Passive Electrical Properties of Microorganisms: I. Conductivity of Escherichia coli and Micrococcus lysodeikticus

Effective conductivities are reported for the bacteria Escherichia coli and Micrococcus lysodeikticus over a range of environmental conductivity. The apparent conductivities of the organisms can be explained in terms of the properties of the cell wall. At low conductivities of the environment, the conductivity of the cell appears to be dominated by the counterions of the fixed charge of the cell wall. At higher conductivities of the suspending medium, evidence suggests that ions from the environment invade the cell wall causing an increase in the effective conductivity of the cell so that it takes on values roughly proportional to that of the environment. The model points to the usefulness of dielectric techniques in studies of the properties of intact cell walls.     

Peptidoglycan turnover and recycling in Gram-positive bacteria

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.     

Transport of outer membrane lipids in mycobacteria

The complex organization of the mycobacterial cell wall poses unique challenges for the study of its assembly. Although mycobacteria are classified evolutionarily as Gram-positive bacteria, their cell wall architecture more closely resembles that of Gram-negative organisms. They possess not only an inner cytoplasmic membrane, but also a bilayer outer membrane that encloses an aqueous periplasm and includes diverse lipids that are required for the survival and virulence of pathogenic species. Questions surrounding how mycobacterial outer membrane lipids are transported from where they are made in the cytoplasm to where they function at the cell exterior are thus similar, and similarly compelling, to those that have driven the study of Gram-negative outer membrane transport pathways. However, little is understood about these processes in mycobacteria. Here we contextualize these questions by comparing our current knowledge of mycobacteria with better-defined systems in other organisms. Based on this analysis, we propose possible models and highlight continuing challenges to improving our understanding of outer membrane assembly in these medically and environmentally important bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Mycoplasma-like sturctures in podzol soils of the southern taiga in the Irtysh region]

Mycoplasm-like organisms were found in taiga podzolic soils of the South Irtish region. They were detected in close contact with soil bacteria more ofter than in a free state. The mycoplasm-like organisms either were absorbed on the bacterial cell walls or grew inside the cytoplasm as filamentous forms; sometimes they filled the whole inner space of the cells, disrupted the cell wall, and were liberated into the surrounding medium.     

Mechanical Consequences of Cell-Wall Turnover in the Elongation of a Gram-Positive Bacterium

A common feature of walled organisms is their exposure to osmotic forces that challenge the mechanical integrity of cells while driving elongation. Most bacteria rely on their cell wall to bear osmotic stress and determine cell shape. Wall thickness can vary greatly among species, with Gram-positive bacteria having a thicker wall than Gram-negative bacteria. How wall dimensions and mechanical properties are regulated and how they affect growth have not yet been elucidated. To investigate the regulation of wall thickness in the rod-shaped Gram-positive bacterium Bacillus subtilis, we analyzed exponentially growing cells in different media. Using transmission electron and epifluorescence microscopy, we found that wall thickness and strain were maintained even between media that yielded a threefold change in growth rate. To probe mechanisms of elongation, we developed a biophysical model of the Gram-positive wall that balances the mechanical effects of synthesis of new material and removal of old material through hydrolysis. Our results suggest that cells can vary their growth rate without changing wall thickness or strain by maintaining a constant ratio of synthesis and hydrolysis rates. Our model also indicates that steady growth requires wall turnover on the same timescale as elongation, which can be driven primarily by hydrolysis rather than insertion. This perspective of turnover-driven elongation provides mechanistic insight into previous experiments involving mutants whose growth rate was accelerated by the addition of lysozyme or autolysin. Our approach provides a general framework for deconstructing shape maintenance in cells with thick walls by integrating wall mechanics with the kinetics and regulation of synthesis and turnover.     

Cell Wall Growth During Differentiation of Proteus Swarmers

This paper describes the process by which the cell wall of Proteus mirabilis, as measured by the presence of the O antigen, develops during the differentiation of swarmers from short cells on an agar surface. The sequence was followed by fluorescent-antibody staining, with both the direct and reverse methods. When the organisms were labeled with fluorescent antibody by the direct method, they showed a progressive diminution of the marker along the cell surface and some increase in the length of the bacteria. However, the label had become completely diluted out before typical swarmers developed. When the bacteria were exposed initially to unlabeled antibody by the reverse technique, and then incubated with fluorescent antibody, they showed a progressive increase both in the intensity of the label along their entire periphery and in cellular length, culminating in the formation of swarmers. It is concluded that in P. mirabilis, as in the few other gram-negative bacteria examined so far, cell wall synthesis takes place diffusely, i.e., by intercalation of new with old components along the length of the cell wall.     

Symbiotic bacteria protect wasp larvae from fungal infestation

Symbiotic associations between different organisms are of great importance for evolutionary and ecological processes [1-4]. Bacteria are particularly valuable symbiotic partners owing to their huge diversity of biochemical pathways that may open entirely new ecological niches for higher organisms [1-3]. Here, we report on a unique association between a new Streptomyces species and a solitary hunting wasp, the European beewolf (Philanthus triangulum, Hymenoptera, Crabronidae). Beewolf females cultivate the Streptomyces bacteria in specialized antennal glands and apply them to the brood cell prior to oviposition. The bacteria are taken up by the larva and occur on the walls of the cocoon. Bioassays indicate that the streptomycetes protect the cocoon from fungal infestation and significantly enhance the survival probability of the larva, possibly by producing antibiotics. Behavioral observations strongly suggest a vertical transmission of the bacteria. Two congeneric beewolf species harbor closely related streptomycetes in their antennae, indicating that the association with protective bacteria is widespread among philanthine wasps and might play an important role in other insects as well. This is the first report on the cultivation of bacteria in insect antennae and the first case of a symbiosis involving bacteria of the important antibiotic-producing genus Streptomyces.     

Anchoring of proteins to lactic acid bacteria

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.     

Effects of mycoplasma infection on the host organism response via p53/NF-κB signaling

Mycoplasmas are bacteria lacking the cell wall, which is the major characteristic of this taxonomic class (Mollicutes). Among bacteria, mycoplasmas possess the smallest genome known for free-living organisms. This feature limits the autonomy of bacteria and makes them increasingly susceptible to changes in the host organism. Many mycoplasmas themselves cause pathological changes in the host organism, often complicated by immune disorders. Infection with certain strains of mycoplasma results in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells, which is the major mediator of the inflammatory response. Furthermore, mycoplasmas can inhibit p53-mediated checkpoint control of cell cycle and apoptosis. Collectively, these properties indicate that mycoplasmas might act as cancer-promoting factors. In this review, we summarize the information known to date on the role of mycoplasmas in the regulation of the host immune response and their functional interactions with p53.     

Nucleoids and coated vesicles of “Epulopiscium” spp.

We describe here aspects of the anatomy of two “Epulopiscium” morphotypes, unusually large bacteria that are not yet cultured and that reproduce by the internal generation of two or more vegetative daughter cells. Two morphotypes, A and B, which are enteric symbionts of several species of herbivorous surgeonfish (Acanthuridae), were collected around the Great Barrier Reef of Australia, preserved there, and later stained for light microscopy. Some samples were examined by electron microscopy. In both morphotypes, countless discrete nucleoplasms or nucleoids were found to occupy a single shallow layer just beneath the surface all around these organisms. At each end of the morphotype B cells, a membrane-bound compartment containing dense cords of chromatin was observed. When these were found at each end of growing daughter cells, no polar compartments were then found in their mother organism. Electron micrographs of sections of morphotype A symbionts show that their outermost region is composed of tightly packed coated vesicles, each surrounded by a thin, dense, spacious capsule. Near the surface of type A organisms the remains of broken vesicles, broken capsules, and a finely fibrous matrix fuse to form a fabric that serves as the cell wall. Morphotype B organisms, however, were observed to have a distinct, morphologically continuous outer wall. Received: 3 December 1997 / Accepted: 11 June 1998     

Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus.

Microbial organisms secrete antibiotics that cause the selective destruction of specific target cells. Although the mode of action is known for many antibiotics, the mechanisms by which these molecules are directed specifically to their target cells hitherto have not been described. Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that cleaves staphylococcal peptidoglycans in general but that is directed specifically to Staphylococcus aureus target cells. The sequence element sufficient for the binding of the bacteriocin as well as of hybrid indicator proteins to the cell wall of S.aureus consisted of 92 C-terminal lysostaphin residues. Targeting to the cell wall of S.aureus occurred either when the hybrid indicator molecules were added externally to the bacteria or when they were synthesized and exported from their cytoplasm by an N-terminal leader peptide. A lysostaphin molecule lacking the C-terminal targeting signal was enzymatically active but had lost its ability to distinguish between S.aureus and S.simulans cells, indicating that this domain functions to confer target cell specificity to the bacteriolytic molecule.     

Electric Conductivity and Internal Osmolality of Intact Bacterial Cells

Intact cells of Streptococcus faecalis and Micrococcus lysodeikticus were found to have high-frequency electric conductivities of 0.90 and 0.68 mho/m, respectively. These measured values, which reflect movements of ions both within the cytoplasm and within the cell wall space, were only about one-third of those calculated on the basis of determinations of the amounts and types of small ions within the cells. Concentrated suspensions of bacteria with damaged membranes showed similarly large disparities between measured and predicted conductivities, whereas the conductivities of diluted suspensions were about equal to predicted values. Thus, the low mobilities of intracellular ions appeared to be interpretable in terms of the physicochemical behavior of electrolytes in concentrated mixtures of small ions and cell polymers. In contrast to the low measured values for conductivity of intact bacteria, values for intracellular osmolality measured by means of a quantitative plasmolysis technique were higher than expected. For example, the plasmolysis threshold for S. faecalis cells indicated an internal osmolality of about 1.0 osmol/kg, compared with a value of only 0.81 osmol/liter of cell water calculated from a knowledge of the cell content and the distribution of small solutes. In all, our results indicate that most of the small ions within vegetative bacterial cells are free to move in an electric field and that they contribute to cytoplasmic osmolality.     

Is the periplasm continuous in filamentous multicellular cyanobacteria?

Filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which individual cells exchange nutrients and, presumably, regulatory molecules. Unknown mechanisms underlie this exchange. Classical electron microscopy shows that filamentous cyanobacteria bear a Gram-negative cell wall comprising a peptidoglycan layer and an outer membrane that are external to the cytoplasmic membrane, and that the outer membrane appears to be continuous along the filament of cells. This implies that the periplasmic space between the cytoplasmic and outer membranes might also be continuous. We propose that a continuous periplasm could constitute a communication conduit for the transfer of compounds, which is essential for the performance of these bacteria as multicellular organisms.     

Protein Phylogenies and Signature Sequences: A Reappraisal of Evolutionary Relationships among Archaebacteria, Eubacteria, and Eukaryotes

The presence of shared conserved insertion or deletions (indels) in protein sequences is a special type of signature sequence that shows considerable promise for phylogenetic inference. An alternative model of microbial evolution based on the use of indels of conserved proteins and the morphological features of prokaryotic organisms is proposed. In this model, extant archaebacteria and gram-positive bacteria, which have a simple, single-layered cell wall structure, are termed monoderm prokaryotes. They are believed to be descended from the most primitive organisms. Evidence from indels supports the view that the archaebacteria probably evolved from gram-positive bacteria, and I suggest that this evolution occurred in response to antibiotic selection pressures. Evidence is presented that diderm prokaryotes (i.e., gram-negative bacteria), which have a bilayered cell wall, are derived from monoderm prokaryotes. Signature sequences in different proteins provide a means to define a number of different taxa within prokaryotes (namely, low G+C and high G+C gram-positive, Deinococcus-Thermus, cyanobacteria, chlamydia-cytophaga related, and two different groups of Proteobacteria) and to indicate how they evolved from a common ancestor. Based on phylogenetic information from indels in different protein sequences, it is hypothesized that all eukaryotes, including amitochondriate and aplastidic organisms, received major gene contributions from both an archaebacterium and a gram-negative eubacterium. In this model, the ancestral eukaryotic cell is a chimera that resulted from a unique fusion event between the two separate groups of prokaryotes followed by integration of their genomes.     

Cell Envelope Morphology of Rumen Bacteria

The cell walls of three species of rumen bacteria (Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii) were studied by a variety of morphological methods. Although all the cells studied were gram-negative and had typical cytoplasmic membranes and outer membranes, great variation was observed in the thickness of their peptidoglycan layers. Megasphaera elsdenii evidenced a phenomenally thick peptidoglycan layer whose participation in septum formation was very clearly seen. All species studied have cell wall "coats" external to the outer membrane. The coat of Bacteroides ruminicola is composed of large (approximately 20 nm) globules that resemble the protein coats of other organisms, whereas the coat of Bacteroides succinogenes is a thin and irregular carbohydrate coat structure. Megasphaera elsdenii displays a very thick fibrillar carbohydrate coat that varies in thickness with the age of the cells. Because of the universality of extracellular coats among rumen bacteria we conclude that the production of these structures is a protective adaptation to life in this particular, highly competitive, environment.     

Experiments with lysis of living cells of Eremothecium ashbyii and of Methanomonas by microbial enzymes

The possibility to use microorganisms as human food is limited by several factors. The intact cell is resistant to digestion, the cell wall is unbalanced in essential amino acids, and the nucleic acids are said to be harmful. For using single cell protein as food it may thus be necessary to disrupt the cell wall and separate the protein from nucleic acid. This paper is concerned with the production and properties of extracellular enzymes able to lyse cell walls of microorganisms. Soil bacteria and actinomycetes have been cultivated and lytic enzymes from these organisms have been used to lyse living cells of the yeast like organism E. ashbyii. Efforts were also made to use these enzymes for lysing cell of a Methanomonas sp.     

Covalent attachment of proteins to peptidoglycan

Bacterial surface proteins are key players in host-symbiont or host-pathogen interactions. How these proteins are targeted and displayed at the cell surface are challenging issues of both fundamental and clinical relevance. While surface proteins of Gram-negative bacteria are assembled in the outer membrane, Gram-positive bacteria predominantly utilize their thick cell wall as a platform to anchor their surface proteins. This surface display involves both covalent and noncovalent interactions with either the peptidoglycan or secondary wall polymers such as teichoic acid or lipoteichoic acid. This review focuses on the role of enzymes that covalently link surface proteins to the peptidoglycan, the well-known sortases in Gram-positive bacteria, and the recently characterized l,d-transpeptidases in Gram-negative bacteria.     

Dorsal-ventral differentiation in Simonsiella and other aspects of its morphology and ultrastructure

The morphology and ultrastructure of the aerobic, Gram-negative multicellular-filamentous bacteria of the genus Simonsiella were investigated by scanning and transmission electron microscopy. The flat, ribbon-shaped, multicellular filaments show dorsal-ventral differentiation with respect to their orientations to solid substrata. The dorsal surface, orientated away from the substrate, is convex and possesses an unstructured capsule. The ventral surface, on which the organisms adhere and glide, is concave and has an extracellular layer with fibrils extending at right angles from the cell wall. The cytoplasm in the ventral region contains a proliferation of intracytoplasmic membranes and few ribosomes in comparison to the cytoplasm in other parts of the cell. Centripetal cell wall formation is asymmetrical and commences preferentially in the ventral region. Quantitative differences in morphology and cytology exist among selected Simonsiella strains. Functional aspects of this dorsalventral differentiation are discussed with respect to the colonization and adherence of Simonsiella to mucosal squamous epithelial cells in its ecological habitat, the oral cavities of warm-blooded vertebrates.List of Abbreviations SEM scanning electron microscope - TEM transmission electron microscope     

Ultrastructal morphology of some prokaryotic microorganisms associated with the hindgut of cockroaches.

Scanning electron microscopy and transmission electron microscopy have been used to visualize the morphology and ultrastructure of two types of microorganisms in the hindgut of the cockroach Blaberus posticus. Both organisms, designated as either short or long rods, are attached to chitinous projections from the gut wall. Micrographs suggest that the organisms are prokaryotic with a cell wall complex characteristic of gram-negative bacteria. However, certain differences were noted between the cell wall complex of the two types. Two forms of the long-rod type were noted, with one form appearing to be a "degenerate" or "transitional" cell. In the degenerate cells, vesicles are observed that often are contiguous with the cytoplasmic membrane. There are indications that the long-rod type may divide by longitudinal fission. Neither the short- nor long-rod type has been cultivated in its respective recognizable form.     

Physiological roles of trehalose in bacteria and yeasts: a comparative analysis

The disaccharide trehalose is widely distributed in nature and can be found in many organisms, including bacteria, fungi, plants, invertebrates and mammals. Due to its particular physical features, trehalose is able to protect the integrity of the cell against a variety of environmental injuries and nutritional limitations. In addition, data available on several species of bacteria and yeast suggest specific functions for trehalose in these organisms. Bacteria can use exogenous trehalose as the sole source of carbon and energy as well as synthesize enormous amounts of the disaccharide as compatible solute. This ability to accumulate trehalose is the result of an elaborate genetic system, which is regulated by osmolarity. Some mycobacteria contain sterified trehalose as a structural component of the cell wall, whereas yeast cells are largely unable to grow on trehalose as carbon source. In these lower eukaryotes, trehalose appears to play a dual function: as a reserve compound, mainly stored in vegetative resting cells and reproductive structures, and as a stress metabolite. Recent findings also point to important biotechnological applications for trehalose.