Unidirectional polar growth of cells of Seliberia stellata and aquatic seliberia-like bacteria revealed by immunoferritin labeling

When grown in a complex peptone-yeast extract culture medium, Seliberia stellata and related morphologically similar aquatic bacterial strains typically divided asymmetrically, giving rise to a motile swarmer and a longer sessile rod. Indirect immunoferritin labeling of these bacteria, followed by incubation during which cell growth occurred, has provided evidence that antigenic cell-surface components are synthesized de novo in a sharply demarcated zone at one pole of the growing parent cells. Cell elongation occurred unidirectionally from the pole showing the de novo surface synthesis; it was this end of the elongating, helically sculptured (i.e., screw-like) rod that became the daughter swarmer cell. The daughter swarmers, produced after polar growth and division of the immunoferritinlabeled parent cells, were not labeled. The immunoferritin label remaining on the parent cell did not appear to be diluted or disturbed by the cell growth and division process. Under the cultural conditions used in this study, the growth and division events which led to production of swarmer cells in the seliberia strains examined met two major criteria of accepted definitions of budding (de novo cell surface synthesis and transverse asymmetry of division). However, the developing daughter cell was not initially narrower than the parent and thus did not increase in cell diameter during growth.In memory: R. Y. Stanier     

Pattern of unequal cell division and development in Caulobacter crescentus

The pattern of asymmetric division has been examined in Caulobacter crescentus (gram-negative aquatic bacteria) by determining the position of the “division site” on cells of different ages. Measurements of cell width and length at this site, which corresponds to the point of eventual cell separation, were made on electron micrographs of cells stained with phosphotungstic acid. The results show that (i) the division site is formed early in the cell cycle and it constitutes the first visible feature on the growing stalked cell to differentiate the incipient swarmer cell, (ii) the division site is located asymmetrically (closer to the swarmer pole than the stalked pole) on the dividing cell, (iii) its position relative to the stalked and swarmer poles does not change during the cell cycle, and (iv) division is consequently unequal, with the swarmer cell always smaller than the stalked cell. The implications of these findings for general models of unequal cell division and stem cell development are discussed.     

Synchronous cell differentiation in Caulobacter crescentus.

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Synchronous Cell Differentiation in Caulobacter crescentus

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Surface-induced swarmer cell differentiation of Vibrio parahaemoiyticus

Vibrio parahaemolyticus distinguishes between life in a liquid environment and life on a surface. Growth on a surface induces differentiation from a swimmer cell to a swarmer cell type. Each cell type is adapted for locomotion under different circumstances. Swimmer cells synthesize a single polar flagellum (Fla) for movement in a liquid medium, and swarmer cells produce an additional distinct flagellar system, the lateral flagella (Laf), for movement across a solid substratum, called swarming. Recognition of surfaces is necessary for swarmer cell differentiation and involves detection of physical signals peculiar to that circumstance and subsequent transduction of information to affect expression of swarmer cell genes (laf). The polar flagellum functions as a tactile sensor controlling swarmer cell differentiation by sensing forces that restrict its movement. Surface recognition also involves a second signal, i.e. nutritional limitation for iron. Studying surface-induced differentiation could reveal a novel mechanism of gene control and lead to an understanding of the processes of surface colonization by pathogens and other bacteria.     

In long bacterial cells,the Min system can act off‐center

In many rod‐shaped bacteria, the Min system is well‐known for generating a cell‐pole to cell‐pole standing wave oscillation with a single node at mid‐cell to align cell division. In filamentous E. coli cells, the single‐node standing wave transitions into a multi‐nodal oscillation. These multi‐nodal dynamics have largely been treated simply as an interesting byproduct of artificially elongated cells. However, a recent in vivo study by Muraleedharan et al. shows how multi‐nodal Min dynamics are used to align non‐mid‐cell divisions in the elongated swarmer cells of Vibrio parahaemolyticus. The authors propose a model where the combined actions of cell‐length dependent Min dynamics, in concert with nucleoid occlusion along the cell length and regulation of FtsZ levels ensures Z ring formation and complete chromosome segregation at a single off‐center position. By limiting the number of cell division events to one per cell at an off‐center position, long swarmer cells are preserved within a multiplying population. The findings unveil an elegant mechanism of cell‐division regulation by the Min system that allows long swarmer cells to divide without the need to ‘dedifferentiate’.     

Nuclear Apparatus of Hyphomicrobium

The nuclear apparatus of Hyphomicrobium sp. strain B-522 is examined by various microscopy and radiolabeling techniques to determine its behavior during the reproductive cycle of these bacteria. The young, swarmer cell contains a single nucleoid comprised of a deoxyribonucleic acid (DNA) molecule with a molecular weight of 3.1 x 10(9). After development of the swarmer into a mature mother cell with a hypha and bud, the nucleoid replicates and separates into two daughter nucleoids during the initial stages of bud formation. After further development of the bud, one of the daughter nucleoids in the mother cell is rapidly transferred through the hypha to the bud. Half of the old DNA strands pass to each consecutive generation of daughter cells, but only 43% of the stable ribonucleic acid is transferred. The role which the hypha plays in the developmental cycle of these bacteria is discussed, and a mechanism for nuclear transfer is proposed.     

Comparative genomic evidence for a close relationship between the dimorphic prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus

The dimorphic prosthecate bacteria (DPB) are alpha-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.     

A cell length‐dependent transition in MinD‐dynamics promotes a switch in division‐site placement and preservation of proliferating elongated Vibrio parahaemolyticus swarmer cells

Vibrio parahaemolyticus exists as swimmer and swarmer cells, specialized for growth in liquid and on solid environments respectively. Swarmer cells are characteristically highly elongated due to an inhibition of cell division, but still need to divide in order to proliferate and expand the colony. It is unknown how long swarmer cells divide without diminishing the population of long cells required for swarming behavior. Here we show that swarmer cells divide but the placement of the division site is cell length‐dependent; short swarmers divide at mid‐cell, while long swarmers switch to a specific non‐mid‐cell placement of the division site. Transition to non‐mid‐cell positioning of the Z‐ring is promoted by a cell length‐dependent switch in the localization‐dynamics of the division regulator MinD from a pole‐to‐pole oscillation in short swarmers to a multi‐node standing‐wave oscillation in long swarmers. Regulation of FtsZ levels restricts the number of divisions to one and SlmA ensures sufficient free FtsZ to sustain Z‐ring formation by preventing sequestration of FtsZ into division deficient clusters. By limiting the number of division‐events to one per cell at a specific non‐mid‐cell position, V. parahaemolyticus promotes the preservation of long swarmer cells and permits swarmer cell division without the need for dedifferentiation.     

Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.     

Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus.

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.     

Differential localization of membrane receptor chemotaxis proteins in the Caulobacter predivisional cell

The methyl-accepting chemotaxis proteins (MCPs) are membrane receptors that initiate signal transduction to the flagellar rotor upon ligand binding. The synthesis of these proteins occurs only in the Caulobacter crescentus predivisional cell coincident with the biosynthesis of the polar flagellum. Both the flagellum and the MCPs are partitioned to only one daughter cell, the swarmer cell, upon division. We report the results of experiments designed to determine the distribution of these MCPs within swarmer cells and predivisional cells. Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. Thus, the MCPs are not specifically localized to the immediate vicinity of the flagellar rotor. The distribution of MCPs was examined in flagellated and non-flagellated vesicles isolated from predivisional cells. The analysis of small predivisional vesicles showed that the MCP content is higher in the flagellated vesicles, and analysis of large flagellated vesicles showed that the MCPs are positioned preferentially in the swarmer cell portion of the predivisional cell. This positional bias of MCPs within predivisional cells could reflect either a large compartment or membrane domain within the incipient swarmer cell, or a gradient of MCPs, with the highest concentration in the vicinity of the flagellum.     

Growth and morphology of Asticcacaulis biprosthecum in defined media

The growth and morphology of cells of Asticcacaulis biprosthecum were studied in defined media to determine the effects of various compounds on the growth rate and on the expression of morphological events of the life cycle. The length of prosthecae could not be controlled by varying the concentration of inorganic phosphate as has been shown for other caulobacters. In defined media, growth was inhibited during conditions favoring rapid metabolism, apparently due to an absolute requirement for cells to complete all stages of the life cycle before cell division could occur. The morphology of cells grown under these conditions was aberrant, i.e., cells appeared elongated and branched and few prosthecae or swarmer cells were produced. Growth of a related bacterium, Asticcacaulis strain S-3, was not inhibited by conditions favoring rapid metabolism. During rapid growth, cell division in this organism occurs in the swarmer stage and prosthecae are not produced. Cell division in S-3 is not obligately coupled to completion of all stages in the complex life cycle, and morphogenesis can be controlled by cultural conditions.     

Utilization of amino acids by Chromatium sp. strain D

Summary The duration of various morphologically distinct phases in the division cycle of the marine heterotroph Cryptothecodinium cohnii was measured in cultures initiated with synchronously excysted swarmer cells. Parent cysts were selectively isolated on plastic surfaces and progeny of a narrow age distribution harvested in a specifically conditioned medium. The swarmer phase, an interval of predivisional encystment, daughter cell formation and excystment were 5.0, 3.0 and 2.0 h respectively. Two major kinds of cytokinesis (production of 2 and 4 daughter cells) were observed resulting in a mean daughter cell number of 2.7 under these conditions. Other growth parameters for this dinoflagellate are described.     

Kinetic model of Proteus mirabilis swarm colony development

 Proteus mirabilis colonies display striking symmetry and periodicity. Based on experimental observations of cellular differentiation and group motility, a kinetic model has been developed to describe the swarmer cell differentiation-dedifferentiation cycle and the spatial evolution of swimmer and swarmer cells during Proteus mirabilis swarm colony development. A key element of the model is the age dependence of swarmer cell behaviour, in particular specifying a minimal age for motility and maximum age for septation and dedifferentiation to swimmer cells. Density thresholds for collective motility by mature swarmer cells serve to synchronize the movements of distinct swarmer cell groups and thus help provide temporal coherence to colony expansion cycles. Numerical computations show that the model fits experimental data by generating a complete swarming plus consolidation cycle period that is robust to changes in parameters which affect other aspects of swarmer cell migration and colony development. The kinetic equations underlying this model provide a different mathematical basis for a temporal oscillator from reaction-diffusion partial differential equations. The modelling shows that Proteus colony geometries arise as a consequence of macroscopic rules governing collective motility. Thus, in this case, pattern formation results from the operation of an adaptive bacterial system for spreading on solid substrates, not as an independent biological function. Kinetic models similar to this one may be applicable to periodic phenomena displayed by other biological systems with differentiated components of defined lifetimes. Received 3 July 1996; received in revised form 9 December 1996     

ZapA, the IgA-degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells

The IgA-degrading metalloprotease, ZapA, of the urinary tract pathogen Proteus mirabilis is co-ordinately expressed along with other proteins and virulence factors during swarmer cell differentiation. In this communication, we have used zapA to monitor IgA protease expression during the differentiation of vegetative swimmer cells to fully differentiated swarmer cells. Northern blot analysis of wild-type cells and beta-galactosidase measurements using a zapA:lacZ fusion strain indicate that zapA is fully expressed only in differentiated swarmer cells. Moreover, the expression of zapA on nutrient agar medium is co-ordinately regulated in concert with the cycles of cellular differentiation, swarm migration and consolidation that produce the bull's-eye colonies typically associated with P. mirabilis. ZapA activity is not required for swarmer cell differentiation or swarming behaviour, as ZapA- strains produce wild-type colony patterns. ZapA- strains fail to degrade IgA and show decreased survival compared with the wild-type cells during infection in a mouse model of ascending urinary tract infection (UTI). These data underscore the importance of the P. mirabilis IgA-degrading metalloprotease in UTI. Analysis of the nucleotide sequences adjacent to zapA reveals four additional genes, zapE, zapB, zapC and zapD, which appear to possess functions required for ZapA activity and IgA proteolysis. Based on homology to other known proteins, these genes encode a second metalloprotease, ZapE, as well as a ZapA-specific ABC transporter system (ZapB, ZapC and ZapD). A model describing the function and interaction of each of these five proteins in the degradation of host IgA during UTI is presented.     

Differentiation of Serratia marcescens 274 into swimmer and swarmer cells

We describe a new sensory response in the enteric bacterium Serratia marcescens. When grown in liquid media, the bacteria were short rods with one to two flagella and displayed classical swimming behavior. Upon transfer to a solid surface (0.7 to 0.8T% agar medium), the bacteria underwent a dramatic change of form. They ceased septation, elongated, and expressed numerous (10 to 100) flagella that covered the lateral sides of the cells. The bacteria now displayed a different form of locomotion--swarming--which allowed them to rapidly move over the top of the solid surface. The differentiation to either swimmer or swarmer cells could be reversed by growth on solid or liquid medium, respectively. To identify conditions that influence this differentiation, the growth environment of S. marcescens was manipulated extensively. The swarming response was monitored by visual and microscopic observation of cell movement on solid surfaces, by immunofluorescent labeling followed by microscopic observation for the presence of elongated, profusely flagellated cells, as well as by estimation of induction of flagellin protein, using Western immunoblot analysis. Conditions that imposed a physical constraint on bacterial movement, such as solid or viscous media, were the most efficient at inducing the swarming response. No chemical constituent of the medium that might contribute to the response could be identified, although the existence of such a component cannot be ruled out. Both swimmer and swarmer cells had flagellin proteins of identical molecular weight, which produced similar proteolysis patterns upon digestion with trypsin.     

First generation synchrony of isolated Hyphomicrobium swarmer populations

A method is described for obtaining synchronously growing swarmer cell populations of Hyphomicrobium sp. strain B-522. This was accomplished by isolating young swarmers from random cultures by centrifugation and filtration. Cell multiplication occurred during 38% of the growth cycle in populations synchronized in this manner. Observations were made of the changes in cellular morphology which occurred during the growth cycle. Of the 14.25 h required for the doubling in cell numbers, an average of 5 h passed before the swarmer cells began to develop their hyphae. This time varied over a range of 10 h. The time interval between the beginning of hyphal development and the beginning of bud formation was 3.5 to 4.5 h. The maturation of the first buds and their separation from the mother cells were completed in 5.5 h. The duration of these steps is compared to those measured previously in agar slide cultures.     

ppGpp and polyphosphate modulate cell cycle progression in Caulobacter crescentus

Caulobacter crescentus differentiates from a motile, foraging swarmer cell into a sessile, replication-competent stalked cell during its cell cycle. This developmental transition is inhibited by nutrient deprivation to favor the motile swarmer state. We identify two cell cycle regulatory signals, ppGpp and polyphosphate (polyP), that inhibit the swarmer-to-stalked transition in both complex and glucose-exhausted media, thereby increasing the proportion of swarmer cells in mixed culture. Upon depletion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or polyP improperly initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell fate determinant DivJ, and develop polar stalks. Furthermore, we show that swarmer cells produce more ppGpp than stalked cells upon starvation. These results provide evidence that ppGpp and polyP are cell-type-specific developmental regulators.