Chinese Journal of Natural Medicines6(2008) 0057 0062
Chinese
Journal of
Natural
Medicines
Activated Antibiotic Production by Inducing Resistance to Capreomycin in Streptomyces lividans and
Streptomyces coelicolor
ZHANG Qin, ZHU Bao-Quan, HU Hai-Feng*
Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China
Available online 20 January 2008
[ABSTRACT]AIM:To develop a novel method for improving antibiotic-producing strains. METHODS: The actinorhodin (Act) and undecylprodigiosin (Red) biosyntheses in Streptomyces lividans 66 and a relA mutant of Streptomyces coelicolor A3(2) were impaired. The mutants with activated/restored Act and Red production were selected by inducing resistance to capreomycin, a basic peptide antibiotic. Western blotting analysis was made to investigate the relationship between activated Act and Red production and the ex-pression of ActII-ORF4 and RedD, the Act and RedD biosynthetic pathway-specific regulators, respectively. RESULTS: the capreo-mycin-resistant (cap) mutations could restore the impaired antibiotic production in Streptomyces coelicolor RelA (relA) without recov-ering the ppGpp synthesis, and activate the Act and Red production in Streptomyces lividans 66, suggesting that the dependence of Streptomyces coelicolor on ppGpp to initiate antibiotic biosynthesis could be bypassed by cap mutations. These cap mutants of Strep-tomyces lividans 66 exhibited different phenotypes with several different mutational types possibly appearing in them, although we have not found the mutational positions so far. CONCLUSION: Our observations demonstrated that cap mutations could activate Act and Red biosyntheses through directly or indirectly upregulating the expression of proteins ActII-ORF4 and RedD, respectively. One novel method to improve antibiotic-producing strains has been found.
[KEY WORDS] Actinorhodin; Undecylprodigiosin; Capreomycin; Resistance
[CLC Number]R978.1 [Document code]A [Article ID]1672-3651(2008) 01-0057-06
1 Introduction
Streptomycetes are filamentous soil bacteria that
undergo a complex cycle of morphological differen-
tiation and produce most of the natural antibiotics
used clinically today[1]. Among them, Streptomyces
coelicolor A3(2), the best genetically studied strep-
tomycete, produces four biochemically and geneti-
cally distinct antibiotics: actinorhodin (Act), unde-
cylprodigiosin (Red), methylenomycin (Mmy) and
calcium-dependent antibiotic (CDA)[2]. Since Act and
Red are two pigmented and typical polyketide antibi-
otics, most previous studies have been focused on
[Received on] 13-Apr-2007
[Foundation Item] This project was supported by the Novel Medi-
cine Discovery Fund from Shanghai Institute of Pharmaceutical
Industry of China (No.200205)
[*Corresponding author]HU Hai-Feng, Prof., Tel: 86-21- 62479808;
Fax: 86-21-62791715; E-mail: haifeng_h@ https://www.doczj.com/doc/c91146796.html,
Copyright?2008, China Pharmaceutical University.
Published by Elsevier B.V. All rights reserved.
their biosynthetic pathways and regulatory mecha-
nisms[3,4]. Therefore, various pathway-specific regu-
latory genes (actII-ORF4, redD, and redZ) have been
identified and characterized[4-6]. In addition, many
pleiotropic regulatory genes (absA, absB, afsR, afsR2,
abaA) in S. coelicolor have been found to regulate
antibiotic biosynthesis[7-9]. Streptomyces lividans 66,
a species closely related to S. coelicolor A3(2), pro-
duces less/no Act and Red under normal conditions,
although it carries the whole gene clusters for Act and
Red biosyntheses (2). Recent studies have demon-
strated that the cutRS signal transduction system and
LysR-type transcriptional regulator negatively regu-
late Act biosynthesis[10,11], and afsR2encoding a
63-amino acid protein stimulates Act and Red produc-
tion in S. lividans[12]. However, the complex regula-
tory mechanisms of Act and Red biosyntheses existing
in S. coelicolor and S. lividans are still not com-
pletely clear.
Recent advances in novel drug discovery and
combinatorial biosyntheses have dramatically im-
proved our ability to produce valuable antibiotics or
other bioactive compounds by microorganisms. How-ever, the utilization of these wild type or engineered strains in industrial processes is often constrained by their limited productivity. Classical strain improve-ment methods rely on UV radiation or chemical mutagenesis. These methods are rather inefficient so that developing efficient and rational strain-improving methods is of considerable importance[13]. Ribosomes are dynamic complexes responsible for translating the genetic information encoded in mRNAs to proteins. Previous studies demonstrated that ribosome is in-volved in regulating the biosynthesis of secondary metabolites in microorganisms[14,15]. Consistent with this, streptomycin, one of translational miscoding agents, is capable of inducing overproducing antibi-otics in various bacteria[16,17].Our previous studies found that other aminoglycoside antibiotics such as gentamicin and geneticin exhibit a similar ability to streptomycin[18]. In addition, capreomycin, a basic peptide antibiotic, is able to inhibit protein synthesis by inducing misreading of ribosome like aminoglyco-sides[19,20]. Here, we examined the ability of capreo-mycin to activate Act and Red biosyntheses in S. livi-dans and restore the impaired Act and Red production in a relA mutant of S. coelicolor A3[2] by inducing resistance to capreomycin. Meanwhile, we analyzed the mechanism of its action by Western blot assay.
2 Materials and Methods
2.1 Strains and preparation of mutants
The S. lividans 66, S. coelicolor M600, a relA mutant of M600 and the capreomycin-resistant mu-tants used in this study are listed in Table 1. After the spore or cell suspensions were spread on GYM agar containing various concentrations of capreomycin and incubated at 30q C for 5-8 days, the spontaneous capreomycin-resistant (cap) mutants occurred. The independent colonies were transferred to new GYM agar and randomly selected for further study after single colony isolation was performed. 2.2 Media and culture conditions
GYM, SMMS and R4 media were prepared as de-scribed previously[2,16,17]. Culture conditions were the same as those described previously for S. coelicolor A3(2)[18]. Determination of antibiotic productivity was always performed using triplicated culture flasks and mean values of the three samples were presented in Table 2 and Figs. 1 and 2. Reproducibility of the results was confirmed at least by two separate ex-periments (Table 1 and Fig. 2).
2.3 Mycelial growth and antibiotic assays
Actinorhodin in R4 medium was determined as described previously[18]. In GYM medium, 10 % (V/V) of 1 mol L1 KOH was added to culture broth and the resultant solution was filtered after standing for 1 h at room temperature. The optical density at OD633 nm of each filtrate was measured for actinorhodin. Mycelial growth was measured by weighing the dry mycelia of each 100 mL culture. After weighing, the dried myce-lia were first extracted with 0.1 mol L 1 KOH to dis-solve actinorhodin, and then extracted with methanol (adjusted pH 2) overnight at room temperature. The amount of undecylprodigiosin was determined by measuring the optical density at 533 nm (H503=100, 500)[2]. 2.4 Western blot analysis
Cultures were grown in GYM liquid medium at 30q C for 84 h. At the denoted time, 1.5 mL of cultures was collected and centrifugated. The resultant pellet was washed by sterilized 0.8 % saline and suspended in 20 mmol L 1 Tris-HCl (pH 7.0) containing 1 mmol L 1 EDTA, 1 mmol dithiothreitol, 10% (V/V) glycerol, protease inhibitor, and 0.5mmol phenylmethylsulfon-ylfuoride. Then the suspension was sonicated as de-scribed previously[21]. Protein concentration was de-termined with dye-binding assay reagent (Bio-Rad, Hercules, Calf.) and bovine serum albumin as the standard. Antibodies against ActII-ORF4 and RedD proteins of Streptomyces coelicolor A3(2) were pre-pared in rabbits by injecting the recombinant proteins and were used as a primary antibody at a dilution of
Table 1 Strains used in this study and resistance analysis
Resistance level (MIC, g mL-1) a) Strain Relevant genotype Resource or reference
Capreomycin Kanamycin Gentamicin 66b)Prototrophic wild type 0.5 0.3 0.1 Cap-1 cap-1 A cap mutant of 66c)30 0.5 0.3 Cap-2 cap-2 A cap mutant of 66 45 0.5 0.3 Cap-3 cap-3 A cap mutant of 66 35 0.2 0.2 M600d) Prototrophic M. J. Bibb (6) 1 0.3 0.3 RelA relA A relA null mutant of M600 (27)0.5 0.3 0.2 Cap-4 cap-4 A cap mutant of RelA 50 0.3 0.2 Cap-5 cap-5 A cap mutant of RelA 45 0.3 0.2 a) MICs were determined by inoculating spores into GYM agar containing various amount of the respective antibiotic and incubating
at 30 q C for 2 days; b) 66, Streptomyces lividans 66; c) All cap mutants are spontaneous; d) M600, Streptomyces coelicolor M600.
1?3 000. Western blots were developed with the en-hanced chemiluminescence Western blotting detection system for chemiluminescent detection as specified by the manufacturer.
Table 2 Mycelial growth and antibiotic production in Streptomyces lividans 66 and the cap mutants
Mycelial dry weight b) (g/100 mL)Act production b)
(OD633)
Red production b)
(mg g 1dry weight)
Strain
GYM R4GYM R4GYM R4
66a)0.1580.181 0.0730.026 0.0938 5.160 Cap-10.179 0.184 0.6500.148 1.407 28.800 Cap-20.203 0.330 0.120 1.842 0.072480.410 Cap-30.169 0.148 0.1480.86 0.118 44.040 a)66, Streptomyces lividans 66; b) Cultures were incubated at 30 q C for 5 days.
3 Results
3.1 Activated antibiotic production in S. lividans.
S. lividans 66 usually produces less/no actinorhodin (Act) and undecylprodigiosin (Red) under some con-ditions. We attempted to activate Act and/or Red pro-duction in S. lividans by inducing resistance to capreomycin. The spontaneous cap mutants were ob-tained at a frequency of 10 7 to 10 9 after the spores of S. lividans 66 were spread and incubated on GYM agar containing 50 and 100 P g mL 1 of capreomycin. About eighty independent cap mutants were randomly selected and examined for the productivity of Act and Red on R4 agar and liquid media. Surprisingly, about 15% (12/80) of mutants exhibited a significant ability to produce Act and/or Red in R4 medium (Table 2 and Fig. 1). Moreover, we examined activated Act and Red production of cap mutants in GYM medium and found that some mutants preferred GYM medium (Cap-1) while other preferred R4 medium (mutants Cap-2 and Cap-3), suggesting the existence of differ-ent mutational types among these cap mutants (Fig.1). MIC analyses revealed that these cap mutants ac-quired different levels of resistance to capreomycin and showed no/weak cross-resistance to kanamycin and gentamicin (aminoglycoside antibiotics, Table 1).
As shown in Fig. 1, aerial mycelium forming and sporulation in some cap mutants were markedly im-paired accompanying activated Act and Red produc-tion, especially on GYM agar. However, mycelial growth in R4 and GYM liquid media was significantly unaffected by cap mutations (Table 2).
3.2 Expression of proteins ActII-ORF4 and RedD
It has been known that proteins ActII-ORF4 and RedD are the positive regulators of Act and Red biosynthe-ses in Streptomyces coelicolor A3(2), respectively[3,5]. Moreover, their expression is growth phase depend-ent[22]. It is essential to analyze the expression pat-terns of these proteins and the correlation with acti-vated Act and Red biosyntheses by cap mutations.
Fig. 1 Aerial mycelial formation, sporulation and anti-biotic production on R4, GYM or SMMS medium. Blue, actinorhodin; red, undecylprodigiosin. (A) Aerial mycelial formation, sporulation and antibiotic production by Par-ent (wild-type), Cap-1 (cap-1) and Cap-2 (cap-2); (B) Ae-rial mycelial formation, sporulation and antibiotic pro-duction by M600 (prototrophic), RelA (relA), Cap-4 (cap-4) and Cap-5 (cap-5).
Therefore, we chose mutants Cap-1 and Cap-2 as the representatives with the wild-type strain of S. lividans 66. As shown in Fig. 2A and B, the upregulation of ActII-ORF4 expression was completely identical to the activated Act biosynthesis. For example, the ex-pression of ActII-ORF4 in mutant Cap-1 was signifi-cantly upregulated from 36 h and reached the maximal level at 60 h accomplishing markedly activated Act biosynthesis from 48 to 72 h compared to those in the wild-type strain. Like ActII-ORF4, protein RedD in Cap-1 was significantly over-expressed from 12 to 36 h accomplishing the markedly activated Red biosyn-thesis from 24 to 72 h compared to those in the wild-type strain (Fig. 2A and B). The low level of protein RedD in Cap-2 resulted in low productivity of Red (Fig. 2A and B). These results indicated that the activated Act and Red biosyntheses by cap mutations evidently correlates with the upregulation of Ac-tII-ORF4 and RedD, the pathway-specific regulators, respectively.
3.3 Restored Act biosynthesis in a relA mutant
It has been proven that a relA mutant of Strepto-myces coelicolor A3(2) loses the ability to produce Act and Red and delays morphological differentiation under conditions of nitrogen limitation owing to the loss of the ability to synthesize the guanine nucleotide ppGpp[23]. It is interesting to examine the ability of cap mutations to restore the impaired Act and Red production in RelA (a relA mutant). Using a similar way described above, we obtained about one hundred independent cap mutants of RelA. After analyzing the Act and Red productivity on SMMS and GYM media, we found that nine mutants exhibited the ability to restore the production of Act and Red in SMMS and
Fig. 2 Western blotting analysis, mycelial growth and antibiotic biosynthetic curves. (A). Mycelial growth, actinorhodin and undecylprodigiosin biosyntheses in GYM liquid medium by Parent (wild-type), Cap-1 (cap-1) and Cap-2 (cap-2). DW, dry weight (B). Western blotting analysis of proteins ActII-ORF4 and RedD in GYM medium by Parent (wild-type), Cap-1 (cap-1) and Cap-2 (cap-2).
GYM media compared to the wild-type strain. More-over, Act biosynthesis was upregulated in GYM me-dium compared to that in M600 (Fig. 1). However, aerial mycelium formed in Cap-4 and Cap-5 was par-tially recovered on GYM agar but not on SMMS com-pared to that of RelA (Fig. 1). These observations demon-strated that cap mutations could not only restore Act and Red production in RelA under conditions of nitrogen limitation, but also overproduce Act and form aerial my-celia under conditions of rich nitrogen.
4 Discussion
In the present study, we found that Act and Red biosyntheses can be activated/restored by inducing high level of resistance to capreomycin (a basic pep-tide antibiotic) in Streptomyces lividans 66 or a relA mutant of Streptomyces coelicolor A3(2), which evi-dently correlates with the upregulation of the expres-sion of proteins ActII-ORF4 and RedD, the Act and Red biosynthetic pathway-specific regulators, respec-tively. Consistent with previous findings[16-18], this method can generate robust antibiotic producers at a surprisingly high frequency (9% to 12%) compared to traditional strain improving methods[13], although the mutational sites have not been found so far.
Most previous studies demonstrated that ribosome is directly or indirectly correlated with regulatory mechanisms of microbial secondary metabolite bio-syntheses[14,24]. For example, aminoglycoside antibi-otics such as streptomycin, gentamicin and geneticin are all able to activate/enhance antibiotic production in Streptomyces and other bacteria by inducing resis-tance to these antibiotics resulting in the mutations in the rpsL gene (encoding ribosomal S12 protein) or other genes[16-18]. Moreover, it has been demonstrated that the structural changes in ribosome induced by miscoding agents (aminoglycosides) correlate with their respective phenotypes[25].Capreomycin, a basic peptide antibiotic, has been characterized as a trans-location inhibitor and can promote translational errors (misreading) in Escherichia coli[19,20]. Therefore, it is not surprising that capreomycin can activate antibiotic production in S. lividans and S. coelicolor A3(2) in a similar way to streptomycin and gentamicin.
Previous studies demonstrated that resistance to capreomycin and viomycin (a structurally similar peptide antibiotic to capreomycin) in M. smegmatis was caused by an altered RNA molecule in the 30S or 50S ribosome subunit and some mutations in 16S RNA gene have been found[19,24]. Nevertheless, we could not detect any mutation in about ten cap mu-tants of S. lividans 66, although we sequenced every 16S rRNA genes and rpsL gene. Another mechanism of resistance to capreomycin has been found to be synthesize modifying enzymes in capreomycin- pro-ducing Streptomyces[26] and is not correlated with
ribosome. The data presented here is not enough to address whether the resistance to capreomycin results from modifying enzymes or other unknown mecha-nisms. Cloning and sequencing analysis of capreomy-cin-resistance determinants in our cap mutants of S. lividans66 is in progress and it helps to elucidate the mechanisms of action.
The guanine nucleotide ppGpp has been proven to be a pivotal signal molecule for the onset of antibiotic production[23,27]. Consistent with this, a relA mutant of S. coelicolor A3(2) losing the ability to synthesize ppGpp also loses the ability to produce antibiotics under conditions of nitrogen limitation[23]. However, our results indicated that the impaired Act and Red biosyntheses in RelA could be partially restored by inducing resistance to capreomycin without recover-ing the synthesis of ppGpp (data not shown). There-fore, the dependence of S. coelicolor on ppGpp to initiate Act production is apparently bypassed by cap mutations, which is consistent with our previous findings for rifampicin-resistant mutations in the rpoB gene encoding RNA polymerase ? subunit [18].
Previous studies demonstrated that transcription of the activator genes actII-ORF4 and redD has been characterized to be growth-phase-dependent, and the transcription of the antibiotic structural genes has been shown to occur after the accumulation of almost maximal levels of actII-ORF4 or redD transcripts[6,22]. It is possible that a threshold concentration of the activator protein is needed for activating the antibi-otic biosynthesis genes. Consistent with this hypothe-sis, the Act and Red biosyntheses in mutant Cap-1 was activated following by upregulating the expres-sion of proteins ActII-ORF4 and RedD, respectively (Fig. 2). The low level of ActII-ORF4 or RedD pro-tein expressed in the wild-type strain apparently was not enough to activate Act and Red biosynthesis (Fig. 2A and 2B). These results indicated that cap muta-tions activate/enhance antibiotic production through indirectly or directly upregulating the expression of the antibiotic biosynthetic pathway-specific regula-tory proteins consistent with gentamicin-resistant and rifampicin-resistant mutations as described in our previous studies[18].
Acknowledgement: We are grateful to Dr. Kozo Ochi for his considerable kind help.
References
[1] Chater KF, Hopwodd DA. Antibiotic biosynthesis in Strep-
tomyces.In Genetics of Bacterial Diversity [J]. Academic
Press London England, 1989: 129-150.
[2] Kesier T, Bibb MJ, Butter MJ, et al. Practical Streptomyces
Genetics [M]. the John Innes Foundation, Norwich, UK, 2000.
[3] Witzorrek A, Bibb M. A novel family of proteins that regu-
lates antibiotic production in streptomycetes appears to con-
tain an OmpR-like DNA-binding fold[J]. Mol Microbiol,
1997, 25 (1): 117-1184.
[4] Fernaandez-Moreno MA, Caballrero JL, Hopwood DA, et al.
The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA
tRNA gene of Streptomyces[J]. Cell, 1991, 66 (2): 769-780.
[5] Narva KE, Feitelson JS. Nucleotide sequence and transcrip-
tional analysis of the redD locus of Streptomyces coelicolor
A3(2) [J]. J Bacteriol, 1990, 172 (1): 326-333.
[6] Takano E, Gramajo HC, Strauch E, et al. Transcriptional
regulation of the redD transcriptional activator gene ac-
counts for growth-phase-dependent production of the antibi-
otic undecylprodigiosin in Streptomyces coelicolor A3(2)[J].
Mol Microbiol, 1992, 6 (19): 2797-2804.
[7] Champness W, Riggle P, Adamidis T. Loci involved in
regulation of antibiotic synthesis[J]. J Cell Biothem, 1990,
14 (1): 88
[8] Adamidis T, Riggle P, Champness W. Mutations in a new
Streptomyces coelicolor locus which globally block antibi-
otic biosynthesis but not sporulation[J]. J Bacteriol, 1990, 172 (6): 2962-2969.
[9] Horinouchi S, Kito M, Nishiyama M, et al. Primary structure
of AfsR, a global regulatory protein for secondary metabolite
formation in Streptomyces coelicolor A3(2)[J]. Gene, 1990,
95 (1): 49-5.
[10] Chang HM, Chen MY, Shieh YT, et al. The cutRS signal
transduction system of Streptomyces lividans represses the biosynthesis of the polyketide antibiotic actinorhodin[J]. Mol
Microbiol, 1996, 21 (5): 1075-1085.
[11] Mart o nez-Costa O, Mart o n-triana AJ, Mart o nez, E, et al. An
additional regulatory gene for actinorhodin production in Streptomyces lividans involves a LysR-type transcriptional regulator?J?. J Bacteriol, 1999, 181 (14): 4353-4364. [12] Vogtli M, Chang C, Cohen SN. afsR2: a previously unde-
tected gene encoding a 63-amino acid protein that stimulates
antibiotic production in Streptomyces lividans [J]. Mol Mi-
crobiol, 1994, 14 (4): 643-653.
[13] Rowlands RT. Industrial strain improvement: mutagenesis
and random screening procedures[J]. EnzymeMicrobiol
Technol, 1984, 6 (1): 3-10.
[14] Martinez-Costa OH, Arias P, Romero NM, et al. A relA/spot
homologous gene from Streptomyces coelicolor A3(2) con-
trols antibiotic biosynthetic genes[J]. J Biol Chem, 1996,
271(18): 10627-10634.
[15] Ochi K, Ohsawa S. Initiation of antibiotic production by the
stringent response of Bacillus subtilis Marburg[J]. J Gen
Microbiol 1984, 130 (10): 2473-2482.
[16] Hosoya Y, Okamoto S, Muramatsu H, et al. Acquisition of
certain streptomycin-resistant (str) mutations enhances anti-
biotic production in bacteria[J]. Antimicrob Agents Chemo-
ther, 1998, 42 (8): 2041-2047.
[17] Shima J, Hesketh A, Okamoto S, et al. Induction of actinor-
hodin production by rpsL(encoding ribosomal protein S12)
mutations that confer streptomycin resistance in Streptomy-
ces lividans and Streptomyces coelicolor A3(2)[J]. J Bacte-
riol, 1996, 178 (24): 7276-7284.
[18] Hu H, Ochi K. Novel approach for improving the productiv-
ity of antibiotic-producing strains by inducing combined re-
sistant mutations[J]. Appl Environ Microbiol, 2001, 67 (4):
1885-1892.
[19] Yamada T, Mizuguchi Y, Nierhaus KH, et al. Resistance to
viomycin conferred by RNA of either ribosomal subunit[J].
Nature, 1978,275 (5): 460-461.
[20] Marrero P, Cabanas MJ, Modolell J. Induction of transla-
tional errors (misreading) by tuberactinomycins and capreomycins[J]. Biochem Biophy Res Commun, 1980, 97
(4): 1047-1052.
[21] Gramajo HC, White J, Hutchinson CR, et al. Overproduction
and localization of components of the polyketide synthase of
Streptomyces glaucescens involved in the production of the
antibiotic tetracenomycin C[J]. J Bacteriol, 1991, 173 (20):
6457-6483.
[22] Gramajo HC, Takano E, Bibb MJ. Stationary-phase produc-
tion of the antibiotic actinorhodin in Streptomyces coelicolor
A3(2) is transcriptionally regulated[J]. Mol Microbiol, 1993,
7 (6): 837-845.
[23] Sun J, Hesketh A, BIBB M. Functional analysis of relA and
rshA, two relA/spoT homologues of Streptomyces coelicolor
A3(2)[J]. J Bacteriol, 2001, 183 (11): 3488-3498.
[24] Taniguchi H, Chang B, Abe C, et al. Molecular analysis of
kanamycin and viomycin resistance in Mycobacterium
smegmatis by use of the conjugation system[J]. J Bacteriol,
1997, 179 (15): 4795-4801.
[25] Cundliffe E. Antibiotic inhibitors of ribosome Function[M].
1981: 402-547, Wiley, New York.
[26] Thiara AS, Cundliffe E. Analysis of two capreo-
mycn-resistance determinants from Streptomyces capreolus
and characterization of the action of their products[J]. Gene,
1995, 167 (1-2): 121-126.
[27] Chakraburtty R, White J, Takano E, et la. Cloning, charac-
terization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2)[J]. Mol Microbiol,
1996, 19 (2): 357-368.
英语广告语集锦 本文是关于经典句子的，仅供参考，如果觉得很不错，欢迎点评和分享。 英语广告语集锦 1.Time is what you make of it.（Swatch） 天长地久。（斯沃奇手表） 2.Make yourself heard.（Ericsson） 理解就是沟通。（爱立信） 3.Engineered to move the human spirit.（Mercedes-Benz） 人类精神的动力。（梅塞德斯-奔驰） 4.Start Ahead.（Rejoice） 成功之路，从头开始。（飘柔） 5.A diamond lasts forever.（De Bierres） 钻石恒久远，一颗永流传。（第比尔斯） 6.Fresh-up with Seven-up.（Seven-up） 提神醒脑，喝七喜。（七喜） 7.Intel Inside.（Intel Pentium） 给电脑一颗奔腾的“芯”。（英特尔奔腾） 8.Connecting People.（Nokia） 科技以人为本。（诺基亚） 9.For the Road Ahead.（Honda）
康庄大道。（本田） 10.Let us make things better.（Philips） 让我们做的更好。（飞利浦） 11.Enjoy Coca-Cola.（Coca-Cola） 请喝可口可乐。（可口可乐） 12.Generation Next.（Pepsi） 新的一代。（百事） 13.The Relentless Pursuit of Perfection.（Lexus） 追求完美永无止境。（凌志汽车） https://www.doczj.com/doc/c91146796.html,munication unlimited.（Motorola） 沟通无极限。（摩托罗拉） 15.Feast your eyes.（Pond’s Cucumber Eye Treatment） 滋润心灵的窗户。（庞氏眼贴片） 16.Focus on life.（Olympus） 瞄准生活。（奥林巴斯） 17.Behind that healthy smile,t here ’s a Crest kid.（Crest toothpaste） 健康笑容来自佳洁士。（佳洁士牙膏） 关于英语的广告词英语培训学校的广告语经典广告词（英语）感谢阅读，希望能帮助您！
英语口语8000句：喜欢上某人时常用口语句子 恋爱和结婚 ●喜欢、爱上…… ◎汤姆是个美男子。 Tom is a lady-killer. *lady-killer直译是“少女杀手”，其实不是杀手，而是指一下子就能迷住女人的男子。 = Tom dates around a lot. (汤姆和好多女人来往。) = Tom is a real playboy. (汤姆真是个花花公子。) ◎汤姆真让我神魂颠倒。 Tom really turns me on. *turn...on“有性方面的吸引力”、“使人着迷”。 I didn't know you felt that way. (我一点儿都不知道你的感觉。) = I'm crazy about Tom. = I have strong feelings for Tom. = I love Tom. = I have the hots for Tom. *俚语。 ◎克里斯长得真帅。 Chris is really a heartbreaker. *用heartbreaker表示“长得很帅，对异性有吸引力的人”。进 一步讲，“heartbreaker”是指给异性带来撕心裂肺的痛苦和失望的人，魅惑他人，让人沉醉的人。Elvis Presley (爱尔维斯·普里斯利)有一首成名曲“Heartbreak Hotel”，指的就是“(因恋爱而)绝望的 人住的饭店”。
Chris breaks a lot of hearts. (克里斯使很多女人尝到了失恋的痛苦。) Chris dates a lot of women. (克里斯和很多女人有来往。) ◎珍妮特真迷人。Janet is a knockout. *如同拳击中的“knock out”一样，表示极具魅力、使对方晕头转向的人，多指女性。 You can say that again! (颇有同感。) = Janet is sexy. = Janet is beautiful. ◎他好像看上你了。I think he has a crush on you. *have a crush on...“看上……”。 Give me a break. (别随便瞎说。) = I think he is infatuated with you. * be infatuated with...“被……迷住，为……神魂颠倒”。 = I think he likes you. ◎简好像喜欢上我了。Jane seems to like me. *seem“好像，看上去像……”。 = I've got the feeling that Jane likes me. = I think Jane likes me. = I have a hunch (that) Jane likes me. ◎戴安娜对杰克有意思。Diana's been coming on to Jack.
英语语法大全 初中英语语法 学习提纲 一、词类、句子成分和构词法： 1、词类：英语词类分十种： 名词、形容词、代词、数词、冠词、动词、副词、介词、连词、感叹词。 1、名词(n.)：表示人、事物、地点或抽象概念的名称。如：boy, morning, bag, ball, class, orange. 2、代词(pron.)：主要用来代替名词。如：who, she, you, it . 3、形容词(adj..)：表示人或事物的性质或特征。如：good, right, white, orange . 4、数词(num.)：表示数目或事物的顺序。如：one, two, three, first, second, third, fourth. 5、动词(v.)：表示动作或状态。如：am, is,are,have,see . 6、副词(adv.)：修饰动词、形容词或其他副词,说明时间、地点、程度等。如：now, very, here, often, quietly, slowly. 7、冠词(art..)：用在名词前,帮助说明名词。如：a, an, the. 8、介词(prep.)：表示它后面的名词或代词与其他句子成分的关系。如in, on, from, above, behind. 9、连词(conj.)：用来连接词、短语或句子。如and, but, before . 10、感叹词(interj..)表示喜、怒、哀、乐等感情。如：oh, well, hi, hello. 2、句子成分：英语句子成分分为七种：主语、谓语、宾语、定语、状语、表语、宾语补足语。 1、主语是句子所要说的人或事物,回答是“谁”或者“什么”。通常用名词或代词担任。如：I’m Miss Green.(我是格林小姐) 2、谓语动词说明主语的动作或状态,回答“做(什么)”。主要由动词担任。如：Jack cleans the room every day. (杰克每天打扫房间) 3、表语在系动词之后,说明主语的身份或特征,回答是“什么”或者“怎么样”。通常由名词、代词 或形容词担任。如：My name is Ping ping .(我的名字叫萍萍) 4、宾语表示及物动词的对象或结果,回答做的是“什么”。通常由名词或代词担任。如：He can spell the word.(他能拼这个词) 有些及物动词带有两个宾语,一个指物,一个指人。指物的叫直接宾语,指人的叫间接宾语。间 接宾语一般放在直接宾语的前面。如：He wrote me a letter . (他给我写了一封信) 有时可把介词to或for加在间接宾语前构成短语,放在直接宾语后面,来强调间接宾语。如： He wrote a letter to me . (他给我写了一封信) 5、定语修饰名词或代词,通常由形容词、代词、数词等担任。如：
经典英语广告语大全 our wheels are always turning. 我们的车轮常转不停。(五十铃汽车) the world smiles with reader’s digest. 《读者文摘》给全世界带来欢笑。(《读者文摘》) one should love animals. they are so tasty. 每个人都应该热爱动物，因为它们很好吃. love the neighbor. but don‘t get caught. 要用心去爱你的邻居，不过不要让她的老公知道. anything is possible. 没有不可能的事。(东芝电子) /take toshiba, take the world. 拥有东芝，拥有世界。（东芝电子） nobody is perfect. 没有一个人的身材是十全十美的。(苗条健身器材) behind every successful man， there is a woman. and behind every unsuccessful man， there are two. 每个成功男人的背后都有一个女人，每个不成功男人的背后都有两个女人。
every man should marry. after all， happiness is not the only thing in life. 再快乐的单身汉迟早也会结婚，幸 福不是永久的嘛. no business too small, no problem too big. 没有不做 的小生意,没有解决不了的大问题。(ibm公司) the wise never marry， and when they marry they become otherwise. 聪明人都是未婚的，结婚的人很难再聪明起来. we’re the dot. in. com. 我们就是网络。(太阳微系统 公司) children in backseats cause accidents. accidents in backseats cause children. 后排座位上的小孩会生出意外，后排 座位上的意外会生出小孩. the new digital era. 数码新时代。(索尼影碟机) love is photogenic. it needs darkness to develop. 爱情就象照片， 需要大量的暗房时间来培养. good to the last drop. 滴滴香浓,意犹未尽。(麦氏咖啡) a kodak moment. 就在柯达一刻。(柯达胶卷)/share moments. share life. (柯达胶卷)
1.在家中 ●从起床到出门 早晨好 Good morning 闹钟响了吗? *go off是闹钟“响”的意思。 Did the alarm clock go off? 该起床了 -It's time to get up! -I don't wanna get up. 快点儿起床! -Get up soon. -I don't want to. 你醒了吗? *get up是动词，表示“起床”、“起”的动作。awake是形容词，表示“醒了”、“没睡”-Are you awake? -I am now. (我刚醒。) 你不舒服吗? -Are you feeling sick? -No, I'm just tired. 睡得好吗? -Did you sleep well? -Yes, I slept very well. / -No, I couldn't fall asleep. ? Would you turn off the alarm clock? -You finally got up. -I'm still sleepy. (我还困着呢!) 今天是个好天! -It's a nice day! -It sure is. ? Did you stay up late last night? 把被子叠好。 Let's fold up the futon. *snore“打呼噜”。 -Did I keep you up? -You were snoring last night. 过去进行时 我做了个可怕的梦。 -I had a nightmare. -It's all right now. 你一直没关灯啊。 You left the light on. I have to go wash my face. 该吃早饭了。 It's time to eat/have breakfast. 我还困着呢。 I'm still sleepy. 我还打哈欠呢。 I'm still yawning. ['j?:ni?] I have a hangover. I'm a night person. 我是用咖啡来提神的。 Coffee wakes me up. 刷牙了吗? Did you brush your teeth? I have to comb my hair. [k?um] vt 梳头发 穿什么好呢 -What should I wear/put on? -The red one. (穿红的吧!) 快换衣服。 Hurry up and get dressed. 把睡衣收好。*put away 收拾，放好-Put those pajamas away! [p?'d?ɑ:m?z] 睡衣 啊，我正要洗呢。) 我走了。妈妈再见! -I'm leaving. Bye mom! -Study hard. 今天我们逃学吧。*play hooky为俚语“逃学”。 -Yeah, let's. 你毛衣穿反了。 You're wearing your sweater inside out. 上下颠倒了。 It's upside down. 可别忘了扔垃圾! ['ɡɑ:bid?] n垃圾，废物 -Don't forget to take out the garbage. -I won't. 今天该你扔垃圾了。 It's your turn to take out the garbage. 今天你们干嘛? -What are you doing today? -We're having a track and field meet. *运动会 你快点儿，我们该迟到了! If you don't hurry, we'll be late. 快点儿，上学该迟到了。 -Hurry or you'll be late for school. -What time is it? 你锁门了吗? Did you lock the door? 没忘了什么东西吧? -Aren't you forgetting something? 都已经8点了! It's already 8:00. 我晚了! I'm late! 我得赶紧走! I have to rush! 你今天会回来得晚吗? -Are you gonna be late today? -No, I'll be home at the usual time. 几点回来? -What time are you coming home? -Around seven o'clock. 饭盒带了吗? -Have you got your lunch box? -Yes, right here. 今天好像要下雨。 -It might rain today. -Take your umbrella with you. [?m'brel?] 出门的时候，可别忘了锁门。 Don't forget to lock the door when you leave. ●从回家到就寝 我回来了。 I'm home. 你回来了。 Welcome home! 今天过得愉快吗? Did you have a good time? 今天怎么样? How did it go today? 我可以出去玩儿会儿吗? -Can I go out to play? -After you finish your homework. 我饿了。 -I'm hungry. -We have some snacks. *[sn?k] 点心 点心在哪儿?
初中英语语法大全汇总 （一） 一.词类( ) 名词英文名称(缩写为n.) 表示人或事物的名称例词等 冠词英文名称(缩写为.) 用在名词前帮助说明名词所指的人和或事物。例词a() 代词英文名称(缩写为) 用来代替名词、形容词或是数词例词 形容词英文名称(缩写为.) 用以修饰名词,表示人或事物的特征 例词. 数词英文名称(缩写为.) 表示数量或是顺序。例词 动词英文名称(缩写为v.) 表示动作或状态。例词() 副词英文名称(缩写为.) 修饰动词、形容词或其他副词。例词 介词英文单词(缩写为.) 表示名词、代词等和句中其他词的关系。例词. 连词英文单词(缩写为.) 用来连接词与词、短语与短语或句与句。例词. 感叹词英文单词(缩写为.) 表示说话时的喜悦、惊讶等情感。例词. 二.名词() 1.总的说来,名词分专有名词和普通名词两类。 专有名词: 表示具体的人,事物,地点或机构的专有名称。 中国亚洲北京。 专有名词的第一个字母要大写。 普通名词: 表示某些人,某类事物,某种物质或抽象概念的名称。例如: 老师茶改革 普通名词又可进一步分为四类 1) 个体名称: 表示单个的人和事物。 马汽车房间苹果风扇照片 2) 集体名称: 表示一群人或一些事物的名称。 人们家庭军队政府集团 3) 物质名词:表示物质或不具备确定形状和大小的个体的物质。 火钢空气水牛奶 4)抽象名词:表示动作,状态,品质或其他抽象概念。 劳动健康生活友情耐力 2.名词按其所表现的事物的性质分为可数名词和不可数名词。 可数名词( )有复数形式,如: a 不可数名词( )一般没有复数形式. 抽象名词, 物质名词和专有名词一般是不可数名词。 沙糖 有少数名词即可作可数名词,也可作不可数名词,但含义不同。 玻璃玻璃杯纸报纸,文件 名词的功能 名词在句中作主语, 宾语,介词宾语,宾语补助语,表语以及名词短语作状语。
附录A Introduction to database information management system The database is stored together a collection of the relevant data, the data is structured, non-harmful or unnecessary redundancy, and for a variety of application services, data storage independent of the use of its procedures, insert new data on the database, revised, and the original data can be retrieved by a common and can be controlled manner. When a system in the structure of a number of entirely separate from the database, the system includes a "database collection". Database management system is a manipulation and large-scale database management software is being used to set up, use and maintenance of the database. Its unified database management and control so as to ensure database security and integrity. Database management system users access data in the database, the database administrator through Database management system database maintenance work. It provides a variety of functions, allows multiple applications and users use different methods at the same time or different time to build, modify, and asked whether the database. It allows users to easily manipulate data definition and maintenance of data security and integrity, as well as the multi-user concurrency control and the restoration of the database. Using the database can bring many benefits: such as reducing data redundancy, thus saving the data storage space; to achieve full sharing of data resources, and so on. In addition, the database technology also provides users with a very simple means to enable users to easily use the preparation of the database applications. Especially in recent years introduced micro-computer relational database management system , intuitive operation, the use of flexible, convenient programming environment to extensive (generally 16 machine, such as IBM / PC / XT, China Great Wall 0520, and other species can run software), data-processing capacity strong. Database in our country are being more and more widely used, will be a powerful tool of economic management. The database is through the database management system (DBMS-DATA BASE
英语广告语集锦 导读: 英语广告语集锦 1.Time is what you make of it.（Swatch） 天长地久。（斯沃奇手表） 2.Make yourself heard.（Ericsson） 理解就是沟通。（爱立信） 3.Engineered to move the human spirit.（Mercedes-Benz） 人类精神的动力。（梅塞德斯-奔驰） 4.Start Ahead.（Rejoice） 成功之路，从头开始。（飘柔） 5.A diamond lasts forever.（De Bierres） 钻石恒久远，一颗永流传。（第比尔斯） 6.Fresh-up with Seven-up.（Seven-up） 提神醒脑，喝七喜。（七喜） 7.Intel Inside.（Intel Pentium） 给电脑一颗奔腾的“芯”。（英特尔奔腾） 8.Connecting People.（Nokia） 科技以人为本。（诺基亚） 9.For the Road Ahead.（Honda） 康庄大道。（本田） 10.Let us make things better.（Philips）
让我们做的更好。（飞利浦） 11.Enjoy Coca-Cola.（Coca-Cola） 请喝可口可乐。（可口可乐） 12.Generation Next.（Pepsi） 新的一代。（百事） 13.The Relentless Pursuit of Perfection.（Lexus） 追求完美永无止境。（凌志汽车） https://www.doczj.com/doc/c91146796.html,munication unlimited.（Motorola） 沟通无极限。（摩托罗拉） 15.Feast your eyes.（Pond’s Cucumber Eye Treatment） 滋润心灵的窗户。（庞氏眼贴片） 16.Focus on life.（Olympus） 瞄准生活。（奥林巴斯） 17.Behind that healthy smile,there ’s a Crest kid.（Crest toothpaste） 健康笑容来自佳洁士。（佳洁士牙膏）
英语口语8000 句 ●从起床到出门 Good morning, mom. 早晨好，妈妈。 Did the alarm clock go off ? Did the alarm clock buzz? Did the alarm clock ring? It's time to get up! 该起床了! I don't wanna get up. 我真不想起。w a n n a＝w a n t t o;g o n n a=b e g o i n g t o美语口语常用说法It's time to get ready. Get up soon. 快点儿起床! I don't want to. 我真不想起。 Are you awake? 你醒了吗? I am now. 我刚醒。 Are you feeling sick? 你不舒服吗? No, I'm just tired. 没有，只是有点儿累 Did you sleep well? 睡得好吗? Yes, I slept very well. 嗯，睡得挺好。 No, I couldn't fall asleep.哪儿啊几乎没睡着Would you turn off the alarm clock? Please turn off the alarm clock. You finally got up. 你终于起来了。 It's a nice day! 今天是个好天! It sure is. 是不错啊。 It's a beautiful day! It's a wonderful day! It's a great day! Did you stay up late last night? Did you go to bed late last night? Let's fold up the futon. 把被子叠好 Let's put the futon away. 把被子收起 You were snoring last night. 打呼噜 Did I keep you up? 影响你睡觉了吗? I had a nightmare. 我做了个可怕的梦 It's all right now. 现在没事了/挺好的 You left the light on. 你一直没关灯啊 You forgot to turn off the light.你忘关灯了 I have to go wash my face. 我得.... It's time to eat breakfast. It's time to have breakfast.
世界名牌英文广告语大全 世界名牌英文广告语举例 1. Everything from head to toe should be coordinated, Wee bok brand clothing and shoes to do this. Wee bok brand clothing 2.Luzon ------ more your style! Luzon brand clothing 3. Put on “Janssen”, you just like to put on a smile. Jansen garments 4. No matter how you look, “Lands'End” swimsuit always make you satisfied, perfect body shape. Lands'End swimsuit 5. Dancing graceful puter promotional advertising language, to develop Italian pride! Cool! Shuai! Italian modern clothing 6. Elegant chic Leiteng Meng! Reiter Mongolian clothing 7. Not seeking the position of the emperor, but the emperor demeanor! Emperor clothing 8. Silver are fashion, international brand! Silver are brand fashion 9. Li Ning slogan: everything is possible.
REVIEW ARTICLE published:28September2011 doi:10.3389/fmicb.2011 .00203 Acquired antibiotic resistance genes:an overview Angela H.A.M.van Hoek1,Dik Mevius2,3,Beatriz Guerra4,Peter Mullany5,Adam Paul Roberts5and Henk J.M.Aarts1* 1Laboratory for Zoonoses and Environmental Microbiology,Centre for Infectious Disease Control,National Institute of Public Health and the Environment,Utrecht, Netherlands 2Central Veterinary Institute of Wageningen UR,Lelystad,Netherlands 3Department of Infectious Diseases and Immunology,Utrecht University,Utrecht,Netherlands 4National Salmonella Reference Laboratory,Federal Institute for Risk Assessment,Berlin,Germany 5Department of Microbial Diseases,University College London Eastman Dental Institute,University College London,London,UK Edited by: Timothy Rutland Walsh,Cardiff University,UK Reviewed by: M.Pilar Francino,Center for Public Health Research,Spain Jun Liu,Mount Sinai School of Medicine,USA *Correspondence: Henk J.M.Aarts,National Institute of Public Health and the Environment, Antonie van Leeuwenhoekla9,3721 MA Bilthoven,Utrecht,Netherlands. e-mail:henk.aarts@rivm.nl In this review an overview is given on antibiotic resistance(AR)mechanisms with special attentions to the AR genes described so far preceded by a short introduction on the dis-covery and mode of action of the different classes of antibiotics.As this review is only dealing with acquired resistance,attention is also paid to mobile genetic elements such as plasmids,transposons,and integrons,which are associated with AR genes,and involved in the dispersal of antimicrobial determinants between different bacteria. Keywords:antimicrobial resistance mechanisms,acquired,antibiotics,mobile genetic elements INTRODUCTION The discovery and production of(synthetic)antibiotics in the?rst half of the previous century has been one of medicine’s greatest achievements.The use of antimicrobial agents has reduced mor-bidity and mortality of humans and contributed substantially to human’s increased life span.Antibiotics are,either as therapeutic or as prophylactic agents,also widely used in agricultural practices. The?rst discovered antimicrobial compound was penicillin (Flemming,1929)aβ-lactam antibiotic.Soon after this very important discovery,antibiotics were used to treat human infec-tions starting with sulfonamide and followed by the aminoglyco-side streptomycin and streptothricin(Domagk,1935;Schatz and Waksman,1944).Nowadays numerous different classes of antimi-crobial agents are known and they are classi?ed based on their mechanisms of action(Neu,1992).Antibiotics can for instance inhibit protein synthesis,like aminoglycoside,chloramphenicol, macrolide,streptothricin,and tetracycline or interact with the syn-thesis of DNA and RNA,such as quinolone and rifampin.Other groups inhibit the synthesis of,or damage the bacterial cell wall asβ-lactam and glycopeptide do or modify,like sulfonamide and trimethoprim,the energy metabolism of a microbial cell. Upon the introduction of antibiotics it was assumed that the evolution of antibiotic resistance(AR)was unlikely.This was based on the assumption that the frequency of mutations generating resistant bacteria was negligible(Davies,1994).Unfortunately, time has proven the opposite.Nobody initially anticipated that microbes would react to this assault of various chemical poi-sons by adapting themselves to the changed environment by developing resistance to antibiotics using such a wide variety of mechanisms.Moreover,their ability of interchanging genes, which is now well known as horizontal gene transfer(HGT)emergence of resistance actually began before the?rst antibiotic, penicillin,was characterized.The?rstβ-lactamase was identi-?ed in Escherichia coli prior to the release of penicillin for use in medical practice(Abraham and Chain,1940).Besidesβ-lactams, the aminoglycoside–aminocyclitol family was also one of the?rst groups of antibiotics to encounter the challenges of resistance (Wright,1999;Bradford,2001).Over the years it has been shown by numerous ecological studies that(increased)antibiotic con-sumption contributes to the emergence of AR in various bacterial genera(MARAN,2005,2007;NethMap,2008).Some examples of the link between antibiotic dosage and resistance development are the rise of methicillin-resistant Staphylococcus aureus(MRSA) and vancomycin-resistant enterococci(VRE).The initial appear-ance of MRSA was in1960(Jevons et al.,1963),whereas VRE were ?rst isolated about20years ago(Uttley et al.,1988).Over the last decades they have remained a reason for concern,but additional public health threats in relation to resistant microorganisms have also arisen(see for example Cantón et al.,2008;Goossens,2009; Allen et al.,2010). Bacteria have become resistant to antimicrobials through a number of mechanisms(Spratt,1994;McDermott et al.,2003; Magnet and Blanchard,2005;Wright,2005): I.Permeability changes in the bacterial cell wall which restricts antimicrobial access to target sites, II.Active ef?ux of the antibiotic from the microbial cell, III.Enzymatic modi?cation of the antibiotic, IV.Degradation of the antimicrobial agent, V.Acquisition of alternative metabolic pathways to those inhib-ited by the drug, VI.Modi?cation of antibiotic targets,
1 (see 、hear 、notice 、find 、feel 、listen to 、 look at (感官动词)+do eg:I like watching monkeys jump 2 （比较级 and 比较级）表示越来越怎么样 3 a piece of cake =easy 小菜一碟（容易） 4 agree with sb 赞成某人 5 all kinds of 各种各样 a kind of 一样 6 all over the world = the whole world 整个世界 7 along with同……一道，伴随…… eg : I will go along with you我将和你一起去 the students planted trees along with their teachers 学生同老师们一起种树 8 As soon as 一怎么样就怎么样 9 as you can see 你是知道的 10 ask for ……求助向…要…（直接接想要的东西） eg : ask you for my book 11 ask sb for sth 向某人什么 12 ask sb to do sth 询问某人某事 ask sb not to do 叫某人不要做某事 13 at the age of 在……岁时 eg：I am sixteen I am at the age of sixteen 14 at the beginning of …………的起初；……的开始 15 at the end of +地点/+时间最后；尽头；末尾 eg : At the end of the day 16 at this time of year 在每年的这个时候 17 be /feel confident of sth /that clause +从句感觉/对什么有信心，自信 eg : I am / feel confident of my spoken English I feel that I can pass the test 18 be + doing 表：1 现在进行时 2 将来时 19 be able to (+ v 原) = can (+ v 原）能够…… eg : She is able to sing She can sing 20 be able to do sth 能够干什么 eg :she is able to sing 21 be afraid to do (of sth 恐惧，害怕…… eg : I'm afraed to go out at night I'm afraid of dog 22 be allowed to do 被允许做什么 eg: I'm allowed to watch TV 我被允许看电视 I should be allowed to watch TV 我应该被允许看电视 23 be angry with sb 生某人的气 eg : Don't be angry with me 24 be angry with(at) sb for doing sth 为什么而生某人的气 25 be as…原级…as 和什么一样 eg : She is as tall as me 她和我一样高 26 be ashamed to 27 be away from 远离 28 be away from 从……离开 29 be bad for 对什么有害 eg : Reading books in the sun is bad for your eyes 在太阳下看书对你的眼睛不好 30 be born 出生于 31 be busy doing sth 忙于做什么事 be busy with sth 忙于…… 32 be careful 当心；小心 33 be different from……和什么不一样 34 be famous for 以……著名 35 be friendly to sb 对某人友好 36 be from = come from 来自 eg ：He is from Bejing He comes from Bejing Is he from Bejing Does he come from Bejing