Characterisation of the cry54Ab1 operon of Bacillus thuringiensis subsp. sichuansis strain MC28

A novel cry gene operon harbouring two open reading frames (ORFs) was discovered in a large plasmid of Bacillus thuringiensis (Bt) subsp. sichuansis MC28 (pMC189). The first ORF encoded 743 amino acids. It had a deduced molecular mass of 84.5 kDa and 73.2% identity with Cry54Aa1 protein as indicated by amino acid homology. This cry gene was designated as cry54Ab1 by the Bt Toxin Nomenclature Committee. The second ORF encoded 503 amino acids and had a deduced molecular mass of 58.2 kDa. It is here called as orf2. The whole cry54Ab1 operon, cry54Ab1 and orf2 were digested with BamHI/SalI and inserted into a shuttle vector, pSTK. The recombinant plasmids were transferred into an acrystalliferous mutant Bt HD73^?. Parasporal inclusions were observed only under expression of the whole cry54Ab1 operon. The bioassay experiments showed that expressed products of the cry54Ab1 and the whole cry54Ab1 operon were all toxic to Culex quinquefasciatus (Diptera).     

青霉素G酰化酶操纵子的负调控因子的研究

青霉素G酰化酶(PA)操纵子的调节基因(pacR)存在于青霉素G酰化酶结构基因(pac)内部Dral-Taql一段约500bp的DNA片段内,此片段内含有2个ORF。2个ORF及其突变体分别克隆到pUC18得到一系列重组质粒,用这些重组质粒转化青霉素G酰化酶产生菌E.coliD816,测定克隆片段对PA表达的影响。如果克隆片段含有具功能的pacR,诱导剂苯乙酸(PAA)不能使由高拷贝却pacR表达的阻抑物全部失活,部分阻抑物结合pac操纵基因,阻碍RNA聚合酶对pac的转录,因此PA的表达量降低。结果表明,阻抑物是由pac结构基因内部的ORF2编码的蛋白因子,pacR即ORF2。RNA—DNA杂交实验证实了pacR在转录水平阻抑pac的表达。     

Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A

This study identified a functional spo0A ORF in enterotoxigenic Clostridium perfringens type A. To evaluate the function of spo0A, an isogenic spo0A knock-out mutant was constructed. The spo0A mutant was unable to form endospores and produce enterotoxin, however, these defects could be restored by complementing the mutant with a recombinant plasmid carrying the wild-type spo0A gene. These results provide evidence that spo0A expression is essential for sporulation and enterotoxin production in C. perfringens.     

The lux genes in Photobacterium leiognathi are closely linked with genes corresponding in sequence to riboflavin synthesis genes.

Three open reading frames (ORFs) have been found in the region downstream of the luxG gene in the Photobacterium leiognathi lux operon. These genes (ORF I, II, and III) are not only closely linked to the lux operon and transcribed in the same direction but also show the same organization and code for proteins homologous in sequence to the gene products of ribB, ribA, and ribH of Bacillus subtilis, respectively. The Photobacterium leiognathi gene (ORF II) corresponding to ribA was expressed in Escherichia coli in the bacteriophage T7 promoter-RNA polymerase system and a 40 kDa 35S-labeled polypeptide has been detected on SDS-PAGE. Expression of DNA extending from luxBEG to ORF II inserted between a strong promoter and a reporter gene and transferred by conjugation into Vibrio harveyi did not affect the expression of the reporter gene. The results provide evidence that neither promoter nor terminator sites were present in the DNA between the luxG and ORF II indicating that these genes might be part of the lux operon.     

Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes.

The hutC gene of Klebsiella aerogenes encodes a repressor that regulates expression of the histidine utilization (hut) operons. The DNA sequence of a region known to contain hutC was determined and shown to contain two long rightward-reading open reading frames (ORFs). One of these ORFs was identified as the 3' portion of the hutG gene. The other ORF was the hutC gene. The repressor predicted from the hutC sequence contained a helix-turn-helix motif strongly similar to that seen in other DNA-binding proteins, such as lac repressor and the catabolite gene activator protein. This motif was located in the N-terminal portion of the protein, and this portion of the protein seemed to be sufficient to allow repression of the hutUH operon but insufficient to allow interaction with the inducer. The presence of a promoterlike sequence and a ribosome-binding site immediately upstream of the hutC gene explained the earlier observation that hutC can be transcribed independently of the other hut operon genes. The predicted amino acid sequence of hut repressor strongly resembled that of the corresponding protein from Pseudomonas putida (S. L. Allison and A. T. Phillips, J. Bacteriol. 172:5470-5476, 1990). An unexpected, leftward-reading ORF extending from about the middle of hutC into the preceding (hutG) gene was also detected. The deduced amino acid sequence of this leftward ORF was quite distinct from that of an unexpected ORF of similar size found immediately downstream of the P. putida hutC gene. The nonstandard codon usage of this leftward ORF and the expression of repressor activity from plasmids with deletions in this region made it unlikely that this ORF was necessary for repressor activity.     

Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens

The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.     

narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes.

In previous studies it has been established that in Escherichia coli the three known subunits of anaerobic nitrate reductase are encoded by the narGHI operon. From the nucleotide sequence of the narI region of the operon we conclude that, in addition to the narG and narH genes, the nar operon contains two other open reading frames (ORFs), ORF1 and ORF2, that encode proteins of 26.5 and 25.5 kilodaltons, respectively. Protein fusions to each of the genes in the operon showed that expression of all four genes was similarly regulated. The reading frames of ORF1 and ORF2 were verified, and the N-terminal sequence for the ORF1 fusion protein was determined. The nar operon therefore contains four genes designated and ordered as narGHJI.