Occurrence and distribution of gas vesicle genes among cyanobacteria.

Gas vesicles (GV) are specialized cell inclusions providing many aquatic procaryotes with buoyancy. In the cyanobacterium Calothrix sp. strain PCC 7601, at least four genes are involved in GV formation. One of those, gvpA1, encodes the major structural GV protein (70 amino acids) and belongs to a multigene family (gvpA1, gvpA2, gvpD). The fourth gene, gvpC, encodes a 162-amino-acid protein, the function of which is still unclear. We used the Calothrix gvpA1 and gvpC genes as probes to perform Southern hybridization experiments with DNA extracted from various cyanobacterial strains. The gvpA gene was found in all the strains that synthesize GV, indicating that its product is an obligatory component of GV. Furthermore, it was found to occur as multiple copies in most of the strains tested. The gvpC gene was only detected in some strains able to synthesize a large amount of GV within a short period. This suggests that the gvpC gene product is a dispensable protein for GV formation and is involved in the efficiency of the assembly process. Based on the occurrence of the gvp genes and on DNA-DNA hybridization patterns, genus assignments are discussed.     

Molecular cloning and nucleotide sequence of a developmentally regulated gene from the cyanobacterium Calothrix PCC 7601: a gas vesicle protein gene.

Since the gas vesicle protein (GVP) is highly conserved among the different gas-vacuolate prokaryotes, a 29-mer oligonucleotide corresponding to a portion of the Anabaena flos-aquae GVP gene was synthesized and used to isolate the GVP structural gene from Calothrix PCC 7601 (= Fremyella diplosiphon). Gas vacuole production in this filamentous cyanobacterium is restricted to hormogonia which occur at a specific stage during the developmental cell cycle. The GVP gene (gvpA) was localized on a 709 bp HindIII-HincII fragment. Nucleotide sequence analysis revealed a 213 bp open reading frame whose deduced amino-acid sequence shows a very high homology with that of the Anabaena flos-aquae GVP. Assuming that the first methionine residue is proteolytically processed, the molecular mass of the Calothrix GVP is 7375 daltons. Sequences resembling the Escherichia coli consensus promoter were found upstream from the gvpA gene. The initiator codon of the gvpA gene is preceded by a polypurine sequence assumed to be the ribosome binding site. Southern hybridizations with a probe specific for the gvpA gene indicated that this gene is not plasmid-borne, and that another homologous gene is present in the Calothrix genome.     

Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranei, comprising 14 genes involved in gas vesicle synthesis (mc-vac-region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc-vac-region produced gas vesicles (Vac+), various deletions in the region 5' to gvpA (encompassing gvpD-gvpM) or 3' to gvpA (containing gvpC, gvpN and gvpO) revealed Vac- transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.     

The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcyctis sp.

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.     

蓝藻伪空胞的特性及浮力调节机制

伪空胞为蓝藻在水体中提供浮力,使其获得适宜的生长条件,最终导致蓝藻水华暴发,了解伪空胞的特征对控制蓝藻水华暴发有重要意义。文章简要回顾了蓝藻伪空胞自1865年被Klebahn发现到1965年被正式命名的研究历程,目前已发现150多种原核生物中含有伪空胞;伪空胞是两末端呈圆锥状的中空圆柱体,伪空胞半径与临界压强遵循方程:Pc=275(r/nm)-1.67MPa;伪空胞气体含量可根据不同原理,利用Walsby伪空胞测定装置、压力浊度计和细胞流式仪测得。总结了伪空胞组成的化学特性,评述了伪空胞gvp基因丛结构功能和GvpA、GvpC的蛋白空间结构。GvpA是伪空胞合成的主要成分,gvpA在伪空胞内存在多个拷贝,其功能仍不清楚;GvpC由33个氨基酸重复单位组成,重复单位越多,伪空胞越不易破裂;概述了伪空胞3种浮力调节机制:镇重物的改变、伪空胞的合成、伪空胞的破裂;归纳了环境因子(光照、温度、氮、磷、钾)参与伪空胞浮力网络调控的途径。提出了目前伪空胞研究面临的困难和问题,对伪空胞的未来研究方向提出探索性的建议。     

Sequence of a genomic DNA clone for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco.

We have cloned and sequenced a gene for the small subunit (SS) of ribulose bis-phosphate carboxylase-oxygenase from Nicotiana tabacum. The tobacco gene is most closely related to the SS genes from the dicots soybean and pea, and less so to the monocots wheat and Lemna; the deduced amino acid sequence of the mature protein is in all cases more closely conserved than is its chloroplast transit sequence. Unlike the genomic sequences of the two monocots, which have one intron, and the two other dicots, which have two introns, the tobacco gene has three introns. The third tobacco intron lies within a highly conserved region of the protein. Its position coincides with the boundary of a 12 amino acid insertion in the SS genes of higher plants, relative to those of blue green algae. The 5' flanking end of the gene carries 67 bp inverted repeats, which flank a series of eight direct repeats; the direct repeats themselves each carry inverted repeats. The 3' untranslated end of this gene differs by only 2 bp from that of an N. sylvestris SS gene.     

Amino acid substitutions in the VanS sensor of the VanA-type vancomycin-resistant Enterococcus strains result in high-level vancomycin resistance and low-level teicoplanin resistance

The vancomycin-resistant enterococci GV1, GV2 and GV3, which were isolated from droppings from broiler farms in Japan have been characterized as VanA-type VRE, which express high-level vancomycin resistance (256 or 512 microg ml(-1), MIC) and low-level teicoplanin resistance (1 or 2 microg ml(-1), MIC). The vancomycin resistances were encoded on plasmids. The vancomycin resistance conjugative plasmid pMG2 was isolated from the GV2 strain. The VanA determinant of pMG2 showed the same genetic organization as that of the VanA genes encoded on the representative transposon Tn1546, which comprises vanRSHAXYZ. The nucleotide sequences of all the genes, except the gene related to the vanS gene on Tn1546, were completely identical to the genes encoded on Tn1546. Three amino acid substitutions in the N-terminal region of the deduced VanS were detected in the nucleotide sequence of vanS encoded on pMG2. There were also three amino acid substitutions in the vanS gene of the GV1 and GV3 strains in the same positions as in the vanS gene of pMG2. Vancomycin induced the increased teicoplanin resistance in these strains.     

The gas vesicle gene cluster from Microcystis aeruginosa and DNA rearrangements that lead to loss of cell buoyancy

Microcystis aeruginosa is a planktonic unicellular cyanobacterium often responsible for seasonal mass occurrences at the surface of freshwater environments. An abundant production of intracellular structures, the gas vesicles, provides cells with buoyancy. A 8.7-kb gene cluster that comprises twelve genes involved in gas vesicle synthesis was identified. Ten of these are organized in two operons, gvpA(I)A(II)A(III)CNJX and gvpKFG, and two, gvpV and gvpW, are individually expressed. In an attempt to elucidate the basis for the frequent occurrence of nonbuoyant mutants in laboratory cultures, four gas vesicle-deficient mutants from two strains of M. aeruginosa, PCC 7806 and PCC 9354, were isolated and characterized. Their molecular analysis unveiled DNA rearrangements due to four different insertion elements that interrupted gvpN, gvpV, or gvpW or led to the deletion of the gvpA(I)-A(III) region. While gvpA, encoding the major gas vesicle structural protein, was expressed in the gvpN, gvpV, and gvpW mutants, immunodetection revealed no corresponding GvpA protein. Moreover, the absence of a gas vesicle structure was confirmed by electron microscopy. This study brings out clues concerning the process driving loss of buoyancy in M. aeruginosa and reveals the requirement for gas vesicle synthesis of two newly described genes, gvpV and gvpW.     

Cellular differentiation in the cyanobacterium Nostoc punctiforme

Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.