Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1

A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.     

嗜麦芽假单胞菌酪氨酸酶基因在大肠杆菌中的克隆与表达

酪氨酸酶基因（ｍｅｌ）编码的酪氨酸酶是合成黑色素的关键酶。用鸟枪法分离嗜麦芽假单胞菌的ｍｅｌ基因：以ｐＵＣ１８为载体,Ｅ．ｃｏｌｉ ＨＢ１０１为受体菌,在加有一定量的Ａｍｐ和Ｌ－ｔｙｒ的酪素平板上筛选到分泌可溶性黑色素的转化子,所含重组质粒ｐＷＳＹ约７００ｂｐ的外源ＤＮＡ片段上携有ｍｅｌ基因,该片段无ＢａｍＨＩ、ＨｉｎｄＩＩＩ、ＥｃｏＲＩ、ＢｃｌＩ等酶的识别位点。Ｓｏｕｔｈｅｒｎ杂交证实此片段确实     

An rpoE-like locus controls outer membrane protein synthesis and growth at cold temperatures and high pressures in the deep-sea bacterium Photobacterium sp. strain SS9

Many deep-sea bacteria have evolved specialized adaptations for life at cold temperatures and high pressures. A locus required for both psychro- and baro- adaptation in the psychrophilic, moderate barophile, Photobacterium species strain SS9 was identified among SS9 transposon mutants. DNA sequence analysis of this locus identified four complete open reading frames (ORFs), which appear to comprise an operon, and a fifth incomplete ORF. All transposon insertions isolated are in ORF3. Extensive sequence similarity exists between the translation products of ORFs 1–3 and a collection of gene products proposed to include alternative RNA polymerase sigma factors and modifiers of sigma factors activity involved in extracytoplasmic sensing and regulation. Based on the similarity between ORF1 and Escherichia coli rpoE , we have tentatively designated this locus the rpoE locus. SS9 rpoE locus ORF3 insertion mutants showed altered abundances of numerous outer membrane proteins and were both baro- and psychro- sensitive. ORF3 mutant revertants that displayed enhanced high-pressure growth also displayed concomitant enhanced low-temperature growth. Most of these revertants possessed DNA rearrangements at the site of the transposon insertion, further demonstrating the importance of the rpoE locus to high-pressure and cold-temperature growth. Complementation analyses indicated that ORF3 functions in OMP synthesis regulation while ORF4 is required for baro- and psychro-adaptation.     

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.     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593

Pseudomonas sp. strain 593, a soil bacterium, is able to use exogenous choline to synthesize phosphatidylcholine via phosphatidylcholine synthase (Pcs). A 2020 bp DNA fragment that hybridized to a Pcs probe was cloned. This fragment contained a large open reading frame (ORF) with two potential ATG start sites that would encode for 293 and 231 amino acid proteins. Fragments containing the two ORFs encoded Pcs when they were inserted into the expression vector pET23a and expressed under the control of the T7 promoter in Escherichia coli BL21(DE3) pLysS. However, when the two ORFs were inserted into the cloning vector pMD18-T and expressed without control of the plasmid promoter in E. coli DH5α, only the larger clone exhibited Pcs activity. This suggested that the larger fragment contained a native promoter driving expression of the smaller ORF. A promoter activity assay, in which DNA fragments were inserted into the promoter-probe plasmid pCB182 and β-galactosidase activity of E. coli transformants was tested, demonstrated that a promoter is indeed present in the DNA region. All results together indicate that the 696 bp ORF, not the larger 897 bp ORF, encodes the Pcs in Pseudomonas sp. strain 593 and carries a promoter in front of its 5' terminus.     

Cloning and sequence analysis of the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus

 Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin-like pigment. An operon, designated mel, containing a gene that encodes tyrosinase, which is involved in the synthesis of melanin pigment, was cloned from the chromosomal DNA of the microorganism into the high-copy plasmid pAK114 and expressed in S. lividans. The tyrosinase activity of the transformed cells was at approximately a 110-fold higher level than that of the same host carrying the plasmid pIJ702, which has the same replication origin as pAK114 and carries the mel operon from S. antibioticus. The sequence analysis of the S. castaneoglobisporus mel operon revealed that an open- reading frame consisting of 378 base pairs(bp), designated ORF378, was found upstream of the tyrosinase gene (TYRC) consisting of 819 bp. In the present study, we constructed a chimeric mel operon consisting of ORF378 from S. castaneoglobisporus and the tyrosinase gene (TYRA) from S. antibioticus. The chimeric mel operon or the S. antibioticus mel operon, which consists of ORF438 and TYRA, expressed the tyrosinase activity in Escherichia coli intracellularly when located under the control of lacZ promoter, and the tyrosinase activity from the former was at a 30-fold higher level than that from the latter. This suggests that the gene contributing to the high expression of the tyrosinase activity in S. castaneoglobisporus is ORF378, rather than TYRC. Received: 12 June 1995/Received revision: 24 July 1995/Accepted: 7 September 1995     

The unstable melC operon of Streptomyces antibioticus is codeleted with a Tn4811-homologous locus.

The melC operon of Streptomyces antibioticus is unstable, undergoing frequent spontaneous deletions. All the delta melC mutants analyzed also lost 2-kb V1 DNA, which contained two open reading frames (ORFs) homologous to ORF4 (a putative oxidoreductase gene) and ORF5 (a putative AraC-type regulatory gene) of Tn4811. The two ORFs may constitute an accessory unit of a different transposon.     

Molecular cloning and functional characterization of a unique multipotent polyphenol oxidase from Marinomonas mediterranea

Marinomonas mediterranea is a recently isolated melanogenic marine bacterium containing laccase and tyrosinase activities. These activities are due to the expression of two polyphenol oxidases (PPOs), a blue multicopper laccase and an SDS-activated tyrosinase. The gene encoding the first one, herein denominated M. mediterranea PpoA, has been isolated by transposon mutagenesis, cloned and expressed in Escherichia coli. Its predicted amino acid sequence shows the existence of a signal peptide and four copper-binding sites characteristic of the blue multicopper proteins, including all fungal laccases. In addition, two additional putative copper-binding sites near its N-terminus are also present. Recombinant expression in E. coli of this protein clearly demonstrates its multipotent capability, showing both laccase-like and tyrosinase-like activities. This is the first prokaryotic laccase sequenced and the first PPO showing such multipotent catalytic activity. The expression of several truncated products indicates that the four copper-binding sites typical of blue multicopper proteins are essential for the laccase activity of this enzyme. However, the last two of these sites are not necessary for tyrosine hydroxylase activity as this activity is retained in a truncated product containing the first two sites as well as the extra histidine-rich clusters close to the N-terminus of the protein.     

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O₂ consumption during B. bufo development.     

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.     

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.