Flagellation Pattern Research Articles

Control of the flagellation pattern in Helicobacter pylori by FlhF and FlhG.

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori. Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the ΔflhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliFN255D, but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliFN225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori, FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly.

Interdependent Polar Localization of FlhF and FlhG and Their Importance for Flagellum Formation of Vibrio parahaemolyticus.

Failure of the cell to properly regulate the number and intracellular positioning of their flagella, has detrimental effects on the cells’ swimming ability. The flagellation pattern of numerous bacteria is regulated by the NTPases FlhF and FlhG. In general, FlhG controls the number of flagella produced, whereas FlhF coordinates the position of the flagella. In the human pathogen Vibrio parahaemolyticus, its single flagellum is positioned and formed at the old cell pole. Here, we describe the spatiotemporal localization of FlhF and FlhG in V. parahaemolyticus and their effect on swimming motility. Absence of either FlhF or FlhG caused a significant defect in swimming ability, resulting in absence of flagella in a ΔflhF mutant and an aberrant flagellated phenotype in ΔflhG. Both proteins localized to the cell pole in a cell cycle-dependent manner, but displayed different patterns of localization throughout the cell cycle. FlhF transitioned from a uni- to bi-polar localization, as observed in other polarly flagellated bacteria. Localization of FlhG was strictly dependent on the cell pole-determinant HubP, while polar localization of FlhF was HubP independent. Furthermore, localization of FlhF and FlhG was interdependent and required for each other’s proper intracellular localization and recruitment to the cell pole. In the absence of HubP or FlhF, FlhG forms non-polar foci in the cytoplasm of the cell, suggesting the possibility of a secondary localization site within the cell besides its recruitment to the cell poles.

Flagellation of Shewanella oneidensis Impacts Bacterial Fitness in Different Environments.

Flagella occur on many prokaryotes, which primarily propel cells to move from detrimental to favorable environments. A variety of species-specific flagellation patterns have been identified. Although it is presumed that for each of these flagellated microorganisms, an evolutionarily fixed flagellation pattern is favored under the normal living conditions, direct evidence is lacking. Here, we use Shewanella oneidensis, a rod-shaped Gram-negative bacterium with a monotrichous polar flagellum (MR-1, the wild-type), as a research model. The investigation has been enabled by multiple mutants with diverse flagellation patterns that had been generated by removing FlhF and FlhG proteins that control flagellar location and number, respectively. Growth assays, as a measure of fitness, revealed that the wild-type strain predominated in spreading on swim plates and in pellicles which form at the air-liquid interface. However, under the pellicles where oxygen is limited, both aflagellated and monotrichous lateral strains showed similar increase in fitness, whereas strains with multiple flagella were less competitive. Moreover, under shaking culturing conditions, the aflagellated strain outcompeted all other strains, including the wild-type, suggesting that cells devoid of flagella would be more likely enriched upon agitation. Overall, these data support the presumption that the monotrichous polar flagellum, as evolutionarily fixed in the wild-type strain, is optimal for the growth fitness of S. oneidensis over any other mutants under most test conditions. However, upon specific changes of environmental conditions, another form could come to predominate. These findings provide insight into the impacts of flagellation patterns and function on bacterial adaptation to differing environments.

A Polar Flagellar Transcriptional Program Mediated by Diverse Two-Component Signal Transduction Systems and Basal Flagellar Proteins Is Broadly Conserved in Polar Flagellates.

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

Synthetic Cystic Fibrosis Sputum Medium Regulates Flagellar Biosynthesis through the flhF Gene in Burkholderia cenocepacia.

Burkholderia cenocepacia belongs to the Burkholderia cepacia complex (Bcc), a group of at least 18 distinct species that establish chronic infections in the lung of people with the genetic disease cystic fibrosis (CF). The sputum of CF patients is rich in amino acids and was previously shown to increase flagellar gene expression in B. cenocepacia. We examined flagellin expression and flagellar morphology of B. cenocepacia grown in synthetic cystic fibrosis sputum medium (SCFM) compared to minimal medium. We found that CF nutritional conditions induce increased motility and flagellin expression. Individual amino acids added at the same concentrations as found in SCFM also increased motility but not flagellin expression, suggesting a chemotactic effect of amino acids. Electron microscopy and flagella staining demonstrated that the increase in flagellin corresponds to a change in the number of flagella per cell. In minimal medium, the ratio of multiple: single: aflagellated cells was 2:3.5:4.5; while under SCFM conditions, the ratio was 7:2:1. We created a deletion mutant, ΔflhF, to study whether this putative GTPase regulates the flagellation pattern of B. cenocepacia K56-2 during growth in CF conditions. The ΔflhF mutant exhibited 80% aflagellated, 14% single and 6% multiple flagellated bacterial subpopulations. Moreover, the ratio of multiple to single flagella in WT and ΔflhF was 3.5 and 0.43, respectively in CF conditions. The observed differences suggest that FlhF positively regulates flagellin expression and the flagellation pattern in B. cenocepacia K56-2 during CF nutritional conditions.

FlhG employs diverse intrinsic domains and influences FlhF GTPase activity to numerically regulate polar flagellar biogenesis in Campylobacter jejuni.

Flagellation in polar flagellates is one of the rare biosynthetic processes known to be numerically regulated in bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. Unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni. Instead, two regions of C. jejuni FlhG that are absent or significantly altered in FlhG orthologs are involved in numerical regulation of flagellar biogenesis. Additionally, we found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. Our work suggests that FlhG ATPases divergently evolved in each polarly flagellated species to employ different intrinsic domains and extrinsic effectors to ultimately mediate a common output - precise numerical control of polar flagellar biogenesis required to create species-specific flagellation patterns optimal for motility.

How bacteria maintain location and number of flagella?

Bacteria differ in number and location of their flagella that appear in regular patterns at the cell surface (flagellation pattern). Despite the plethora of bacterial species, only a handful of these patterns exist. The correct flagellation pattern is a prerequisite for motility, but also relates to biofilm formation and the pathogenicity of disease-causing flagellated bacteria. However, the mechanisms that maintain location and number of flagella are far from being understood. Here, we review our knowledge on mechanisms that enable bacteria to maintain their appropriate flagellation pattern. While some peritrichous flagellation patterns might occur by rather simple stochastic processes, other bacterial species appear to rely on landmark systems to define the designated flagellar position. Such landmarks are the Tip system of Caulobacter crescentus or the signal recognition particle (SRP)-GTPase FlhF and the MinD/ParA-type ATPase FlhG (synonyms: FleN, YlxH and MinD2). The latter two proteins constitute a regulatory circuit essential for diverse flagellation patterns in many Gram-positive and negative species. The interactome of FlhF/G (e.g. C-ring proteins FliM, FliN, FliY or the transcriptional regulator FleQ/FlrA) seems evolutionary adapted to meet the specific needs for a respective pattern. This variability highlights the importance of the correct flagellation pattern for motile species.

MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly.

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

From molecular evolution to biobricks and synthetic modules: a lesson by the bacterial flagellum

The bacterial flagellum is a motility structure and represents one of the most sophisticated nanomachines in the biosphere. Here, we review the current knowledge on the flagellum, its architecture with respect to differences between Gram-negative and Gram-positive bacteria and other species-specific variations (e.g. the flagellar filament protein, Flagellin). We further focus on the mechanism by which the two nucleotide-binding proteins FlhF and FlhG ensure the correct reproduction of flagella place and number (the flagellation pattern). We will finish the review with an overview of current biotechnological applications, and a perspective of how understanding flagella can contribute to developing modules for synthetic approaches.

Spatial and numerical regulation of flagellar biosynthesis in polarly flagellated bacteria

Control of surface organelle number and placement is a crucial aspect of the cell biology of many Gram-positive and Gram-negative bacteria, yet mechanistic insights into how bacteria spatially and numerically organize organelles are lacking. Many surface structures and internal complexes are spatially restricted in the bacterial cell (e.g. type IV pili, holdfasts, chemoreceptors), but perhaps none show so many distinct patterns in terms of number and localization as the flagellum. In this review, we discuss two proteins, FlhF and FlhG (also annotated FleN/YlxH), which control aspects of flagellar assembly, placement and number in polar flagellates, and may influence flagellation in some bacteria that produce peritrichous flagella. Experimental data obtained in a number of bacterial species suggest that these proteins may have acquired distinct attributes influencing flagellar assembly that reflect the diversity of flagellation patterns seen in different polar flagellates. Recent findings also suggest FlhF and FlhG are involved in other processes, such as influencing the rotation of flagella and proper cell division. Continued examination of these proteins in polar flagellates is expected to reveal how different bacteria have adapted FlhF or FlhG with specific activities to tailor flagellar biosynthesis and motility to fit the needs of each species.

Polar Flagellar Biosynthesis and a Regulator of Flagellar Number Influence Spatial Parameters of Cell Division in Campylobacter jejuni

Spatial and numerical regulation of flagellar biosynthesis results in different flagellation patterns specific for each bacterial species. Campylobacter jejuni produces amphitrichous (bipolar) flagella to result in a single flagellum at both poles. These flagella confer swimming motility and a distinctive darting motility necessary for infection of humans to cause diarrheal disease and animals to promote commensalism. In addition to flagellation, symmetrical cell division is spatially regulated so that the divisome forms near the cellular midpoint. We have identified an unprecedented system for spatially regulating cell division in C. jejuni composed by FlhG, a regulator of flagellar number in polar flagellates, and components of amphitrichous flagella. Similar to its role in other polarly-flagellated bacteria, we found that FlhG regulates flagellar biosynthesis to limit poles of C. jejuni to one flagellum. Furthermore, we discovered that FlhG negatively influences the ability of FtsZ to initiate cell division. Through analysis of specific flagellar mutants, we discovered that components of the motor and switch complex of amphitrichous flagella are required with FlhG to specifically inhibit division at poles. Without FlhG or specific motor and switch complex proteins, cell division occurs more often at polar regions to form minicells. Our findings suggest a new understanding for the biological requirement of the amphitrichous flagellation pattern in bacteria that extend beyond motility, virulence, and colonization. We propose that amphitrichous bacteria such as Campylobacter species advantageously exploit placement of flagella at both poles to spatially regulate an FlhG-dependent mechanism to inhibit polar cell division, thereby encouraging symmetrical cell division to generate the greatest number of viable offspring. Furthermore, we found that other polarly-flagellated bacteria produce FlhG proteins that influence cell division, suggesting that FlhG and polar flagella may function together in a broad range of bacteria to spatially regulate division.

PflI, a Protein Involved in Flagellar Positioning in Caulobacter crescentus

The bacterial flagellum is important for motility and adaptation to environmental niches. The sequence of events required for the synthesis of the flagellar apparatus has been extensively studied, yet the events that dictate where the flagellum is placed at the onset of flagellar biosynthesis remain largely unknown. We addressed this question for alphaproteobacteria by using the polarly flagellated alphaproteobacterium Caulobacter crescentus as an experimental model system. To identify candidates for a role in flagellar placement, we searched all available alphaproteobacterial genomes for genes of unknown function that cluster with early flagellar genes and that are present in polarly flagellated alphaproteobacteria while being absent in alphaproteobacteria with other flagellation patterns. From this in silico screen, we identified pflI. Loss of PflI function in C. crescentus results in an abnormally high frequency of cells with a randomly placed flagellum, while other aspects of cell polarization remain normal. In a wild-type background, a fusion of green fluorescent protein (GFP) and PflI localizes to the pole where the flagellum develops. This polar localization is independent of the flagellar protein FliF, whose oligomerization into the MS ring is thought to define the site of flagellar synthesis, suggesting that PflI acts before or independently of this event. Overproduction of PflI-GFP often leads to ectopic localization at the wrong, stalked pole. This is accompanied by a high frequency of flagellum formation at this ectopic site, suggesting that the location of PflI is a sufficient marker for a site for flagellar assembly.

Use of Computer-Assisted Motion Analysis for Quantitative Measurements of Swimming Behavior in Peritrichously Flagellated Bacteria

An assay was developed which identifies individual bacterial tumbles and so allows rapid, quantitative measurements of tumble frequency in free-swimming bacteria. Tumble frequency is modulated by cells to enable chemotaxis. Mutations in the chemotaxis signal transduction pathway typically have phenotypes of altered tumble frequency. The purpose of this assay is to quantitatively measure steady-state tumble frequency to enable comparisons of mutant strain phenotypes. It was developed usingEscherichia colibut should be applicable to other species with a peritrichous flagellation pattern, such asSalmonella typhimurium.Tumbles are defined by a combination of the parameters rate of change of direction and swimming speed, with a rapid change of direction defining the beginning of a tumble and increased swimming speed defining the end. These parameters have previously been shown to be correlated with tumbles in general but not used to identify discrete tumble events. The computer assay was validated by comparing its results with manual observations by eye. The assay was intended to be most sensitive to swimming patterns similar to wild type so as to resolve subtle changes which would result from partial-function mutations. It quantitatively detects extreme behavioral phenotypes as well and can be modified to increase resolution at either extreme if necessary.

Functions of bacterial flagella.

Many bacterial species are motile by means of flagella. The structure and implantation of flagella seems related to the specific environments the cells live in. In some cases, the bacteria even adapt their flagellation pattern in response to the environmental conditions they encounter. Swarming cell differentiation is a remarkable example of this phenomenon. Flagella seem to have more functions than providing motility alone. For many pathogenic species, studies have been performed on the contribution of flagella to the virulence, but the result is not clear in all cases. Flagella are generally accepted as being important virulence factors, and expression and repression of flagellation and virulence have in several cases been shown to be linked. Providing motility is always an important feature of flagella of pathogenic bacteria, but adhesive and other properties also have been attributed to these flagella. In nonpathogenic bacterial colonization, flagella are important locomotive and adhesive organelles as well. In several cases where competition between several bacterial species exists, motility by means of flagella is shown to provide a specific advantage for a bacterium. This review gives an overview of studies that have been performed on the significance of flagellation in a wide variety of processes where flagellated bacteria are involved.

Telluria mixta (Pseudomonas mixta Bowman, Sly, and Hayward 1988) gen. nov., comb. nov., and Telluria chitinolytica sp. nov., Soil-Dwelling Organisms Which Actively Degrade Polysaccharides

Pseudomonas mixta (type strain, ACM 1762 [= ATCC 49108], an actively dextranolytic species that possesses both lateral and polar flagella, was compared with the strictly aerobic, rod-shaped, chitinolytic bacterium "Pseudomonas chitinolytica" ACM 3522T (= CNCM I-804) (T = type strain), which has a similar flagellation pattern, by performing phenotypic characterization and DNA-DNA hybridization studies and by analyzing DNA base compositions and 16S rRNA sequences. Our results indicated that "P. chitinolytica" ACM 3522T was phenotypically and genotypically distinct from P. mixta and other phenotypically analogous Pseudomonas spp., Xanthomonas maltophilia, and other aerobic chitin degraders. The 16S rRNA sequences of strains ACM 1762T and ACM 3522T were found to be very similar (97%) to each other and indicated that these organisms are proteobacteria that belong to the beta subclass. The strains were deeply branched in the beta subclass and were distinct from other pseudomonads, including Pseudomonas cepacia, and from Comamonas testosteroni. On the basis of phenotypic, genotypic, and phylogenetic evidence, it is proposed that P. mixta and "P. chitinolytica" ACM 3522T represent two distinct species in a new genus called Telluria. Thus, the genus Telluria gen. nov. contains Telluria mixta comb. nov. and Telluria chitinolytica sp. nov., which are strictly aerobic, rod-shaped, soil-dwelling bacteria that are active polysaccharide degraders.

Isolation and characterization of behavioural mutants and genes of Agrobacterium tumefaciens

Agrobacterium tumefaciens exhibits an unusual flagellation pattern in the electron microscope. The typical pattern is of two flagella in a polar or sub-polar tuft at one pole only, plus one or two lateral flagella on each side. Twenty independent behavioural mutants were isolated, after Tn5 mutagenesis, and divided into several classes. Fourteen non-motile mutants were differentiable into non-flagellate and flagellate subclasses, one non-chemotactic mutant appeared to have a defect in the intracellular signalling pathway, and one other mutant displayed an unusual pattern of polar cell pairing. Four mutants with impaired swarming ability were also isolated. Root colonization ability was impaired in the non-motile mutant for which this property was tested. From all but three of the mutants, the Tn5 insertion sites, including flanking sequences, have been cloned and used as probes to isolate a set of seven cosmids from a wild-type A. tumefaciens library. No hybridization to Escherichia coli or Pseudomonas DNA was detected for the A. tumefaciens mutant flanking sequences, but varying degrees of similarity to Rhizobium meliloti behavioural genes were detected. By complementation of A. tumefaciens behavioural mutants and hybridization of mutant flanking sequences, one cosmid insert was shown to contain at least nine behavioural loci, and another, linked to it on the chromosome, at least three. This suggests that, as in other organisms, behavioural genes are clustered in the A. tumefaciens genome.

Rhizobia in tropical legumes: Cultural characteristics of Bradyrhizobium and Rhizobium sp.

We studied the cultural characteristics (carbohydrate utilization and associated pH changes in the media, temperature and pH optima, as well as flagellation patterns) of 51 Rhizobium sp. and 27 Bradyrhizobium sp. symbionts of tropical legumes. Both fast- and slow-growing rhizobia were isolated from nodules of Desmodium heterophyllum and D. uncinatum. Contrary to previous data, two-thirds of the bradyrhizobia could use disaccharides as the sole carbon-source. Twice as many Rhizobium sp. as Bradyrhizobium sp. utilized inositol and trehalose, suggesting that they might have some use in differentiating between the genera. The extent to which bradyrhizobia raised the pH of media was inversely correlated with the initial pH (no change at pH 7). A number of bradyrhizobia could, in combination with various carbohydrates, lower the pH, suggesting that some Bradyrhizobium sp. are able to modify their excretion products in response to environmental signals. Some Rhizobium sp. possessed only a single, subpolar flagellum. Our data thus demonstrate that Rhizobium sp. associated with tropical legumes are as heterogeneous as bradyrhizobia of the “cowpea miscellany”.

Variations in the number of centrioles, the number of microtubule organizing centers 1 and the percentage of mitotic abnormalities inPhysarum polycephalum amoebae

Several, stable amoebal strains which differ phenotypically from the diploid parental amoebal strain have been obtained in the MyxomycetePhysarum polycephalum. They were detected using their flagellation pattern as a discriminating parameter. This approach is valid since the number of flagella by phase contrast microscopy correlates with the number of anterior centrioles obtained using three-dimensional reconstructions of the nucleo-flagellar complexes from serial thin sections. The complexity of the structures of the various nucleo-flagellar complexes suggests that in these strains the duplication time of centrioles is not strictly regulated as it is in haploid amoebae. In agreement with this hypothesis, several pro-centrioles were observed in interphase amoebae. Although the anterior centrioles are linked to the mtoc 1 during interphase, the number of mtoc 1 cannot regulate the number of centrioles since some strains possess two mtoc 1 but only one pair of centrioles. Neither the number of centrioles nor the number of mtoc 1 are related to ploidy. Stable strains with one (all haploid strains), two (some diploid strains) and three (some diploid strains) mtoc 1 have been observed. Thus each mtoc 1 is duplicated once per cell cycle implying that it must possess some information which plays a role in the morphogenesis of the new mtoc 1. Except in one case, the number of mitotic abnormalities increases exponentially with the number of mtoc 1. This observation suggests that the mtoc 1 could correspond to the interphase state of the mitotic center.