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Uptake and concentration of inorganic ions are part of the complex cellular processes required for cell homeostasis, as well as for mineral formation by organisms. These ion transport mechanisms include distinct cellular compartments and chemical phases that play various roles in the physiology of organisms. Here, the prominent cases of dense ion pools in unicellular organisms are briefly reviewed. The specific observations that were reported for different organisms are consolidated into a wide perspective that emphasizes general traits. It is suggested that the intracellular ion pools can be divided into three types: a high cytoplasmic concentration, a labile storage compartment that hosts dense ion-rich phases, and a mineral-forming compartment in which a stable long-lived structure is formed. Recently, many labile pools were identified in various organisms using advanced techniques, bringing many new questions about their possible roles in the formation of the stable mineralized structures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The bacterial peptidoglycan layer forms a complex mesh-like structure that surrounds the cell, imparting rigidity to withstand cytoplasmic turgor and the ability to tolerate stress. As peptidoglycan has been the target of numerous clinically successful antimicrobials such as penicillin, the biosynthesis, remodeling and recycling of this polymer has been the subject of much interest. Herein, we review recent advances in the understanding of peptidoglycan biosynthesis and remodeling in a variety of different organisms. In order for bacterial cells to grow and divide, remodeling of cross-linked peptidoglycan is essential hence, we also summarize the activity of important peptidoglycan hydrolases and how their functions differ in various species. There is a growing body of evidence highlighting complex regulatory mechanisms for peptidoglycan metabolism including protein interactions, phosphorylation and protein degradation and we summarize key recent findings in this regard. Finally, we provide an overview of peptidoglycan recycling and how components of this pathway mediate resistance to drugs. In the face of growing antimicrobial resistance, these recent advances are expected to uncover new drug targets in peptidoglycan metabolism, which can be used to develop novel therapies., (© 2020 Elsevier Inc. All rights reserved.)

Bacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how cells cope with stressful conditions and how this may lead to dramatic morphological changes. These changes may not only allow harmless cells to withstand environmental insults but can also benefit pathogenic bacteria by enabling them to escape from the immune system and the activity of antibiotics. A better understanding of stress-induced morphogenesis will help us to develop new approaches to combat such harmful pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Facile, eco-friendly synthesis of metal nanoparticles has been proposed as a cost effective method. In the present study, we propose the facile synthesis of silver-silver chloride (Ag-AgCl) nanoparticles (NPs) using the medicinally important Agrimonia pilosa plant extract without addition of capping or stabilizing agents. The Ag-AgCl NPs synthesis was observed at 40 °C after 10 min incubation; the synthesis of Ag-AgCl NPs was indicated by color change and confirmed by UV-vis spectroscopic peak at 454 nm. TEM analysis confirmed Ag-AgCl NPs were 10-20 nm in size and spherical, and oval in shape. Elemental composition was determined by energy dispersive X-ray analysis, and crystalline structure was confirmed by X-ray diffraction spectroscopy. Different phytocomponents present in the plant extract were analyzed by Gas Chromatography-Mass spectrometry, and the interaction of biomolecules in reduction process was analyzed by Fourier transform infrared spectroscopy studies. The synthesized Ag-AgCl NPs showed significant antibacterial efficiency, analyzed by well diffusion assay against pathogenic bacteria including Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus saprophyticus, Escherichia coli, Pseudomonas putida. Minimum inhibitory concentration and minimum bactericidal concentration were evaluated by microbroth dilution, and spread plate method, respectively. The possible mechanism of bacterial growth inhibition is due to changes in bacterial cell wall morphology that was studied by FE-SEM analysis., (Copyright © 2018. Published by Elsevier Ltd.)

Antimicrobial molecules have been used for more than 50 years now and are the basis of modern medicine. No surgery can nowdays be imagined to be performed without antibiotics; dreadful diseases like tuberculosis, leprosis, siphilys, and more broadly all microbial induced diseases, can be cured only through the use of antimicrobial treatments. However, the situation is becoming more and more complex because of the ability of microbes to adapt, develop, acquire, and share mechanisms of resistance to antimicrobial agents. We choose to introduce this review by briefly drawing the panorama of antimicrobial discovery and development, but also of the emergence of microbial resistance. Then we describe how Atomic Force Microscopy (AFM) can be used to provide a better understanding of the mechanisms of action of these drugs at the nanoscale level on microbial interfaces. In this section, we will address these questions: (1) how does drug treatment affect the morphology of single microbes?; (2) do antimicrobial molecules modify the nanomechanical properties of microbes, or do the nanomechanical properties of microbes play a role in antimicrobial activity and efficiency?; and (3) how are the adhesive abilitites of microbes affected by antimicrobial drugs treatment? Finally, in a second part of this review we focus on recent studies aimed at changing the paradigm of the single molecule/cell technology that AFM typically represents. Recent work dealing with the creation of a microbe array which can be explored by AFM will be presented, as these developments constitute the first steps toward transforming AFM into a higher throughput technology. We also discuss papers using AFM as NanoMechnanicalSensors (NEMS), and demonstrate the interest of such approaches in clinical microbiology to detect quickly and with high accuracy microbial resistance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The ability to encapsulate cells individually in droplets has many potential applications, for example for observing the heterogeneity of behaviors within a population. However, implementing operations on moving droplets require feedback control and instruments that provide precise timing. These technical difficulties impede the adoption of droplet microfluidic protocols in nonspecialist labs. In this chapter we describe an approach to produce and manipulate droplets that remain stationary within a microfluidic chamber, by fabricating a microfluidic device having three-dimensional topography. The method uses microchannels that confine the fluids everywhere except in predefined regions where the channels have a large height, a technique known as "rails and anchors." By relying on the natural tendency of droplets to minimize their surface area, the approach provides a wide range of droplet manipulation tools. This chapter shows how this can be used to produce droplets, and several biological applications are demonstrated., (© 2018 Elsevier Inc. All rights reserved.)

Atomic force microscopy is nowadays a well-established technique that permits the investigation of numerous parameters of living matter. In particular, it allows the exploration of the mechanical properties of living organisms in almost physiological conditions. Here, we focus on the use of this technology to review recent contributions that relates the physiology and pathology of bacteria, yeast, plant and mammalian cells to their nano-mechanical properties., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The hok/sok locus has been shown to enhance the growth of bacteria in adverse growth conditions such as high temperature, low starting-culture densities and antibiotic treatment. This is in addition to their well-established plasmid-stabilization effect via post-segregational killing of plasmid-free daughter cells. It delays the onset of growth by prolonging the lag phase of bacterial culture, and increases the rate of exponential growth when growth eventually begins. This enables the cells adapt to the prevailing growth conditions and enhance their survival in stressful conditions. These effects functionally complement defective SOS response mechanism, and appear analogous to the growth effects of FtsZ in the SOS pathway. In this study, the role of FtsZ in the hok/sok-induced changes in bacterial growth and cell division was investigated. Morphologic studies of early growth-phase cultures and cells growing under temperature stress showed elongated cells typical of FtsZ inhibition/deficiency. Both ftsZ silencing and over-expression produced comparable growth effects in control cells, and altered the growth changes observed otherwise in the hok/sok + cells. These changes were diminished in SOS-deficient strain containing mutant FtsZ. The involvement of FtsZ in the hok/sok-induced growth changes may be exploited as drug target in host bacteria, which often propagate antibiotic resistance elements., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited. These problems can be circumvented by developing biosensors in Bacillus spores, which can be engineered to address all of these concerns. In its sporulated state, a whole cell-based biosensor has a remarkably long life span and is exceptionally resistant to environmental insult. When these spores are germinated for use in analytical techniques, they show no loss in performance, even after long periods of storage under harsh conditions. In this chapter, we will discuss the development and use of whole cell-based sensors, their adaptation to spore-based biosensors, their current applications, and future directions in the field., (© 2017 Elsevier Inc. All rights reserved.)

Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Genetically encoded small-molecule sensors are important tools for revealing the dynamics of metabolites and other small molecules in live cells over time. We recently developed RNA-based sensors that exhibit fluorescence in proportion to a small-molecule ligand. One class of these RNA-based sensors are termed Spinach riboswitches. These are RNAs that are based on naturally occurring riboswitches, but have been fused to the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, producing fluorescence upon binding the cognate small-molecule analyte. Here, we describe how to design and optimize these sensors by adjusting critical sequence elements, guided by structural insights from the Spinach aptamer. We provide a stepwise procedure to characterize sensors in vitro and to express sensors in bacteria for live-cell imaging of metabolites. Spinach riboswitch sensors offer a simple method for fluorescence measurement of a wide range of metabolites for which riboswitches exist, including nucleotides and their derivatives, amino acids, cofactors, cations, and anions., (© 2016 Elsevier Inc. All rights reserved.)

Along with discovery of new antibacterial agents, it is important to develop novel drug delivery systems to effectively deliver drugs within bacterial cells for enhanced therapeutic activity. Liposomes have been extensively investigated as pharmaceutical carriers for improvement of therapeutic index of antimicrobial agents. The aim of this present study was to evaluate the antibacterial activity of free and liposomal formulation of apigenin, a plant based isoflavone and elucidate the mode of action. Distearoylphosphatidylcholine liposomes were prepared having nano-range particle size (104.3±1.8 nm), narrow particle distribution (0.204) and high encapsulation efficiency of apigenin (89.9±2.31%). Antibacterial activity of apigenin and efficacy of liposome-mediated apigenin delivery were determined from minimum inhibitory concentration values. Interaction studies using electron microscopy revealed adherence and fusion of liposomal apigenin with the bacteria causing membrane perturbation through reactive oxygen species generation which was evaluated by epi-fluorescence microscopy and fluorescence activated cell sorting. The interaction of apigenin liposomes with bacterial membrane increased intracellular drug concentration and thus, can be employed to deliver apigenin within cells to augment its antibacterial activity. Increased efficacy and hemocompatibility of this formulation paves way for future evaluation of underlying molecular mechanisms and in vivo testing for enhanced therapeutic effects., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Viability PCR uses cell membrane integrity to differentiate live cells from dead. Our new approach improves viability PCR by enabling it to also discriminate between cells with an intact cell membrane and the ability to actively maintain bacterial homeostasis and cells that have an intact membrane but are metabolically inactive., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In bacteria, animals, fungi, and many single celled eukaryotes, division is initiated by the formation of a ring of cytoskeletal protein at the nascent division site. In bacteria, the tubulin-like GTPase FtsZ serves as the foundation for the cytokinetic ring. A conserved feature of FtsZ is an intrinsically disordered peptide known as the C-terminal linker. Chimeric experiments suggest the linker acts as a flexible boom allowing FtsZ to associate with the membrane through a conserved C-terminal domain and also modulates interactions both between FtsZ subunits and between FtsZ and modulatory proteins in the cytoplasm., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Unraveling the structure of microbial cells is a major challenge in current microbiology and offers exciting prospects in biomedicine. Atomic force microscopy (AFM) appears as a powerful method to image the surface ultrastructure of live cells under physiological conditions and allows real-time imaging to follow dynamic processes such as cell growth, and division and effects of drugs and chemicals. The following chapter introduces different methods of sample preparation to gain insights into the microbial cell organization. Successful strategies to immobilize microorganisms, including physical entrapment and chemical attachment, are described. This step is a key step and a prerequisite of any analysis and persists as an important limitation to the application of AFM to microbiology due to the wide diversity of microorganisms. Finally, some applications are depicted which underlie the ability of AFM to explore living microbes with unprecedented resolution., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To understand the underlying mechanisms of bacterial infections, researchers have for long addressed the molecular interactions occurring when the bacterium interacts with host target cells. In these studies, primarily based on in vitro systems, molecular details have been revealed along with increased knowledge regarding the general infection process. With the recent advancements in in vivo imaging techniques, we are now in a position to bridge a transition from classical minimalistic in vitro approaches to allow infections to be studied in its native complexity-the live organ. Techniques such as multiphoton microscopy (MPM) allow cellular-level visualization of the dynamic infection process in real time within the living host. Studies in which all interplaying factors, such as the influences of the immune, lymphatic, and vascular systems can be accounted for, are likely to provide new insights to our current understanding of the infection process. MPM imaging becomes extra powerful when combined with advanced surgical procedure, allowing studies of the illusive early hours of infection. In this chapter, our intention is to provide a general view on how to design and carry out intravital imaging of a bacterial infection. While exemplifying this using a spatiotemporally well-controlled uropathogenic Escherichia coli (UPEC) infection in rat kidneys, we hope to provide the reader with general considerations that can be adapted to other bacterial infections in organs other than the kidney., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The goal of nanomaterials' surface modification using a biomaterial is to preserve the materials' bulk properties while modifying only their surface to possess desired recognition and specificity. Here, we have developed a phase-assisted, modified Brust-Schiffrin methodological synthesis of metallo nanocomplexes anchored by a peptide, N,N'-(1,3-propylene)-bis-hippuricamide. The spectral, thermal and morphological characterizations assure the formation of nanocomplexes. Therapeutic behavior of all the nanocomplexes has been well sighted by evaluating their DNA unpacking skills. In addition, we demonstrate their biological inspiration by targeting few bacterial and fungal strains. The in vitro antimicrobial investigation reports that all the nanocomplexes disrupt microbial cell walls/membranes efficiently and inhibit the growth of microbes. These sorts of nanocomplexes synthesized in large quantities and at low cost, deliver versatile biomedical applications, and can be used to treat various diseases which may often cause high mortality., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bacterial cell growth is a complex process consisting of two distinct phases: cell elongation and septum formation prior to cell division. Although bacteria have evolved several different mechanisms for cell growth, it is clear that tight spatial and temporal regulation of peptidoglycan synthesis is a common theme. In this review, we discuss bacterial cell growth with a particular emphasis on bacteria that utilize tip extension as a mechanism for cell elongation. We describe polar growth among diverse bacteria and consider the advantages and consequences of this mode of cell elongation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Using food and commensal lactic acid bacteria (LAB) as vehicles for DNA delivery into epithelial cells is a new strategy for vaccine delivery or gene therapy. However, present methods for DNA delivery with LAB have suffered low efficiency. Our goal was to develop a new system to deliver DNA into epithelial cells with high efficiency using food and commensal LAB. An Escherichia coli-LAB shuttle plasmid, pLKV1, for DNA delivery into eukaryotic cells was constructed. Two reporter plasmids with green and red fluorescent protein genes were also constructed to monitor the uptake of protein and DNA, respectively. Bacteria delivering these reporter plasmids into Caco-2 cells were monitored by fluorescence microscopy. Several methods that weaken the bacterial cell wall prior to co-culture with Caco-2 cells were evaluated for their role in the improvement of gene transfer efficiency. Treating Streptococcus gordonii with penicillin and lysozyme greatly increased its rate of gene delivery to mammalian cells compared to untreated control bacteria, while glycine pretreatment promoted the highest gene transfer rate for Lactococcus lactis. Uptake of green fluorescent bacteria by Caco-2 cells showed that the cell wall-weakening treatment promoted the internalization of the noninvasive bacteria into Caco-2 cells. In conclusion, we have developed a noninvasive system using LAB as a vehicle for vaccine delivery or gene therapy, and tested this system in vitro with Caco-2 cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aerobic methanotrophs are metabolically unique bacteria that are able to utilize methane and some other C1-compounds as sole sources of carbon and energy. A defining characteristic of these organisms is the use of methane monooxygenase (MMO) enzymes to catalyze the oxidation of methane to methanol. For a long time, all methanotrophs were considered to be obligately methylotrophic, that is, unable to grow on compounds containing C-C bonds. This notion has recently been revised. Some members of the genera Methylocella, Methylocystis, and Methylocapsa are now known to be facultative methanotrophs, that is, capable of growing on methane as well as on some multicarbon substrates. The diagnosis of facultative methanotrophy in new isolates requires a great degree of caution since methanotrophic cultures are frequently contaminated by heterotrophic bacteria that survive on metabolic by-products of methanotrophs. The presence of only a few satellite cells in a culture may lead to false conclusions regarding substrate utilization, and several early reports of facultative methanotrophy are likely attributable to impure cultures. Another recurring mistake is the misidentification of nonmethanotrophic facultative methylotrophs as facultative methanotrophs. This chapter was prepared as an aid to avoid both kinds of confusion when examining methanotrophic isolates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A critical and irreversible step in the cell division cycle is cytokinesis which physically separates the two daughter cells. This event is consequently subject to tight spatial and temporal regulation. This review focuses on the spatial regulatory mechanisms controlling the position of the division plane. Studies performed in prokaryotic and eukaryotic systems have revealed that various signal-emitting spatial cues - mitotic spindle, nucleus, nucleoid or cell tips - can favour or inhibit the assembly of the cytokinetic apparatus in their vicinity. Most often, several mechanisms operate in parallel to integrate spatial information and promote faithful genome segregation as well as proper cytoplasmic division. We primarily describe the spatial regulatory mechanisms operating in the fission yeast model system, where a detailed molecular understanding of cytokinesis has been achieved. In this system, spatial regulations target a major factor controlling the position of the division plane, the anillin-like protein Mid1. These mechanisms are then compared to spatial regulatory mechanisms prevailing in animal cells and rod-shaped bacteria., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Immunoaffinity is an established chromatographic method for isolating macromolecules independently on the presence of specific tags while the tight interaction between antigen and antibody has been exploited to stabilize proteins during crystallization trials. Therefore, it seems reasonable to try to combine the two protocols, namely to co-express the target proteins together with their specific antibodies to obtain stable complexes suitable for direct purification and further analyses. Using the variable region of single domain llama antibodies, we showed that the co-expression of antigen-antibody pairs is feasible in both the periplasm and the cytoplasm of bacteria. Moreover, the complexes that were formed in vivo could be purified using a tag fused to the recombinant antibody and remained stable during gel-filtration. The co-expression and co-purification strategy significantly increased the final protein yields promoting the accumulation of functional intrabodies. The described method may offer a suitable alternative for the purification of proteins intended for crystallization trials and it may also be used as a general purification protocol for both antigens and recombinant antibodies., (Copyright 2010 Elsevier Inc. All rights reserved.)

Living cells can be considered as a highly efficient molecular engines spatially enclosed, remaining however fragile. By combining cells with silica materials in an appropriate way, novel living hybrid material technologies can be designed. After showing the real interplay between silica species and living organisms in nature, this featuring article summarizes the considerable progress in cell encapsulation into silica matrixes. Generally speaking, bioencapsulation allows protecting cells from harsh environment and controlling their surrounding as well as their concentration. This combination produces ultimately a device that can be oriented to drive the desired biochemical reactions. Particularly, this article highlights that functional living matters are very promising in the development of new eco-friendly processes. Compared to conventional chemical process, these hybrid systems would be enabled to use greater and in more efficient way renewable resources (i.e. solar energy) to produce a vast array of chemicals. Additionally, encapsulated cell technology has opened the possibility to design various other kinds of bioactive materials such as cleaning systems, biosensors and artificial organs. Through different examples, including the immobilization of microorganisms, photosynthetic organelles, plant cells and animal cells, the interests and the preparation methods of these living hybrid materials are discussed., (Copyright 2009 Elsevier Inc. All rights reserved.)

L-forms are "cell wall-deficient" bacteria which are able to grow as spheroplasts or protoplasts. They can be differentiated into four types depending on their ability to revert to the parental, cell-walled form and to the extent of their cell-wall modification. L-forms are significant in modern science because of their contributions to an improved understanding of principal questions and their interactions with eukaryotes. This review particularly focuses on research using stable protoplast-type L-forms which have contributed to a better understanding of the structural and functional organisation of the cytoplasmic membrane and of cell division. These L-forms, which have only a single surrounding bilayer membrane, also represent a unique expression system for production of recombinant proteins. A large proportion of L-form publications concern their putative role in human disease and its therapy, a topic which is discussed briefly. L-forms have also been used to form intracellular associations with plant cells and have been shown to elicit induced disease resistance offering a novel method for plant protection. The recent decline in active research on L-forms is a concern as knowledge and experience, as well as unique L-form strains which have been maintained for decades, are being lost.

The recent development of cell biology techniques for bacteria to allow visualization of fundamental processes in time and space, and their use in synchronous populations of cells, has resulted in a dramatic increase in our understanding of cell division and its regulation in these tiny cells. The first stage of cell division is the formation of a Z ring, composed of a polymerized tubulin-like protein, FtsZ, at the division site precisely at midcell. Several membrane-associated division proteins are then recruited to this ring to form a complex, the divisome, which causes invagination of the cell envelope layers to form a division septum. The Z ring marks the future division site, and the timing of assembly and positioning of this structure are important in determining where and when division will take place in the cell. Z ring assembly is controlled by many factors including negative regulatory mechanisms such as Min and nucleoid occlusion that influence Z ring positioning and FtsZ accessory proteins that bind to FtsZ directly and modulate its polymerization behavior. The replication status of the cell also influences the positioning of the Z ring, which may allow the tight coordination between DNA replication and cell division required to produce two identical newborn cells.

Energy-filtering transmission electron microscopy (EFTEM) allows the determination of elemental distributions out of a sequence of energy filtered images. Combined with electron tomography, EFTEM is a powerful tool to obtain three-dimensional chemical maps from sub-cellular structures. However, there is no existing software in the public-domain for the computation and analysis of 3D-chemical maps. Here, we present a Java-based program to compute 3D-elemental distribution. This program is available as a set of plug-ins for the public-domain Java image processing program Image J inspired by NIH Image. Its implemented algorithms have been successfully applied to the three-dimensional localization of iron granules in semi thin (200 nm) epon sections from the vent worm Riftia pachyptalia.

This work aims at developing a more accurate measurement of the physical parameters of fractal dimension and the size distribution of large fractal aggregates by small-angle light scattering. The theory of multiple scattering has been of particular interest in the case of fractal aggregates for which Rayleigh theory is no longer valid. The introduction of multiple scattering theory into the interpretation of scattering by large bacterial aggregates has been used to calculate the fractal dimension and size distribution. The fractal dimension is calculated from the form factor F(q) at large scattering angles. At large angles the fractal dimension can also be computed by considering only the influence of the very local environment on the optical contrast around a subunit. The fractal dimensions of E. coli strains flocculated with two different cationic polymers have been computed by two techniques: static light scattering and confocal image analysis. The fractal dimensions calculated with both techniques at different flocculation times are very similar: between 1.90 and 2.19. The comparison between two completely independent techniques confirms the theoretical approach of multiple scattering of large flocs using the Mie theory. Size distributions have been calculated from light-scattering data taking into account the linear independence of the structure factor S(q) relative to each size class and using the fractal dimension measured from F(q) in the large-angle range or from confocal image analysis. The results are very different from calculations made using hard-sphere particle models. The size distribution is displaced toward the larger sizes when multiple scattering is considered. Using this new approach to the analysis of very large fractal aggregates by static light multiple scattering, the fractal dimension and size distribution can be calculated using two independent parts of the scattering curve.

In the turgid cells of plants, protists, fungi, and bacteria, walls resist swelling; they also confer shape on the cell. These two functions are not unrelated: cell physiologists have generally agreed that morphogenesis turns on the deformation of existing wall and the deposition of new wall, while turgor pressure produces the work of expansion. In 1990, I summed up consensus in a phrase: "localized compliance with the global force of turgor pressure." My purpose here is to survey the impact of recent discoveries on the traditional conceptual framework. Topics include the recognition of a cytoskeleton in bacteria; the tide of information and insight about budding in yeast; the role of the Spitzenkörper in hyphal extension; calcium ions and actin dynamics in shaping a tip; and the interplay of protons, expansins and cellulose fibrils in cells of higher plants., (Copyright 2002 Elsevier Science (USA))

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations., (Copyright 2000 Academic Press.)

Membrane potential (delta psi) is generated and maintained by concentration gradients of ions such as sodium, potassium, chloride, and hydrogen. Changes in cytoplasmic delta psi in the course of surface-receptor-mediated processes related to the development, function, and pathology of many cell types often play a role in transmembrane signaling. Cytoplasmic delta psi is also reduced to zero when the membrane is ruptured by chemical or physical agents. Mitochondrial delta psi is reduced when energy metabolism is disrupted, notably in apoptosis. In bacteria, which lack mitochondria, delta psi reflects both the state of energy metabolism and the physical integrity of the cytoplasmic membrane. Flow cytometry can be used to estimate membrane potential in eukaryotic cells, mitochondria in situ, isolated mitochondria, and bacteria. Older methods, using lipophilic cationic dyes such as the cyanines and rhodamine 123 or lipophilic anionic dyes such as the oxonols can detect relatively large changes in delta psi and identify heterogeneity of response in subpopulations comprising substantial fractions of a cell population. Newer ratiometric techniques allow precise measurement of delta psi to within 10 mV or less. Among other factors, action of efflux pumps, changes in membrane structure, and changes in protein or lipid concentration in the medium in which cells are suspended can produce changes in cellular fluorescence which may be misinterpreted as changes in delta psi. Techniques for estimation and measurement of Delta Psi therefore typically require careful control of cell and reagent concentrations and incubation times and selection of appropriate controls if they are to provide accurate information., (Copyright 2000 Academic Press.)

Renewed interest in the relationships between viability and culturability in bacteria stems from three sources: (1) the recognition that there are many bacteria in the biosphere that have never been propagated or characterized in laboratory culture; (2) the proposal that some readily culturable bacteria may respond to certain stimuli by entering a temporarily non-culturable state termed 'viable but non-culturable' (VBNC) by some authors; and (3) the development of new techniques that facilitate demonstration of activity, integrity and composition of non-culturable bacterial cells. We review the background to these areas of interest emphasizing the view that, in an operational context, the term VBNC is self-contradictory (Kell et al., 1998) and the likely distinctions between temporarily non-culturable bacteria and those that have never been cultured. We consider developments in our knowledge of physiological processes in bacteria that may influence the outcome of a culturability test (injury and recovery, ageing, adaptation and differentiation, substrate-accelerated death and other forms of metabolic self-destruction, prophages, toxin-antitoxin systems and cell-to-cell communication). Finally, we discuss whether it is appropriate to consider the viability of individual bacteria or whether, in some circumstances, it may be more appropriate to consider viability as a property of a community of bacteria.

The bacterial flagellum is a complex multicomponent structure which serves as the propulsive organelle for many species of bacteria. Rotation of the helical flagellar filament, driven by a proton-powered motor embedded in the cell wall, enables the flagellum to function as a screw propeller. It seems likely that almost all of the genes required for flagellar formation and function have been identified. Continuing analysis of the portions of the genome containing these genes may reveal the existence of a few more. Transcription of the flagellar genes is under the control of the products of a single operon, and so these genes constitute a regulon. Other controls, both transcriptional and post-transcriptional, have been identified. Many of these genes have been sequenced, and the information obtained will aid in the design of experiments to clarify the various regulatory mechanisms of the flagellar regulon. The flagellum is composed of several substructures. The long helical filament is connected via the flexible hook to the complex basal body which is located in the cell wall. The filament is composed of many copies of a single protein, and can adopt a number of distinct helical forms. Structural analyses of the filament are adding to our understanding of this dynamic polymer. The component proteins of the hook and filament have all been identified. Continuing studies on the structure of the basal body have revealed the presence of several hitherto unknown basal-body proteins, whose identities and functions have yet to be elucidated. The proteins essential for energizing the motor, the Mot and switch proteins, are thought to exist as multisubunit complexes peripheral to the basal body. These complexes have yet to be identified biochemically or morphologically. Not surprisingly, flagellar assembly is a complex process, occurring in several stages. Assembly occurs in a proximal-to-distal fashion; the basal body is assembled before the hook, and the hook before the filament. This pattern is also maintained within the filament, with monomers added at the distal end of the polymer; the same is presumably true of the other axial components. An exception to this general pattern is assembly of the Mot proteins into the motor, which appears to be possible at any time during flagellar assembly. With the identification of the genes encoding many of the flagellar proteins, the roles of these proteins in assembly is understood, but the function of a number of gene products in flagellar formation remains unknown.(ABSTRACT TRUNCATED AT 400 WORDS)