Annual Review of Microbiology - Volume 52, 1998

A plethora of novel gene-encoded antimicrobial peptides from animals, plants and bacteria has been described during the last decade. Many of the bacterial peptides possess modified building blocks such as thioethers and thiazoles or unsaturated and stereoinverted amino acids, which are unique among ribosomally made peptides. Genetic and biochemical studies of many of these peptides, mostly the so-called lantibiotics, have revealed the degree to which cells are capable of transforming peptides by posttranslational modification. The biosynthesis follows a general scheme: Precursor peptides are first modified and then proteolytically activated; the latter may occur prior to, concomitantly with or after export from the cell. The genes for the biosynthetic machinery are organized in clusters and include information for the antibiotic prepeptide, the modification enzymes and accessory functions such as dedicated proteases and ABC transporters as well as immunity factors and regulatory proteins. These fundamental aspects are discussed along with the biotechnological potential of the peptides and of the biosynthesis enzymes, which could be used for construction of novel, peptide-based biomedical effector molecules.

It has been a decade since multicellularity was proposed as a general bacterial trait. Intercellular communication and multicellular coordination are now known to be widespread among prokaryotes and to affect multiple phenotypes. Many different classes of signaling molecules have been identified in both Gram-positive and Gram-negative species. Bacteria have sophisticated signal transduction networks for integrating intercellular signals with other information to make decisions about gene expression and cellular differentiation. Coordinated multicellular behavior can be observed in a variety of situations, including development of E. coli and B. subtilis colonies, swarming by Proteus and Serratia, and spatially organized interspecific metabolic cooperation in anaerobic bioreactor granules. Bacteria benefit from multicellular cooperation by using cellular division of labor, accessing resources that cannot effectively be utilized by single cells, collectively defending against antagonists, and optimizing population survival by differentiating into distinct cell types.

The body plan of modular organisms is based on an indeterminate structure composed of iterated units or modules arrayed at various levels of complexity (such as leaves, twigs, and branches). Examples of modular organisms include plants and many sessile benthic invertebrates. In contrast, the body of unitary organisms is a determinate structure consisting usually of a strictly defined number of parts (such as legs or wings) established only during embryogenesis. Mobile animals are examples. Unlike that of unitary creatures, the form of a modular organism derives from a characteristic pattern of branching or budding of modules, which may remain attached or become separated to live physiologically independent lives as parts of a clone. Modular organisms tend to be sessile or passively mobile and, as genetic individuals, have the capacity for exponential increase in size. They do not necessarily undergo systemic senescence, and do not segregate somatic from germ line cells. It is argued here that bacteria are essentially modular organisms where the bacterial cell, microcolony, and macrocolony are modules of different levels of complexity analogous to modules of macroorganisms. This interpretation provides a broad conceptual basis for understanding the natural history of bacteria, and may illuminate the evolutionary origins and developmental biology of modular creatures.

Growth of enteric bacteria on acetate as the sole source of carbon and energy requires operation of a particular anaplerotic pathway known as the glyoxylate bypass. In this pathway, two specific enzymes, isocitrate lyase and malate synthase, are activated to divert isocitrate from the tricarboxylic acid cycle and prevent the quantitative loss of acetate carbons as carbon dioxide. Bacteria are thus supplied with the metabolic intermediates they need for synthesizing their cellular components. The channeling of isocitrate through the glyoxylate bypass is regulated via the phosphorylation/dephosphorylation of isocitrate dehydrogenase, the enzyme of the tricarboxylic acid cycle which competes for a common substrate with isocitrate lyase. When bacteria are grown on acetate, isocitrate dehydrogenase is phosphorylated and, concomitantly, its activity declines drastically. Conversely, when cells are cultured on a preferred carbon source, such as glucose, the enzyme is dephosphorylated and recovers full activity. Such reversible phosphorylation is mediated by an unusual bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase, which contains both modifying and demodifying activities on the same polypeptide. The genes coding for malate synthase, isocitrate lyase, and isocitrate dehydrogenase kinase/phosphatase are located in the same operon. Their expression is controlled by a complex dual mechanism that involves several transcriptional repressors and activators. Recent developments have brought new insights into the nature and mode of action of these different regulators. Also, significant advances have been made lately in our understanding of the control of enzyme activity by reversible phosphorylation. In general, analyzing the physiological behavior of bacteria on acetate provides a valuable approach for deciphering at the molecular level the mechanisms of cell adaptation to the environment.

There was a long-held belief that the gram-positive soil bacterium Bacillus subtilis is a strict aerobe. But recent studies have shown that B. subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron acceptor, or by fermentation. How B. subtilis alters its metabolic activity according to the availability of oxygen and alternative electron acceptors is but one focus of study. A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, occupies an early stage in the regulatory pathway governing anaerobic respiration. One of the essential roles of ResD and ResE in anaerobic gene regulation is induction of fnr transcription upon oxygen limitation. FNR is a transcriptional activator for anaerobically induced genes, including those for respiratory nitrate reductase, narGHJI. B. subtilis has two distinct nitrate reductases, one for the assimilation of nitrate nitrogen and the other for nitrate respiration. In contrast, one nitrite reductase functions both in nitrite nitrogen assimilation and nitrite respiration. Unlike many anaerobes, which use pyruvate formate lyase, B. subtilis can carry out fermentation in the absence of external electron acceptors wherein pyruvate dehydrogenase is utilized to metabolize pyruvate.

The last decade has seen significant advances in our understanding of the physiology, ecology, and molecular biology of chemoautotrophic bacteria. Many ecosystems are dependent on CO2 fixation by either free-living or symbiotic chemoautotrophs. CO2 fixation in the chemoautotroph occurs via the Calvin-Benson-Bassham cycle. The cycle is characterized by three unique enzymatic activities: ribulose bisphosphate carboxylase/oxygenase, phosphoribulokinase, and sedoheptulose bisphosphatase. Ribulose bisphosphate carboxylase/oxygenase is commonly found in the cytoplasm, but a number of bacteria package much of the enzyme into polyhedral organelles, the carboxysomes. The carboxysome genes are located adjacent to cbb genes, which are often, but not always, clustered in large operons. The availability of carbon and reduced substrates control the expression of cbb genes in concert with the LysR-type transcriptional regulator, CbbR. Additional regulatory proteins may also be involved. All of these, as well as related topics, are discussed in detail in this review.

A mechanism for regulating gene expression at the level of transcription utilizes an antagonist of the sigma transcription factor known as the anti-sigma (anti-σ) factor. The cytoplasmic class of anti-σ factors has been well characterized. The class includes AsiA form bacteriophage T4, which inhibits Escherichia coli σ⁷⁰; FlgM, present in both gram-positive and gram-negative bacteria, which inhibits the flagella sigma factor σ²⁸; SpoIIAB, which inhibits the sporulation-specific sigma factors, σ^F and σ^G, of Bacillus subtilis; RbsW of B. subtilis, which inhibits stress response sigma factor σ^B; and DnaK, a general regulator of the heat shock response, which in bacteria inhibits the heat shock sigma factor σ³². In addition to this class of well-characterized cytoplasmic anti-sigma factors, a new class of homologous, inner-membrane–bound anti-σ factors has recently been discovered in a variety of eubacteria. This new class of anti-σ factors regulates the expression of so-called extracytoplasmic functions, and hence is known as the ECF subfamily of anti-sigma factors. The range of cell processes regulated by anti-σ factors is highly varied and includes bacteriophage phage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport, and virulence.

The persistence of chloroaromatic compounds can be caused by various bottlenecks, such as incomplete degradative pathways or inappropriate regulation of these pathways. Patchwork assembly of existing pathways in novel combinations provides a general route for the development of strains degrading chloroaromatics. The recruitment of known complementary enzyme sequences in a suitable host organism by conjugative transfer of genes might generate a functioning hybrid pathway for the mineralization of some chloroaromatics not degraded by the parent organisms. The rational combination uses (a) peripheral, funneling degradation sequences originating from aromatics-degrading strains to fulfill the conversion of the respective analogous chloroaromatic compound to chlorocatechols as the central intermediates; (b) a central chlorocatechol degradation sequence, the so-called modified ortho pathway, which brings about elimination of chlorine substituents; and (c) steps of the 3-oxoadipate pathway to reach the tricarboxylic acid cycle. The genetic organization of these pathway segments has been well characterized. The specificity of enzymes of the xylene, benzene, biphenyl, and chlorocatechol pathways and the specificity of the induction systems for the chlorinated substrates are analyzed in various organisms to illustrate eventual bottlenecks and to provide alternatives that are effective in the conversion of the “new” substrate. Hybrid pathways are investigated in “new” strains degrading chlorinated benzoates, toluenes, benzenes, and biphenyls. Problems occurring after the conjugative DNA transfer and the “natural” solution of these are examined, such as the prevention of misrouting into the meta pathway, to give a functioning hybrid pathway. Some examples clearly indicate that patchwork assembly also happens in nature.

Clostridium perfringens causes human gas gangrene and food poisoning as well as several enterotoxemic diseases of animals. The organism is characterized by its ability to produce numerous extracellular toxins including α-toxin or phospholipase C, θ-toxin or perfringolysin O, κ-toxin or collagenase, as well as a sporulation-associated enterotoxin. Although the genes encoding the α-toxin and θ-toxin are located on the chromosome, the genes encoding many of the other extracellular toxins are located on large plasmids. The enterotoxin gene can be either chromosomal or plasmid determined. Several of these toxin genes are associated with insertion sequences. The production of many of the extracellular toxins is regulated at the transcriptional level by the products of the virR and virS genes, which together comprise a two-component signal transduction system.

In collaboration with the National Cancer Institute, Bristol-Myers Squibb has developed paclitaxel for treatment of various cancers; it has been approved by the Food and Drug Administration for the treatment of ovarian and metastatic breast cancer. Originally paclitaxel was isolated and purified from the bark of Pacific yew trees. This source of paclitaxel was considered to be economically and ecologically unsuitable as it required the destruction of the yew trees. This review article describes alternate methods for the production of paclitaxel, specifically, a semisynthetic approach and the application of biocatalysis in enabling the semisynthesis of paclitaxel. Three novel enzymes were discovered in our laboratory that converted the variety of taxanes to a single molecule, namely 10-deacetylbaccatin III (paclitaxel without C-13 side chain and C-10 acetate), a precursor for paclitaxel semisynthesis. These enzymes are C-13 taxolase (catalyzes the cleavage of C-13 side chain of various taxanes), C-10 deacetylase (catalyzes the cleavage of C-10 acetate of various taxanes), and C-7 xylosidase (catalyzes the cleavage of C-7 xylose from various xylosyltaxanes). Using a biocatalytic approach, paclitaxel and a variety of taxane in extracts of a variety of Taxus cultivars were converted to a 10-deacetylbaccatin III. The concentration of 10-deacetylbaccatin III was increased by 5.5- to 24-fold in the extracts treated with the enzymes, depending upon the type of Taxus cultivars used. Biocatalytic processes have also been described for the preparation of C-13 paclitaxel side chain synthons. The chemical coupling of 10-deacetylbaccatin III or baccatin III to C-13 paclitaxel side chain has been summarized to prepare paclitaxel by semisynthesis.

This review describes a mechanism of viral transformation involving activation of cellular signaling pathways. We focus on four viral oncoproteins: the E5 protein of bovine papillomavirus, which activates the platelet-derived growth factor β receptor; gp55 of spleen focus forming virus, which activates the erythropoietin receptor; polyoma virus middle T antigen, which resembles an activated receptor tyrosine kinase; and LMP-1 of Epstein-Barr virus, which mimics an activated tumor necrosis factor receptor. These examples indicate that diverse viruses induce cell transformation by activating cellular signal transduction pathways. Study of this mechanism of viral transformation will provide new insights into viral tumorigenesis and cellular signal transduction.

A variety of microbial dechlorination mechanisms have been demonstrated in laboratory microcosms, pure cultures, and in situ sedimentary environments. New perspectives on in situ processes from these efforts allow the design of more realistic bioremediation strategies that complement natural processes regardless of whether the strategy used is one of engineered accelerated bioremediation or natural attenuation. Since 1994 the scientific community has acquired considerable knowledge regarding natural attenuation of organochlorine compounds. Natural attenuation of chlorinated solvents has been documented at a number of field sites. Reductive dechlorination driven by co-contaminants or naturally occurring organics as substrates in combination with aerobic or co-metabolic degradation contains certain chlorinated solvent plumes. Although natural attenuation is not a panacea, at sites where it is applicable, it offers a scientifically sound, cost-effective method to remediate groundwater contaminated with chlorinated solvents.

Nosocomial outbreaks and pseudo-outbreaks caused by the nontuberculous mycobacteria (NTM) have been recognized for more than 20 years and continue to be a problem. Most of these outbreaks have involved the rapidly growing mycobacterial species Mycobacterium fortuitum and M. abscessus. The reservoir for these outbreaks is generally municipal and (often separate) hospital water supplies. These mycobacterial species and others are incredibly hardy, able to grow in municipal and distilled water, thrive at temperatures of 45°C or above (M. xenopi and M. avium complex), and resist the activity of organomercurials, chlorine, 2% concentrations of formaldehyde and alkaline glutaraldehyde, and other commonly used disinfectants. Disease outbreaks usually involve sternal wound infections, plastic surgery wound infections, or postinjection abscesses. Pseudo-outbreaks most commonly relate to contaminated bronchoscopes and endoscopic cleaning machines (M. abscessus) and contaminated hospital water supplies (M. xenopi). Knowledge of the reservoir of these species, their great survival capabilities within the hospital, and newer molecular techniques for strain comparison have helped control and more quickly identify current nosocomial outbreaks or pseudo-outbreaks caused by the NTM.

The nuclear export of intron-containing HIV-1 RNA is critically dependent on the activity of Rev, a virally encoded sequence-specific RNA-binding protein. Rev shuttles between the nucleus and the cytoplasm and harbors both a nuclear localization signal and a nuclear export signal. These essential peptide motifs have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export. HIV-1 Rev therefore represents an excellent system with which to study aspects of transport across the nuclear envelope.

The budding yeast Saccharomyces cerevisiae divides asymmetrically, giving rise to a mother cell and a smaller daughter cell. Individual mother cells produce a finite number of daughter cells before senescing, undergoing characteristic changes as they age such as a slower cell cycle and sterility. The average life span is fixed for a given strain, implying that yeast aging has a strong genetic component. Genes that determine yeast longevity have highlighted the importance of such processes as cAMP metabolism, epigenetic silencing, and genome stability. The recent finding that yeast aging is caused, in part, by the accumulation of circular rDNA molecules has unified many seemingly disparate observations.

Plasmodium falciparum is an obligate human parasite that is the causative agent of the most lethal form of human malaria. Transmission of P. falciparum to a new human host requires a mosquito vector within which sexual replication occurs. P. falciparum replicates as an intracellular parasite in man and as an extracellular parasite in the mosquito, and it undergoes multiple developmental changes in both hosts. Changes in the environment and the activities of parasites in these various life-cycle stages are likely to be reflected in changes in the metabolic needs and capabilities of the parasite. Most of our knowledge of the metabolic capabilities of P. falciparum is derived from studies of the asexual erythrocytic cycle of the parasite, the portion of the parasite life cycle found in infected humans that is responsible for malarial symptoms. Efforts to control transmission and to understand the sometimes unique biology of this parasite have led to information about the metabolic capabilities of sexual and/or sporogonic stages of these parasites. This review focuses on comparing and contrasting the carbohydrate, nucleic acid, and protein synthetic capabilities of asexual erythrocytic stages and sexual stages of P. falciparum.

For many years it has been known that thymine auxotrophic microorganisms undergo cell death in response to thymine starvation [thymineless death (TLD)]. This effect is unusual in that deprivation of many other nutritional requirements has a biostatic, but not lethal, effect. Studies of numerous microbes have indicated that thymine starvation has both direct and indirect effects. The direct effects involve both single- and double-strand DNA breaks. The former may be repaired effectively, but the latter lead to cell death. DNA damaged by thymine starvation is a substrate for DNA repair processes, in particular recombinational repair. Mutations in recBCD recombinational repair genes increase sensitivity to thymineless death, whereas mutations in RecF repair protein genes enhance the recovery process. This suggests that the RecF repair pathway may be critical to cell death, perhaps because it increases the occurrence of double-strand DNA breaks with unique DNA configurations at lesion sites. Indirect effects in bacteria include elimination of plasmids, loss of transforming ability, filamentation, changes in the pool sizes of various nucleotides and nucleosides and in their excretion, and phage induction. Yeast cells show effects similar to those of bacteria upon thymine starvation, although there are some unique features. The mode of action of certain anticancer drugs and antibiotics is based on the interruption of thymidylate metabolism and provides a major impetus for further studies on TLD. There are similarities between TLD of bacteria and death of eukaryotic cells. Also, bacteria have “survival” genes other than thy (thymidylate synthetase), and this raises the question of whether there is a relationship between the two. A model is presented for a molecular basis of TLD.

Genome and pre-genome replication in all animal DNA viruses except poxviruses occurs in the cell nucleus (Table 1). In order to reproduce, an infecting virion enters the cell and traverses through the cytoplasm toward the nucleus. Using the cell's own nuclear import machinery, the viral genome then enters the nucleus through the nuclear pore complex. Targeting of the infecting virion or viral genome to the multiplication site is therefore an essential process in productive viral infection as well as in latent infection and transformation. Yet little is known about how infecting genomes of animal DNA viruses reach the nucleus in order to reproduce. Moreover, this nuclear locus for viral multiplication is remarkable in that the sizes and composition of the infectious particles vary enormously. In this article, we discuss virion structure, life cycle to reproduce infectious particles, viral protein's nuclear import signal, and viral genome nuclear targeting.