Gary R. Jacobson

Uptake of Succinate and Malate in Cultured Cells and Bacteroids of TWO Slow-growing Species of Rhizobium

SUMMARY: The uptake of succinate and malate has been compared in cultured cells and bacteroids of two species of slow-growing Rhizobium: R. japonicum (USDA 1-1 10) and cowpea Rhizobium(USDA 3278). Cultured cells of both organisms actively accumulated both compounds, and uptake was abolished by KCN and 2,4-DNP, but not by arsenate. Kinetic studies using cultured cells showed that succinate competitively inhibited malate uptake, and vice versa, implying a common step in the uptake of these dicarboxylic acids. Uptake of both of these compounds was inhibited by osmotic shock and N-ethylmaleimide in cultured cells of both species. Purified bacteroids accumulated succinate in a process that was sensitive to 2,4-DNP and KCN, but at a rate significantly slower than for cultured cells. No detectable malate uptake was observed in purified symbiotic cells. Furthermore, succinate uptake was insensitive to osmotic shock in bacteroids of both strains. These results show that although bacteroids of both strains are competent in succinate uptake, significant differences exist in the expression and/or stability of dicarboxylate uptake systems between free-living and symbiotic cells.

Carbohydrate uptake in the oral pathogen Streptococcus mutans: mechanisms and regulation by protein phosphorylation

Streptococcus mutans is the primary etiological agent of dental caries in man and other animals. This organism and other related oral streptococci use carbohydrates almost exclusively as carbon and energy sources, fermenting them primarily to lactic acid which initiates erosion of tooth surfaces. Investigations over the past decade have shown that the major uptake mechanism for most carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), although non-PTS systems have also been identified for glucose and sucrose. Regulation of sugar uptake occurs by induction/repression and inducer exclusion mechanisms in S. mutans, but apparently not by inducer expulsion as is found in some other streptococci. In addition, ATP-dependent protein kinases have also been identified in S. mutans and other oral streptococci, and a regulatory function for at least one of these has been postulated. Among a number of proteins that are phosphorylated by these enzymes, the predominant soluble protein substrate is the general phospho-carrier protein of the PTS, HPr, as had previously been observed in a variety of Gram-positive bacteria. Recent results have provided evidence for a role for ATP-dependent phosphorylation of HPr in the coordination of sugar uptake and its catabolism in S. mutans. In this review, these results are summarized, and directions for future research in this area are discussed.

31P-NMR studies of the oral pathogen Streptococcus mutans: observation of lipoteichoic acid

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.

TheEscherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and theEscherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.

Embryology and the Study of Microbial Development

The study of embryology, and consequently of biological development, was initiated by Aristotle in 340 b.c. He followed and recorded the development of a chick embryo within the egg, noting that the developing embryo went through distinct chronological stages that resembled those of other organisms. From this observation stemmed the postulate that ontogeny is a recapitulation of phylogeny (Baer’s Law).

Genetic Control of Development, Mating Type Determination, and Programmed Death in Ciliated Protozoa

Somatic cells within a multicellular organism are linearly programmed to differentiate and die either during embryological development, or subsequently after maturation of the organism (Chapter 2). This fact necessitated, during evolutionary history, the separation of the soma from the germ line as well as the establishment of sexuality to provide organismal rejuvenation and species continuity (Chapter 3). Similar processes of differentiation, sex, and programmed death have evolved in the unicellular ciliated protozoa, but because the entire program of differentiation occurs within a single cell, somewhat different mechanisms and structural elements must underlie these processes. This chapter deals primarily with development in the ciliates. These organisms provide the simplest microbial system in which programmed death is known to occur as the terminal step in a sequence of developmental events.

Cellular Mortality, Growth Regulation, and the Phenomenon of Cancerous Transformation

Over the past few decades a huge research effort has been devoted to understanding the mechanisms of cancerous transformation. This effort has led to recognition of the fact that the unrestricted growth of most cancer cells is caused by release of normal cells within the body from the regulatory constraints imposed upon them. An understanding of cancerous transformation therefore leads to knowledge of the events controlling normal cell proliferation. In this chapter we shall first discuss the possible evolutionary origins of differentiated animal cells, both somatic (tissue) cells and germ (reproductive) cells. Subsequently, we shall consider the events that occur when normal cells become cancerous. It will become apparent that two distinct events frequently (but not always) accompany the transformation process: loss of sensitivity to growth regulation, and loss of the constraints of cellular mortality. Further, these two events are clearly distinct from each other, as well as from the genetic events that determine expression of the differentiated state. Although the phenomenon of cell mortality sometimes referred to as programmed cell death is presently poorly understood, it seems to have evolved as the terminal step in a sequence of irreversible differentiation events.

Molecular Events Accompanying Morphogenesis

Morphogenesis, the development of shape, can be discussed both at the organismal level and the cellular level. Within a multicellular embryo several distinct morphogenetic processes occur simultaneously. First, differential mitosis results in selective stimulation of the growth of certain cell types relative to others. Selective growth can be due to any one of several cellular or extracellular factors, or to the localization of a cell type within a part of the embryo: (1) differential nutrient supplies or differential abilities of the cell types to transport and accumulate nutrients; (2) the presence of cell-specific hormones and growth factors which stimulate (or inhibit) growth of those cells that possess the requisite receptor proteins; or (3) bioelectric activities, due to the presence of ion selective channel proteins in the plasma membranes of certain cells, which may influence growth rate either by controlling the cytoplasmic ion composition or by creating a transcellular electrical potential. Second, programmed cell death plays an important role in morphogenetic processes during embryogenesis. In the developing tadpole, the Rohan-beard cells, which are important constituents of the nervous system, are programmed to die at a certain developmental stage so that other conducting cells can replace them. Loss of the tadpole tail, loss of the webbing between the fingers of the developing human hand, and regression of the Mullerian or Wolfian duct in developing male or female mammals, respectively, represent other examples of programmed cell death allowing embryonic morphogenesis. Third, cell migration and changes in cell shape occur continuously during development.

Mechanisms of Chemoreception, Electrical Signal Transduction, and Biological Response

Living organisms receive and respond to a variety of chemicals and energy stimuli, and these interactions influence differentiation and regulate sexual activities. Well-characterized examples include responses of coelenterates to peptide morphogens (Chapter 5) and of haploid yeast cells to sexual pheromones (Chapter 9). In the developing embryo as in developing microbial systems, cell migration and elongation may be governed by chemotactic processes (Chapters 1, 2, and 4). Bioelectric stimuli also play an important role in developmental processes. Examples include the development of cell polarity and tissue regeneration, as discussed in Chapter 2, and fertilization of sea urchin eggs (Chapter 10). Thus, it is crucial to an understanding of differentiation that the mechanisms of chemoreception and electrical signal transmission be elucidated. In this chapter we discuss chemotactic and mechanotactic processes in Gram-negative bacteria and selected eukaryotic microorganisms.

Cellular Recognition: Mechanisms and Consequences of Homotypic and Heterotypic Adhesions

It is widely accepted that cell surface macromolecules must mediate recognition phenomena that allow like cells within a tissue to adhere to one another (homotypic adhesion) as well as to cells of other types (heterotypic adhesion). These macromolecular interactions occur by multistep processes that are important in the formation and maintenance of tissues both in the adult organism and during embryonic development. Moreover, we now know that specific cell-cell interactions regulate many physiological processes. Contact inhibition of growth is a well-documented example of negative growth regulation induced by homotypic cellular adhesions. Cellular motility may also be regulated in a negative sense as observed in the phenomenon of contact inhibition of motion. And highly specific intercellular adhesions can induce the syntheses of enzymes and proteins responsible for the expression of tissue-specific traits.