几个重要的实验方法

0 代谢进化路径的描述

*Due to its bioresistant nature and the limited amount of time that microbial communities have been exposed to it, 4CNB is an ideal compound for the identification of possible evolutionary events of novel catabolic pathways in microorganisms.

*Spontaneous point mutations occur at low frequency; therefore, it would likely take an extraordinarily long time to accumulate adequate mutations for the evolution of an efficient enzyme. 1降解菌株突变株的获得方法

A plasmid-cured mutant, Comamonas sp. strain 2B, was obtained through subculturing in medium lacking bromoxynil. *A

mutant

MJZ-1

defective

in

the

degradation

of

3-phenoxybenzoate was obtained by successive streaking on LB agar.

*Intriguingly, a spontaneous mutant of strain ZWLR2-1, designated Pseudomonas sp. strain ZWLR2-1D, was obtained in LB medium.

从这篇文章中摘抄的语句如下：

Spontaneous mutants of strain CPE3 that were not able to grow on

chlorobenzoates were isolated at a frequency of approximately 10-3 cell-1 generation-1 on nonselective media. Reversion of these mutants was tested by plating 1010 cells on agar containing 3-chlorobenzoate, and under these conditions reversion could not be detected, indicating that the cbaABC genes had been deleted. *2菌株降解性能稳定性的描述

The capabilities of JS667 and JS668 to grow on DPA were relatively stable. After seven transfers in 0.75% (wt/vol) tryptic soy broth, small portions of JS667 (20.8%) and JS668 (5.5%) lost the ability to degrade DPA.

3 取样间隔时间的描述

*Samples were collected at appropriate intervals to monitor reaction progress by nitrite analysis. 4 ORF分析的描述

*Analyses of open reading frames (ORFs) and an amino acid identity search were performed using ORF finder programs and BLASTX on NCBI website.

BLASTp analysis of the predicted open reading frames (orfs) against the NCBI database revealed a potential bromoxynil-degrading gene cluster (denoted bhb)

5如果一个基因的行使功能需要一个拍档，目前存在两个候选

者，如何判定其中一个是最终人选？

Since TcpA is an FADH2-dependent monooxygenase, it requires a flavin reductase to provide FADH2. Sequence analysis indicates that both tcpX and tcpB encode potential flavin reductases. However, TcpX is likely to generate FADH2 for TcpA due to its similarity to several

partner

flavin

reductases

of

FADH2-dependent

monooxygenases and its gene location proximity with tcpA.

6 如何判断一个gene cluster 中某个特定“ORF”功能

*Among the products encoded by these genes, BagX, BagI, and BagK exhibit moderate identities with the enzymes involved in the gentisate pathway from different bacterial genera. Interestingly, no obvious homology was found between BagL and any functionally identified proteins. Given the fact that only the gene encoding the maleylpyruvate isomerase of known functions is ―missing‖ in the bag cluster, bagL is likely to encode a novel type of maleylpyruvate isomerase. 7 蛋白异源纯化

*Recombinant BagL was overexpressed in E. coli BL21(DE3) as an N-terminal His6-tagged fusion protein for easy purification.

8 在体外验证基因的功能

*The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH. 补充对照实验的描述

no activity was detected during the same procedure performed with cell extracts of E. coli BL21(DE3) harboring pET22b with no insert. Maleylpyruvate hydrolase activity was measured by the decrease of absorption at 330 nm. 9 酶活最适条件的表达

*Maximum activity was exhibited at pH 8.3 in the range of pH 7.0 to 8.9 in 50 mM Tris-HCl buffer.

*Maximumactivity of HbzF-His6 was observed at pH7.6 when tested in 50mMTris-HCl buffer and its optimal activity exhibited at 30°C.

*The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively.

10 质谱/液相检测的表达方法

*GC/MS analysis of the unknown compound extracted from the culture fluids after growth of JS667 on 1mM DPA revealed a characteristic mass fragment [M+]at m/z 185 with major fragment

ions at m/z 168, 156, and 139, which are similar to those of 3- and 4-hydroxydiphenylamines.

*Both maleate and pyruvate were identified as products of maleylpyruvate hydrolysis in the system containing purified HbzF-His6 by comparison with standards on a high-performance liquid chromatography (HPLC) system as described previously , with 5mM H2SO4 as the eluant at a flow rate of 0.6 ml/min. Their retention times were 8.3 and 9.2 min, respectively, which could be easily distinguished from that of fumarate (retention time, 15.6 min). In a quantitative-analysis system containing 200 Mmaleylpyruvate and 10 ug purified HbzF-His6, almost equimolar amounts of maleate (178 uM) and pyruvate (1 uM) were produced following the completion

of

maleylpyruvate

hydrolysis

determined

by

spectrophotometer, after incubation at 30°C for 30min. This indicated the stoichiometric production of maleate and pyruvate from maleylpyruvate.

*During the analysis by liquid chromatography-mass spectrometry (LC-MS), performed with an UltiMate 3000 RSLC series system (Dionex, Sunnyvale, CA) coupled with a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics, Bremen, Germany), two compounds appeared at retention times of 1.4 and 2.3min, giving an [M-H]- ion at m/z 87.0086 and an [M-H]-ion at 115.0040,

corresponding to pyruvate and maleate standards, at m/z 87.0096 and 115.0038, respectively.

11利用基因文库筛选基因的一段描述（The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library.）

*A genomic library of strain KSM1 was constructed by using the broad-host-range cosmid pKS13S (31)in E. coli XL-1 Blue MR (Stratagene) and S. japonicum UT26 according to previously described methods .The cosmid-based genomic library of strain KSM1 was screened for a clone with DON conversion activity by using HPLC, as described below. Approximately 108 cells of each clone were suspended in 1.5 ml of the assay solution (MM containing 20 mM DON). After incubation for 1 to 2 days at 28°C, the cells were removed by centrifugation and filtration. The remaining DON in each filtrated supernatant was examined by HPLC analysis , and a clone with DON conversion activity was obtained. The cosmid clone (pKSM1007) was isolated from the host cell and reintroduced into strain UT26 to confirm the DON-degrading activity derived from the cosmid insert.

12 构建基因文库筛选阳性克隆子

A library of PstI and EcoRI fragments from Flavobacterium

plasmid pPDL2 was created by Mulbry et al. (20), and the subclones

pWWM1079 and pWWM44, which had been shown to encompass and flank the

opd gene, were used as the starting material for sequencing. *An EcoRI-SacI-digested genomic DNA library of strain ZWLR2-1 was constructed using pBluescript II SK (Stratagene, La Jolla, CA). The library was screened for positive clones by PCR using primers nitro-dioF (5-ACCCACCTTCAAGCACTCTG-3) and nitro-dioR (5-CGAWGGCATACGTCCAAWCC-3), which were derived from a fragment encoding a portion of the ring-hydroxylating dioxygenase αsubunit in strain ZWLR2-1 (12).

The insert from positive clone pZWL01 was sequenced, and the upstream sequence was obtained by a genome-walking strategy (16). 13 SEFA-PCR 扩增的表达方法（结合PSC123自杀质粒） Some clones lost the ability to transform DPA or catechol and the regions flanking the insertions were sequenced via primer walking outward from the transposon.

*A genome-walking strategy for both directions was used to obtain the flanking sequence of this fragment, as shown in Fig. 1B. *Subsequently, a farther upstream ~7-kb DNA fragment was obtained by genome walking.

14使用NCBI 网站进行BLAST的相关描述

*To retrieve the sequences of ferredoxins (Fdxs) and ferredoxin reductases (FdRs) in bacteria closely related to strain KSM1, BLAST searches were performed on the NCBI website against the complete genome sequences of four KSM1 relatives , using the sequences of terpedoxin (Tdx) and terpedoxin reductase (TdR) as queries for Fdxs and FdRs, respecively. 15 IPTG诱导的相关表述

*E. coli BL21(DE3) strains carrying the recombinant plasmids were grown in LB, supplemented with kanamycin/ml, at 37°C to an optical density at 600 nm of 0.6 and then induced with 0.1 mM IPTG for 6 h at 16°C.

*When the optical density at 600 nm (OD600) of the culture had reached 0.6, the incubation temperature was decreased to 15°C. After incubation for 1 h, 0.5mM IPTG (and 0.5mM FeCl3 for Kdx expression) was added, and the culture was incubated for an additional 24 h at 15°C.

*E. coli BL21(DE3) strains carrying the resulting plasmids were grown in LB at 37°C to an optical density at 600 nm (OD600) of 0.6 and then induced for4hby addition of 0.4 mM (IPTG) at 30°C. 16 异源表达（heterologous expression）

*When E. coli was used as a host for heterologous expression, the screen did not result in the isolation of any clone with the target activity; however, when Sphingobium japonicum strain UT26 was used, the screen enabled the isolation of a clone that transformed DON to an unknown metabolite (tentatively named compound A) detectable by UV .

Notably, E. coli cells harboring pKSM1007 did not decrease the amount of DON or produce compound A, indicating that pKSM1007 cannot be obtained through activity-based screening of the library using E. coli as a host. 17 PCR 方法筛选文库

*When the KSM1 genomic library was screened by PCR, the follow ing degenerate primer sets were used as forward and reverse primers: Fdxd-f and a mixture of Fdxd-r1 and Fdxd-r2 for amplification of the Fdx-encoding gene, and a mixture of Fdrd-f1 and Fdrd-f2 and a mixture of Fdrd-r1 and Fdrd-r2 for amplification of the FdR-encoding gene. 18 酶切表示

*The PCR product was purified, treated with NdeI and BamHI, and ligated into pET-28a(+) which had been digested with the same restriction enzymes. The resulting plasmid, designated pETcnbZ, was used to transform E. coli BL21(DE3).

19 BLAST 比对时出现“相似”情形的描述

*The N-terminal sequence and peptide fingerprints of this deaminase were determined, and BLAST searches revealed no match with significant similarity to any functionally characterized proteins. *The N-terminal amino acid sequence for the first eight residues was determined to be PDAVVSFP. BLASTN and BLASTP searches at the GenBank and PDB databases revealed no match with significant similarity.

*The most striking match from the data bank was a hypothetical protein in strain JS42 with 100% identity to Orf1 in strain CNB-1. In addition, a homolog (OrfJ) to Orf2 was also identified from a degradative genetic fragment of C. testosteroni TA441.

A BLAST search showed these proteins to have significant homology to the IstA and IstB proteins encoded by members of the IS21 family of transposons (13, 23). As is frequently observed in IS21-like sequences, the two genes are translationally

coupled, indicating that their products are functionally related. Alignment of the predicted amino acid sequences with other IS21 family IstA and IstB proteins showed them to be most closely related to the Ist proteins from two other IS21-like transposons, IS1326 from Pseudomonas aeruginosa (5) and an insertion sequence found in the chromosome of Agrobacterium tumefaciens C58 .

20 Recombinat protein 进行酶活测试时的表述

When purified H6-BagLwas incubated with maleylpyruvate, no change in the absorption spectrum between 250 to 400 nm21 took place.

21 酶活稳定性的描述

After 20 days storage at -80°C with 50% glycerol, the enzyme retained 62%of its original activity, and 27%of the activity remained after 50 days.

22 Southern hybridization 的表述

Southern hybridization of total DNA from strain JS705 with a gene probe for the intB13 integrase gene was indeed positive (Fig. 6). Positively hybridizing cosmids to the intB13 gene probe were retrieved from the JS705 cosmid library, and physical maps were constructed. 23 接合实验的描述

*In mating experiments between strain JS705 as donor and R. eutropha JMP2 as recipient, we tested whether the clc and mcb genes were transmissible. Upon selection for growth on chlorobenzene, transconjugants of R. eutropha JMP2 were detected at a frequency of 2.5 ×10-8 per recipient. 24 引物中酶切位点的引入

The hbzF gene was amplified from strain NCIMB 9867 genomic DNA using the primer pair F-22b-F (5-GACCATATGCAA TCGAGT CGGTTGCTC C-3) and F-22b-R (5-AAACTCGAG CAC CGA GCG GAT GGC AAT-3), with NdeI and XhoI sites incorporated into their respective 5 ends.

The nicA gene was PCR amplified from genomic DNA of strain S16

with Prime STAR HS DNA polymerase (TaKaRa Co. Ltd., People’s Republic of China). Primers were designed so that the forward primer contained an EcoRI site and the reverse primer contained a SalI site. The primer sequences (restriction sites are underlined) were as follows:

forward primer, 5󰀀-GGCGAATTCTATGCGCGATGCAGAAC-3󰀀 (corresponding to positions 4,077 to 4,061 in the nic gene cluster, as previously reported [18]); and reverse primer, 5󰀀-AATGTCGA CTGCTGCTGCCGTGTGA-3󰀀 (positions 2,223 to 2,238 in the nic gene cluster, as previously reported [18]).

*Purified PCR products were treated with restriction enzymes and then ligated into the similarly treated pET-21a(+), except for cnbH, which was cloned into pET-28a(+).The PCR products were purified, treated with EcoRI and SalI, and ligated into pET-27b(+) (Novagen Corp., Germany) which had been digested with the same restriction enzymes.

Primers were designed to incorporate an EcoRI site in the forward primer and an XhoI site in the reverse primer. The primers for hsp, with restriction sites underlined, were as follows: forward, 5-GCGGAATTCA ATGCAGAGAAAGCTT-3 (corresponding to positions 1080 to 1095), and reverse, 5-TTACTCGAGGGGCAACTCCTCTTG-3 (positions 2016 to 2002). The PCR product of hsp was digested with EcoRI and XhoI and ligated into pET-27b(+) (Novagen).

补充： 克隆一个基因

Cloning and overexpression of orf243. The product of orf243 was overex-

pressed by cloning the gene in expression vector pET-15b. Since there are no

suitable restriction sites to facilitate cloning, the gene was amplified by PCR

using a forward primer (5󰀀 GCACCATGGCGCATGCCCGCGTTC 3󰀀) contain-

ing an NcoI site (shown in bold type) overlapping the initiating methionine codon

and a reverse primer (5󰀀 CTGTGACTAACCCGGCGCGGTTC 3󰀀) correspond-

ing to the 3󰀀 end of the gene. Since there is a BamHI site downstream of the stop

codon of orf243, the PCR-amplified fragment was digested with NcoI and BamHI

and cloned in pET-15b digested with similar enzymes. The resulting recombinant

plasmid was designated pSM4.

To examine the expression of orf243 in both the presence and absence

of opd,

the opd gene was cloned under the control of the lacZ promoter in the broad-

host-range plasmid vector pMMB206. The opd gene was amplified by PCR from

pSM12, which carries opd as an NdeI-XhoI fragment cloned in pET23b, using vector-specific

primers.

The

forward

primer

(GCCAGAATTCAGGGAGACCA

CAACGGTTTCACT) contained an EcoRI site (shown in bold type) upstream

of the opd ribosomal binding site and the reverse primer (GCCAGGATCC

CAAAAAACCCCTCAAGACCC) contained a BamHI site (shown in bold type)

downstream of the region specifying the C-terminal His tag encoded in pET23b.

The resultant 1.2-kb PCR product was cloned in pMMB206 as an EcoRI-BamHI fragment to give pSM5.

25 描述敲除策略的效率

Of 200 colonies tested, 8.5% (17/200) of streptomycin-resistant

clones were kanamycin resistant, indicating that spontaneous streptomycin resistance is a minor concern in the counterselection step. Overall, 62.7% (94/150), .3% (30/84), 30.6% (22/72), and 42.7% (50/117) of kanamycin-sensitive, streptomycin-resistant clones carried the ecfG, phyR, crtI, and crtY deletions, respectively, while the remaining had reverted to thewild type. These values are close to the theoretical 1:1 ratio of the wild type to the mutant genotype and thus prove the efficiency of the system.

25 酶活力的表述

One unit of maleylpyruvate hydrolase activity was defined as the amount required for the disappearance of 1 umol of maleylpyruvate permin at 23°C. Specific activities are expressed as units per milligram of protein, and the values are expressed as means standard + deviations calculated from triplicate assays. 26 利用RT-PCR 反应诱导物对基因簇表达效果的描述

（*RT-PCR was performed with RNA derived from the succinate-grown cultures of strain NyZ101, with or without the induction of 3-hydroxybenzoate.）

The effect of nicotine on nic2 operon expression was determined by comparing the mRNA levels of all six genes (hspB, orf5, iso, nfo, hpo and ami) in strain S16 grown in the presence or

absence of nicotine using quantitative RT-PCR (RT-qPCR). In the absence of nicotine, all transcript levels were relatively low, but were significantly increased in response to nicotine (hspB, a 112.5-fold increase; orf5, a 96.5-fold increase; iso,a .4-fold increase; nfo, a 31.4-fold increase; hpo,a 62.0-fold increase; and ami, an 18.8-fold increase). 27 降解途径的描述

nicA encodes an oxidoreductase, which converts nicotine to 3-succinoylpyridine through pseudooxynicotine. Based on enzymatic reactions and direct evidence obtained using H218O labeling, the process may consist of enzyme-catalyzed dehydrogenation, followed by spontaneous hydrolysis and then repetition of the dehydrogenation and hydrolysis steps.

convert nicotine to 2,5-dihydroxypyridine (DHP) and succinic

acid

through

N-methylmyosmine,

pesudooxynicotine,

and 3-succinoylpyridine (SP)

*In this pathway, gentisate 1,2-dioxygenase (GDO) catalyzes the ring-cleavage oxidation of gentisate to yield maleylpyruvate, which is further degraded by either isomerization to fumarylpyruvate and subsequent hydrolysis to fumarate and pyruvate (1) or direct hydrolysis tomaleate and pyruvate.

We have shown that orf243 encodes a polypeptide of 27 kDa, which plays a role in the degradation of p-nitrophenol and is likely to act in concert with opd in the degradation of parathion.

*The metabolic pathway and the genes involved in HCH degradation have been studied in great detail in S. paucimobilis strain UT26 (a mutant of SS86 resistant to nalidixic acid) and to a lesser extent in B90 (Fig. 1). *In this study, an aerobic strain of Comamonas sp. 7D-2 was shown to degrade the brominated aromatic herbicide bromoxynil completely and release two equivalents of bromides under aerobic conditions.

These results indicate that two successive

reductive dehalogenation reactions occurred instead of a reductive plus oxygenic dehalogenation in the DBHB dehalogenation process (Fig. 1A).

Thus, a wide diversity of aromatics are channeled (activated) via different peripheral pathways to a few key central intermediates that suffer dearomatization and further conversion to intermediary metabolites, such as acetyl-CoA, succinyl-CoA or pyruvate, via some central pathways that are conserved in evolution and function.

*A second aerobic strategy for cleaving the aromatic ring relies on the use of oxygenases, but solely to form a non-aromatic epoxide. Since these aerobic pathways share features of anaerobic atabolism, they are called aerobic hybrid pathways. Thus, in these hybrid pathways, as in the anaerobic catabolism, all metabolites are activated to CoA thioesters through the action of an initial CoA ligase,the ring cleavage is carried out hydrolytically rather than oxygenolytically.

*Recent works have shown that lignin can be the substrate of bacterial dedicated peripheral pathways releasing low molecular weight phenolic products that finally lead to protocatechuate or some derivates of the latter（指原儿茶酸）.

Degradation of aromatic compounds in the ecosystem is usually accomplished by microbial consortia where syntrophic interactions between species involve interchange of byproducts.

In some cases, the degradation of a single compound can take place through multiple pathways. This is the case for toluene which can be broken down via five alternative aerobic pathways and by at least one other pathway that operates under anoxic conditions. Many pathways are still only partially described and, therefore, significant advances are expected in the field.

The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty.

The nic2 gene cluster, which contains five crucial genes (hspB, iso, nfo, hpo and ami) that are responsible for the late steps of nicotine degradation from

6-hydroxy-3-succinoyl-pyridine

(HSP)

through

2,5-dihydroxypyridine (DHP) to the TCA cycle, was recently discovered in P. putida S16 .

The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxy-lamine, followed by conversion of DCA to Krebs cycle intermediates.

The lower pathway reactions and genes involved in conversion of PDC into oxaloacetic and pyruvic acids in Comamonas species are still hypothetical and have not been experimentally identified. *Differential

proteomic

analysis

showed

a

linuron-dependent

upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and

enzymes associated with a modified chlorocatechol ortho-cleavage pathway.

*Interestingly, in the aniline catabolic Delftia sp. strain AN3, the association of an aniline multicomponent dioxygenase with a catechol meta-cleavage pathway (61), typical for degradation of nonchlorinated aromatics (31), was reported.

In contrast to SRS16,WDL1 can only efficiently degrade linuron in synergy with other DCA-degrading bacteria (10).

The consumption of 2C4NP was accompanied by concomitant increase in cell growth that reached a maximum growth equivalent to OD600of 0.39±0.016.

Also, as observed, 2C4NP degradation pathway in strain SJ98 merges in metabolic pathway characterized for degradation of PNP.

To our surprise, chloro-1,4-benzoquinone (CBQ) and CHQ, rather than PNP, 4-nitrocatechol (4-NC), and BT, as previously proposed, were identified during 2C4NP degradation.

In a biotransformation time course, 2C4NPconsumption (230uM) was approximately equivalent to the total accumulation of both CHQ (201uM) and CBQ (12 uM), indicating nearly stoichiometric formation of CBQ

and CHQfrom 2C4NP.

28 挖掘基因组

The spreadability of PCA 4,5-cleavage pathway in bacteria was explored via data- mining of microbial genomes.

29 质粒去除菌株的表达

C. testosteroni strain CNB-2 is a plasmid-curing derivative from C. testosteroni strain CNB-1.

30 multicomponent dioxygenase 系统的描述

The irst enzymes of the pathway, the chlorobenzene dioxygenase

and

the

cis-chlorobenzene

dihydrodiol

dehydrogenase, are encoded on a plasmid-located transposon Tn5280. Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for he NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB. 31 介绍ORF tentative的功能

Sequence homologies with other known dioxygenases allowed the assignment of putative protein functions to each of the ORFs (Table I). We propose to designate the genes as follows: tcbAa,

coding for the large subunit of the terminal oxygenase; tcbAb, encoding the small terminal oxygenase subunit; tcbAc, the ferre- doxin; tcbAd, the NADH reductase; and tcbB, the dihydrodiol dehydrogenase.

Here we have shown that the genes for the chlorobenzene dioxygenase are contiguous on the DNA and indeed code for four protein subunits, two of which make up the terminal oxygenase, one the ferredoxin, and the last one the NADH reductase.

*Four other genes (cnbR, cnbE, cnbF, and cnbI) were tentatively（试探性地;不确定地） identified according to their high sequence identities to other functionally identified genes.

32 两个蛋白差异之间的比较

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase（= non-heme iron-containing dioxygenases）exhibit substantial sequence similarity yet have different substrate specificities.

*show how the nitrotoluene-responsive regulator NtdR can be generated from a NagR-like ancestor by just a few mutations. NtdR and NagR differ by only five amino acids, at positions 74, 169, 1, 227 and 232.

To elucidate the contributions of the five amino acid differences between NtdR and NagR to effector specificity, each amino acid

was selectively substituted either separately or in combination with other substitutions.

NagR recognizes only five of 63 tested compounds (salicylate, gentisate, 4-hydroxybenzoate, 4-isopropylbenzoate and methyl salicylate) and in particular, does not recognize nitroaromatic compounds. NtdR, in contrast, activates the expression of 2-nitrotoluene degradation genes not only when nitroaromatic compounds are present, but also in the presence of a wide range of aromatic acids and analogues, in total 41 chemicals.

33 如何判断重组酶的活性

*The increase in A235, due to the formation of 2-aminophenol, was used for estimation of the activity of hydroxylaminobenzene mutase.

34 如何在体外验证一个基因的功能

*The decarboxylation and hydration (from compound VII to compound VIII) was catalyzed by CnbE and CnbF, which was confirmed in recombinant E. coli by simultaneous expression of the two genes (data not shown).

35 获取基因通过基因水平转移

*The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by

IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.

36 酶活测试时及结果分析

When HSP was added (0.5-5 mM), the amount of NicR2–DNA complex decreased, and complete inhibition was observed at 4 mM HSP.

The resulting plasmid, designated pET27b-nicA, was used to transform E. coli BL21(DE3).

*A strict anaerobic bacterium, Desulfitobacterium sp. Strain Y51, isolated in our laboratory exhibits a strong dehalogenating activity against PCE at concentrations as high as 960 M and as low as 0.6 M, converting it to cis-DCE via TCE (29).

*The PceA dehalogenase activity of strain Y51 was highly enhanced when PCE or TCE was added to the culture medium;in particular the addition of TCE at a concentration of 0.6 mM led to 25-fold-higher enzyme activity compared to the control, although the reason remains to be elucidated.

*Recombinant BagL was purified to homogeneity as a His-tagged protein and likely a dimer by gel filtration.

*Addition of hemin (dissolved in dimethyl sulfoxide) dramatically increased OnpA activity in a concentration-dependent manner.

*However, truncated NCgl2918 without a C-terminal domain did not show any activities against maleylpyruvate (34).

*In Gram-negative species, GSH is considered to play a key role against oxygen toxicity.

*Expression of the small subunit of a multicomponent aniline dioxygenase was also observed, although it was not significantly different from the control culture.

*P. putida PaW340 containing mhbRTDHIM could not grow on 3-hydroxybenzoate; however, supplying mhbT in trans allowed the bacteriumto grow on the substrate.

*Thus, the substrate specificity of the PceA dehalogenase of strain Y51 is relaxed toward highly chlorinated alkanes such as the tetra-, penta-, and hexachloroethanes but rather strict for non-highly chlorinated ones, as seen for the cis-DCE and chloroalkanes with three chlorines.

Cofactors, such as NAD, NADH,

NADP, and NAD 2󰀀-phosphate reduced tetrasodium salt, were separately added to the enzymatic reaction buffer containing purified HSP hydroxylase (0.5 󰀀g). The enzyme was activated only upon the addition of NADH, while other cofactors could not contribute to the reaction.

*The reaction mixture consisted of 50 mM sodium phosphate buffer (pH

7.0), 10 mM Fe(NH4)2(SO4)2·6H2O, and ~5 mg of cell-free lysate containing soluble protein, in the total reaction volume of 1 ml. Reactions were initiated by the addition of 50μM of benzenetriol (BT) to the reaction mixture, and enzyme activity was monitored with wavelength scan over a range of 220–360 nm at a regular interval of 1 min.

37 前后处理方法不同导致结果差异的比较

*Isotope-coded protein labeling (ICPL) was used to detect differentially expressed proteins in Variovorax sp. SRS16 when grown in the presence of linuron compared to growth without linuron.

*SDS-PAGE profiles confirmed the increased expression of a protein of ~55 kDa in the IPTG-supplemented cultures compared to noninduced cultures (data not shown).

Further evidence is provided by the increased 2,4,6-TCP degradation upon coexpression of cloned tcpA and tcpX compared to that with tcpA expression alone.

*Real-time quantitative PCR analysis showed a 57-fold change in gene

transcription

of

the

bag

cluster

from

the

3-hydroxybenzoate-induced samples compared to the data from the noninduced samples.

*Addition of FAD gave a 10% increase in activity of the

ortho-nitrophenol 2-monooxygenase from strain B2, whereas with OnpA, activity was stimulated by about 60%, perhaps due to the overexpression of OnpA in E. coli.

*H6-BagL exhibited approximately one-eighth and 2-fold of the activity when GSH and Cys-Gly replacing L-cysteine, respectively. *The calculated result from real-time quantitative PCR suggested that, 2 h after 3-hydroxybenzoate induction, hbzF was transcribed at a level 16-fold higher than in the noninduced cells.

*By phylogenetic comparisons, they concluded that the allele for the nahAc gene in seven isolates was incongruent to that for 16S rRNA, suggesting recent horizontal transfer of the nah genes. 38 比较差异的描述

*In addition, resting cells of hspB gene deletion mutant could not degrade HSP, different from hspA gene deletion mutant and strain S16.

*BagL forms an independent clade with a closer relationship to BSH S-transferases or MSH-dependent maleylpyruvate isomerase than to MSH S-transferases.

*Evolutionary analysis indicated that CnbH was more related to A than to B.

*In addition, real-time qPCR analysis showed a 34-fold increase in gene transcription of the pnp cluster from the 2C4NP-induced samples, in

comparison with the data from the non induced samples.

*Comparison of sesquiterpene production in E. coli expressing native sesquiterpene synthase genes and E. coli expressing the synthetic ADS gene showed from a 10- to 300-fold improvement in terpene synthesis in the latter.

*Metabolic engineering of the DXP pathway increased the flux in carotenoid accumulation by 2- to 40-fold over incubation periods of 20–50 h.

*DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26.

*A higher number of differentially downregulated proteins compared to upregulated proteins was observed in the linuron-amended cultures.

*In contrast to E. coli BL21(DE3) control strains, IPTG-induced cells of E. coli containing pJexpress416_libA showed linuron degradation (80 % after 7 h; 100 % in

20 h) and the formation of DCA. Without IPTG induction, a slow linuron degradation was observed (50 and 60% after 24 and 48 h,

respectively [data not shown]).

*TftC has a higher catalytic efficiency for FAD than for FMN , while HpaC uses FMN with higher efficiency than FAD (5). With regard to the flavin preference, TcpXH is similar to HpaC.

The affinities of DdnA toward these toxins are comparable to those of other P450s for bacterial carbon utilization.

Regardless of glucose or resorcinol, the expressions of rolH, rolM, and rolD in the mutant RES167 rolR were much higher than in RES167 grown with glucose (Fig. 2A), indicating that RolR indeed negatively regulated the expression of rolHMD gene. The ProlR promoter showed lower transcriptional activities in the presence of RolR than in the absence of RolR (Fig. 2B), indicating that RolR negatively regulated the transcription of its own gene.

*The biochemistry and genetics of microorganisms capable of degrading simple hydrocarbons (monoaromatic compounds or n-alkanes) has been nearly fully elucidated although thus far only a few pathways of the genus Azoarcus, Thauera and related microorganisms have been thoroughly described compared with those known for aerobic biodegradation.

*The use of pre-induced cells as inoculum resulted in the improved kinetics of degradation as observed by the consumption of 300 uM substrate in 20 h compared to 38–40 h with non-induced cells.

39 超声波破碎 Sonication was

performed using 99 cycles consisting of 200 W for 6 s with 6-s intervals between cycles in an ice bath. Cell debris and unbroken cells were removed by centrifu- gation at 14,000 󰀀 g for 20 min.

These observations show that ORF1 contains a

gene necessary for nicotine degradation, presumably in the first several steps.

TLC analysis of the products formed by incubation of nicotine and whole cells of transformant pMD18-ORF1 at pH 7.0 from 0 to 4 h.

To obtain purified protein for enzymatic analysis and to verify the prediction for the novel gene, the nicA gene from strain S16 was

cloned and expressed in E. coli BL21(DE3) cells to obtain a C-terminal His-tagged fusion protein. After IPTG induction,

large amounts of proteins with molecular masses of approxi- mately 65 kDa were found in the E. coli lysate, whereas no such band was observed for uninduced cells or for cells containing the vector alone (Fig. 4C). ESI-Q-TOF-MS

analysis showed that the molecular ion peaks [(M󰀀H) 󰀀]of N-

methylmyosmine (C10H13N2), pseudooxynicotine (C10H15N2O), and SP (C9H10NO3) were at m/z 161.10732, 179.117, and 180.06552, respectively

A resting cell reaction of E. coli DH5󰀀 (harboring pUC19 plasmids) was also subjected to the same conditions as a con- trol. After 20 h, the concentration of nicotine or HSP in the mixture remained constant, and no product was detected. Antibiotics were used, when

required, at the following concentrations: ampicillin (100

󰀀g/ml or 5 󰀀g/ml for E. coli or S. macrogolitabida strains, re- spectively), kanamycin (20 󰀀g/ml), gentamicin (10 󰀀g/ml), and chloramphenicol (15 󰀀g/ml).

Non-quantitative reverse transcription polymerase

chain reaction (RT-PCR) showed that the 4 genes appeared to be upregulated in 6 mM nicotine or 1 mM DHP addition separately, whereas no significant effects in the 4 genes expressions were observed with the individual addition of 1 mM NFM, 6 mM maleamic acid, or 6 mM maleic acid (Fig. S2A and Fig. S2B).

To our

knowledge, this is the first report of a quantitative study of nicotine- induced changes in global protein and mRNA expression in a Pseudomonas strain. Our approach of combining comparative

proteomics data with molecular genetics reveals interesting Pseudomo- nas-specific proteins that could be involved in nicotine degradation.

蛋白质组差异的描述

To uncover molecular pathways in nicotine degradation, we

obtained a global overview of protein expression changes between a reference condition (glycerol as the sole carbon source and

(NH4)2SO4 as nitrogen source) and a treatment condition (nicotine as the sole carbon and nitrogen source). Only proteins with.= 3-fold changes and

p-values,0.05 were reported as differentially expressed proteins. With these predefined criteria, 126 putative proteins showed a

significant change in protein abundance between the nicotine and glycerol mediums

Nicotine exposure caused

several changes in the abundance of proteins related to carbohy- drate metabolism in the proteome of strain S16 (Table S2). NicA2 (PPS_4081), HspB (PPS_4061) (13), Pnao (PPS_4080) [18], and Sapd (PPS_4079) were more abundant in the nicotine condition than in the glycerol condition, suggesting that these enzymes are

required for nicotine degradation。Interestingly, the two most likely enzymes (PPS_4077 and

PPS_4078) sets for nicotine degradation are significantly up- regulated in the nicotine condition, making them the probable genes responsible for nicotine degradation. The expression of

enzyme NicA1 (PPS_0381) in nicotine culture was about 5 times as in glycerol culture. Furthermore, the protein HspA (PPS_0380) was still differently expressed from HspB (PPS_4061) in the nicotine condition as previous reported [13] (Figure 3).

Culturing P.

putida S16 cells in 1 g l 21

nicotine resulted in an altered content of

several outer membrane proteins, especially in the increased abundance of porin and RND efflux system.

RND family efflux transporter,MFP subunit

(PPS_2905) were found to increase following nicotine exposure.

There is, therefore, a need to identify other

proteins and pathways capable of degrading nicotine that are not evident from homology modeling, and it should be possible to use a proteomic approach to search for proteins involved in regulatory and molecular mechanisms involving nicotine catabolism.

蛋白组学的优越性

In the last few years there have been significant efforts to

identify the key genes for the hydroxylation of SP through genome library screening and wild-type enzyme purification. Unfortunate- ly, these efforts did not result in identifying any genes related to SP hydroxylation. Progress is likely to require the development of integrated experimental approaches. With a proteomics approach, it is possible to observe the entire set of protein products

synthesized and utilized by an organism [17]. It can help us to accurately determine the boundaries and enumeration of ORFs, and verify unknown ORFs that cannot be well established on the basis of homology.

Verification of gene transcriptional expressions

In order to validate the proteomic data and assess relative

transcriptional level of genes related or supposedly related to nicotine degradation in P. putida S16, we utilized RT-PCR and

RT-qPCR to compare mRNA levels of genes nicA2, pnao, sapd, mfs, spmA, spmC, porin, nicA1, and hspA, supposedly related to nicotine degradation with or without nicotine induction (Figure 4). The RT-qPCR analysis revealed that all target genes appeared to be up-regulated in nicotine-induced P. putida S16. The mRNAs of the genes relative to the pathway of nicotine degradation were 18.8- to 90.3- fold more highly expressed in P. putida S16 with nicotine relative to non-nicotine induction among which the 90.3-fold

upregulated mRNA of nicA2 was the greatest difference in mRNA levels observed for the tested genes. The mRNAs of spmA and spmC were also 13.6- and 4.5-fold upregulated, respectively (Figure 4A). Similar results were also observed using semi-quantitative RT- PCR, each of these genes was induced after exposure in nicotine, indicating a role in nicotine degradation of strain S16 (Figure 4B). Overall these data indicate that mRNA levels determined by RT- qPCR were consistent with protein levels measured by 2D LC-

MS/MS, and the differentially expressed proteins were regulated at transcriptional level.

The 2D LC-MS/MS and RT-qPCR analysis revealed that the

protein and mRNA levels of spmA and spmC were highly expressed in P. putida S16 with nicotine induction, and the fact that the spmA,

酶活的测量

HspB activity was determined at 25 °C by measuring NADH oxidation in a quartz cuvette at 340 nm (NADH, 󰀀340 󰀀 6220

The product of orf243 was overex-

pressed by cloning the gene in expression vector pET-15b.

The HspB sequence was submitted to the mGenThreader

server for fold assignment, and the protein structures with sig- nificant hits were chosen as templates for further modeling (Table S1). Five models were generated, and the one with the lowest discrete optimized protein energy score was chosen for visualization (Table S2). The three-dimensional structure is the NCBI-curated FAD domain in pfam01494 (supplemental Fig. S2, A and B). Further experimental evidences supported the HspB Reaction Product ―Succinic Semialdehyde‖ Detection— After derivatization by silanization, the product of the HspB reaction was analyzed by GC-MS. The molecular ion peak is at m/z 174, and featured fragments are at m/z 29 ([CHO] ), 45

([COOH]

), ([Si(CH3)3O]

), and 73 ([(CH2)2COOH] )

(Fig. 5).

紫外检测

*HspB UV-visible maximum absorbance at 382 and 452 nm with a mini-mum at 410 nm is characteristic of a flavoprotein. *supernatant of the boiled enzyme solution exhibited absorption maxima at 265, 375, and 450 nm, identical to FAD. 基因定位

*The DNA sequence of this gene (designated opdA)is 87% identical to the Flavobacterium gene, but the genomic location of the Agrobacterium gene, i.e., chromosomal or plasmid borne, has not been reported. A sequence

alignment of PHHY to pHBH showed very little sequence identity [4] and the chain lengths are very different, 6

and 391 residues, respectively. Although the two enzymes catalyse similar reactions, no common substrates have so far been identified.

Phenol hydroxylase accepts phenol and simple hydroxyl-, amino-, halogen- or methyl-derivatives of phenol among its substrates [1,2,7] and catalyses the hydroxylation of these compounds in the ortho position

The phenol hydroxylase from

Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best

substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or

methyl-substituted phenols. Strain 7D-2 completely

degraded 0.3 mM bromoxynil within 28 h with the cellular OD600 increasing from 0.05 to 0.135.

The nitrilase encoded by bxn2, when cloned and expressed in Escherichia coli DH5α (pUC-bxn2), effi- ciently hydrolysed bromoxynil to an equivalent amount of DBHB (see Supplementary Fig. S1), as confirmed by comparison with the standard DBHB compound and by

high-performance liquid chromatography (HPLC)-mass spectrometry (MS) identification

Although the complementary expression of bhbA alone resulted in dehalogenation activity in resting cells, its activ- ity was only approximately 10% that of bhbAB (Fig. 4A).

To investigate

the interaction between BhbB and BhbA, the dehaloge- nase activities in the cytoplasmic and membrane fractions fromstrain 2B-bhbAB and strain 2B-bhbA(only expressing bhbA) were analysed respectively

The dehalogenase from strain 2B-bhbAB was enriched from the membrane fraction

Upon exposure

to air at 4°C, approximately 100% of the BhbA activity was retained after 24 h, and approximately 60% activity remained after 48 h.

In addition to DBHB and BHB, the chlorinated hydroxy- benzoate counterparts 3,5-dichloro-4-hydroxybenzoate (DCHB) and 3-chloro-4-hydroxybenzoate (CHB) were also dehalogenated, as summarized in Table 3. Dehalo- genation occurred exclusively in the ortho position with respect to the hydroxyl group.

Methyl parathion hydrolase activity was determined at 35 ◦C by measuring the increase in absorbance at 400 nm resulting from the p-nitrophenol released by the hydrolysis of methyl parathion. Enzyme samples (20 µl) were added to an assay mixture containing 974 µl 200mM phosphate-buffered saline (PBS, pH 8) and 6 µl methyl parathion (10 mg ml −1). Samples

boiled for 5 min were used as controls. One unit (U) of MPH activity was defined as the amount of enzyme required to hydrolyze 1 µmol methyl parathion in 1 min at 35 ◦C.

Debromination

is the critical step for the mineralization of bromoxynil because the product bearing fewer halogen substituents is more susceptible to aerobic biodegradation Bromoxynil has been reported to be

reductively dehalogenated to as far as the product 4- hydroxybenzonitrile by the organohalide respiring bacte- rium of D. chlororespirans under anaerobic conditions

BhbB expression significantly enhanced the

dehalogenation activity in resting cells and contributed more than 90% of BhbA activity to the membrane fraction

Amylase activity detection

Amylase expression by Bacillus colonies was detected by growing colonies overnight on an LB plate containing 1% starch and then staining the plate with iodine.

Using E.coli JM109 chromosomal DNA as the template, the promoter functional region of the E.coli lpp gene was PCR amplified using the primer pair P1/P2, with the SacI site introduced by the forward primer; the mazE gene was ampli- fied using the primer pair P3/P4, with the ClaI site introduced by the reverse primer. Primers P2 and P3 were designed as completely reverse complements, and the two PCR fragments of the lpp promoter and the mazE gene were spliced by overlap extension; this generated the mazE over-expression cassette, which was cloned into the corresponding sites of the integration vector pDG1730, thereby generating pDGE.

基因无痕敲除的优势

In these cases, the resulting strains

were free of selection markers, thus allowing the repeated use of the method for further manipulations of the Bacillus chromosome. No prerequisite strain is needed for this newly developed method, so it will have wide applications.

The multiple and tandem promoters, and the signal peptide-encoding

sequence of the cell-wall protein-encoding gene (cwp), were amplified from Brevibacillus brevis B15 chromosomal DNA, using the primer pair P9/P10, with the BamHI and PstI sites

introduced by the forward and reverse primers, respectively. The PCR product was cloned into the corresponding site of pP43NMK to generate pP15MK, which made the promoters and peptide-encoding sequence fused in-frame with the mpd gene in pP43NMK.

The two primer pairs P13/P14 and P15/P16 were used to amplify the upstream (bpr-front) and downstream (bpr-back) DNA sequences of the bpr gene (encoding bacil- lopeptidase F) from B.subtilis 1A751 chromosomal DNA; the XhoI-HpaI-SbfI-AscI/ClaI-NheI-NcoI-SalI and XmaI-FseI- MfeI-NsiI/AgeI-AvrII-AsiSI-SpeI sites were introduced by

the two primer pairs, respectively.

When NADPH was used as the cofactor, the

relative activity was 2.82-fold higher than that of NADH (Table 2).

There were no significant differences between the BhbA activities when the dehalogenation reactions were per- formed under anaerobic or aerobic conditions, although the dehalogenation activity of resting cells was approxi- mately 10-fold higher in presence of oxygen than in the absence.

Complementation of a

desulfurization (dsz) mutant provided the genes from Rhodococcus sp. strain IGTS8 responsible for desulfu-

rization. Expression of this fragment in E. coli also conferred the ability to desulfurize DBT. Neither the

nucleotide sequences nor the deduced amino acid sequences of the enzymes exhibited significant similarity to

sequences obtained from the GenBank, EMBL, and Swiss-Prot databases, indicating that these enzymes are novel enzymes.

gene products of dszA and dszB act in concert to convert

DBT-sulfone to 2-HBP.

As much as 70% of the sulfur in these

fuels may be in the form of heterocyclic organic compounds, such as benzothiophene, dibenzothiophene (DBT), and more complex thiophenes (4). These bacteria, however, suffer

from the disadvantage that they metabolize DBT via a ring- destroying oxidative pathway (11) that results in a net carbon loss and reduction in fuel value. In addition, it has been ob- served that the pathway for naphthalene metabolism (5) closely resembles the ring-destroying DBT-degrading pathway, raising the possibility that further undesired metabolism of structurally related non-sulfur-containing fuel components may occur.

pathway involves the sequential metabolism of DBT to DBT- sulfoxide, DBT-sulfone, DBT-sulfonate, and finally 2-HBP and sulfite. A modification of this pathway in which 29-hydroxybi- phenyl-2-sulfinate and 29-hydroxybiphenyl-2-sulfonate are ad- ditional intermediates has recently been described In

the NTG mutagenesis experiments, cell suspensions were treated with 500 mgof NTG per ml for enough time so that 30 to 50% of the

cells were killed. Com-

bination mutagenesis in which we used both NTG and UV light was also per-

formed; in these experiments an overall mortality rate of more than 99.9% was

obtained. Colonies that survived the mutagenesis treatments were transferred to

RM plates and screened for the Dsz2 phenotype by replica plating them onto

BSM agarose plates supplemented with 1.2 ml of a saturated ethanol solution of

DBT per liter. After 24 h, production of 2-HBP was visualized under UV

illumination as fluorescent zones in the agarose surrounding Dsz1 colonies.

A second HSP hydoxylase (HspB), a more active form than HspA with only 11% amino acid sequence homology, was purified and

characterized. 紫外扫描的描述： Enzyme Assay

Chlorohydroquinone (CHQ) dioxygenase activity was determined by spectrophotometric method using Lambda EZ 201 UV–Visible spectrophotometer (Perkin-Elmer Inc, Massachusetts, USA). The reaction mixture contained (in a final volume of 1 ml) 20 mM phosphate buffer, 0.1 mM CHQ, 0.1 mM ferrous sulfate, and 05–1.0 mg of cell free supernatant containing soluble proteins. CHQ dioxygenase activity was monitored with wavelength scan over a range of 220–340 nm at a regular interval of 1 min.

Enzyme Assay and Detection of Maleylacetate

To identify the product of ring cleavage for CHQ, enzyme assay for CHQ dioxygenase activity was carried out. The spectrophotometric analysis showed a peak at (k=) 291 nm, designated to be CHQ, disappearing gradually giving rise to that peak of the malelylacetate (MA) at 243 nm (Fig.5).