Functional complementation of pyran ring formation in actinorhodin biosynthesis in Streptomyces coelicolor A3(2) by ketoreductase genes for granaticin biosynthesis

A mutation in actVI-ORF1, which controls C-3 reduction in actinorhodin biosynthesis by Streptomyces coelicolor, was complemented by gra-ORF5 and -ORF6 from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22. It is hypothesized that, while gra-ORF5 alone is a ketoreductase for C-9, gra-ORF6 gives the enzyme regiospecificity also for C-3.     

Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain <Emphasis Type="Italic">Streptomyces lincolnensis</Emphasis> ATCC 25466

A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to ≈1–3 % of the production in the ATCC 25466 strain.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Construction and characteristics of mini-Mu phages

The paper reports on the principles of construction, physical characterization and results of preliminary genetic investigation of hybrid plasmids containing Mu DNA sequences or deletion derivatives of phage Mu, the so-called mini-Mu phages. The mini-Mu were obtained by joining both phage ends within one plasmid in a regular orientation. A collection obtained by in vitro manipulations included 14 recombinant plasmids containing different DNA fragments of the Mu genome. Seven plasmids have both ends of phage Mu, three plasmids containing regularly oriented ends, i.e. mini-phages of different size: the mini-Mu5 (11 kb) within pRM8 plasmid, the mini-Mu4 Ap (18 kb) within pRM6 and the mini-mini-Mu (4.4 kb) within pRM5. The collection comprises mini-Mu phages with the gene kil inactivated after treatment with hydroxylamine. Biological properties of the hybrid plasmids have been preliminary studied.     

Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis

Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.     

Differential binding of RNA polymerase to the pRM and pR promoters of bacteriophage lambda

Escherichia coli RNA polymerase binding to the promoters pR and pRM of bacteriophage lambda was visualized and quantitated by electron microscopy. Although the two promoters are located close together in the phage genome, their proximity to the end of an 889-bp HaeIII DNA fragment made it possible to position binary complexes within 18 bp (2%) intervals. Thus, polymerase binding to pR and pRM could be distinguished by comparing the locations of binary complexes formed with wild-type and mutant (prm-) DNA at 37 degrees and 15 degrees C. We found that at 37 degrees C, RNA polymerase bound primarily to pR, while at 15 degrees C the efficiency of binding was the same at pRM as at pR. In addition, at 15 degrees C the overall efficiency of binding was significantly reduced relative to that at 37 degrees C. When the enzyme was incubated with prm- DNA, binding to pRM was reduced at both temperatures, as expected. Reduced binding to pRM was accompanied by an increase in binding to pR, apparently as a consequence of the low enzyme-to-DNA ratios used in these experiments.     

Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis

Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.     

Evidence that the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate is a product of the methylreductase reaction in Methanobacterium

When 7-mercaptoheptanoylthreonine phosphate (HS-HTP) was used as the sole source of electrons for reductive demethylation of 2-(methylthio)-ethanesulfonic acid (CH3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain delta H, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH3-S-CoM----CH4 + CoM-S-S-HTP. CH4 and CoM-S-S-HTP were produced stoichiometrically in a ratio of 1:1. Coenzyme M (HS-CoM) inhibited HS-HTP driven methanogenesis indicating that CH3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.     

Role of CH/pi interactions in substrate binding by Escherichia coli beta-galactosidase

Interactions between carbohydrates and aromatic amino-acid residues are often observed in structures of carbohydrate-protein complexes. They are characterized by an orientation of the pyranose or furanose ring parallel with the aromatic ring of amino-acid residues. An important role in the formation of these complexes is supposed to be played by CH/pi interactions. This paper presents an ab initio quantum chemistry study of CH/pi interactions between beta-galactosidase from E. coli and its substrates and products. The energy stabilizing the interaction between Trp999 residue and substrate bound in the shallow binding mode was calculated at the MP2/6-31+G(d) level as 5.2kcalmol(-1) for the glucose moiety of allolactose, 2.4kcalmol(-1) for the galactose moiety of allolactose and 5.0kcalmol(-1) for the glucose moiety of lactose. The energy stabilizing the interaction between Trp568 residue and galactose in the deep binding mode was calculated as 2.7kcalmol(-1). Interaction energies at the HF/6-31+G(d) and B3LYP/6-31+G(d) levels were small or repulsive; therefore, highly correlated ab initio methods were necessary to study these interactions. These unexpectedly strong interactions give a rationale for allolactose formation and illustrate the role of the Trp999 residue. In addition, this illustrates the importance of CH/pi interactions for the function of carbohydrate-binding proteins and carbohydrate-processing enzymes.     

Establishment of a gene transfer system for Rhodothermus marinus

Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB^– mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7±1 and 42±4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3±0.7×10⁶ cfu/g DNA at 22.5 kV/cm, 200 and 25 F. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.     

盐生盐杆菌RM07 DNA片段在大肠杆菌中的定点诱变和启动子功能分析

将来源于嗜盐古生菌——盐生盐杆菌(Halobacterium halobium)基因组的RM07 DNA片段以正反两个方向分别插入大肠杆菌启动子探针载体pKK232-8携带的报告基因——氯霉素抗性基因(cat)的上游,得到RM07-cat融合的质粒pRM07-1( )和pRM07-1(-),将其分别转入大肠杆菌HB101,进而检测了不同转化子菌株的氯霉素抗性水平和细胞内氯霉素乙酰转移酶蛋白质浓度。结果表明：正向的RM07片段在真细菌(大肠杆菌)中具有启动子活性,能够驱动cat报告基因的表达;而反向的RM07片段在大肠杆菌中不具有启动子活性。对RM07片段进行了定点诱变分析,检测了特定核苷酸突变对启动子活性的影响,结果进一步精确定位了RM07片段中对在大肠杆菌中的启动子功能有重要作用的关键碱基,并且通过改造RM07片段的碱基组成成分大幅提高了其在大肠杆菌中的启动子活性。     

Mini-Mu transposition of bacterial genes on the transmissible plasmid

Using the pRM30 plasmid, an Ap^s deletion derivative of broad host range plasmid RP4 with integrated new miniMu 5 (11 kb), we followed the transfer ofEscherichia coli chromosomal genes to the recipient strain. The miniMu 5-mediated transposition of chromosomal genes occurs onto the plasmid with integrated miniMu5 rather than onto the “recipient” plasmid pNH602. The plasmid DNA in recipient cells was detected by electrophoresis. One of the acquired hybrid plasmids pTB2 was analyzed genetically and by restriction endodeoxyribonuclease digestion. A structure consisting of miniMu-chromosomal segment-miniMu as a product of Mu-mediated transposition was detected.     

苏云金杆菌在华北果园土壤中消长动态的研究

本文报道了产生核黄素的苏云金杆菌CH菌株在果园土壤中的消长动态。研究结果表明 ,将CH菌株制剂喷洒于苹果园土壤表面后 1个月内 ,CH菌株的菌数基本上维持在喷洒后的 10 5/ g水平 ,其后菌数急剧减少 ,2个月后从 10 5/ g的水平减少到 10 4 / g的水平 ,并长时间维持 10 4 / g这一水平 ;CH菌株制剂喷洒于果园土壤后对土壤中固有的Bacillus属细菌数的消长没有明显的影响 ,Bacillus属细菌数长时间维持在 10 6～ 10 5/ g之间 ,处于一种相对稳定的状态 ;在土壤透水性和灌水条件良好的土壤条件下 ,喷洒于土壤表面的苏云金杆菌可缓慢向深处移动。     

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.