Several types of domain occur in beta-1, 4-glycanases. The best characterized of these are the catalytic domains and the cellulose-binding domains. The domains may be joined by linker sequences rich in proline or hydroxyamino acids or both. Some of the enzymes contain repeated sequences up to 150 amino acids in length. The enzymes can be grouped into families on the basis of sequence similarities between the catalytic domains. There are sequence similarities between the cellulose-binding domains, of which two types have been identified, and also between some domains of unknown function. The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences.
The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.
The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS)
The demarcation of protist kingdoms is reviewed, a complete revised classification down to the level of subclass is provided for the kingdoms Protozoa, Archezoa, and Chromista, and the phylogenetic basis of the revised classification is outlined. Removal of Archezoa because of their ancestral absence of mitochondria, peroxisomes, and Golgi dictyosomes makes the kingdom Protozoa much more homogeneous: they all either have mitochondria and peroxisomes or have secondarily lost them. Predominantly phagotrophic, Protozoa are distinguished from the mainly photosynthetic kingdom Chromista (Chlorarachniophyta, Cryptista, Heterokonta, and Haptophyta) by the absence of epiciliary retronemes (rigid thrust-reversing tubular ciliary hairs) and by the lack of two additional membranes outside their chloroplast envelopes. The kingdom Protozoa has two subkingdoms: Adictyozoa, without Golgi dictyosomes, containing only the phylum Percolozoa (flagellates and amoeboflagellates); and Dictyozoa, made up of 17 phyla with Golgi dictyosomes. Dictyozoa are divided into two branches: (i) Parabasalia, a single phylum with hydrogenosomes and 70S ribosomes but no mitochondria, Golgi dictyosomes associated with striated roots, and a kinetid of four or five cilia; and (ii) Bikonta (16 unicellular or plasmodial phyla with mitochondria and bikinetids and in which Golgi dictyosomes are not associated with striated ciliary roots), which are divided into two infrakingdoms: Euglenozoa (flagellates with discoid mitochondrial cristae and trans-splicing of miniexons for all nuclear genes) and Neozoa (15 phyla of more advanced protozoa with tubular or flat [usually nondiscoid] mitochondrial cristae and cis-spliced spliceosomal introns). Neozoa are divided into seven parvkingdoms: (i) Ciliomyxa (three predominantly ciliated phyla with tubular mitochondrial cristae but no cortical alveoli, i.e., Opalozoa [flagellates with tubular cristae], Mycetozoa [slime molds], and Choanozoa [choanoflagellates, with flattened cristae]); (ii) Alveolata (three phyla with cortical alveoli and tubular mitochondrial cristae, i.e., Dinozoa [Dinoflagellata and Protalveolata], Apicomplexa, and Ciliophora); (iii) Neosarcodina (phyla Rhizopoda [lobose and filose amoebae] and Reticulosa [foraminifera; reticulopodial amoebae], usually with tubular cristae); (iv) Actinopoda (two phyla with axopodia: Heliozoa and Radiozoa [Radiolaria, Acantharia]); (v) Entamoebia (a single phylum of amoebae with no mitochondria, peroxisomes, hydrogenosomes, or cilia and with transient intranuclear centrosomes); (vi) Myxozoa (three endoparasitic phyla with multicellular spores, mitochondria, and no cilia: Myxosporidia, Haplosporidia, and Paramyxia); and (vii) Mesozoa (multicells with tubular mitochondrial cristae, included in Protozoa because, unlike animals, they lack collagenous connective tissue).
Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this area. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified. The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review, we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in several instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins embedded in the membrane are affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed. Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering, change in lipid/protein ratio) that compensate for the changes in membrane structure are treated.
Nickel plays an essential role in many microorganisms. The hydrolysis of urea by microbial ureases is facilitated by enzyme-bound nickel in an as yet unknown fashion. A large number of microorganisms possess uptake hydrogenases which contain nickel. In methane biogenesis, the reductive cleavage of the thioether bond of methyl coenzyme M is catalyzed by a nickel-containing enzyme. The synthesis of acetyl coenzyme A in acetogenic bacteria and in some methanogens is carried out by a nickel-containing enzyme which is usually termed carbon monoxide dehydrogenase, but may be more aptly named acetyl coenzyme A synthetase. Acetoclastic methanogens appear to utilize this same enzyme operating in reverse to obtain energy from acetate. The list of microorganisms which require or use nickel is sure to expand, and other roles for this biological metal may be demonstrated as microbial nickel requirements are explored. The specific transport of nickel has only been studied in a few microorganisms, and much work remains to be done to understand this important field. An important question which has not yet been considered in this paper is why the four nickel-containing groups of enzymes require nickel. The enzymes may make use of the intrinsic chemical reactivity of nickel ion. In the case of urease catalysis, chemical model studies have shown that ethanolysis and hydrolysis of N-(2-pyridylmethyl) urea was specifically promoted by nickel(II) ion. Also, nickel is well known for its reactivity with hydrogen and is widely used in chemical reductions. The role of nickel in hydrogenase and methyl coenzyme M reductase may be related to these properties. Similarly, the affinity of nickel for carbon monoxide and the capacity of nickel to catalyze carbonylation reactions may have led to the utilization of nickel in CO dehydrogenase. Thus, the nickel chemistry observed in microorganisms predates, but is analogous to, the nickel chemistry used by modern day inorganic chemists.
The L-A double-stranded RNA virus of Saccharomyces cerevisiae has a 4.6- kb single-segment genome encapsidated in an icosahedral T - 1 particle whose asymmetric element is a dimer of Gag, the major coat protein. L-A encodes Gag and its RNA-dependent RNA polymerase (Pol), the latter formed as a Gag-Pol fusion protein produced by a -1 ribosomal frameshift. The efficiency of ribosomal frameshifting is important for viral propagation and is controlled by the host MOF genes. In vitro template-dependent, viral RNA-specific. RNA- binding, replication, and transcription systems have been used to study the mechanisms of these reactions. The packaging site on viral plus strands and the part of the Pol domain of the Gag-Pol fusion protein that recognizes it have been defined. The viral transcripts lack both 5' cap structure and 3' poly(A). The host SK12, SK13, and SK18 genes limit viral propagation by blocking the translation of non-poly(A) mRNA. The host SK11/XRN1 gene encodes a 5' → 3' exoribonuclease specific for uncapped mRNA and known to be involved in mRNA turnover. Its action on the uncapped viral mRNA is limited by the cap removal activity of the viral Gag protein acting on cellular mRNAs. M double-stranded RNAs, satellites of I.-A. encode secreted 'killer' toxins as a preprotein. The processing of the preprotoxin is carried out by the Kex1p and Kex2p proteases. The discovery of these genes in this system led to the discovery of mammalian prohormone processing enzymes. Yeast genetics enables detailed dissection of host-virus interactions, most of which probably have homologs in mammalian systems.
Strictly anaerobic halophiles, namely fermentative, sulfate-reducing, homoacetogenic, phototrophic, and methanogenic bacteria are involved in the oxidation of organic carbon in hypersaline environments. To date, six anaerobic fermentative genera, containing nine species, have been described. Two of them are homoacetogens. Six species belong to the family Haloanaerobiaceae, as indicated by their unique 16S rRNA oligonucleotide sequences. Desulfohalobium retbaense and Desulfovibrio halophilus represent the only two moderately halophilic sulfate reducers so far reported. Among anoxygenic phototrophic anaerobes, a few purple bacteria with optimal growth at salinities between 6 and 11% NaCl have been isolated from hypersaline habitats. They belong to the genera Rhodospirillum, Chromatium, Thiocapsa, and Ectothiorhodospira. The commonest organisms isolated so far are Chromatium salexigens, Thiocapsa halophila, and Rhodospirillum salinarum. Extremely halophilic purple bacteria have most commonly been isolated from alkaline brines and require about 20 to 25% NaCl for optimal growth. They belong to the family Ectothiorodhospiraceae. Their osmoregulation involves synthesis or uptake of compatible solutes such as glycine-betaine that accumulate in their cytoplasm. The existence of methanogens in hypersaline environments is related to the presence of noncompetitive substrates such as methylamines, which originate mainly from the breakdown of osmoregulatory amines. Methanogenesis probably does not contribute to the mineralization of carbohydrates at NaCl concentrations higher than 15%. Above this concentration, sulfate reduction is probably the main way to oxidize H2 (although at rates too low to use up all the H2 formed) and occupies a terminal function kn the degradation of carbohydrates. Three genera and five species of halophilic methylotrophic methanogens have been reported. A bloom of phototrophic bacteria in the marine salterns of Salins-de-Giraud, located on the Mediterranean French coast in the Rhone Delta, is also described.
Frankia strains are N2-fixing actinomycetes whose isolation and cultivation were first reported in 1978. They induce N2-fixing root nodules on diverse nonleguminous (actinorhizal) plants that are important in ecological successions and in land reclamation and remediation. The genus Frankia encompasses a diverse group of soil actinomycetes that have in common the formation of multilocular sporangia, filamentous growth, and nitrogenase-containing vesicles enveloped in multilaminated lipid envelopes. The relatively constant morphology of vesicles in culture is modified by plant interactions in symbiosis to give a diverse array of vesicles shapes. Recent studies of the genetics and molecular genetics of these organisms have begun to provide new insights into higher-plant-bacterium interactions that lead to productive N2-fixing symbioses. Sufficient information about the relationship of Frankia strains to other bacteria, and to each other, is now available to warrant the creation of some species based on phenotypic and genetic criteria.
Artemisinin and its derivatives are endoperoxide-containing compounds which represent a promising new class of antimalarial drugs. In the presence of intraparasitic iron, these drugs are converted into free radicals and other electrophilic intermediates which then alkylate specific malaria target proteins. Combinations of available derivatives and other antimalarial agents show promise both as first-line agents and in the treatment of severe disease.
The members of the family Parvoviridae are among the smallest of the DNA viruses, with a linear single-stranded genome of about 5 kilobases. Currently the family is divided into three genera, two of which contain viruses of vertebrates and a third containing insect viruses. This review concentrates on the vertebrate viruses, with emphasis on recent advances in our insights into the molecular biology of viral replication. Traditionally the vertebrate viruses have been distinguished by the presence or absence of a requirement for a coinfection with a helper virus before productive infection can occur, hence the notion that the dependoviruses (adeno-associated viruses [AAV]) are defective. Recent data would suggest that not only is there a great deal of structural and genetic organizational similarity between the two types of vertebrate viruses, but also there is significant similarity in the molecular biology of productive replication. What differs is the physiological condition of the host cell that renders it permissive. Healthy dividing cells are permissive for productive replication by autonomous parvoviruses; such cells result in latent infection by dependoviruses. For a cell to become permissive for productive AAV replication, it must have been exposed to toxic conditions which activate a latent AAV genome. Such conditions can be caused by helper-virus infection or exposure to physical (UV light) or chemical (some carcinogens) agents. In this paper the molecular biology of replication is reviewed, with special emphasis on the role of the host and the consequences of viral infection for the host.
The enigmatic coexistence of O2-sensitive nitrogenase and O2-evolving photosynthesis in diazotrophic cyanobacteria has fascinated researchers for over two decades. Research efforts in the past 10 years have revealed a range of O2 sensitivity of nitrogenase in different strains of cyanobacteria and a variety of adaptations for the protection of nitrogenase from damage by both atmospheric and photosynthetic sources of O2. The most complex and apparently most efficient mechanisms for the protection of nitrogenase are incorporated in the heterocysts, the N2-fixing cells of cyanobacteria. Genetic studies indicate that the controls of heterocyst development and nitrogenase synthesis are closely interrelated and that the expression of N2 fixation (nif) genes is regulated by pO2.
Bioremediation is defined as the act of adding or improving the availability of materials (e.g., nutrients, microorganisms, or oxygen) to contaminated environments to cause an acceleration of natural biodegradative processes. The results of field experiments and trials following actual spill incidents have been reviewed to evaluate the feasibility of this approach as a treatment for oil contamination in the marine environment. The ubiquity of oil-degrading microorganisms in the marine environment is well established, and research has demonstrated the capability of the indigenous microflora to degrade many components of petroleum shortly after exposure. Studies have identified numerous factors which affect the natural biodegradation rates of oil, such as the origin and concentration of oil, the availability of oil-degrading microorganisms, nutrient concentrations, oxygen levels, climatic conditions, and sediment characteristics. Bioremediation strategies based on the application of fertilizers have been shown to stimulate the biodegradation rates of oil in aerobic intertidal sediments such as sand and cobble. The ratio of oil loading to nitrogen concentration within the interstitial water has been identified to be the principal controlling factor influencing the success of this bioremediation strategy. However, the need for the seeding of natural environments with hydrocarbon-degrading bacteria has not been clearly demonstrated under natural environmental conditions. It is suggested that bioremediation should now take its place among the many techniques available for the treatment of oil spills, although there is still a clear need to set operational limits for its use. On the basis of the available evidence, we have proposed preliminary operational guidelines for bioremediation on shoreline environments.
This review presents a comparison between the complex genetic regulatory networks that control nitrogen fixation in three representative rhizobial species, Rhizobium meliloti, Bradyrhizobium japonicum, and Azorhizobium caulinodans. Transcription of nitrogen fixation genes (nif and fix genes) in these bacteria is induced primarily by low-oxygen conditions. Low-oxygen sensing and transmission of this signal to the level of nif and fix gene expression involve at least five regulatory proteins, FixL, FixJ, FixK, NifA, and RpoN (sigma 54). The characteristic features of these proteins and their functions within species-specific regulatory pathways are described. Oxygen interferes with the activities of two transcriptional activators, FixJ and NifA. FixJ activity is modulated via phosphorylation-dephosphorylation by the cognate sensor hemoprotein FixL. In addition to the oxygen responsiveness of the NifA protein, synthesis of NifA is oxygen regulated at the level of transcription. This type of control includes FixLJ in R. meliloti and FixLJ-FixK in A. caulinodans or is brought about by autoregulation in B. japonicum. NifA, in concert with sigma 54 RNA polymerase, activates transcription from -24/-12-type promoters associated with nif and fix genes and additional genes that are not directly involved in nitrogen fixation. The FixK proteins constitute a subgroup of the Crp-Fnr family of bacterial regulators. Although the involvement of FixLJ and FixK in nifA regulation is remarkably different in the three rhizobial species discussed here, they constitute a regulatory cascade that uniformly controls the expression of genes (fixNOQP) encoding a distinct cytochrome oxidase complex probably required for bacterial respiration under low-oxygen conditions. In B. japonicum, the FixLJ-FixK cascade also controls genes for nitrate respiration and for one of two sigma 54 proteins.
These data provide convincing, albeit circumstantial, evidence that S. mutans, possibly S. sobrinus, and lactobacilli are human odontopathogens. As such, dental caries is a diagnosable and treatable infection. Aciduricity appears to be the most consistent attribute of S. mutans that can be associated with both its selection in stagnant areas and its cariogenicity. Other aciduric species such as S. sobrinus appear to be important primarily in smooth-surface decay and, as such, may be a cariogenic determinant when rampant decay occurs. Colonization by S. mutans occurs after tooth eruption, and if the fissures become colonized in their depths, then decay may be inevitable. However, if this colonization is delayed until the fissure depths are occupied by other bacteria, there is the possibility that decay will not occur or its occurrence will be greatly reduced. This understandingg of the ecology of S. mutans suggests that treatment strategies which interfere with the colonization of S. mutans may have a profound effect on the incidence of dental decay in human populations.
Heat shock proteins (Hsps) were first identified as proteins whose synthesis was enhanced by stresses such as an increase in temperature. Recently, several of the major Hsps have been shown to be intimately involved in protein biogenesis through a direct interaction with a wide variety of proteins. As a reflection of this role, these Hsps have been referred to as molecular chaperones. Hsp70s interact with incompletely folded proteins, such as nascent chains on ribosomes and proteins in the process of translocation from the cytosol into mitochondria and the endoplasmic reticulum. Hsp60 also binds to unfolded proteins, preventing aggregation and facilitating protein folding. Although less well defined, other Hsps such as Hsp90 also play important roles in modulating the activity of a number of proteins. The function of the proteolytic system is intertwined with that of molecular chaperones. Several components of this system, encoded by heat-inducible genes, are responsible for the degradation of abnormal or misfolded proteins. The budding yeast Saccharomyces cerevisiae has proven very useful in the analysis of the role of molecular chaperones in protein maturation, translocation, and degradation. In this review, results of experiments are discussed within the context of experiments with other organisms in an attempt to describe the current state of understanding of these ubiquitous and important proteins.
In a variety of fungal species, mating between haploid cells is initiated by the action of peptide pheromones. The identification and characterization of several fungal pheromones has revealed that they have common structural features classifying them as lipopeptides. In the course of biosynthesis, these pheromones undergo a series of posttranslational processing events prior to export. One common modification is the attachment of an isoprenoid group to the C terminus of the pheromone precursor. Genetic and biochemical investigations of this biosynthetic pathway have led to the elucidation of genes and enzymes which are responsible for isoprenylation of other polypeptides including the nuclear lamins, several vesicular transport proteins, and the oncogene product Ras. The alpha-factor of Saccharomyces cerevisiae serves as a model for studying the biosynthesis, export, and bioactivity of lipopeptide pheromones. In addition to being isoprenylated with a farnesyl group, the alpha-factor is secreted by a novel peptide export pathway utilizing a yeast homolog of the mammalian multidrug resistance P-glycoprotein. The identification of putative lipopeptide-encoding loci within other fungi, including the human immunodeficiency virus-associated opportunistic pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis, has stimulated much interest in understanding possible roles for pheromones in fungal proliferation and pathogenicity. Knowledge of variations within the processing, export, and receptor-mediated signal transduction pathways associated with different fungal lipopeptide pheromones will continue to provide insights into similar mechanisms which exist in higher eukaryotes.
Knowledge of the structure and function of the genes and proteins of the rotaviruses has expanded rapidly. Information obtained in the last 5 years has revealed unexpected and unique molecular properties of rotavirus proteins of general interest to virologists, biochemists, and cell biologists. Rotaviruses share some features of replication with reoviruses, yet antigenic and molecular properties of the outer capsid proteins, VP4 (a protein whose cleavage is required for infectivity, possibly by mediating fusion with the cell membrane) and VP7 (a glycoprotein), show more similarities with those of other viruses such as the orthomyxoviruses, paramyxoviruses, and alphaviruses. Rotavirus morphogenesis is a unique process, during which immature subviral particles bud through the membrane of the endoplasmic reticulum (ER). During this process, transiently enveloped particles form, the outer capsid proteins are assembled onto particles, and mature particles accumulate in the lumen of the ER. Two ER-specific viral glycoproteins are involved in virus maturation, and these glycoproteins have been shown to be useful models for studying protein targeting and retention in the ER and for studying mechanisms of virus budding. New ideas and approaches to understanding how each gene functions to replicate and assemble the segmented viral genome have emerged from knowledge of the primary structure of rotavirus genes and their proteins and from knowledge of the properties of domains on individual proteins. Localization of type-specific and cross-reactive neutralizing epitopes on the outer capsid proteins is becoming increasingly useful in dissecting the protective immune response, including evaluation of vaccine trials, with the practical possibility of enhancing the production of new, more effective vaccines. Finally, future analyses with recently characterized immunologic and gene probes and new animal models can be expected to provide a basic understanding of what regulates the primary interactions of these viruses with the gastrointestinal tract and the subsequent responses of infected hosts.
The state of intracellular water has been a matter of controversy for a long time for two reasons. First, experiments have often given conflicting results. Second, hitherto, there have been no plausible grounds for assuming that intracellular water should be significantly different from bulk water. A collective behavior of water molecules is suggested here as a thermodynamically inevitable mechanism for generation of appreciable zones of abnormal water. At a highly charged surface, water molecules move together, generating a zone of water perhaps 6 nm thick, which is weakly hydrogen bonded, fluid, and reactive and selectively accumulates small cations, multivalent anions, and hydrophobic solutes. At a hydrophobic surface, molecules move apart and local water becomes strongly bonded, inert, and viscous and accumulates large cations, univalent anions, and compatible solutes. Proteins and many other biopolymers have patchy surfaces which therefore induce, by the two mechanisms described, patchy interfacial water structures, which extended appreciable distances from the surface. The reason for many conflicting experimental results now becomes apparent. Average values of properties of water measured in gels, cells, or solutions of proteins are often not very different from the same properties of normal water, giving no indication that they are averages of extreme values. To detect the operation of this phenomenon, it is necessary to probe selectively a single abnormal population. Examples of such experiments are given. It is shown that this collective behavior of water molecules amounts to a considerable biological force, which can be equivalent to a pressure of 1,000 atm (1.013 x 10(5) kPa). It is suggested that cells selectively accumulate K+ ions and compatible solutes to avoid extremes of water structure in their aqueous compartments, but that cation pumps and other enzymes exploit the different solvent properties and reactivities of water to perform work of transport or synthesis.
The amounts of bacterial toxins and of some plant and animal proteins that kill humans, monkeys, mice, guinea pigs, and rabbits are tabulated and are discussed in the light of guidelines for the cloning of genes coding for toxins.
Biomass formation represents one of the most basic aspects of bacterial metabolism. While there is an abundance of information concerning individual reactions that result in cell duplication, there has been surprisingly little information on the bioenergetics of growth. For many years, it was assumed that biomass production (anabolism) was proportional to the amount of ATP which could be derived from energy-yielding pathways (catabolism), but later work showed that the ATP yield (YATP) was not necessarily a constant. Continuous-culture experiments indicated that bacteria utilized ATP for metabolic reactions that were not directly related to growth (maintenance functions). Mathematical derivations showed that maintenance energy appeared to be a growth rate-independent function of the cell mass and time. Later work, however, showed that maintenance energy alone could not account for all the variations in yield. Because only some of the discrepancy could be explained by the secretion of metabolites (overflow metabolism) or the diversion of catabolism to metabolic pathways which produced less ATP, it appeared that energy-excess cultures had mechanisms of spilling energy. Bacteria have the potential to spill excess ATP in futile enzyme cycles, but there has been little proof that such cycles are significant. Recent work indicated that bacteria can also use futile cycles of potassium, ammonia, and protons through the cell membrane to dissipate ATP either directly or indirectly. The utility of energy spilling in bacteria has been a curiosity. The deprivation of energy from potential competitors is at best a teleological explanation that cannot be easily supported by standard theories of natural selection. The priming of intracellular intermediates for future growth or protection of cells from potentially toxic end products (e.g., methylglyoxal) seems a more plausible explanation.
A wide variety of compounds can be biodegraded via reductive removal of halogen substituents. This process can degrade toxic pollutants, some of which are not known to be biodegraded by any other means. Reductive dehalogenation of aromatic compounds has been found primarily in undefined, syntrophic anaerobic communities. We discuss ecological and physiological principles which appear to be important in these communities and evaluate how widely applicable these principles are. Anaerobic communities that catalyze reductive dehalogenation appear to differ in many respects. A large number of pure cultures which catalyze reductive dehalogenation of aliphatic compounds are known, in contrast to only a few organisms which catalyze reductive dehalogenation of aromatic compounds. Desulfomonile tiedjei DCB-1 is an anaerobe which dehalogenates aromatic compounds and is physiologically and morphologically unusual in a number of respects, including the ability to exploit reductive dehalogenation for energy metabolism. When possible, we use D. tiedjei as a model to understand dehalogenating organisms in the above-mentioned undefined systems. Aerobes use reductive dehalogenation for substrates which are resistant to known mechanisms of oxidative attack. Reductive dehalogenation, especially of aliphatic compounds, has recently been found in cell-free systems. These systems give us an insight into how and why microorganisms catalyze this activity. In some cases transition metal complexes serve as catalysts, whereas in other cases, particularly with aromatic substrates, the catalysts appear to be enzymes.
The life cycle of the budding yeast Saccharomyces cerevisiae has two broad aspects, cell proliferation and transitions between haploid and diploid cell types. Haploids mate to form diploids, and diploids undergo meiosis to form haploids. The life cycle of S. cerevisiae has an additional aspect beyond proliferation, mating, and meiosis: haploid yeast cells (of appropriate genotype) can exhibit a 'homothallic' life cycle, one in which a haploid cell can give rise to diploid cells capable of meiosis and spore formation. Yeast strains of other genotypes exhibit a 'heterothallic' life cycle, in which a haploid cell is unable to yield diploid cells. Studies of S. cerevisiae have provided a molecular understanding of (i) the different types of yeast cells that participate in mating and meiosis (haploid types a and α and the diploid a/α cell) and (ii) the mechanism for homothallism. Cell specialization in S. cerevisiae is governed by a master regulatory locus, the mating-type locus (MAT), whose two alleles (MATa and MATα) code for regulatory proteins (one activator and two repressor activities). One of the repressor activities (a1-α2) requires products coded by both MAT alleles and thus acts as a molecular monitor for diploidy. These regulatory proteins govern transcription of different gene sets, including a-specific genes (expressed only in a cells), α-specific genes (expressed only in α cells), and haploid-specific genes (expressed in both a and α cells). The homothallic life cycle (ability of haploid cells to produce diploid cells) occurs because of mating-type interconversion: cells first change from one mating type to the other by a programmed genetic rearrangement. Then sibling cells mate to form an a/α diploid cell. Mating-type interconversion is thus a process in which the master regulatory locus, MAT, is itself regulated. This review presents an overview of the mating-type locus and how it regulates transcription of other genes, as well as a description of the different methods used for assaying mating and associated phenomena. The molecular mechanism of mating-type interconversion ('cassette' transposition) is summarized, and biological aspects of the switching process, genetic variations that lead to a heterothallic life cycle, and different possible mechanisms for homothallism are discussed. The review concludes with a description of features of the life cycles of other organisms (the fission yeast Schizosaccharomyces pombe, filamentous fungi such as Neurospora crassa, and basidiomycetes such as Schizophyllum commune and Ustilago maydis, as well as ciliates and algae).
The accurate replication and segregation of Saccharomyces cerevisiae chromosomes require participation of at least three cis-acting dfeoxyribonucleic acid (DNA) elements as well as the numerous proteins that form the replication and segregation complexes. At least some and possibly all chromosomal replication origins can be assayed in plasmids by their ability to direct autonomous replication of plasmid DNA. Function of these autonomously replicating sequences (ARSs) requires an 11-base-pair consensus sequence and flanking DNA. While the proteins that interact with the ARS consensus sequence have not been identified, the genes encoding many proteins necessary for DNA replication are known. These genes have been identified by screening collections of conditional mutants as well as by 'reverse genetic' approaches. Chromosome segregation requires binding of the mitotic spindle to chromosomes at their centromere. Functional yeast centromeres are contained within a 120- to 130-base-pair sequence composed of three conserved sequence elements. Isolated fragments of centromeric DNA are being used as tools to isolate proteins that bind to these sequences. Telomeres are dynamic structures that lengthen and shorten during vegetative growth. Several genes that influence telomere length have been identified. The unusual C1-3A repeat found in yeast telomeres is believed to stabilize as well as to facilitate the replication of chromosomal ends. Artificial chromosomes constructed from cloned structural genes, ARS elements, centromeres, and telomeres are 2 to 3 orders of magnitude less stable than natural chromosomes. Chromosome length is clearly an important determinant of chromosome stability, but other, as yet unidentified, elements are almost certainly required for chromosome replication and segregation.
Oxidative stress is strongly implicated in a number of diseases, such as rheumatoid arthritis, inflammatory bowel disorders, and atherosclerosis, and its emerging as one of the most important causative agents of mutagenesis, tumorigenesis, and aging. Recent progress on the genetics and molecular biology of the cellular responses to oxidative stress, primarily in Escherichia coli and Salmonella typhimurium, is summarized. Bacteria respond to oxidative stress by invoking two distinct stress responses, the peroxide stimulon and the superoxide stimulon, depending on whether the stress is mediated by peroxides or the superoxide anion. The two stimulons each contain a set of more than 30 genes. The expression of a subset of genes in each stimulon is under the control of a positive regulatory element; these genes constitute the OxyR and SoxRS regulons. The schemes of regulation of the two regulons by their respective regulators are reviewed in detail, and the overlaps of these regulons with other stress responses such as the heat shock and SOS responses are discussed. The products of Oxy-R- and SoxRS-regulated genes, such as catalases and superoxide dismutases, are involved in the prevention of oxidative damage, whereas others, such as endonuclease IV, play a role in the repair of oxidative damage. The potential roles of these and other gene products in the defense against oxidative damage in DNA, proteins, and membranes are discussed in detail. A brief discussion of the similarities and differences between oxidative stress responses in bacteria and eukaryotic organisms concludes this review.
Sporulation of the yeast Saccharomyces cerevisiae is restricted to one type of cell, the a/alpha cell, and is initiated after starvation for nitrogen in the absence of a fermentable carbon source. More than 25 characterized genes are expressed only during sporulation and are referred to as meiotic genes or sporulation-specific genes. These genes are in the early, middle, and late expression classes. Most early genes have a 5' regulatory site, URS1, and one of two additional sequences, UASH or a T4C site. URS1 is required both to repress meiotic genes during vegetative growth and to activate these genes during meiosis. UASH and the T4C site also contribute to meiotic expression. A different type of site, the NRE, is found in at least two late genes. The NRE behaves as a repression site in vegetative cells and is neutral in meiotic cells. Many regulatory genes that either repress or activate meiotic genes have been identified. One group of regulators affects the expression of IME1, which specifies a positive regulator of meiotic genes and is expressed at the highest levels in meiotic cells. A second group of regulators acts in parallel with or downstream of IME1 to influence meiotic gene expression. This group includes UME6, which is required both for repression through the URS1 site in vegetative cells and for IME1-dependent activation of an upstream region containing URS1 and T4C sites. IME1 may activate meiotic genes by modifying a UME6-dependent repression complex at a URS1 site. Several additional mechanisms restrict functional expression of some genes to meiotic cells. Translation of IME1 has been proposed to occur only in meiotic cells; several meiotic transcripts are more stable in acetate medium than in glucose medium; and splicing of MER2 RNA depends on a meiosis-specific gene, MER1.
The removal of cell-bound water through air drying and the addition of water to air-dried cells are forces that have played a pivotal role in the evolution of the prokaryotes. In bacterial cells that have been subjected to air drying, the evaporation of free cytoplasmic water (Vf) can be instantaneous, and an equilibrium between cell-bound water (Vb) and the environmental water (vapor) potential (psi wv) may be achieved rapidly. In the air-dried state some bacteria survive only for seconds whereas others can tolerate desiccation for thousands, perhaps millions, of years. The desiccated (anhydrobiotic) cell is characterized by its singular lack of water--with contents as low as 0.02 g of H2O g (dry weight)-1. At these levels the monolayer coverage by water of macromolecules, including DNA and proteins, is disturbed. As a consequence the mechanisms that confer desiccation tolerance upon air-dried bacteria are markedly different from those, such as the mechanism of preferential exclusion of compatible solutes, that preserve the integrity of salt-, osmotically, and freeze-thaw-stressed cells. Desiccation tolerance reflects a complex array of interactions at the structural, physiological, and molecular levels. Many of the mechanisms remain cryptic, but it is clear that they involve interactions, such as those between proteins and co-solvents, that derive from the unique properties of the water molecule. A water replacement hypothesis accounts for how the nonreducing disaccharides trehalose and sucrose preserve the integrity of membranes and proteins. Nevertheless, we have virtually no insight into the state of the cytoplasm of an air-dried cell. There is no evidence for any obvious adaptations of proteins that can counter the effects of air drying or for the occurrence of any proteins that provide a direct and a tangible contribution to cell stability. Among the prokaryotes that can exist as anhydrobiotic cells, the cyanobacteria have a marked capacity to do so. One form, Nostoc commune, encompasses a number of the features that appear to be critical to the withstanding of a long-term water deficit, including the elaboration of a conspicuous extracellular glycan, synthesis of abundant UV-absorbing pigments, and maintenance of protein stability and structural integrity. There are indications of a growing technology for air-dried cells and enzymes. Paradoxically, desiccation tolerance of bacteria has virtually been ignored for the past quarter century. The present review considers what is known, and what is not known, about desiccation, a phenomenon that impinges upon every facet of the distributions and activities of prokaryotic cells.
Chlamydiae are medically important bacteria responsible for a wide range of human infections and diseases. Repeated episodes of infection promote chronic inflammation associated with detrimental immune system-mediated pathologic changes. However, the true nature of chlamydial pathogenesis may encompass repeated infection superimposed upon persistent infection, which would allow for heightened immune reactivity. During the course of chlamydial infection, numerous host elaborated factors with inhibitory or modifying effects may cause alterations in the chlamydia-host cell relationship such that the organism is maintained in a nonproductive stage of growth. Abnormal or persistent chlamydiae have been recognized under a variety of cell culture systems. The numerous factors associated with altered growth suggest an innate flexibility in the developmental cycle of chlamydiae. This review evaluates in vitro studies of chlamydial persistence and correlates these model systems to features of natural chlamydial disease.
The examination of fungal chromosomes by pulsed-field gel electrophoresis has revealed that length polymorphism is widespread in both sexual and asexual species. This review summarizes characteristics of fungal chromosome-length polymorphism and possible mitotic and meiotic mechanisms of chromosome length change. Most fungal chromosome-length polymorphisms are currently uncharacterized with respect to content and origin. However, it is clear that long tandem repeats, such as tracts of rRNA genes, are frequently variable in length and that other chromosomal rearrangements are suppressed during normal mitotic growth. Dispensable chromosomes and dispensable chromosome regions, which have been well documented for some fungi, also contribute to the variability of the fungal karyotype. For sexual species, meiotic recombination increases the overall karyotypic variability in a population while suppressing genetic translocations. The range of karyotypes observed in fungi indicates that many karyotypic changes may be genetically neutral, at least under some conditions. In addition, new linkage combinations of genes may also be advantageous in allowing adaptation of fungi to new environments.
The large number of different proteins synthesized by the mycobacterial cell are currently classified and studied in terms of groups of proteins with certain common properties such as physical and chemical characteristics, function, and localization in the mycobacterial cell. Proteins that are actively secreted during culture on synthetic media represent a particular group of great current interest. At least eight proteins secreted by Mycobacterium tuberculosis have been isolated and characterized to various extents. The genes coding for five proteins secreted from M. tuberculosis and/or Mycobacterium bovis BCG have been cloned and sequenced. All of them contain typical signal sequences. The proteins of the antigen 85 complex, which form the main subject of this review, are often the most common proteins in M. tuberculosis culture fluid. The constituents denoted 85A, 85B, and 85C are encoded by three genes located at different sites in the mycobacterial genome and show extensive cross-reactivity as well as homology at amino acid and gene levels. The proteins differ slightly in molecular mass in the 30- to 31-kDa region, and all of them are fibronectin-binding proteins, but the significance of the latter observation and the role of these proteins in mycobacterial physiology and interaction with the infected host remain to be elucidated. The antigen 85 complex proteins are strongly immunogenic in natural and experimental mycobacterial infections in terms of both induction of antibody synthesis and T-cell-mediated reactions. The well-recognized difference in the efficacy of live and dead mycobacterial vaccines should be considered in relation to the group of secreted antigens. After inoculation, live bacteria in vaccines such as BCG multiply in the host, probably releasing several constituents belonging to the class of secreted proteins and hence resulting in more efficient stimulation of the immune system. Secreted mycobacterial antigens are expected to be of particular significance in induction of various immune responses that are responsible for development of protective immunity in some individuals and for clinical symptoms and complications of the ensuing disease in others.
The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review.
Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli.
All living cells must conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from gene to finished protein product. One crucial "quality control" point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. During selection of amino acids, synthetases very often have to distinguish the cognate substrate from a homolog having just one fewer methyl group in its structure. The binding energy of a methyl group is estimated to contribute only a factor of 100 to the specificity of binding, yet synthetases distinguish such closely related amino acids with a discrimination factor of 10,000 to 100,000. Examples of this include methionine versus homocysteine, isoleucine versus valine, alanine versus glycine, and threonine versus serine. Many investigators have demonstrated in vitro the ability of certain aminoacyl-tRNA synthetases to edit, that is, correct or prevent incorrect attachment of amino acids to tRNA molecules. Several major editing pathways are now established from in vitro data. Further, at least some aminoacyl-tRNA synthetases have recently been shown to carry out the editing function in vivo. Editing has been demonstrated to occur in both Escherichia coli and Saccharomyces cerevisiae. Significant energy is expended by the cell for editing of misactivated amino acids, which can be reflected in the growth rate. Because of this, cellular levels of aminoacyl-tRNA synthetases, as well as amino acid biosynthetic pathways which yield competing substrates for protein synthesis, must be carefully regulated to prevent excessive editing. High-level expression of recombinant proteins imposes a strain on the biosynthetic capacity of the cell which frequently results in misincorporation of abnormal or wrong amino acids owing in part to limited editing by synthetases. Unbalanced amino acid pools associated with some genetic disorders in humans may also lead to errors in tRNA aminoacylation. The availability of X-ray crystallographic structures of some synthetases, combined with site-directed mutagenesis, allows insights into molecular details of the extraordinary selectivity of synthetases, including the editing function.