Cloning and regulation of Erwinia herbicola pigment genes.

The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes.     

Mapping of a carotenogenic gene cluster from Erwinia herbicola and functional identification of six genes

Six genes have been mapped and identified by hybridization or by carotenoid analysis of deletion mutants on a 9 kb chromosomal fragment originating from Erwinia herbicola Eho 10. These genes include crtB, E and I which have been formerly described for Rhodobacter, and three new ones: crtZ encoding lycopene cyclase, crtH for beta-carotene hydroxylase, and crtG for zeaxanthin glycosilase. Their arrangement on the plasmid has been elucidated.     

Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1.

The pheromone-responsive conjugative plasmid pPD1 (59 kb) of Enterococcus faecalis encodes the bacteriocin 21 (bac21) determinant. Cloning, transposon insertion mutagenesis and sequence analysis of the bac21 determinant showed that an 8.5-kb fragment lying between kb 27.1 and 35.6 of the pPD1 map is required for complete expression of the bacteriocin. The 8.5-kb fragment contained nine open reading frames (ORFs), bacA to bac1, which were oriented in the same (upstream-to-downstream) direction. Transposon insertions into the bacA to bacE ORFs, which are located in the proximal half of bac21, resulted in defective bacteriocin expression. Insertions into the bacF to bac1 ORFs, which are located in the distal half of bac21, resulted in reduced bacteriocin expression. Deletion mutant analysis of the cloned 8.5-kb fragment revealed that the deletion of segments between kb 31.6 and 35.6 of the pPD1 map, which contained the distal region of the determinant encoding bacF to bac1, resulted in reduced bacteriocin expression. The smallest fragment (4.5 kb) retaining some degree of bacteriocin expression contained the bacA to bacE sequences located in the proximal half of the determinant. The cloned fragment encoding the 4.5-kb proximal region and a Tn916 insertion mutant into pPD1 bacB trans-complemented intracellularly to give complete expression of the bacteriocin. bacA encoded a 105-residue sequence with a molecular mass of 11.1 kDa. The deduced BacA protein showed 100% homology to the broad-spectrum antibiotic peptide AS-48, which is encoded on the E. faecalis conjugative plasmid pMB2 (58 kb). bacH encoded a 195-residue sequence with a molecular mass of 21.9 kDa. The deduced amino acid sequence showed significant homology to the C-terminal region of HlyB (31.1% identical residues), a protein located in the Escherichia coli alpha-hemolysin operon that is a representative bacterial ATP-binding cassette export protein.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

螺旋霉素3-O-酰基转移酶基因的删除和主要产生螺旋霉素组分Ⅰ菌株的获得

螺旋霉素(SP)为16元环大环内酯类抗生素,含有螺旋霉素Ⅰ、Ⅱ和Ⅲ个组分,其结构的差异为16元内酯环的C3上分别连接羟基(SPⅠ)、乙酰基(SPⅡ)和丙酰基(SPⅢ);SPⅡ和SPⅢ是在相同的3-O-酰基转移酶催化下SPⅠ进一步酰化的产物。SPⅠ、SPⅡ和SPⅢ在生物学活性方面无大差异。为简化螺旋霉素组分,便于今后对其结构进行进一步改造,根据碳霉素和麦迪霉素生物合成中的3-O-酰基转移酶序列,设计了简并性PCR引物,并采用SON-PCR(single oligonucleotide nested PCR)方法,从螺旋霉素产生菌S.spiramyceticus F21中进行特异性扩增,获得了螺旋霉素3-O-酰基转移酶基因(sspA)及其侧翼序列,共约4.3kb(其中的3457nt DNA序列已被Genbank收录,DQ642742)。采用DNA同源双交换技术对S.spiramyceticus F21中的sspA进行了删除。对螺旋霉素原株和sspA缺失变株进行发酵产物提取和HPLC分析表明:原株中SPⅠ、SPⅡ和SPⅢ的相对含量分别为7.8%、67%和25%,变株中则分别为72%、18%和9.6%;变株主要组分为SPⅠ。螺旋霉素sspA缺失变株的获得为螺旋霉素组分简化及其衍生物的结构改造奠定了基础。     

Tn5 mutagenesis of Rhizobium trifolii host-specific nodulation genes result in mutants with altered host-range ability

Summary A 14 kb DNA fragment from the Sym plasmid of the Rhizobium trifolii strain ANU843, known to carry common nodulation nod and host specific nodulation hsn genes, was extensively mutagenised with transposon Tn5. A correlation between the site of Tn5 insertion and the induced nodulation defect led to the identification of three specific regions (designated I, II, III) which affected nodulation ability. Twenty-three Tn5 insertions into region I (ca. 3.5 kb) affected normal root hair curling ability and abolished infection thread formation. The resulting mutants were unable to nodulate all tested plant species. Tn5 insertions in regions II and III resulted in mutants which showed an exaggerated root hair curling (Hac⁺⁺) response on clover plants. Ten region II mutants which occurred over a 1.1 kb area showed a greatly reduced nodulation ability on clovers and produced aborted, truncated infection threads. Tn5 insertions into region III (ca. 1.5 kb) altered the outcome of crucial early plant recognition and infection steps by R. trifolii. Seven region III mutants displayed host-range properties which differed from the original parent strain. Region III mutants were able to induce marked root hair distortions, infection threads, and nodules on Pisum sativum including the recalcitrant Afghanistan variety. In addition region III mutants showed a poor nodulation ability on Trifolium repens even though the ability to induce infection threads was retained on this host. The altered host-range properties of region III mutants could only be revealed by mutation and the mutant phenotype was shown to be recessive.     

Cloning and expression of the rfe–rff gene cluster of Escherichia coli

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.     

The large-sized plasmids of enterohemorrhagic Escherichia coli O157 strains encode hemolysins which are presumably members of the E. coliα-hemolysin family

Abstract Most enterohemorrhagic Escherichia coli O157:H7 strains harbor a large-sized (90 kb) plasmid designated pO157 and show an enterohemolytic phenotype. In this study the hemolytic activity of E. coli O157:H7 strain EDL933 was investigated. Curing of strain EDL933 from pO157 resulted in loss of its hemolytic activity. By transformation with Tn801-tagged pO157 (pSK3), the hemolysin-negative E. coli K-12 strains C600 and DH5 _α became positive for hemolysin production. By transformation of recombinant plasmids carrying a 11.9 kb Bam HI fragment and a 5.3 kb Sal I fragment of pSK3 hemolytic activity is revealed when tranformed in E. coli C600 or DH5α DNA-hybridization of pO157 and subclones with the α-hemolysin specific DNA probe was only found under conditions of low stringency. No hybridization was found with enterohemolysin I (EHly1) and enterohemolysin II (EHly2) probes. Our results indicate that a hitherto not described hemolysin belonging to the α-hemolysin family is encoded by the 90 kb plasmid of E. coli O157 strains.     

Isolation of DNA fragment containing phoS gene of Escherichia coli K-12

The DNA fragment containing the phoS gene, a regulatory gene for alkaline phosphatase, has been isolated from Escherichia coli K-12 chromosomal DNA by cutting off the DNA with Hind III restriction enzyme and by cloning the gene with plasmid vector pTP 4 which was constructed in this study. The isolated fragment was of about 12.3 kbp and seemed to contain the phoT, glmS, and bgl genes. The 12.3 kbp Hind III fragment was subjected to restriction enzymes EcoR I, BamH I, Sal I, and Pst I, and was found to possess two EcoR I, no BamH I, a Sal I, and four Pst I sites. Partial deletion using these restriction enzymes suggested that the about 6 kbp Hind III-Pst I fragment contained the phoS and phoT genes. Further analysis with other restriction enzymes revealed that the 6 kbp Hind III-Pst I fragment contained a BstE II, two Mlu I and four Hpa I sites. The deletion of these restriction sites using single-strand-specific nuclease S1 suggested that the BstE II and one of Mlu I sites were in the phoT gene, and the BstE II and two Mlu I sites were not in the phoS gene.     

Overlapping deletion in two spontaneous phase variants of Coxiella burnetii

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.     

Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli

Pigment was produced by Escherichia coli cells carrying recombinant plasmids pNIL100, pNIL200 and pNIL400 containing DNA from Rhodococcus sp. E. coli cells containing pNIL100 or pNIL200 (with DNA inserts from Rhodococcus sp. JL10 and Rhodococcus sp. ATCC 21145 respectively) produced both blue and pink pigments, while cells containing pNIL400 (with a DNA insert from Rhodococcus sp. ATCC 21145) produced only pink pigment. Colonies of E. coli(pNIL100) and E. coli(pNIL200) were dark blue, whereas E. coli(pNIL400) colonies were pink. No pigment was detected in Streptomyces griseus transformants containing pNIL100, pNIL200 or pNIL400. Restriction endonuclease mapping indicated that the cloned DNA fragments were different. The pigment gene(s) in pNIL200 producing both the blue and pink pigments were contained within a 2.8 kb DNA fragment. The pigments produced by E. coli transformants containing pNIL200 were characterized by visible and UV spectroscopy. No similar pigments were detected in Rhodococcus sp. ATCC 21145.     

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.     

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).     

草生欧文氏菌(E.herbicola)CSH1065质粒的重组标记及Fib基因的遗传转移

利用DNA分子克隆技术及遗传重组方法,在E.herbicola CSH1065质粒上插入了Km_R及Mob基因,利用细菌间的接合转移,使E.herbicola CSH1065质粒转移到E.coli HB101中,从而获得了E.herbicola CSH1065的Fib(fungi inhibition)基因在E.coli HB101中的表达,进一步证明E.herbicola CSH1065 Fib基因功能只涉及其细胞内的大质粒,与其染色体基因组无关,其黄色色素基因与Fib功能无关。这些结果还为DNA分子杂交方法所证实。     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

Mapping of the SLA complex of miniature swine: mapping of the SLA gene complex by pulsed field gel electrophoresis.

The overall order of the regions of the swine major histocompatibility complex (MHC), the SLA complex, was determined by pulsed field gel electrophoresis (PFGE). It was found that the order of the regions is class II-class III-class I. A class I probe hybridized to a 420 kb Mlu I and a 420 kb Not I fragment as did a class III probe for C2. None of the class II probes hybridized to these fragments. Thus, linkage of class I to class III was shown. The class III C2, Bf, and C4 genes were found to residue in a 190 kb Not I fragment. Linkage of class III and class II genes was shown when both the class III C4 and the class II DR probes hybridized to the same 195 kb Sac II and 340 kb Not I fragments. The class I probe did not hybridize to these fragments. The order of the regions, class II-class III-class I, is similar to that of human MHC genes and may have been conserved in evolution so that coordinated expression of MHC genes could be achieved.