Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.     

Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.

Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.     

Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120.

Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp. strain PCC 7120 genomic DNA. One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherichia coli and Anabaena sp. strain PCC 7119. This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins. One gene was identified in a library and was subcloned into a pUC vector and used to transform E. coli strains lacking functional thioredoxin. The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E. coli. Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and f1. Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids. The Anabaena strain 7120 thioredoxin gene was expressed in E. coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate. The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin. It is a small heat-stable redox protein and an efficient protein disulfide reductase. It is not a substrate for E. coli thioredoxin reductase. Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E. coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts. The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin. However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown.     

迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白在蓝藻中的表达

在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。     

Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding

The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.     

蓝细菌断裂DNA聚合酶DnaE的体内/外反式剪接分析

蓝细菌Anabaena PCC7120的DNA聚合酶DnaE由2个断裂基因dnaENI和dnaECI编码.通过纯化它们的部分基因在大肠杆菌中的表达产物DnaENI′和DnaECI′来免疫家兔获得抗体,用于对断裂的DnaE在体内和体外的反式剪接活性进行鉴定.在体外实验中,将2个断裂的DNA聚合酶DnaENI′和DnaECI′混合,进行反应,并利用它们的抗体对反应产物进行鉴定;在体内实验中,利用以上抗体对Anabaena PCC7120细胞总蛋白进行免疫杂交分析.实验结果表明,蓝细菌AnabaenaPCC7120中断裂的2个DnaE多肽在体内和体外均能进行反式剪接.体外实验中,纯化的DnaENI′和DnaECI′的混合反应产生新的蛋白产物,既有成熟的反式剪接产物DnaEN′-,又有剪切了内含肽后的副产物DnaEN′和DnaE,且DTT的存在对剪接反应具有促进作用;此外体内实验中,在Anabaena PCC7120细胞中既能检测到完整的DnaE,也能检测到未作用的DnaENI,但未能检测到DnaECI.     

Characterization of an insertion sequence (IS891) of novel structure from the cyanobacterium Anabaena sp. strain M-131.

When recombinant plasmids that were transferred to the cyanobacterium Anabaena sp. strain M-131 were transferred back to Escherichia coli, some of the transformants contained inserts. One of the insertion sequences (ISs) was characterized by sequencing. This 1,351-base-pair IS contained an open reading frame that was capable of encoding a peptide of 310 amino acids and had terminal sequences with distinctive structures, but it lacked terminal inverted repeats and did not duplicate target DNA upon insertion. The element bore no significant sequence homology to any sequence stored in the GenBank data base. Restriction analysis of the genomes of Anabaena sp. strain M-131 and Anabaena sp. strain PCC 7120 showed those strains to be closely related. Sequences homologous to the IS element were also present in the DNA of Anabaena strain PCC 7120, but the copy numbers and chromosomal locations of such sequences differed in the two strains. The largest visualized plasmid was 425 kilobases (kb) in M-131 and 410 kb in PCC 7120; at least the former plasmid contained multiple copies of the element, as did a 115-kb plasmid in M-131.     

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.     

Tn5 mutagenesis of Anabaena sp. strain PCC 7120: isolation of a new mutant unable to grow without combined nitrogen.

Methylation by Ava methylases in Escherichia coli increases the efficiency to transfer of Tn5 in pBR322bla:: Tn5 from E. coli to Anabaena sp. strain PCC 7120 by conjugation. Following conjugation, Tn5 but not pBR322 sequences were found at many different positions in the Anabaena chromosome. This procedure was used to mutagenize, tag, and clone a previously unrecognized gene required for nitrogen fixation in this Anabaena sp.     

The structural nif genes of four symbiotic Anabaena azollae show a highly conserved physical arrangement

Cloned DNA from Anabaena sp. PCC 7120 was used to determine the distribution of restriction sites around NifHDK genes of endosymbionts extracted from Azolla species. Although many of the restriction sites of the symbiotic Anabaena nifHDK genes differed from those of the free-living Anabaeba sp. PCC 7120, three apparently identical restriction sites were found in and around the nifD region. The arrangement of A. azollae and Anabaena sp. PCC 7120 nif H,D,K genes appears similar, with nifH and nifD linked and nifK some distance away from nifD. The A. azollae nifHDK genes appear strongly conserved among the Azolla species examined, regardless of the geographical origin of the ferns.     

鱼腥藻PCC7120基因组中一个影响异形胞发育和图式形成的新区域

以Tn5 10 87b诱变鱼腥藻PCC712 0 ,筛选不能利用氮气生长、异形胞图式发生变化的突变株。突变株 # 180 1经缺氮诱导 2 4h后无异形胞形成 ,48h后沿藻丝形成少量成熟异形胞 ,占藻细胞总数比例为 2 .8% ,其异形胞发育速度和形成频率均与野生型有显著区别。以ClaⅠ酶切该突变株总DNA、自环化后以电泳冲法转化大肠杆菌 ,回收带有插入位点两侧DNA片段的转座子Tn5 10 87b。经测序确定转座子位于未知功能基因alr0 0 99第 14 8和 14 9碱基之间。为证明突变性状确由alr0 0 99内的插入突变引起而不是由于其他二次突变 ,通过同源双交换将含新霉素抗性基因的C .K2片段定向插入基因组中alr0 0 99的EcoRV位点 ,获得重构的鱼腥藻突变株DR2 14 ,其表型与原突变株 # 180 1相同。以上结果表明alr0 0 99或其下游基因是鱼腥藻PCC712 0基因组中与异形胞发育和图式形成相关的一个新区域。     

Conjugative transfer of a plasmid from Escherichia coli to various strains of the order Actinomycetales

The conjugal transfer of autonomous and integrative plasmids from the donor strain Escherichia coli S17-1 to strains of genera Actinomadura, Arthrobacter, Kitasatoa, Micromonospora, Nocardia, Rhodococcus, Saccharopolyspora, and to 16 strains of the genus Streptomyces was demonstrated. The status of plasmids in recipient strains and the stability of their inheritance were analyzed. Plasmids constructed for strains of the genus Streptomyces were shown to function in a large number of strains belonging to the order Actinomycetales. The well-developed system of Streptomyces vector molecules and cloned genes of antibiotic biosynthesis allows their transfer to those microorganisms for which conventional techniques of plasmid transfer by regenerated protoplast transformation or electroporation have not been developed or are inefficient.     

DNA restriction dependent on two recognition sites: activities of the SfiI restriction-modification system in Escherichia coli

In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4-6 bp long, the SfiI endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the SfiI endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the SfiI restriction-modification system were transformed with plasmids carrying SfiI sites. The SfiI system often failed to restrict the survival of a plasmid with one SfiI site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated SfI sites were not restricted. No rearrangements of the plasmids carrying SfiI sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, SfiI displays all of the hallmarks of a restriction-modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro. For both restriction in vivo and DNA cleavage in vitro, SfiI operates best with two recognition sites on the same DNA.     

Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.     

Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis.

Conjugal transposons play an important role in the dissemination of antibiotic resistance determinants in the streptococci and have been postulated to exist in Bacteroides fragilis. To investigate the presence of conjugal transposons in B. fragilis, we employed a Tra- derivative of the transfer factor pBFTM10 contained in the chimeric plasmid pGAT400 delta BglII. We attempted to restore transferability to this plasmid from a series of transconjugants generated by crossing B. fragilis TMP230 containing the TET transfer factor with B. fragilis TM4000, a standard recipient. Transconjugant TM4.2321 transferred pGAT400 delta BglII to Escherichia coli HB101 at almost the same frequency as did the Tra+ parental plasmid, pGAT400. Analysis of the transferred plasmids revealed the presence of 9.6 kilobases of additional DNA in every case but at different positions in independent isolates. The presence of this DNA, designated Tn4399, allowed the pGAT400 delta BglII derivatives to retransfer from the TM4000 background to B. fragilis or E. coli recipients. DNA hybridization studies demonstrated the presence of one copy of Tn4399 in TMP230 and three copies at new sites in TM4.2321. Tn4399 is a new B. fragilis transposon with unique transfer properties that may play a role in the dissemination of drug resistance genes. It differs from previously described conjugal transposons by its ability to mobilize nonconjugal plasmids in cis.     

Chemoheterotrophic Growth of the Cyanobacterium Anabaena sp. Strain PCC 7120 Dependent on a Functional Cytochrome c Oxidase

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium commonly used as a model organism for studying cyanobacterial cell differentiation and nitrogen fixation. For many decades, this cyanobacterium was considered an obligate photo-lithoautotroph. We now discovered that this strain is also capable of mixotrophic, photo-organoheterotrophic, and chemo-organoheterotrophic growth if high concentrations of fructose (at least 50 mM and up to 200 mM) are supplied. Glucose, a substrate used by some facultatively organoheterotrophic cyanobacteria, is not effective in Anabaena sp. PCC 7120. The gtr gene from Synechocystis sp. PCC 6803 encoding a glucose carrier was introduced into Anabaena sp. PCC 7120. Surprisingly, the new strain containing the gtr gene did not grow on glucose but was very sensitive to glucose, with a 5 mM concentration being lethal, whereas the wild-type strain tolerated 200 mM glucose. The Anabaena sp. PCC 7120 strain containing gtr can grow mixotrophically and photo-organoheterotrophically, but not chemo-organoheterotrophically with fructose. Anabaena sp. PCC 7120 contains five respiratory chains ending in five different respiratory terminal oxidases. One of these enzymes is a mitochondrial-type cytochrome c oxidase. As in almost all cyanobacteria, this enzyme is encoded by three adjacent genes called coxBAC1. When this locus was disrupted, the cells lost the capability for chemo-organoheterotrophic growth.     

Conjugal Transfer of Plasmid R6K γ <Emphasis Type="Italic">ori</Emphasis> Minireplicon Derivatives from <Emphasis Type="Italic">Escherichia coli</Emphasis> to Various Genera of Pathogenic Bacteria

Three R6K-derived γ ori minireplicons were successfully transferred by conjugation from Escherichia coli to several species of pathogenic bacteria. The pFL129 replicon encodes the wild-type initiation replication protein π, while plasmids pFL130 and pAG101 encode mutant forms of the π protein conferring the plasmid copy-up phenotype. Plasmids could be transferred to all recipient species tested, although high efficiency conjugal transfer was only obtained with genera of the Enterobacteriaceae. The efficiency of plasmid transfer to all recipients was lower for the copy-up derivatives, pFL130 and pAG101, than for pFL129. The three γ ori replicons were stably maintained in all transconjugants except pFL129 in Listeria monocytogenes. The two mutant plasmids retained their copy-up phenotype in the new bacterial hosts.     

解毒酶基因的克隆及其在大肠杆菌和蓝藻中的表达(英文)

近几年来 ,在明确了杀虫药剂抗性机理的基础上 ,从杀虫药剂抗性的昆虫中分离出高抗性基因 (即解毒酶基因 ) ,将该基因克隆到表达载体 pRL 4 39上 ,得到表达载体 pRL B1,将其转化大肠杆菌HB10 1,获得了可以表达解毒酶基因的转基因工程菌株。同时构建了穿梭表达载体 pDC B1,并转化大肠杆菌HB10 1后 ,在抗生素氨卞霉素 (30 μg/mL)和卡那霉素 (30 μg/mL)平板上挑选阳性克隆 ,将阳性克隆的细胞、蓝藻和结合质粒以三亲结合转移的方式转入蓝藻。斑点杂交、Southern分析结果表明已经获得了Synechococcussp .PCC 794 2转基因工程藻。     

Modification of the N-Terminal Nucleotide Sequence of Mature hGM-CSF Results in High Expression in the Foreign Host, Anabaena sp. Strain PCC 7120

Cloning and high foreign expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene were achieved in Anabaena sp. strain PCC 7120 cells. To promote high expression of hGM-CSF in cyanobacterial cells, PCR primers were designed to modify the N-terminal cDNA sequence of mature hGM-CSF, including a GC rich region and some discriminating against codons according to the degeneracy codon rules, selecting for prokaryotic usage codons. The PCR product encoding the modified hGM-CSF was inserted downstream of the promoter, PpsbA of the shuttle vector pRL439, then ligated with pDC-08 to generate the shuttle expression plasmid, pDC-GM1. The resulting shuttle expression plasmid was transferred into the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120 using the tri-parental conjugation transfer method. The results of PCR amplification of wild type and transgenic cells indicated that the hGM-CSF gene was successfully cloned into Anabaena sp. strain PCC 7120 cells. Western blot analysis showed that the protein expression of modified hGM-CSF in transgenic cells harboring pDC-GM1 was 136% higher than that of non-modified hGM-CSF in transgenic cells harboring pDC-GM0. Additionally, there were similar rate of growth and content of Chl a as compared to controls, suggesting that foreign hGM-CSF did not impair the photosynthetic activity of host cells. Taken together, the results indicate that modification of the N-terminal nucleotide sequence of mature hGM-CSF results in high expression in the transgenic cells.     

Expression of the Anabaena sp. strain PCC 7120 xisA gene from a heterologous promoter results in excision of the nifD element.

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.