Introduction

The gram-positive, soil-inhabiting, filamentous bacterial genus Streptomyces is characterized by its ability to produce a wide variety of secondary metabolites such as antibiotics, parasiticides, herbicides, and pharmacologically active substances. Another characteristic feature of the genus is its complex multicellular development. On an agar medium, spores germinate to form a branched, multinucleoid substrate mycelium, which then produces an aerial mycelium. After septa have been formed at regular intervals along the aerial hyphae, long chains of uninucleoid spores are formed. On the other hand, some Streptomyces species, including the streptomycin producer Streptomyces griseus, can make submerged spores in liquid medium. In S. griseus, sporogenic hyphae, which seem to be equivalent to aerial hyphae on solid culture, are formed from vegetative hyphae and subsequently undergo septation to form chains of unicellular submerged spores.

Peptideglycan (PG), which comprises alternating copolymer of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with pentapeptide side-chains branching from the MurNAc residues, is a primary constituent of the Gram-positive bacterial cell wall. It functions in maintaining cell shape and cytoplasmic turgor pressure. PG is a dynamic macromolecule that is actively remodeled to enable cell growth and morphological differentiation. For the remodeling of cell wall, synthesis and cleavage of PG must be tightly regulated, with the activities of biosynthetic and hydrolytic enzymes coordinated in both space and time. Cell wall hydrolases are diverse enzymes that are typically grouped on the basis of substrate specificity and resulting cleavage products. N-acetylmuramoyl-L-alanine amidase, which cleaves between MurNAc and the first residue (L-Ala) of the peptide side chain, is one of the major cell wall hydrolases. Although significant remodeling of the cell wall must accompany the filamentous growth and morphological changes associated with the different stages of the Streptomyces life cycle, little is known about cell wall remodeling in Streptomyces. In this study, to investigate cell wall remodeling in S. griseus, the spatial localization and function of the five N-acetylmuramoyl-L-alanine amidases were examined. The spatial localization and function of a novel component of the bacterial core morphogenic apparatus, RodZ, were also examined.

1. The spatial localization of five N-acetylmuramovl-L-alanine amidases during filamentous growth and sporulation in S. griseus

Although N-acetylmuramoyl-L-alanine amidase has an important role in cleaving the septum to release daughter cells after cell division in Escherichia coli, the function of it in a filamentous bacterial genus Streptomyces was unknown. In silico analysis of the S. griseus genome revealed that S. griseus has five secreted N-acetylmuramoyl-L-alanine amidases, designated as AmiA to AmiE (Fig. 1). Each protein was produced as a fusion protein carrying a 3 x FLAG tag at its C-terminus in S. griseus and its localization in hyphae cultured in a liquid medium was examined by immunofluorescence microscopy.

All of the five N-acetylmuramoyl-L-alanine amidases showed an interesting localization. The foci were observed at (i) tips of sporogenic hyphae and (ii) several spots arranged spirally with almost regular intervals along the sporogenic hyphae (Fig. 2). The foci appeared to be dispersed after septa were formed. Because AmiC and AmiD have a Twin-arginine translocation (Tat)-dependent signal peptide, a fusion protein carrying GFP at the C-terminus (AmiC-GFP or AmiD-GFP) was also produced in S. griseus and localization of them in living hyphae cultured on an agar medium was examined by fluorescence microscopy. The pattern of localization of AmiC-GFP and AmiD-GFP in aerial hyphae was very similar to that of AmiC-FLAG and AmiD-FLAG in sporogenic hyphae. This result suggested that AmiC and AmiD were transported across the cell membrane by the Tat system, which is dedicated to the translocation of folded proteins, and localized at their destination even as GFP fusion proteins. Then, AmiD was fused with RFP in a similar manner and localization of AmiC-GFP and AmiD-RFP was examined in a same recombinant strain. AmiC-GFP and AmiD-RFP were almost colocalized. This result suggested that not only AmiC and AmiD but also other three N-acetylmuramoyl-L-alanine amidases were colocalized all together at specific positions in the surface of sporogenic or aerial hyphae before septation.

2. Functional analysis of five N-acetylmuramoyl-L-alanine amidases in S. griseus

The course of transcription of the five N-acetylmuramoyl-L-alanine amidase genes in liquid culture was examined by semi-quantitative RT PCR. Transcription of amiA, amiC, and amiE was greatly increased in the late growth phase, while transcription of amiB and amiD was almost constant during growth. This result suggested that AmiA, AmiC, and AmiE should be involved in morphological development. To reveal the function of the N-acetylmuramoyl-L-alanine amidases, each ami gene was deleted from the chromosome. Among five amidase-mutants, only the AamiA mutant showed a distinct phenotype, as determined by scanning electron microscope. In the AamiA mutant, approximately 40% of aerial hyphae produced spores with abnormal length because of septaion with irregular intervals. The other mutants showed a similar disturbance in the position of septation with a very low frequency (approximately 1%). Then, a AamiAC double mutant and a AamiACE triple mutant were also constructed. In the AamiAC and AamiACE mutants, approximately 70% and 90% of aerial hyphae, respectively, produced abnormal spores. This result indicated that N-acetylmuramoyl-L-alanine amidases, especially AmiA, AmiC, and AmiE, had an important role in the formation of septa. Localization of the amidases at spots with almost regular intervals along sporogenic hyphae also supported this notion. Because the aerial hyphae of the AamiACE mutant had a rough appearance and sometimes swelled in the middle part, the amidases may have some influence on biogenesis of cell wall. Localization of the amidases at hyphal tips, where cell walls are generated and the hyphae extend, may support this idea.

3. The spatial localization and function of RodZ in S. griseus

Recently, a new common player, RodZ, in bacterial cell morphogenesis was discovered. RodZ is a membrane protein with bitopic topology such that the N-terminal region including a helix-turn-helix motif is in the cytoplasm, whereas the C-terminal region is exposed to periplasm. In E. coli, RodZ forms helical structures associated with the cell membrane, similar to the MreB bacterial actin. The N-terminal cytoplasmic domain containing a helix-turn-helix motif may mediate important interactions between RodZ and MreB. In contrast, the C-terminal region of RodZ probably interacts with enzymes that contribute to PG synthesis in the periplasm. Thus, RodZ probably mediates spatial information from cytoskeletal proteins in the cytoplasm to a PG synthesis machinery in the periplasm. By a Blast search, only one RodZ homolog (SGR1770) was found in S. griseus. To investigate the function of the RodZ homolog, the rodZ homolog gene (named rodZ hereafter) was deleted from the S. griseus chromosome. Interestingly, the ErodZ mutant showed similar phenotypes as the AamiACE triple mutant; the aerial hyphae of the ArodZ mutant produced spores with abnormal length, had a rough appearance, and sometimes swelled in the middle part. On the other hand, RodZ was produced as a fusion protein carrying GFP at its C-terminus in S. griseus and its localization in living hyphae cultured on an agar medium was examined by fluorescence microscopy. The pattern of localization of RodZ-GFP was very similar to that of the N-acetylmuramoyl-L-alanine amidases; the foci were observed at both tips and several spots arranged spirally with almost regular intervals along the aerial hyphae.

4. Colocalization of RodZ and AmiC in S. griseus

Because functional and spatial relationships between RodZ and the N-acetylmuramoyl-L-alanine amidases were suggested as described above, RodZ-GFP and AmiC-RFP were simultaneously produced in a same recombinant strain. Expectedly, exact colocalization of RodZ-GFP and AmiC-RFP was observed. However, RodZ seemed to be unnecessary for the localization of N-acetylmuramoyl-L-alanine amidases, because AmiC and AmiD could localize at specific positions in the surface of aerial hyphae even in the ArodZ mutant. Further work is needed to reveal the molecular mechanism for localization of N-acetylmuramoyl-L-alanine amidases.

Figure 1. Schematic representation of the domain structure of five N-acetylmuramoyl-L-alanine amidases in S. griseus. N-acetylmuramoyl-L-alanine amidase has an amidase_2 or amidase_3 domain (indicated by a black box). AmiE has an amidase_3 domain, while others have an amidase_2 domain. AmiA, AmiB, and AmiE have signal peptides (SP-Sec) for secretion by the Sec pathway, while AmiC and AmiD have signal peptides (SP-Tat) for secretion by the Tat system. AmiA has a transglycosylase-like domain (grey box) and two PG-binging domains (hatched box), as well as an amidase_2 domain.

Figure 2. Localization of AmiA-FLAG in the surface of sporogenic hyphae in S. griseus as determined by immunofluorescence microscopy.

放線菌は菌糸状に生育するが栄養菌糸から形成された特殊な菌糸が胞子に分化する。放線菌の菌糸成長と胞子形成においては、菌糸先端や隔壁形成部位において細胞壁の合成と分解が起こると考えられるが、これらについてはほとんど研究がなされていなかった。本論文はペプチドグリカン分解酵素の1つであるN-アセチルムラモイル-L-アラニンアミダーゼの局在と機能に関する解析により、放線菌の菌糸成長と胞子形成に関して新たな知見を得ることを目的としており、7章からなる。

第1章では、放線菌、ペプチドグリカン分解酵素、バクテリアの細胞骨格タンパク質などについて、これまでの知見をまとめるとともに、本研究の目的と本論文の構成について述べている。

第2章では、細胞内タンパク質局在を指標にした標的タンパク質の探索について述べている。最初に、固体培地で気中菌糸・胞子形成時期に強く転写されることがトランスクリプトーム解析によって示されていた遺伝子群から、局在予測や機能予測により、97個の遺伝子を選択した。これらの遺伝子産物を蛍光タンパク質(RFP)との融合タンパク質として生産させ、気中菌糸での局在をスライド培養法により観察した。しかしながら、興味ある局在を示すタンパク質はなかった。次に、細胞壁に局在すると考えられるタンパク質に注目し、15種の分泌タンパク質の液体培養菌糸での局在について、免疫蛍光顕微鏡観察により解析した。その結果、ペプチドグリカン結合ドメインをもったN-アセチルムラモイル-L-アラニンアミダーゼAmiAが、興味深い局在を示すことがわかった。AmiAはスポット状に局在したが、このスポットはsporogenic hypha上、らせん状にほぼ等間隔に観察された。また、sporogenic hyphaの先端部には、このスポットが必ず観察された。

第3章では、S.griseusゲノムにコードされるAmiAを含む5つのN-アセチルムラモイル-L-アラニンアミダーゼ(AmiA,AmiB,AmiC,AmiD,AmiE)の局在解析について述べている。AmiA以外の4つのアミダーゼは、いずれも、上述したAmiAと同様の局在を示した。一方、AmiC,AmiDについてはGFP(あるいはRFP)との融合タンパク質を生産する組換え放線菌株を構築し、気中菌糸での局在がsporogenic hyphaでの局在と同様であることを示した。また、AmiCとAmiDが共局在することを示し、5つのアミダーゼが同一の部位に局在すると推測した。

第4章では、5つのアミダーゼのin vivoでの機能に関して、遺伝子破壊により解析した結果を述べている。いずれの遺伝子破壊株でも、菌糸隔壁が形成される位置に異常が生じ、アブノーマルな長さをもつ胞子が形成されたが、その頻度はamiA破壊株が最も高く、おおよそ40%の気中菌糸で異常が観察された。その他の遺伝子破壊株では頻度がずっと低く、数%程度であった。amiAとamiCの二重破壊株、amiA、amiC、amiE三重破壊株も作製したが、これらの株では、アブノーマルな長さの胞子を作る頻度は、それぞれ70%、90%と大幅に上昇するとともに、細胞壁強度が著しく低下することが示唆された。以上の結果などから、N-アセチルムラモイル-L-アラニンアミダーゼは胞子形成時の隔壁形成および細胞壁合成全般に重要な機能をもつことが明らかになった。

第5章では、大腸菌などで新たに見出された細胞骨格形成の鍵タンパク質であるRodZの、放線菌でのホモログタンパク質(RodZと命名)の局在と機能について解析した結果を述べている。気中菌糸での隔壁形成の初期において、RodZはAmiAと同様の局在を示した。また、rodZ破壊株は、amiA、amiC、amiE三重破壊株と似た表現型を示した。

第6章では、RodZとAmiCの局在について調べた結果を述べている。RodZ、AmiCをそれぞれGFP、RFPと融合させたタンパク質を同時に生産する組換え放線菌株を用いて、両タンパク質が共局在することが示された。一方、rodZ破壊株においてもAmicは正常に局在したことから、RodZはAmicの局在には必須ではないことが示唆された。

第7章では、本研究を総括するとともに、今後の研究の展望について述べている。

以上、本論文は、放線菌のN-アセチルムラモイル-L-アラニンアミダーゼの局在と機能を明らかにし、放線菌の菌糸成長と胞子形成に関する新たな知見をもたらすものであり、学術上ならびに応用上貢献するところが少なくない。よって、審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。