Gas Vesicle Genes Identified in Bacillus megaterium and Functional Expression in Escherichia coli

Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea. They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light. In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function). In cyanobacteria, the number of genes involved has not been determined. We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (gvp) from Bacillus megaterium VT1660 and their functional expression in Escherichia coli. Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of E. coli strains that carry this gvp gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-constrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy. In B. megaterium, the gvp region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and gvpA, -P, -Q, -B, -R, -N, -F, -G, -L, -S, -K, -J, -T, and -U, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in E. coli. To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.     

Genetic analysis of the gas vesicle gene cluster in haloarchaea

Gas vesicles are buoyant intracellular organelles composed of a rigid proteinaceous membrane surrounding a gas-filled space. Many prokaryotic microorganisms including photosynthetic and heterotrophic bacteria and halophilic and methanogenic archaea produce gas vesicles. In the majority of cases gas vesicles function in providing vertical motility to cells in aquatic environments. Recent genetic analysis of several halophilic archaeal (haloarchaeal) species has shown that a large cluster of genes [gvpMLKJIHGFEDACN(O)] is necessary for gas vesicle formation.     

丝状体蓝藻藻殖段的分化及其调节机制

本文介绍了丝状体蓝藻(亦称蓝细菌) 的藻殖段的分化及其调节机制。藻殖段与正常藻丝体的区别在于细胞形状、细胞内存有气囊和可移动的短而直的藻丝链等。本文对许多环境因子包括光和营养因素等促进或抑制藻殖段的分化进行了讨论;还介绍了念珠藻(Nostoc) ,单歧藻(Tolypothrix) 和眉藻(Calothrix)所具有复杂的细胞发育过程,即具气囊又可移动的藻殖段分化,异形胞分化以及营养细胞的补偿性色适应。这三种细胞类型的适应形成取决于两种不同的光受体系统。藻殖段和异形胞两者的分化可能取决于光合电子传递链;而营养细胞的补偿性色适应则受光敏色素的调节。此外,谷酰胺合成酶合成和活性调节的PII蛋白,在协同藻殖段分化、异形胞分化及营养细胞的补偿色适应中起重要作用。由于蓝藻藻殖段分化及其调节机制是一个新的研究领域,关于它的知识尚不完整,亟待人们加强研究。     

丝状体蓝藻藻殖段的分化及其调节机制

本文介绍了丝状体蓝藻（亦称蓝细菌）的藻殖段的分化及其调节机制。藻殖段与正常藻丝体的区别在于细胞开状、细胞内存有气囊和可移动的短而真的藻丝链等。本文对许多环境因子包括光和营养因素等促进或抑制藻殖段的分化进行一讨论;还介绍了含球藻（Ｎｏｓｔｏｃ）,单歧藻（Ｔｏｌｙｐｏｔｈｒｉｘ）和眉藻（Ｃａｌｏｔｈｒｉｘ）所具有复杂的细胞发育过程,即具气囊又可移动的藻殖段分化,异形胞分化以及营养细胞的被偿性色适应。这     

Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-gvpDE genes

Gas-vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p-vac region consisting of 14 different gvp genes that are arranged in two clusters: p-gvpACNO and, oppositely oriented, p-gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p-gvpDE and p-gvpF–M. The 5′-terminus of the p-gvpF–M mRMA was located 169 nucleotides upstream of p-gvpF within p-gvpE. The p-gvpG and p-gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas-vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p-gvp gene expression for gas-vesicle synthesis was determined by transformation experiments using the Vac^? species Haloferax volcanii as recipient. Construct ΔA containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac^? transformants, but the addition of p-gvpA on a second vector restored gas-vesicle synthesis to wild-type level (Vac⁺⁺). Similarly, double transformants containing p-gvpD–M plus p-gvpACNO, or p-gvpG–M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p-gvpFED–ACNO were Vac⁺⁺. Transformants containing the p-vac region either lacking gvpA, gvpF, or gvpGHI were Vac^?, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas-vesicle formation. Double transformants containing the constructs p-gvpF–M plus p-gvpACNO (ΔDE) accumulated gas vesicles (Vac⁺) but synthesized fewer than the wild type, showing that the p-gvpDE genes are not necessary for gas-vesicle assembly. A repressor function affecting the synthesis of the p-gvpF–M mRNA could be suggested for p-gvpD and the 5′- region of its mRNA.     

发状念珠藻藻殖段的分化及其光合特性的研究

Hormogonia of Nostoc flagelliforme is one of the developmental stages in the life cycle of cyanobacterium. High yields of pure hormogonia were obtained by weak light (the filaments were covered by sterilized sand for blocking light), red light, white light plus DCMU (3, 4-dichlorophenyl-1, 1-dimethylurea) in the culture. These pure fractions of hormogonia allowed the study of physiological measurements in comparison to vegetative filaments. The photosynthesis in the hormogonia and the vegetative filaments was characterized by fluorescence emission spectra at 77 K, absorption spectrum and oxygen evolution. Absorption spectrum of the hormogoia and vegetative filaments did not reveal difference. The data indicated the similarity of pigment contents between hormogonia and vegetative filaments. Some differences were observed in oxygen evolution of vegetative filaments and hormogonia in the temperature range of 15 ℃ to 45 ℃ and light intensity around 110 μmol·m-2·s-1 to 1200 μmol·m-2·s-1. The fluorescence emission spectra showed that energy distribution between the two photosystems in mature colonies was more balance than in hormogonia. The absorption of light energy in phycobilisomes and the transfer to the two photosystems in the hormogonia were more effective than in the mature colonies. It may be concluded that the formation of hormogonia affected on the structure and function of phytosynthesis.     

Electron Transport Regulates Cellular Differentiation in the Filamentous Cyanobacterium Calothrix

Differentiation of the filamentous cyanobacteria Calothrix sp strains PCC 7601 and PCC 7504 is regulated by light spectral quality. Vegetative filaments differentiate motile, gas-vacuolated hormogonia after transfer to fresh medium and incubation under red light. Hormogonia are transient and give rise to vegetative filaments, or to heterocystous filaments if fixed nitrogen is lacking. If incubated under green light after transfer to fresh medium, vegetative filaments do not differentiate hormogonia but may produce heterocysts directly, even in the presence of combined nitrogen. We used inhibitors of thylakoid electron transport (3-[3,4-dichlorophenyl]-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) to show that the opposing effects of red and green light on cell differentiation arise through differential excitations of photosystems I and II. Red light excitation of photosystem I oxidizes the plastoquinone pool, stimulating differentiation of hormogonia and inhibiting heterocyst differentiation. Conversely, net reduction of plastoquinone by green light excitation of photosystem II inhibits differentiation of hormogonia and stimulates heterocyst differentiation. This photoperception mechanism is distinct from the light regulation of complementary chromatic adaptation of phycobilisome constituents. Although complementary chromatic adaptation operates independently of the photocontrol of cellular differentiation, these two regulatory processes are linked, because the general expression of phycobiliprotein genes is transiently repressed during hormogonium differentiation. In addition, absorbance by phycobilisomes largely determines the light wavelengths that excite photosystem II, and thus the wavelengths that can imbalance electron transport.