Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.     

Surface viscoelasticity of individual gram-negative bacterial cells measured using atomic force microscopy

The cell envelope of gram-negative bacteria is responsible for many important biological functions: it plays a structural role, it accommodates the selective transfer of material across the cell wall, it undergoes changes made necessary by growth and division, and it transfers information about the environment into the cell. Thus, an accurate quantification of cell mechanical properties is required not only to understand physiological processes but also to help elucidate the relationship between cell surface structure and function. We have used a novel, atomic force microscopy (AFM)-based approach to probe the mechanical properties of single bacterial cells by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of the AFM tip due to the viscoelastic properties of the cell. For these experiments, we chose a representative gram-negative bacterium, Pseudomonas aeruginosa PAO1, and we used regular V-shaped AFM cantilevers with pyramid-shaped and colloidal tips. We find that the cell response is well described by a three-element mechanical model which describes an effective cell spring constant, k(1), and an effective time constant, tau, for the creep deformation. Adding glutaraldehyde, an agent that increases the covalent bonding of the cell surface, produced a significant increase in k(1) together with a significant decrease in tau. This work represents a new attempt toward the understanding of the nanomechanical properties of single bacteria while they are under fluid conditions, which could be of practical value for elucidating, for instance, the biomechanical effects of drugs (such as antibiotics) on pathogens.     

Differential Lipopolysaccharide Core Capping Leads to Quantitative and Correlated Modifications of Mechanical and Structural Properties in Pseudomonas aeruginosa Biofilms

Bacterial biofilms are responsible for the majority of all microbial infections and have profound impact on industrial and geochemical processes. While many studies documented phenotypic differentiation and gene regulation of biofilms, the importance of their structural and mechanical properties is poorly understood. Here we investigate how changes in lipopolysaccharide (LPS) core capping in Pseudomonas aeruginosa affect biofilm structure through modification of adhesive, cohesive, and viscoelastic properties at an early stage of biofilm development. Microbead force spectroscopy and atomic force microscopy were used to characterize P. aeruginosa biofilm interactions with either glass substrata or bacterial lawns. Using isogenic migA, wapR, and rmlC mutants with defined LPS characteristics, we observed significant changes in cell mechanical properties among these strains compared to wild-type strain PAO1. Specifically, truncation of core oligosaccharides enhanced both adhesive and cohesive forces by up to 10-fold, whereas changes in instantaneous elasticity were correlated with the presence of O antigen. Using confocal laser scanning microscopy to quantify biofilm structural changes with respect to differences in LPS core capping, we observed that textural parameters varied with adhesion or the inverse of cohesion, while areal and volumetric parameters were linked to adhesion, cohesion, or the balance between them. In conclusion, this report demonstrated for the first time that changes in LPS expression resulted in quantifiable cellular mechanical changes that were correlated with structural changes in bacterial biofilms. Thus, the interplay between architectural and functional properties may be an important contributor to bacterial community survival.Biofilms are sessile microbial communities growing on a surface or at an interface, often enmeshed in polymeric substances. Being the predominant mode of microbial growth in nature, bacterial biofilms are particularly problematic in the context of human health, accounting for up to 80% of all bacterial infections. In industrial processes, bacterial biofilms cause corrosion and biofouling, resulting in considerable loss of productivity. In the natural environment, biofilms play a role in modulating worldwide geochemical cycles. Given the impact of biofilms in these diverse areas, the need for developing effective strategies to control them is of paramount importance. Since bacterial cell surface structures are convenient targets for control agents, their roles in influencing biofilm function and architecture warrant in-depth investigations. To date, most studies of biofilms have focused on genetic regulation, phenotypic differentiation and their contribution to antibiotic resistance. In contrast, the mechanical and structural properties that link the genotypes to phenotypes of bacterial biofilms are not well understood and rarely studied in a quantitative and correlated manner.Pseudomonas aeruginosa is a gram-negative opportunistic pathogen implicated in serious infections in patients with cystic fibrosis and immunocompromised patients. This bacterium has a relatively large genome (6.3 Mb) consistent with its propensity to utilize versatile metabolic pathways, thereby developing antibiotic resistance and producing an arsenal of virulence factors, including lipopolysaccharide (LPS) present on the cell surface. LPS is localized in the outer leaflet of the outer membrane of all gram-negative bacteria, forming the first point of contact between the bacterial cell and any surface that it colonizes or therapeutic agents. The LPS of P. aeruginosa consists of three regions: lipid A, core oligosaccharide (core OS), and O antigen. The O antigen is synthesized as two distinct forms with overlapping pathways: the shorter A-band homopolymer is the so-called “common polysaccharide antigen” among this species and consists of repeating d-rhamnose (d-Rha) subunits, while the longer B-band heteropolymer is composed of repeating tri- to pentasaccharide subunits that vary among the 20 serotypes of P. aeruginosa (42). The core OS is conceptually divided into the highly conserved inner core and the more variable outer core. Depending on the linkage of l-rhamnose (l-Rha) with two distinct d-glucose (d-Glc) residues, two main glycoforms of the core OS exist (see Fig. ​Fig.1A).1A). In the “capped” glycoform, l-Rha is α-1,3 linked to a d-Glc and acts as the acceptor molecule for O antigen, resulting in the production of smooth LPS. In the “uncapped” glycoform, l-Rha is α-1,6 linked to a different d-Glc and is not substituted with O antigen, resulting in the production of rough LPS (39). In addition, the presence or absence of the α-1,6 linked l-Rha substituted with a terminal d-Glc gives rise to the so-called intact or truncated outer core, respectively. The functional significance of this terminal glucose is unclear at present, although a role in host cell binding has been proposed (57).Open in a separate windowFIG. 1.Comparison of LPSs from Pseudomonas aeruginosa wild-type strain PAO1 and mutant strains with LPS core variants. (A) Schematic diagram illustrating the chemical structures of smooth LPS and rough LPS. The gray and black arrows point to the position of outer core truncation and position of O-antigen capping, respectively. (B) Silver-stained SDS-polyacrylamide gels illustrating LPS profiles of planktonic and biofilm cells. The two gray arrows and the black arrows point to the position of O antigen and position of core-plus-one entities, respectively. Planktonic cells (lanes 1 to 4) and biofilm cells (lanes 5 to 8) of strain PAO1 (P), migA mutant (M), wapR mutant (W), and rmlC mutant (R) are shown.Mechanical processes that are important in the biofilm life cycle include bacterial adhesion, cohesion, and viscoelasticity. Bacterial adhesion is a prerequisite for surface colonization and the most important functional determinant in the early stages of biofilm development. Accurate measurement of adhesion is therefore essential for monitoring the tendency of bacteria to attach to surfaces and to switch from a planktonic lifestyle to a biofilm lifestyle. Data accumulated in previous studies suggest that LPS is involved in bacterial cell adhesion to both abiotic (2, 8, 20, 32, 35, 54, 56) and biotic (17, 38, 49, 56, 57) surfaces. Moreover, environmental factors, such as growth temperature, pH, ionic strength, nutrient availability, and oxygen levels, may influence cell adhesion via modification of LPS expression and conformation (16, 36, 46, 47, 53). The effect of LPS on bacterial adhesion to various types of surfaces apparently involves distinct and complex mechanisms that remain to be elucidated.Bacterial cohesion, herein defined as cell-to-cell adherence, is crucial to the formation of microbial flocs and the growth and detachment of established bacterial biofilms. Quantification of cohesion is important for understanding biofilm biology, and such data are crucial for modeling and forecasting biofilm development so that better control strategies can be developed (55). Previous studies of biofilm cohesiveness have characterized it as highly stratified (3, 4, 18, 43), influenced by ionic strength (14, 34), proportional to shear rate (37), and often variable over 3 orders of magnitude (40, 50). Although an earlier study by Spiers and Rainey (48) provided semiquantitative measurements of the role of LPS on bacterial cohesion within a biofilm, a truly quantitative account of the effect of LPS on biofilm cohesion has not been demonstrated.Bacterial viscoelasticity refers to the combined liquid-like and solid-like characteristics in the behavior of polymeric systems such that when deformed under stress, their strain can increase over time (i.e., creep) and their original shape may be only partially restored upon stress relief (19). Although earlier reports suggested that LPS modulates bacterial cell compressibility and helps prevent catastrophic structural failure due to mechanical stress (1, 52), no direct physical evidence of its involvement in these processes has yet been presented. Therefore, monitoring biofilm viscoelasticity is crucial for demonstrating how well biofilms resist stresses, due to, for instance, fluid shear and antimicrobial peptides (5, 6). To date, quantitative data on how LPS affects viscoelastic properties of biofilms are lacking, and existing studies have merely focused on elasticity measurements (7, 52). Recently, our group has developed an atomic force microscopy (AFM)-based technique called microbead force spectroscopy (MBFS) to measure the adhesive forces and viscoelastic properties of cells within bacterial biofilms (28). In this study, we expand the application of this MBFS method to measure cohesive forces between cells at an early stage of biofilm development.Biofilm structure refers to the distribution of biomass or carbonaceous materials associated with cells (including all viable and nonviable cells and their extracellular polymeric substances) within the space occupied by a biofilm. It is known to be very heterogeneous and highly stratified, typically composed of a cohesive basal layer and a relatively fragile top layer (15, 18, 43). Using confocal laser scanning microscopy (CLSM), biofilm structure can be quantitatively described in terms of textural and volumetric parameters (11, 29). Textural parameters characterize the pattern of cell clusters and interstitial voids in a biofilm, whereas volumetric parameters describe the morphological characteristics of bacterial biofilms in three dimensions (3-D) (11). Biomass distribution is affected by the surrounding environment and may reflect fundamental processes occurring within biofilms, such as nutrient transport, accumulation rate, microbial physiology, and mechanical behavior (29). Therefore, quantifying biofilm structure by CLSM will allow us to understand the underlying processes and the relationship between biofilm architecture and behavior (15).To examine the effects of differential LPS core capping on the mechanical properties of early biofilms (here defined as confluent bacterial lawns that have just begun to develop into full-fledged biofilms) and the structural properties of mature biofilms, we compare P. aeruginosa wild-type strain PAO1 with those of its isogenic migA, wapR, and rmlC mutant strains with defects in the respective genes affecting LPS core biosynthesis (22, 31, 39, 41). The migA gene (PA0705) encodes the putative α-1,6-rhamnosyltransferase necessary for the attachment of the terminal d-Glc to the outer core (39). The wapR gene (PA5000) encodes the putative α-1,3-rhamnosyltransferase crucial to the capping of the core with O antigen (39). The rmlC gene (PA5164) encodes a dTDP-4-dehydrorhamnose 3,5-epimerase essential in the biosynthesis of TDP-l-Rha, which is the precursor for the l-Rha in the LPS core (41). Defects in migA, wapR, and rmlC mutants result in the expression of different LPS phenotypes, including a truncated outer core and/or a lack of capping by O antigen (Table ​(Table1).1). In this study, we test the hypothesis that LPS contributes to biofilm function and architecture through modulation of cellular mechanics and microcolony structures, thereby contributing to bacterial community survival. By correlating quantitative mechanical changes in early P. aeruginosa biofilms and structural changes in mature biofilms due to differences in LPS chemistry, we aim to elucidate how the properties of these important bacterial cell surface molecules can alter the physical nature of biofilms.

TABLE 1.

Pseudomonas aeruginosa strains used in this study

ISOLATION AND CHARACTERIZATION OF DOMINANT TUNICAMYCIN RESISTANCE MUTATIONS IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Seven independent mutations that confer resistance to the nucleoside antibiotic tunicamycin in the unicellular green alga Chlamydomonas reinhardtii were isolated in wild-type diploid cells. Upon resolution to haploidy, all the mutations segregated as single mutations and were semi-dominant when retested in heterozygous diploids. In addition, the TUN1-3 allele affects the ability of cells to agglutinate during conjugation in the absence of tunicamycin. The seven mutations map to a single locus on linkage group VIII. These mutations may be useful as a selectable marker in transformation studies of Chlamydomonas and in studies of processes that require asparagine-linked glycosylation.     

Contribution of primary aromatic amines to the mutagenicity of gasifier tars and coal oils

Two reactions that chemically alter primary aromatic amines (PAA) were used to assess the contribution of these compounds to the indirect bacterial mutagenicity of tar from an experimental low Btu gasifier. The first reaction, nitrosation, effectively eliminated the mutagenicity of several PAA standards and a coal oil when run in a low pH media (1.2). When applied to gasifier tar, extensive direct (not requiring metabolic activity) mutagenicity was generated. This direct mutagenicity limited the interpretation of results. When the pH of the reaction media was raised to 2.5, the mutagenicity of PAA standards and the coal oil were still greater than 90% eliminated, however, no direct mutagenicity was observed for the gasifier tar. Furthermore, only 61% of the indirect (requiring metabolic activation) mutagenicity was eliminated. Acetylation reduced the indirect activity of most primary amine standards by greater than 79%. Acetylation of the tar likewise eliminated part, but not all, of the activity, whereas most of the activity of the coal oil was eliminated. These results indicated that a much lower percentage of the mutagenic activity of low Btu coal tar samples was due to primary aromatic amines than was the case for coal oil.     

Flagella in prokaryotes and lower eukaryotes.

During the past year, significant advances have been made in the understanding of both prokaryotic and eukaryotic flagella. About 50 genes are dedicated to the assembly and operation of bacterial flagella. Recent discoveries have advanced our understanding of how these genes are regulated and how their products assemble into a functional, rotating organelle. The dynein arms of eukaryotic flagella are now also better understood. Several genes that are found in the mechanochemical macroassemblies have been cloned, and other loci have been identified, suggesting that there is even greater complexity than first expected.     

Tryptophan Analog Resistance Mutations in Chlamydomonas Reinhardtii

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.     

Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. C hlamydomonas reinhardtii flagella use an asymmetric beat stroke, similar to a breast stroke, to propel cells forward. To generate the asymmetric beat stroke, dynein activity must be regulated both along the length and around the circumference of the flagella. If all dyneins were active at the same time, the flagella would exist in a state of rigor. The dyneins are located in two rows along the length of the doublet microtubules. The inner dynein arms are heterogeneous in composition with at least eight heavy chains and various intermediate and light chains arranged in an elaborate morphology that repeats every 96 nm (Kagami and Kamiya, 1992; Mastronarde et al., 1992). In contrast, the outer dynein arms are biochemically and morphologically homogeneous (Huang et al., 1979; Mitchell and Rosenbaum, 1985; Kamiya, 1988); each outer dynein arm contains three dynein heavy chains and 10 intermediate and light chains. The inner and outer arms appear to have different functions in the formation of the beat stroke; the inner arms generate the waveform of the beat stroke, whereas the outer arms provide additional force to the waveform (Brokaw and Kamiya, 1987).Previous workers had shown that dynein regulation is imposed, in part, by activities of the radial spokes and the central pair complex. Mutant strains that are missing or have altered radial spokes or central pair complexes are paralyzed even if they have a full complement of dyneins (Adams et al., 1981; Piperno et al., 1981). Many extragenic suppressors of this paralysis phenotype do not restore the missing structures, but rather suppress by altering either inner arm or outer arm region structures (Huang et al., 1982a ; Piperno et al., 1992; Porter et al., 1992, 1994). These data suggest that direct or indirect interactions exist between the dynein arms and the radial spokes or central pair complexes.Over 80 proteins in Chlamydomonas flagella are phosphorylated (Piperno et al., 1981), which makes dynein regulation by phosphorylation an attractive model. Hasegawa et al. (1987) showed that a higher percentage of demembranated axonemes reactivate with ATP after treatments that lower cAMP levels or inhibit cAMP-dependent protein kinase (cAPK)¹. In flagella from other organisms, cAMP has an opposite role (for reviews see Tash and Means, 1983; Tash, 1989). An increased frequency of reactivation also occurs after the NP-40–soluble components are extracted from the axonemes, which suggests that the cAPK, target phosphoproteins, and endogenous phosphatases are all integral axonemal components (Hasegawa et al., 1987). In quantitative sliding disintegration assays, the inner dynein arm activity of axonemes that are missing the radial spokes is increased in the presence of pharmacological or specific peptide inhibitors of cAPK (Smith and Sale, 1992; Howard et al., 1994). Reconstitution experiments with axonemes that are missing the radial spokes suggest that radial spokes normally function to activate the inner dynein arms by inhibiting a cAPK (Smith and Sale, 1992; Howard et al., 1994). It is not known if the cAPK directly phosphorylates inner dynein arm components or phosphorylates another axonemal component that then acts on the inner dynein arms (Howard et al., 1994).The f (originally called I1) inner arms are biochemically the best studied inner dynein arm complex. This complex is comprised of two dynein heavy chains and three intermediate chains of 140, 138, and 110 kD; it can be purified by sucrose density centrifugation (Piperno and Luck, 1981; Smith and Sale, 1991; Porter et al., 1992) or ion-exchange chromatography (Kagami and Kamiya, 1992). The purified complex has low ATPase activity and only rarely translocates microtubules in vitro (Smith and Sale, 1991; Kagami and Kamiya, 1992). Deep-etch EM of the purified f inner arm shows a two-headed complex that is connected to a common base by thin stalks (Smith and Sale, 1991). Longitudinal EM image analyses have shown that this complex is located just proximally of the first radial spoke in each 96-nm repeating unit (Piperno et al., 1990; Mastronarde et al., 1992). Mutations at three different loci (PF9/ IDA1, IDA2, and IDA3) result in the complete loss of the f complex (Kamiya et al., 1991; Kagami and Kamiya, 1992; Porter et al., 1992). The PF9/IDA1 locus encodes a dynein heavy chain that is believed to be one of the two heavy chains that are components of the f complex (Porter, 1996).We undertook a new approach to identify axonemal components involved in dynein regulation; we isolated and characterized mutant strains that were unable to perform phototaxis. In Chlamydomonas, phototaxis is a behavior by which cells orient to the direction of incident light. Light direction is detected by the eyespot, an asymmetrically located organelle, and a signal is transmitted to the flagella using voltage-gated ion channels (Harz and Hegemann, 1991). For cells to perform phototaxis, the waveforms of the two flagella are altered coordinately. The trans flagellum, which is located farther from the eyespot, beats with a larger front amplitude than the cis flagellum to turn the cell toward the light (Rüffer and Nultsch, 1991). It seemed likely that the alterations in the beat amplitudes needed for correct phototactic behavior could be caused by differential dynein regulation in the cis and trans flagella. Therefore, we hypothesized that there should be a class of phototactic mutant strains that is not able to perform phototaxis because of defects in the regulation of dyneins. Three of the eight phototactic mutant strains that we characterized had biochemical defects in the f class of inner dynein arms. One of these strains, pf9-4, was missing the entire f complex, and the other two strains, mia1-1 and mia2-1, exhibited a novel f class inner dynein arm biochemical phenotype. These observations suggest that the f inner dynein arm is a target for regulation during phototaxis.