The O-specific chain structure of the major component from the lipopolysaccharide fraction of Halomonas magadii strain 21 MI (NCIMB 13595)

An O-specific polysaccharide containing D-galactose and D-glucose, was isolated from the water-soluble lipopolysaccharide fraction of the alkaliphilic bacterium Halomonas magadii. The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a trisaccharide repeating unit, as shown below: [structure: see text]     

Characterization of Natronobacterium magadii phage ΦCh1, a unique archaeal phage containing DNA and RNA

A novel archaeal bacteriophage, ΦCh1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii (L13) was used to demonstrate infectivity of phage ΦCh1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that ΦCh1 is a temperate phage. The phage morphology resembles other members of Myoviridae -infecting Halobacterium species. In solution below 2 M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS–PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that ΦCh1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80–700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and ΦCh1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage ΦCh1 is the first virus described for the genus Natronobacterium , and the first phage containing DNA and RNA in mature phage particles.     

A new 'paralyzed' flagella mutant, OC-10, in Chlamydomonas reinhardtii (Volvocales, Chlorophyceae) that can be reactivated with adenosine triphosphate

A new ‘paralyzed’ mutant. OC–10, was isolated in Chlamydomonas reinhardtii Dangeard. OC-10 cannot swim and generally shows very little flagellar movement. However, when OC-10 was demembranated, axonemal motility was reactivated in the presence of adenosine triphosphate (ATP) or adenosine diphosphate (ADP). The beating form of the reactivated axonemes was almost the same as that of the wild-type axonemes. Flagellar regeneration of OC-10 was slower than that of the wild-type. Electron microscopic examination showed no abnormality in OC-10 flagella, but SDS/PAGE revealed that mobility of a flagellar membrane protein was changed and a few bands disappeared in OC-10 flagella, When the mutant was crossed to wild-type to form temporary dikaryon cells with 4 flagella, OC-10 flagella did not regain motility. Tetrad analysis of crosses between OC–10 and wild-type demonstrated a 1:1 segregation on the basis of flagellar motility. From these results, we suppose that OC-10 may be limited in ATP availability inside the flagella, or altered in flagellar membrane proteins important for motility.     

Lipid rafts function in Ca2+ signaling responsible for activation of sperm motility and chemotaxis in the ascidian Ciona intestinalis

Lipid rafts are specialized membrane microdomains that function as signaling platforms across plasma membranes of many animal and plant cells. Although there are several studies implicating the role of lipid rafts in capacitation of mammalian sperm, the function of these structures in sperm motility activation and chemotaxis remains unknown. In the ascidian Ciona intestinalis, egg-derived sperm activating- and attracting-factor (SAAF) induces both activation of sperm motility and sperm chemotaxis to the egg. Here we found that a lipid raft disrupter, methyl-β-cyclodextrin (MCD), inhibited both SAAF-induced sperm motility activation and chemotaxis. MCD inhibited both SAAF-promoted synthesis of intracellular cyclic AMP and sperm motility induced by ionophore-mediated Ca(2+) entry, but not that induced by valinomycin-mediated hyperpolarization. Ca(2+)-imaging revealed that lipid raft disruption inhibited Ca(2+) influx upon activation of sperm motility. The Ca(2+)-activated adenylyl cyclase was clearly inhibited by MCD in isolated lipid rafts. The results suggest that sperm lipid rafts function in signaling upstream of cAMP synthesis, most likely in SAAF-induced Ca(2+) influx, and are required for Ca(2+)-dependent pathways underlying activation and chemotaxis in Ciona sperm.     

Flagellar radial spoke protein 3 is an A-kinase anchoring protein (AKAP)

Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144-180. Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.     

Ion-coupling determinants of Na+-driven and H+-driven flagellar motors

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7(E)) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7(E)) supported sodium-driven motility in motA or motB E.coli cells, and the swimming speed was even higher than with the original stator of E.coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7(E)) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor.     

Domains in the 1alpha dynein heavy chain required for inner arm assembly and flagellar motility in Chlamydomonas.

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1alpha Dhc of the I1 inner dynein arm. The 1alpha Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH(2)- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1alpha Dhcs were recovered. The truncated 1alpha Dhc fragments lacked the dynein motor domain, but still assembled with the 1beta Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1alpha motor domain in the I1 structure and further revealed the role of the 1alpha Dhc in flagellar motility and phototactic behavior.     

Characterization of a Chlamydomonas Insertional Mutant that Disrupts Flagellar Central Pair Microtubule-associated Structures

Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke–defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.     

The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution

Chondrichthyes possess an evolved type of spermatozoa. Their flagellar apparatus is characterized by the presence of flagellar roots which form the axis of the midpiece, and the existence of one or two lateral elements associated with the axoneme. Osteichthyes, mainly teleosteans, show a great diversity of spermatic forms. The primitive spermatozoon with a 9 + 2 pattern flagellum is common. The primitive spermatozoon has evolved along different lines. The spermatic diversity which results from this is mainly evident in the structure of the flagellar apparatus. In the animal kingdom the primitive spermatozoon with a 9 + 2 pattern flagellum, present in primitive metazoa, is retained in phyla where external fertilization is maintained. The main evolutionary tendencies--elongation, aflagellarity or biflagellarity--are generally connected with the acquisition of internal fertilization. These evolutionary tendencies are found in teleosteans. It is not possible to link aflagellarity or biflagellarity of the gamete in certain fishes to this method of fertilization. Only the elongation of the spermatozoon is connected, in certain cases, with internal fertilization, but this cannot be taken as general.     

Chymotrypsin-like protease activity associated with demembranated sperm of chum salmon.

Our previous study suggested that a chymotrypsin-like protease was involved in the motility of chum salmon sperm (Inaba K, Morisawa M, Biomed Res (1991) 12, 435-437). In this study, we examined the peptidase activity of demembranated sperm of chum salmon using ten synthetic peptides. When spermatozoa were treated with 0.04% Triton X-100 for extracting the plasma membrane and the suspension was separated into the Triton-soluble and insoluble fractions by centrifugation, only the hydrolytic activity towards succinyl (Suc)-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (MCA), a typical substrate for chymotrypsin-like protease, was mostly retained in the insoluble fraction. The bulk of the activities toward other substrates was detected in the soluble fraction. Flagellar axonemes isolated from demembranated sperm showed considerable hydrolytic activity toward Suc-Leu-Leu-Val-Tyr-MCA and the activity was still retained in the axoneme even after further washing. The hydrolysis was activated by a low concentration of SDS, suggesting that the protease associated with the axonemes is a multicatalytic ATP-dependent proteinase (proteasome). Motility of demembranated sperm was inhibited by Suc-Leu-Leu-Val-Tyr-MCA in an ATP-concentration-dependent manner. These results suggest that proteasomes associated with flagellar axoneme regulate flagellar motility.     

Swimming and twitching motility are essential for attachment and virulence of Pantoea ananatis in onion seedlings

Pantoea ananatis is a widespread phytopathogen with a broad host range. Despite its ability to infect economically important crops, such as maize, rice and onion, relatively little is known about how this bacterium infects and colonizes host tissue or spreads within and between hosts. To study the role of motility in pathogenicity, we analysed both swimming and twitching motility in P. ananatis LMG 20103. Genetic recombineering was used to construct four mutants affected in motility. Two flagellar mutants were disrupted in the flgK and motA genes, required for flagellar assembly and flagellar rotation, respectively. Similarly, two twitching motility mutants were generated, impaired in the structure (pilA) and functioning (pilT) of the type IV pili. The role of swimming and twitching motility during the infection cycle of P. ananatis in onion seedlings was determined by comparing the mutant‐ and wild‐type strains using several in vitro and in planta assays. From the results obtained, it was evident that flagella aid P. ananatis in locating and attaching to onion leaf surfaces, as well as in pathogenicity, whereas twitching motility is instrumental in the spread of the bacteria on the surface once attachment has occurred. Both swimming and twitching motility contribute towards the ability of P. ananatis to cause disease in onions.     

Intracellular motility apparatus of halobacteria: Electron microscopic study

The intracellular disc-like lamellar structure (DLS) earlier detected in the motility apparatus of Halobacterum salinarum and details of insertion of proximal ends of flagella into DLS were studied using electron microscopy. Analysis of ultrathin sections obtained after fixation with potassium permanganate established that DLS, absent in bacteria, contains a membrane-like structure. Electron microscopic studies of cell ghosts obtained by mild cytolysis in low-NaCl solutions shed additional light as on details of DLS structure and so on localization of flagellar proximal ends. Structural organization of the motility apparatus of bacteria and archaebacteria as representatives of two distinct taxonomic domains is discussed.     

The archaeal halophilic virus-encoded Dam-like methyltransferase M. phiCh1-I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli

The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100-800 nucleotides. A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.phiCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of phiCh1. After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the phiCh1 progeny genomes after infection of N. magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3'. Additionally, expression of M.phiCh1-I in dam- E. coli cells led to a complete substitution of the function of M.Dam in DNA mismatch repair.     

Exploitation of a new flagellatropic phage of Erwinia for positive selection of bacterial mutants attenuated in plant virulence: towards phage therapy

Aims: To isolate and characterize novel bacteriophages for the phytopathogen, Erwinia carotovora ssp. atroseptica (Eca), and to isolate phage‐resistant mutants attenuated in virulence. Methods and Results: A novel flagellatropic phage was isolated on the potato‐rotting bacterial species, Eca, and characterized using electron microscopy and restriction analysis. The phage, named ΦAT1, has an icosahedral head and a long, contractile tail; it belongs to the Myoviridae family. Partial sequencing revealed the presence of genes with homology to those of coliphages T4, T7 and Mu. Phage‐resistant transposon mutants of Eca were isolated and studied in vitro for a number of virulence‐related phenotypes; only motility was found to be affected. In vivo tuber rotting assays showed that these mutants were attenuated in virulence, presumably because the infection is unable to spread from the initial site of inoculation. Conclusions: The Eca flagellum can act as a receptor for ΦAT1 infection, and resistant mutants are enriched for motility and virulence defects. Significance and Impact of the Study: ΦAT1 is the first reported flagellatropic phage found to infect Eca and has enabled further study of the virulence of this economically important phytopathogen.     

MITOSIS IN DUNALIELLA BIOCULATA (CHLOROPHYTA): CENTRIN BUT NOT BASAL BODIES ARE AT THE SPINDLE POLES

Centrin, a 20 kDa calmodulin-like protein, is located in various basal body-associated fibers in protists. We used indirect immunofluorescence of isolated cytoskeletons or methanol-fixed cells to analyze the distribution of centrin during mitosis of the biflagellate green alga Dunaliella bioculata (Butcher). The distance between the nucleus and the basal apparatus decreased in late interphase, presumably caused by the contraction of the two centrin-containing nucleus–basal body connectors (NBBCs). During prophase, centrin accumulated on the new basal bodies as shown by postembedding immunogold labeling of serial thin sections. The new basal bodies were in close contact with plaque-like structures on the nuclear envelope. In mitotic cells, basal body pairs were separated and positioned at a considerable distance from the poles of the mitotic spindle. At this stage, we observed four separated centrin dots, two associated with the pairs of basal bodies and two located at the spindle poles as shown by double immunofluorescence, including anti-tubulin staining. The latter signals corresponded to an accumulation of centrin between the plasma membrane and the nuclei, indicating that centrin could be involved in mitotic movements of the nuclei. In telophase, centrin was observed along the nuclear surface and one new NBBC developed in each cell half. Our results demonstrate that centrin is present at the acentriolar spindle poles of Dunaliella independently from its localization in the basal apparatus.     

Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.     

Haloarchaeal myovirus φCh1 harbours a phase variation system for the production of protein variants with distinct cell surface adhesion specificities

The φCh1 myovirus, which infects the haloalkaliphilic archaeon Natrialba magadii, contains an invertible region that comprises the convergent open reading frames (ORFs) 34 and 36, which code for the putative tail fibre proteins gp34 and gp36 respectively. The inversion leads to an exchange of the C-termini of these proteins, thereby creating different types of tail fibres. Gene expression experiments revealed that only ORF34 is transcribed, indicating that φCh1 produces tail fibre proteins exclusively from this particular ORF. Only one of the two types of tail fibres encoded by ORF34 is able to bind to Nab. magadii in vitro. This is reflected by the observation that during the early phases of the infection cycle, the lysogenic strain L11 carries its invertible region exclusively in the orientation that produces that specific type of tail fibre. Obviously, Nab. magadii can only be infected by viruses carrying this particular type of tail fibre. By mutational analysis, the binding domain of gp34 was localized to the C-terminal part of the protein, particularly to a galactose-binding domain. The involvement of galactose residues in cell adhesion was supported by the observation that the addition of α-D-galactose to purified gp34 or whole virions prevented their attachment to Nab. magadii.     

THE MITOTIC APPARATUS : Fine Structure of the Isolated Unit

The fine structure of the mitotic apparatus isolated from the sea urchin egg has been investigated. The isolation was accomplished by lysis of metaphase eggs in a 1 M solution of hexanediol, buffered at pH 6. The fine structure of the isolated apparatus was studied after fixation with osmium tetroxide directly in the isolation medium. The spindle is composed of fine fibrils, approximately 20 mµ in diameter, which appear tubular. Similar fibrils, radially oriented, are found in the aster. If the isolated mitotic apparatus is exposed to water at pH 6 before fixation, the structure is considerably modified. The most pronounced effects are an increase in the number of large membrane-bounded vesicles and in the amount of free granular material present. The conditions necessary for the fixation of the mitotic apparatus in dividing cells are discussed in the light of these observations on the isolated unit.     

Ultrastructure of the spermatozoid in Halimeda tuna (chlorophyceae)

The mature Halimeda tuna spermatozoid was studied under the electron microscope. It is pear-shaped and biflagellate. The nucleus is an uncondensed structure except for a few opaque (chromatin) aggregations and shows a rounded profile. The endoplasmic reticulum is a rather well-developed system of rough cisternae engaged in synthesis and storage of proteins. Free ribosomes are numerous. A large mitochondrial apparatus shaped like a horseshoe lies in the anterior gamete region. Only one single plastid is found, and it exhibits a deeply indented outline, a partially structured matrix, osmiophilic globules, and three to four starch grains. The axoneme pattern is 9 + 2. B tubules show septate lumina. A peculiar structure provided with a three-layered shell covering materials of varying electrondensity lies on the upper surface of both basal bodies. The flagellar root system exhibits a cruciate pattern and sets having an inconstant number of microtubules – ie, three, four, or five.