American Society for Microbiology

We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae.

This review focuses on the gene-enzyme relationships and the regulation of different levels of the aromatic amino acid biosynthetic pathway in a simple eukaryotic system, the unicellular yeast Saccharomyces cerevisiae. Most reactions of this branched pathway are common to all organisms which are able to synthesize tryptophan, phenylalanine, and tyrosine. The current knowledge about the two main control mechanisms of the yeast aromatic amino acid biosynthesis is reviewed. (i) At the transcriptional level, most structural genes are regulated by the transcriptional activator GCN4, the regulator of the general amino acid control network, which couples transcriptional derepression to amino acid starvation of numerous structural genes in multiple amino acid biosynthetic pathways. (ii) At the enzyme level, the carbon flow is controlled mainly by modulating the enzyme activities at the first step of the pathway and at the branch points by feedback action of the three aromatic amino acid end products. Implications of these findings for the relationship of S. cerevisiae to prokaryotic as well as to higher eukaryotic organisms and for general regulatory mechanisms occurring in a living cell such as initiation of transcription, enzyme regulation, and the regulation of a metabolic branch point are discussed.

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.

Nhiễm trùng đường hô hấp do Pseudomonas aeruginosa và Burkholderia cepacia đóng vai trò chính trong sinh bệnh học của xơ nang (CF). Bài tổng quan này tóm tắt những tiến bộ mới nhất trong việc hiểu mối tương tác giữa vật chủ và mầm bệnh trong CF với sự nhấn mạnh vào vai trò và kiểm soát của sự chuyển đổi thành dạng nhầy trong P. aeruginosa, hiện tượng này biểu hiện sự thích ứng của loại mầm bệnh cơ hội này với quá trình nhiễm trùng mãn tính trong CF, và sức đề kháng tự nhiên với kháng sinh của B. cepacia, sự lây lan giữa người với người, và đôi khi gây tử vong nhanh chóng do loại vi khuẩn này gây ra. Mặc dù việc hiểu cơ chế chuyển đổi thành dạng nhầy trong P. aeruginosa đã tiến đến mức mà hiện tượng này đã trở thành một hệ thống mô hình để nghiên cứu phản ứng căng thẳng của vi khuẩn trong sinh bệnh học vi sinh, thách thức gần đây hơn với B. cepacia, vốn nổi lên như một mầm bệnh thực sự mạnh mẽ của CF, được thảo luận trong bối cảnh các vấn đề lâm sàng, phân loại, truyền nhiễm và các phương thức tiềm năng của kiểu bệnh lý.