Kasai Sabu, Sumimoto Takumi
Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, Japan.
Eur J Biochem. 2002 Dec;269(23):5851-60. doi: 10.1046/j.1432-1033.2002.03304.x.
Photobacterium phosphoreum IFO 13896 emits light strongly when cultured in medium containing 3% NaCl, but only weakly in medium containing 1% NaCl. It is known that dim or dark mutants appear frequently and spontaneously from this parent strain. To confirm that riboflavin biosynthesis is stimulated when the lux operon is active, the amount of light emitted and flavins synthesized under strongly or weakly light emitting conditions was determined. In comparison with the parent strain cultured in 3% NaCl, the same strain cultured in 1% NaCl emitted 1/36 the light and produced 1/4 the flavins, while three dim or dark mutants, M1, M2 and M3 cultured in 3% NaCl, emitted almost no light, 1/58 the light and 1/10 the light and produced 1/8, 1/5 and 1/3 the amount of flavins, respectively. From these results, we deduced that the genes for riboflavin synthesis, rib genes, are organized in an operon in this strain. In P. phosphoreum NCMB 844, it has been reported that a rib gene cluster is present just downstream of the lux operon. However, among rib genes, the gene for pyrimidine deaminase/pyrimidine reductase, ribD, was not found in this cluster. Because a complete rib operon seems to be necessary for efficient regulation at the transcriptional level, we expected ribD to be present downstream of this cluster and sequenced this region, using SUGDAT, Sequencing Using Genomic DNA As a Template. We could not find this gene but found a gene for hybrid-cluster protein (prismane protein). To find ribD in a different region, a partial ribD sequence was amplified and sequenced using a PCR-based method, and subsequently the genomic DNA was sequenced in both directions from this partial sequence using SUGDAT. Because ribC was found just downstream of ribD, we sequenced further downstream of ribC and confirmed that another complete set of rib genes, ribD, ribC, ribBA, and ribE, is present in P. phosphoreum. The presence of a complete rib operon in P. phosphoreum explains why this species can synthesize flavins at enhanced levels to sustain a strong light emission. Furthermore, we sequenced the rib operon in Vibrio fischeri, another representative luminous bacterium, in a manner similar to that described above, and confirmed that a complete operon is present also in this species. The organization of rib genes in an operon in the Proteobacteria gamma-subdivision is discussed.
磷光发光杆菌IFO 13896在含3%氯化钠的培养基中培养时强烈发光，但在含1%氯化钠的培养基中仅微弱发光。已知该亲本菌株经常自发出现暗或弱发光突变体。为了证实当lux操纵子活跃时核黄素生物合成受到刺激，测定了在强发光或弱发光条件下发出的光量和合成的黄素量。与在3%氯化钠中培养的亲本菌株相比，在1%氯化钠中培养的同一菌株发出的光为前者的1/36，产生的黄素为前者的1/4，而在3%氯化钠中培养的三个暗或弱发光突变体M1、M2和M3，几乎不发光、发出的光分别为前者的1/58和1/10，产生的黄素分别为前者的1/8、1/5和1/3。根据这些结果，我们推断该菌株中核黄素合成基因rib基因以操纵子形式存在。在磷光发光杆菌NCMB 844中，据报道在lux操纵子下游正好存在一个rib基因簇。然而，在这个基因簇中未发现嘧啶脱氨酶/嘧啶还原酶基因ribD。由于完整的rib操纵子似乎是转录水平有效调控所必需的，我们预期ribD存在于该基因簇下游，并使用以基因组DNA为模板的测序方法SUGDAT对该区域进行了测序。我们未找到该基因，但发现了一个杂交簇蛋白（棱柱烷蛋白）基因。为了在不同区域找到ribD，使用基于PCR的方法扩增并测序了部分ribD序列，随后使用SUGDAT从该部分序列向两个方向对基因组DNA进行测序。由于在ribD下游正好发现了ribC，我们对ribC下游进一步测序，并证实磷光发光杆菌中存在另一套完整的rib基因，即ribD、ribC、ribBA和ribE。磷光发光杆菌中完整rib操纵子的存在解释了该物种为何能以增强水平合成黄素以维持强烈发光。此外，我们以与上述类似方法对另一种代表性发光细菌费氏弧菌中的rib操纵子进行了测序，并证实该物种中也存在完整的操纵子。讨论了γ-变形菌纲中rib基因在操纵子中的组织形式。
