……多，比……更 He has more books than me. 他的书比我多。 He is more careful than the others. 他比其他人更仔细。 2. 与其……不如 He is more lucky than clever. 与其说他聪明，不如说他幸运。 He is more （a）scholar than （a）teacher. 与其说他是位教师，不如说他是位学者。 注：该句型主要用于同一个人或物在两个不同性质或特征等方面的比较，其中的比较级必须用加more 的形式，不能用加词尾-er 的形式。 No more than/not more than 1. no more than 的意思是“仅仅”“只有”“最多不超过”，强调少。如： --This test takes no more than thirty minutes. 这个测验只要30分钟。 --The pub was no more than half full. 该酒吧的上座率最多不超过五成。-For thirty years，he had done no more than he （had）needed to. 30年来，他只干了他需要干的工作。 2. not more than 为more than （多于）的否定式，其意为“不多于”“不超过”。如：Not more than 10 guests came to her birthday party. 来参加她的生日宴会的客人不超过十人。 比较： She has no more than three hats. 她只有3顶帽子。（太少了） She has not more than three hats. 她至多有3顶帽子。（也许不到3顶帽子） I have no more than five yuan in my pocket. 我口袋里的钱最多不过5元。（言其少） I have not more than five yuan in my pocket. 我口袋里的钱不多于5元。（也许不到5元） more than, less than 的用法 1. （指数量）不到，不足 It’s less than half an hour’s drive from here. 开车到那里不到半个钟头。 In less than an hour he finished the work. 没要上一个小时，他就完成了工作。 2. 比……（小）少 She eats less than she should. 她吃得比她应该吃的少。 Half the group felt they spent less than average. 半数人觉得他们的花费低于平均水平。 more…than,/no more than/not more than (1)Mr．Li is ________ a professor; he is also a famous scientist． (2)As I had ________ five dollars with me, I couldn’t afford the new jacket then． (3)He had to work at the age of ________ twelve． (4)There were ________ ten chairs in the room．However, the number of the children is twelve． (5)If you tel l your father what you’ve done, he’ll be ________ angry． (6)－What did you think of this novel? －I was disappointed to find it ________ interesting ________ that one． 倍数表达法 1. “倍数+形容词（或副词）的比较级+than+从句”表示“A比B大（长、高、宽等）多少倍” This rope is twice longer than that one.这根绳是那根绳的三倍（比那根绳长两倍）。The car runs twice faster than that truck.这辆小车的速度比那辆卡车快两倍（是那辆卡车的三倍）。 2. “倍数+as+形容词或副词的原级+as+从句”表示“A正好是B的多少倍”。
“tｈｅwａｙ+从句”结构的意义及用法 首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。 下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句 例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法
不对。 Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.
初中英语比较级和最高级讲解与练习 形容词比较级和最高级 一．绝大多数形容词有三种形式,原级,比较级和最高级, 以表示形容词说明的性质在程度上的不同。 1. 形容词的原级: 形容词的原级形式就是词典中出现的形容词的原形。例如: poor tall great glad bad 2. 形容词的比较级和最高级: 形容词的比较级和最高级形式是在形容词的原级形式的基 础上变化的。分为规则变化和不规则变化。 二．形容词比较级和最高级规则变化如下: 1) 单音节形容词的比较级和最高级形式是在词尾加-er 和-est 构成。 great (原级) greater(比较级) greatest(最高级) 2) 以-e 结尾的单音节形容词的比较级和最高级是在词尾加-r 和-st 构成。 wide (原级) wider (比较级) widest (最高级) 3) 少数以-y, -er, -ow, -ble结尾的双音节形容词的比较级和最高级是在词尾加 -er 和-est构成。 clever(原级) cleverer(比较级) cleverest(最高级)， slow(原级) slower(比较级) slowest (最高级) 4) 以-y 结尾,但-y 前是辅音字母的形容词的比较级和最高级是把-y 去掉,加上-ier 和-est 构成. happy (原形) happier (比较级) happiest (最高级) 5) 以一个辅音字母结尾其前面的元音字母发短元音的形容词的比较级和最高级是双写该 辅音字母然后再加-er和-est。 原形比较级最高级原形比较级最高级 big bigger biggest hot hotter hottest red redder reddest thin thinner thinnest 6) 双音节和多音节形容词的比较级和最高级需用more 和most 加在形容词前面来构 成。 原形比较级最高级 careful careful more careful most careful difficult more difficult most difficult delicious more delicious most delicious 7)常用的不规则变化的形容词的比较级和最高级: 原级比较级最高级 good better best 好的 well better best 身体好的 bad worse worst 坏的 ill worse worst 病的 many more most 许多 much more most 许多 few less least 少数几个 little less least 少数一点儿 （little littler littlest 小的） far further furthest 远（指更进一步，深度。亦可指更远） far farther farthest 远（指更远，路程）
表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。 如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如： 我不喜欢他说话的态度。 正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。 一、in a way的用法 表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。 二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。 三、by the way的用法
形容词比较级和最高级的形式 一、形容词比较级和最高级的构成 形容词的比较级和最高级变化形式规则如下 构成法原级比较级最高级 ①一般单音节词末尾加 er 和 est strong stronger strongest ②单音节词如果以 e结尾，只加 r 和 st strange stranger strangest ③闭音节单音节词如末尾只有一个辅音字母， 须先双写这个辅音字母，再加 er和 est sad big hot sadder bigger hotter saddest biggest hottest ④少数以 y, er(或 ure), ow, ble结尾的双音节词， 末尾加 er和 est(以 y结尾的词，如 y前是辅音字母， 把y变成i，再加 er和 est，以 e结尾的词仍 只加 r和 st) angry Clever Narrow Noble angrier Cleverer narrower nobler angriest cleverest narrowest noblest ⑤其他双音节和多音节词都在前面加单词more和most different more different most different 1) The most high 〔A〕mountain in 〔B〕the world is Mount Everest，which is situated 〔C〕in Nepal and is twenty nine thousand one hundred and fourty one feet high 〔D〕 . 2) This house is spaciouser 〔A〕than that 〔B〕white 〔C〕one I bought in Rapid City，South Dakota 〔D〕last year. 3) Research in the social 〔A〕sciences often proves difficulter 〔B〕than similar 〔C〕work in the physical 〔D〕sciences. 二、形容词比较级或最高级的特殊形式：
比较级和最高级 1.在形容词词尾加上―er‖ ―est‖ 构成比较级、最高级： bright（明亮的）—brighter—brightest broad（广阔的）—broader—broadest cheap（便宜的）—cheaper—cheapest clean（干净的）—cleaner—cleanest clever（聪明的）—cleverer—cleverest cold（寒冷的）—colder—coldest cool（凉的）—cooler—coolest dark（黑暗的）—darker—darkest dear（贵的）—dearer—dearest deep（深的）—deeper—deepest fast（迅速的）—faster—fastest few（少的）—fewer—fewest great（伟大的）—greater—greatest hard（困难的,硬的）—harder—hardest high（高的）—higher—highest kind（善良的）—kinder—kindest light（轻的）—lighter—lightest long（长的）—longer—longest loud（响亮的）—louder—loudest low（低的）—lower—lowest near（近的）—nearer—nearest new（新的）—newer—newest poor（穷的）—poorer—poorest quick（快的）—quicker—quickest quiet（安静的）—quieter—quietest rich（富裕的）—richer—richest short（短的）—shorter—shortest slow（慢的）—slower—slowest small（小的）—smaller—smallest smart（聪明的）—smarter—smartest soft（柔软的）—softer—softest strong（强壮的）—stronger—strongest sweet（甜的）—sweeter—sweetest tall（高的）-taller-tallest thick（厚的）—thicker—thickest warm（温暖的）—warmer—warmest weak（弱的）—weaker—weakest young（年轻的）—younger—youngest 2.双写最后一个字母,再加上―er‖ ―est‖构成比较级、最高级： big（大的）—bigger—biggest fat（胖的）—fatter—fattest hot（热的）—hotter—hottest red（红的）—redder—reddest sad（伤心的）—sadder—saddest thin（瘦的）—thinner—thinnest wet（湿的）—wetter—wettest mad（疯的）—madder—maddest 3.以不发音的字母e结尾的形容词,加上―r‖ ―st‖ 构成比较级、最高级：able（能干的）—abler—ablest brave（勇敢的）—braver—bravest close（接近的）—closer—closest fine（好的,完美的）—finer—finest large（巨大的）—larger—largest late（迟的）—later—latest nice（好的）—nicer—nicest ripe（成熟的）—riper—ripest
The way的用法及其含义（一） 有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析： 一、the way作先行词，后接定语从句 以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.
英语语法---比较级和最高级的用法 在英语中通常用下列方式表示的词:在形容词或副词前加more(如 more natural,more clearly )或加后缀 -er(newer,sooner )。典型的是指形容词或副词所表示的质、量或关系的增加。英语句子中，将比较两个主体的方法叫做“比较句型”。其中，像“A比B更……”的表达方式称为比较级；而“A最……”的表达方式则称为最高级。组成句子的方式是将形容词或副词变化成比较级或最高级的形态。 一、形容词、副词的比较级和最高级的构成规则 1．一般单音节词和少数以-er，-ow结尾的双音节词，比较级在后面加-er，最高级在后面加-est； （1）单音节词 如：small→smaller→smallest short→shorter→shortest tall→taller→tallest great→greater→greatest （2）双音节词 如：clever→cleverer→cleverest narrow→narrower→narrowest 2．以不发音e结尾的单音节词，比较在原级后加-r，最高级在原级后加-st； 如：large→larger→largest nice→nicer→nicest able→abler→ablest 3．在重读闭音节（即：辅音＋元音＋辅音）中，先双写末尾的辅音字母，比较级加-er，最高级加-est； 如：big→bigger→biggest hot→hotter→hottest fat→fatter→fattest 4．以“辅音字母＋y”结尾的双音节词，把y改为i，比较级加-er，最高级加-est； 如：easy→easier→easiest heavy→heavier→heaviest busy→busier→busiest happy→happier→happiest 5．其他双音节词和多音节词，比较级在前面加more，最高级在前面加most； 如：beautiful→more beautiful→most beautiful different→more different→most different easily→more easily→most easily