A novel approach to cell immobilization with filamentous Escherichia coli

Summary The presence of 18-crown-6 in growth medium produces filamentous Escherichia coli of up to 200 times the normal cell length. The immobilization of these filamentous cells by confinement enables a choice of membranes of larger pore size which in turn speeds up the flow rate of the system. A novel approach of using filamentous cells to facilitate cell immobilization by confinement is proposed.     

Role of microtubules in tip growth of fungi

Polarized cell growth is observed ubiquitously in all living organisms. Tip growth of filamentous fungi serves as a typical model for polar growth. It is well known that the actin cytoskeleton plays a central role in cellular growth. In contrast, the role of microtubules in polar growth of fungal tip cells has not been critically addressed. Our recent study, using a green fluorescent protein (GFP)-labeled tubulin-expressing strain of the filamentous fungus Aspergillus nidulans and treatment with an anti-microtubule reagent, revealed that microtubules are essential for rapid hyphal growth. Our results indicated that microtubule organization contributes to continuous tip growth throughout the cell cycle, which in turn enables the maintenance of an appropriate mass of cytoplasm for the multinucleate system. In filamentous fungi, the microtubule is an essential component of the tip growth machinery that enables continuous and rapid growth. Recent research developments are starting to elucidate the components of the tip growth machinery and their functions in many organisms. This recent knowledge, in turn, is starting to enhance the importance of fungal systems as simple model systems to understand the polar growth of cells.     

Mixed culture biooxidation of phenol. I. Determination of kinetic parameters

A mixed culture derived from soil and activated sludge organisms was used to degrade phenol which was inhibitory to microorganisms at higher concentrations. The purpose of the experiments was to determine the kinetic parameters governing growth of the organisms by measuring growth rates in batch culture. To maintain a constant inoculum for the experiments inoculum was taken from a continuously operating continuous culture. Two populations were studied corresponding to two separate residence times in the continuous culture apparatus. One contained predominantly filamentous organisms, the other nonfilamentous. Five kinetic models were applied to the data and the best kinetic parameters for each model were determined by nonlinear least squares techniques. The models were then evaluated for best relative fit to the data. No significant differences were found between the models on the basis of fit and so a choice was made on the grounds of simplicity. A model proposed by Haldane was chosen as the best. No function however gave a satisfactory fit at the highest growth rates obtained. This experimental maximum in the plot of growth rate against substrate concentration was very sharp.     

Growth During the Bacterial Cell Cycle: Analysis of Cell Size Distribution

Cell volume distributions were determined electronically for steady-state cultures of Escherichia coli, Bacillus megaterium, Bacillus subtilis, and Salmonella typhimurium by use of a Coulter transducer-multichannel analyzer system of good resolution. All of the cell volume distributions had the same general shape, even though cultures were grown at widely different rates. Some results were independent of any particular growth model. Both the variability in the volumes of dividing cells and the fraction of constricted and unseparated doublet cells increased with growth rate. The greater separation to single cells at slow growth rates is in agreement with the general finding that filamentous and hyphal forms are greatly reduced in slowly growing chemostat cultures. The distributions were fitted equally well by simple models which assumed that cell growth was either linear or exponential throughout the entire cell cycle. It is concluded that methods of determining growth rate by analysis of distributions of bacterial volumes do not yet have sufficient resolution to distinguish between a variety of alternative models for growth of bacteria.     

The Role of Light in the Interrelated Processes of Morphogenesis and Photosynthesis in the Fern Gametophyte

The interrelationship of photosynthesis and photomorphogenesis in the gametophyte of the fern Pteridium aquilinum was examined in detail. High energy red light produces filamentous gametophytes composed of greatly elongated cells. Blue light of equal energy produces normal cordate gametophytes composed of isodiametric cells. The assimilative rates of the two forms are identical during the initial stages of development. However, the filamentous gametophytes eventually exhibit a decline in assimilation not matched by their normal counterparts. Although this decrease in assimilation of gametophytes exposed to red light may be traced to a decrease in the photo-synthetic rate due to a decrease in chlorophyll synthesis, these events occur subsequent to the establishment of the filamentous mode of growth and are therefore a result, not a cause, of filamentous development. Gametophyte assimilation is also impeded under low energy blue light, due to a reduction in the photosynthetic rate at this low energy; and the initial filamentous stage, in which the cells remain normally isodiametric, is not exceeded. The addition of extra exogenous sucrose restores the capacity for growth and normal cordate prothalli result. It may therefore be concluded that the ultimate form of the gametophyte is under the control of a photoreceptor that is sensitive to blue light, and activated at a relatively low energy level. Red light at any level is ineffective in promoting normal cordate development, but is effective in supporting growth through vigorous photosynthesis, yielding a filament composed of greatly elongated cells. The eventual photosynthetic decline of the filamentous form is due directly to this particular growth mode. Low energy blue light is sufficient to effect form (the cells remain isodiametric), but insufficient to provide the necessary energy for growth through vigorous photosynthesis. Therefore, in order for the gametophyte to reach maturity it must be supplied with light energy that not only satisfies the requirement for form but the requirement for growth as well. The implications of these requirements are discussed in the text.     

Growth mechanisms and growth kinetics of filamentous microorganisms

Filamentous microorganisms are of major biotechnological importance, being responsible for production of the majority of secondary metabolites, particularly antibiotics. Two main groups are involved, filamentous fungi and filamentous actinomycetes, particularly the streptomycetes. In terms of cellular growth mechanisms, these groups differ greatly. Eukaryotic fungi possess subcellular organelles and cytoskeletal structures directing growth while prokaryotic streptomycetes have no such cellular organization. Despite these fundamental differences, both groups exhibit similar morphologies, growth patterns, growth forms, and hyphal and mycelial growth kinetics on solid media and in liquid culture both grow as dispersed mycelia and pellets. The article therefore discusses the relationship between cellular growth mechanisms and vegetative growth in both filamentous fungi and actinomycetes, the conceptual and theoretical models applicable to both groups, and the significance of such models in industrial fermentation processes.     

A study of the response properties of retinula cells of flies using nonlinear identification theory

The functional properties of retinula cells of the fly Calliphora crythrocephala (wild type) have been determined through the application of nonlinear identification theory using white-gaussian stimulus functions. These results are also compared to similiar recordings of lamina cells. The accuracy of the resulting models is shown by the fact that those obtained from just 30 sec tests predict the actual total responses to the white-gaussian stimuli with a mean square error of about 5% and for a 2200 sec test the error is reduced to 2%. It has been shown that the second order kernels define all of the nonlinear properties and are the primary terms in the models for describing the variations in functional responses with illumination level, changing adaptation conditions and even variations in the conditions of the intracellular preparations.     

An updated perspective on the role of environmental autocorrelation in animal populations

Ecological theory predicts that the presence of temporal autocorrelation in environments can considerably affect population extinction risk. However, empirical estimates of autocorrelation values in animal populations have not decoupled intrinsic growth and density feedback processes from environmental autocorrelation. In this study, we first discuss how the autocorrelation present in environmental covariates can be reduced through nonlinear interactions or by interactions with multiple limiting resources. We then estimated the degree of environmental autocorrelation present in the Global Population Dynamics Database using a robust, model-based approach. Our empirical results indicate that time series of animal populations are affected by low levels of environmental autocorrelation, a result consistent with predictions from our theoretical models. Claims supporting the importance of autocorrelated environments have been largely based on indirect empirical measures and theoretical models seldom anchored in realistic assumptions. It is likely that a more nuanced understanding of the effects of autocorrelated environments is necessary to reconcile our conclusions with previous theory. We anticipate that our findings and other recent results will lead to improvements in understanding how to incorporate fluctuating environments into population risk assessments.     

Isolation of UmRrm75, a gene involved in dimorphism and virulence of Ustilago maydis

Ustilago maydis displays dimorphic growth, alternating between a saprophytic haploid yeast form and a filamentous dikaryon, generated by mating of haploid cells and which is an obligate parasite. Induction of the dimorphic transition of haploid strains in vitro by change in ambient pH has been used to understand the mechanisms governing this differentiation process. In this study we used suppression subtractive hybridization to generate a cDNA library of U. maydis genes up-regulated in the filamentous form induced in vitro at acid pH. Expression analysis using quantitative RT-PCR showed that the induction of two unigenes identified in this library coincided with the establishment of filamentous growth in the acid pH medium. This expression pattern suggested that they were specifically associated to hyphal development rather than merely acid pH-induced genes. One of these genes, UmRrm75, encodes a protein containing three RNA recognition motifs and glycine-rich repeats and was selected for further study. The UmRrm75 gene contains 4 introns, and produces a splicing variant by a 3'-alternative splicing site within the third exon. Mutants deleted for UmRrm75 showed a slower growth rate than wild type strains in liquid and solid media, and their colonies showed a donut-like morphology on solid medium. Interestingly, although ΔUmRrm75 strains were not affected in filamentous growth induced by acid pH and oleic acid, they exhibited reduced mating, post-mating filamentous growth and virulence. Our data suggest that UmRrm75 is probably involved in cell growth, morphogenesis, and pathogenicity in U. maydis.     

A model for one-dimensional morphoelasticity and its application to fibroblast-populated collagen lattices

The mechanical behaviour of solid biological tissues has long been described using models based on classical continuum mechanics. However, the classical continuum theories of elasticity and viscoelasticity cannot easily capture the continual remodelling and associated structural changes in biological tissues. Furthermore, models drawn from plasticity theory are difficult to apply and interpret in this context, where there is no equivalent of a yield stress or flow rule. In this work, we describe a novel one-dimensional mathematical model of tissue remodelling based on the multiplicative decomposition of the deformation gradient. We express the mechanical effects of remodelling as an evolution equation for the effective strain, a measure of the difference between the current state and a hypothetical mechanically relaxed state of the tissue. This morphoelastic model combines the simplicity and interpretability of classical viscoelastic models with the versatility of plasticity theory. A novel feature of our model is that while most models describe growth as a continuous quantity, here we begin with discrete cells and develop a continuum representation of lattice remodelling based on an appropriate limit of the behaviour of discrete cells. To demonstrate the utility of our approach, we use this framework to capture qualitative aspects of the continual remodelling observed in fibroblast-populated collagen lattices, in particular its contraction and its subsequent sudden re-expansion when remodelling is interrupted.     

Intracellular development of Bdellovibrio bacteriovorus

The intracellular growth of Bdellovibrio bacteriovorus, a bacterial parasite, was studied by a light-optical method using time-lapse cinemicrography. The organism was found to be capable of growth in the periplasmic space of filamentous cells of the host bacterium Pseudomonas fluorescens without any contact with the cytoplasmic membrane. Several B. bacteriovorus cells could grow simultaneously in the bdelloplasm.     

The relationship between neuronal calcium concentration and firing rate during stochastic synaptic inputs

We propose a novel, nonlinear theory about reading neuronal information using intracellular calcium concentrations, which includes the linear theory already developed in the literature as a special case. The theory is numerically confirmed using the Pinsky-Rinzel and integrate-and-fire models with constant rate Poisson inputs. Applying the theory to models with non-constant inputs, we find that there is a time lag equal to the calcium buffering time constant between the instantaneous firing rate and the firing rate estimated using calcium concentrations.     

Single live cell imaging of chromosomes in chloramphenicol-induced filamentous Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading opportunistic pathogen in human infections, and it is renowned for its intrinsic resistance to structurally and functionally unrelated antibiotics. Filamentation induced by antibiotics appears to trigger bacteria to depart from a normal growth phase and enter a stationary growth phase. As antibiotic concentrations decline below a therapeutic range, filamentous bacteria begin to divide normally, leading to a more rapid regrowth of the bacteria. Furthermore, filamentous bacteria are associated with an increase in endotoxin release. Moreover, the immune system of a patient needs to cope with uncharacteristic filamentous bacteria. Thus, it is biologically and clinically significant to study and understand bacterial filamentation. In this study, we investigate the frequencies, conditions, and characteristics of a filamentous P. aeruginosa at single cell and single chromosome resolutions. Our results show that filamentous cells (elongated rods) contain multiple copies of the cell's chromosome. It appears that the unsuccessful segregation of replicated chromosomes in an individual cell accompanies the formation of undivided filamentous cells. The quantity of chromosomes and the length of the filamentous wild-type cells increase as the chloramphenicol concentration increases to 50 and 250 microg/mL, suggesting that chloramphenicol induces the filamentation. Filamentation in three strains of P. aeruginosa depends on the expression level of efflux pump (MexAB-OprM) and the minimum inhibitory concentration of chloramphenicol. This study also opens up the new possibility of real-time monitoring of modes of actions of antibiotics in live cells with both temporal and spatial resolution.     

Overexpression of autophagy-related genes inhibits yeast filamentous growth

Under conditions of nitrogen stress, the budding yeast S. cerevisiae initiates a cellular response involving the activation of autophagy, an intracellular catabolic process for the degradation and recycling of proteins and organelles. In certain strains of yeast, nitrogen stress also drives a striking developmental transition to a filamentous form of growth, in which cells remain physically connected after cytokinesis. We recently identified an interrelationship between these processes, with the inhibition of autophagy resulting in exaggerated filamentous growth. Our results suggest a model wherein autophagy mitigates nutrient stress, and filamentous growth is responsive to the degree of this stress. Here, we extended these studies to encompass a phenotypic analysis of filamentous growth upon overexpression of autophagy-related (ATG) genes. Specifically, overexpression of ATG1, ATG3, ATG7, ATG17, ATG19, ATG23, ATG24 and ATG29 inhibited filamentous growth. From our understanding of autophagy in yeast, overexpression of these genes does not markedly affect the activity of the pathway; thus, we do not expect that this filamentous growth phenotype is due strictly to diminished nitrogen stress in ATG overexpression mutants. Rather, these results highlight an additional undefined regulatory mechanism linking autophagy and filamentous growth, possibly independent of the upstream nitrogen-sensing machinery feeding into both processes.     

Stochastic nucleoid segregation dynamics as a source of the phenotypic variability in E. coli

Segregation of the replicating chromosome from a single to two nucleoid bodies is one of the major processes in growing bacterial cells. The segregation dynamics is tuned by intricate interactions with other cellular processes such as growth and division, ensuring flexibility in a changing environment. We hypothesize that the internal stochasticity of the segregation process may be the source of cell-to-cell phenotypic variability, in addition to the well-established gene expression noise and uneven partitioning of low copy number components. We compare dividing cell lineages with filamentous cells, where the lack of the diffusion barriers is expected to reduce the impact of other factors on the variability of nucleoid segregation dynamics. The nucleoid segregation was monitored using time-lapse microscopy in live E. coli cells grown in linear grooves. The main characteristics of the segregation process, namely, the synchrony of partitioning, rates of separation, and final positions, as well as the variability of these characteristics, were determined for dividing and filamentous lineages growing under the same conditions. Indeed, the gene expression noise was considerably homogenized along filaments as determined from the distribution of CFP and YFP stochastically expressed from the chromosome. We find that 1) the synchrony of nucleoid partitioning is progressively decreasing during consecutive cell cycles, but to a significantly lesser degree in filamentous than in dividing cells; 2) the mean partitioning rate of nucleoids is essentially the same in dividing and filamentous cells, displaying a substantial variability in both; and 3) nucleoids segregate to the same distances in dividing and filamentous cells. Variability in distances is increasing during successive cell cycles, but to a much lesser extent in filamentous cells. Our findings indicate that the variability of the chromosome segregation dynamics is reduced upon removal of boundaries between nucleoids, whereas the remaining variability is essentially inherent to the nucleoid itself.     

A nonlinear structured population model of tumor growth with quiescence

A nonlinear structured cell population model of tumor growth is considered. The model distinguishes between two types of cells within the tumor: proliferating and quiescent. Within each class the behavior of individual cells depends on cell size, whereas the probabilities of becoming quiescent and returning to the proliferative cycle are in addition controlled by total tumor size. The asymptotic behavior of solutions of the full nonlinear model, as well as some linear special cases, is investigated using spectral theory of positive simigroup of operators. Supported in part by the National Science Foundation under Grant No. DMS-8722947     

Lipid-induced filamentous growth in Ustilago maydis

The phytopathogenic fungus Ustilago maydis is obligately dependent on infection of maize to complete the sexual phase of its life cycle. Mating interactions between haploid, budding cells establish an infectious filamentous cell type that invades the host, induces large tumours and eventually forms large masses of black spores. The ability to switch from budding to filamentous growth is therefore critical for infection and completion of the life cycle, although the signals that influence the transition have not been identified from the host or the environment. We have found that growth in the presence of lipids promotes a filamentous phenotype that resembles the infectious cell type found in planta. In addition, the ability of the fungus to respond to lipids is dependent on both the cAMP signalling pathway and a Ras/MAPK pathway; these pathways are known to regulate mating, filamentous growth and pathogenesis in U. maydis. Overall, these results lead us to hypothesize that lipids may represent one of the signals that promote and maintain the filamentous growth of the fungus in the host environment.     

Nonlinear relativistic quantum theory of Cherenkov emission of longitudinal Langmuir waves in a plasma

A nonlinear relativistic quantum theory of stimulated Cherenkov emission of longitudinal waves by a relativistic monoenergetic electron beam in a cold isotropic plasma is presented. The theory makes use of a quantum model based on the Klein-Gordon equation. The instability growth rates are obtained in the linear approximation and are shown to go over to the familiar growth rates in the classical limit. The mechanisms for the nonlinear saturation of relativistic Cherenkov beam instabilities are described with allowance for quantum effects, and the corresponding analytic solutions are derived.     

Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p

The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Delta mutants for growth at 37 degrees C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans.