Margaret C. Henk

Extensive Diversity of Ionizing-Radiation-Resistant Bacteria Recovered from Sonoran Desert Soil and Description of Nine New Species of the Genus Deinococcus Obtained from a Single Soil Sample

ABSTRACT The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a 60Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus, Geodermatophilus, and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.

Evidence that acetyl phosphate functions as a global signal during biofilm development

We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüß and Wolfe, 1994, Mol Microbiol 12: 973–984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild‐type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co‐ordinate the expression of surface structures and cellular processes involved in the initial stages of wild‐type biofilm development.

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO2 concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO2-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO2 conditions. These growth conditions also partly inhibit the induction of the CO2-concentrating mechanism. When cells are grown with acetate in the dark, the CO2-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO2-concentrating mechanism under low-CO2 conditions. We suggest that this ultrastructural reorganization under lowCO2 conditions plays a role in the CO2-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.

Beggiatoa in microbial mats at hydrocarbon vents in the Gulf of Mexico and Warm Mineral Springs, Florida

Microbial mats were collected from a variety of sites near hydrocarbon vents along the slope in the northern Gulf of Mexico and, for comparison, from Warm Mineral Springs, Florida, USA. A predominant microorganism in each of the mats was the giant bacterium,Beggiatoa. Diameters of the bacterial filaments ranged from about 6 µm to approximately 200 µm. The latter organisms are the largest prokaryotic organisms yet found. All filaments over about 10 µm in diameter contained a large central vacuole, producing a cell with the cytoplasm as a cylindrical tube underlying the cytoplasmic membrane. Sulfur globules were confined to this peripheral layer. Push cores often contained pyrite tubules whose appearance is suggestive of aBeggiatoa origin. Determinations ofδ13C inBeggiatoa mats from vents along the Louisiana slope yielded values in the range of −26.6 to −27.9‰ (PDB), suggesting an unusually high degree of isotope fractionation (−24.9‰) relative to the carbon source in the ambient seawater, which is typical of sulfur-oxidizing chemoautotrophs. The presence of SO (elemental sulfur) within cells ofBeggiatoa resulting from oxidation of H2S supports the importance of bacterial sulfate reduction processes in the underlying vents for the sustenance of theBeggiatoa mats.

Chlamydomonas reinhardtii mutants without ribulose-1,5-bisphosphate carboxylase-oxygenase lack a detectable pyrenoid

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The most prominent protein in the pyrenoid is the enzyme ribulose-1,5-bisphosphate carboxylaseoxygenase (Rubisco). We have investigated whether the pyrenoid is present in strains of Chlamydomonas reinhardtii Dangeard containing mutations in the chloroplast rbcL gene. The mutants examined include a nonsense mutant lacking Rubisco, 18-7G, a missense mutant with an inactive Rubisco, 10-6C, and a temperature-sensitive mutant, 68-4PP, which contains Rubisco at room temperature but lacks the protein at 35°C. Normally, each C. reinhardtii cell has one chloroplast containing one large pyrenoid. In the nonsense mutant and 68-4PP at the non-permissive temperature no pyrenoid was observed. In the other strains, even those with an inactive Rubisco, the pyrenoid appeared normal. These results indicate that the presence of the Rubisco protein is necessary for the formation of a normal pyrenoid in C. reinhardtii. It is also clear that the Rubisco does not have to be active for normal pyrenoid formation, as strains 10-C and F-60 had morphologically normal pyrenoids.

Filamentous sulfide-oxidizing bacteria at hydrocarbon seeps of the Gulf of Mexico.

Mats consisting of the large sulfide‐oxidizing bacterium, Beggiatoa, were collected from the sediment/water interface at several locations in the Gulf of Mexico. The collection sites were associated with the presence of petroleum hydrocarbons or the microbial breakdown products of the hydrocarbons. The morphologies of the mats varied with the nature of the underlying sediments, and some mats were pigmented either yellow or orange instead of the usual white. At one site, beggiatoas were found that had a diameter of nearly 200 μm, making them the largest prokaryotic organism known. In filaments with a diameter of over approximately 10 μm the cytoplasm was restricted to a thin layer immediately underlying the cell membrane, and the majority of the cell consisted of a vacuole with unknown contents. Beggiatoa filaments often rotated as they moved by gliding. Parallel rows of 15 nm diameter pores were found on the surface of the beggiatoas. The pores may have been wound in a spiral fashion around the cell. These pores may be involved in the gliding motility of the bacteria by the motion imparted by the excretion of slime through the pores. Several structures with the typical morphology of prokaryotic cells but lacking a cell wall were found within the vacuolar and cytoplasmic portions of the hollow beggiatoas. Some of these internal “symbionts” ultrastructurally resembled methanotrophic bacteria like those that have been seen in animals taken from vent areas. Other symbionts ultrastructurally resembled autotrophic bacteria with carboxysome‐like structures. These internal symbionts may enable the Beggiatoa to grow in different environments on different carbon sources. They also provide important evidence for the endosymbiotic theory of the evolution of internal organelles of eukaryotic organisms.

Method for Collecting Air-Water Interface Microbes Suitable for Subsequent Microscopy and Molecular Analysis in both Research and Teaching Laboratories

ABSTRACT A method has been developed for collecting air-water interface (AWI) microbes and biofilms that enables analysis of the same sample with various combinations of bright-field and fluorescence light microscopy optics, scanning and transmission electron microscopy (TEM), and atomic force microscopy. The identical sample is then subjected to molecular analysis. The sampling tool consists of a microscope slide supporting appropriate substrates, TEM grids, for example, that are removable for the desired protocols. The slide with its substrates is then coated with a collodion polymer membrane to which in situ AWI organisms adhere upon contact. This sampling device effectively separates the captured AWI bacterial community from the bulk water community immediately subtending. Preliminary data indicate that the AWI community differs significantly from the water column community from the same sample site when both are evaluated with microscopy and with 16S ribosomal DNA sequence-based culture-independent comparisons. This microbe collection method can be used at many levels in research and teaching.

PHRAGMOPLASTS IN CYTOKINESIS OF CEPHALEUROS PARASITICUS (CHLOROPHYTA) VEGETATIVE GELLS 1

Cytokinesis in apical cells of actively growing cultures of Cephaleuros parasiticus Karsten sporangiate thalli was examined with transmission electron microscopy. A massive, interzonal cytokinetic microtubule spindle is anchored at its poles to the medial surfaces of the daughter nuclei at telophase. Later, the daughter nuclei are widely separated and no longer associated with the interzonal spindle; however, the spindle retains its shape and becomes a distinct phragmoplast with an array of vesicles, presumably derived from dictyosomes, aligned in the division plane. Fusion of the vesicles gives rise to a thin cell plate. Some bundles of microtubules in the spindle appear to mark the sites of plasmodesmata formation, but no endoptasmic reticulum is directly involved in plasmodesmata formation. No infurrowing or phycoplast array of microtubules is involved in the cytokinesis. The relationship, if any. between the metaphase‐anaphase mitotic microtubule system and the interzonal cytokinetic spindle has not been determined. Cephaleuros parasiticus isone of only four green algae now known to contain a higher plant‐like phragmoplast and cytokinetic process. The observations reported can be interpreted as very strong evidence for a phylogenetic affinity between the Trentepohliaceae and the Charophyceae, but consideration of ulvophycean features of the Trentepohliaceae such as motile cell ultrastructure and life histories precludes unequivocal assignment of the family to either the Charophyceae or Ulvophyceae.

Phragmoplast-mediated cytokinesis in trentepohlia : results of TEM and immunofluorescence cytochemistry

This paper reports transmission electron microscopy (TEM) and immunofluorescence evidence for phragmoplast-mediated cytokinesis in the green alga, Trentepohlia (Ulvophyceae, Chlorophyta). This type of cytokinesis is normally found only in land plants and some charophycean green algae. Earlier TEM work documented a phragmoplast in another trentepohlialean alga, Cephaleuros. Numerous molecular studies have shown that both Trentepohlia and Cephaleuros are clearly not in the charophycean (streptophyte) lineage of green algae. Also, details of vegetative cytokinesis in Trentepohlia and Cephaleuros indicate differences from the processes and structures observed in the streptophytes. Parallel evolution could be the explanation for a phragmoplast-mediated cytokinesis in both the chlorophycean Trentepohliales and the streptophyte lineage of charophycean green algae and land plants.

Low-CO2-inducible protein synthesis in the green alga Dunaliella tertiolecta

In the green marine alga Dunaliella tertiolecta, a CO2-concentrating mechanism is induced when the cells are grown under low-CO2 conditions (0.03% CO2). To identify proteins induced under low-CO2 conditions the cells were labelled with 35SO42−, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO2 (5% CO2 in air) were switched to low CO2, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO2-grown cells and an aditional 49-kDa band exclusively in low-CO2-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO2 cell types as well as an immunoreactive 100-kDa band occurring only in low-CO2-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO2 concentration.