ATP level variations in heterotrophic bacteria during attachment on hydrophilic and hydrophobic surfaces.

A survey of the extracellular ATP levels of 86 heterotrophic bacteria showed that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter secreted elevated amounts of extracellular ATP, ranging from 6.0 to 9.8 pM ATP/colony forming unit (cfu), and that gram-positive bacteria of the genera Kocuria and Planococcus secreted up to 4.1 pM ATP/cfu. Variations in the levels of extracellular and intracellular ATP-dependent luminescence were monitored in living cells of Sulfitobacter mediterraneus ATCC 700856T and Planococcus maritimus F 90 during 48 h of attachment on hydrophobic (poly[tert-butyl methacrylate], PtBMA) and hydrophilic (mica) surfaces. The bacteria responded to different polymeric surfaces by producing either intracellular or extracellular ATP. The level of intracellular ATP in S. mediterraneus ATCC 700856T attached to either surface was as high as 50-55 pM ATP/cfu, while in P. maritimus F 90 it was 120 and 250 pM ATP/cfu on PtBMA and mica, respectively. S. mediterraneus ATCC 700856T generated about 20 and 50 pM of extracellular ATP/cfu on PtBMA and mica, respectively, while the amount generated by P. maritimus F 90 was about the same for both surfaces, 6 pM ATP/cfu. The levels of extracellular ATP generated by S. mediterraneus during attachment on PtBMA and mica were two to five times higher than those detected during the initial screening. High-resolution atomic force microscopy imaging revealed a potentially interesting correlation between the porous cell-surface of certain alpha- and gamma-proteobacteria and their ability to secrete high amounts of ATP.     

Digital image analysis of growth and starvation responses of a surface-colonizing Acinetobacter sp.

Surface growth of an Acinetobacter sp. cultivated under several nutrient regimens was examined by using continuous-flow slide culture, phase-contrast microscopy, scanning confocal laser microscopy, and computer image analysis. Irrigation of attached coccoid stationary-phase Acinetobacter sp. cells with high-nutrient medium resulted in a transition from coccoid to bacillar morphology. Digital image analysis revealed that this transition was biphasic. During phase I, both the length and the width of cells increased. In contrast, cell width remained constant during phase II, while both cell length and cell area increased at a rate greater than in phase I. Cells were capable of growth and division without morphological transition when irrigated with a low-nutrient medium. Rod-shaped cells reverted to cocci by reduction-division when irrigated with starvation medium. This resulted in conservation of cell area (biomass) with an increase in cell number. In addition, the changes in cell morphology were accompanied by changes in the stability of cell attachment. During phase I, coccoid cells remained firmly attached. Following transition in high-nutrient medium, bacillar cells displayed detachment, transient attachment, and drifting behaviors, resulting in a spreading colonization pattern. In contrast, cells irrigated with a low-nutrient medium remained firmly attached to the surface and eventually formed tightly packed microcolonies. It is hypothesized that the coccoid and bacillar Acinetobacter sp. morphotypes and associated behavior represent specialized physiological adaptations for attachment and colonization in low-nutrient systems (coccoid morphotype) or dispersion under high-nutrient conditions (bacillar morphotype).     

Tateyamaria omphalii gen. nov., sp. nov., an alpha-Proteobacterium isolated from a top shell Omphalius pfeifferi pfeifferi

A Gram-negative, strictly aerobic, coccoid to short rod-shaped marine bacterium strain MKT107(T) was isolated from the molluscan top shell Omphalius pfeifferi pfeifferi collected on the coast of Japan. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain MKT107(T) constitutes a new lineage in alpha-Proteobacteria related to the genera Nereida, Roseobacter, Staleya, Oceanibulbus and Sulfitobacter. Strain MKT107(T) was found to require salt for its growth and to be mesophilic. It contained 18:1omega7c and 16:0 as major cellular fatty acids and 3-OH 10:0 and 3-OH 12:0 as hydroxy fatty acids. The DNA base composition of the isolate was 61.6 mol% G+C. The major quinone was Q-10. Sufficient differences existed to distinguish this strain from currently recognized bacterial genera. Therefore, the isolate is classified as representing a new genus and species, Tateyamaria omphalii gen. nov., sp. nov. (type strain MKT107(T) =IAM 15108(T) =KCTC 12333(T); GenBank accession no. AB193438).     

Competition for polymers among heterotrophic bacteria, isolated from particles of the Equatorial Atlantic.

Three heterotrophic bacterial strains, isolated from organic particles of the upper water column of the Equatorial Atlantic, taken during a cruise on the R/V METEOR (1997), were investigated concerning their physiological and phylogenetic properties using classic microbiological and modern molecular-biological methods. All isolates are gram-negative rods able to use polymers such as cellulose, chitin or starch as sole carbon source. The phylogeny of these isolates was investigated by fluorescence in situ hybridization (FISH) and 16S rDNA sequencing. The three isolated strains belong to the Cytophaga/Flavobacteria, gamma-Proteobacteria (Marinobacter sp.), and alpha-Proteobacteria (Sulfitobacter pontiacus). In order to study succession during growth on polymers naturally occurring in marine habitats, FISH was used as a new approach to detect cells from different phylogenetic clusters in the course of a single growth experiment. Mixed cultures consisting of the isolated strains in equal amounts were incubated with cellulose, chitin or starch. Isolate 4301-10/2, a member of the gamma-Proteobacteria, dominated in mixed cultures growing on cellulose, chitin, or starch after only 10 days, with 55, 60, and 95%, respectively, of cells hybridizing with 4,6-diamidino-2-phenylindole (DAPI).     

Role of AmiA in the morphological transition of Helicobacter pylori and in immune escape

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-beta(1,4)-N-acetylmuramyl-L-Ala-D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-kappaB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection.     

Factors affecting production of coccoid forms by Campylobacter jejuni on solid media during incubation

Conditions influencing the conversion of oxygen into toxic derivatives in media were investigated for their effects on production of coccoid forms in cultures of the enteric pathogen Campylobacter jejuni. Compared with stored media, production of coccoid forms was less on freshly prepared media. Whether freshly prepared or stored before use, brucella agar media produced the fewest coccoid forms under the test conditions. Addition of supplements used as detoxifying agents minimized production of these forms on media but antibiotic formulations used in selective media did not influence production of coccoid forms. Furthermore, the type of incubation atmosphere and the strain of C. jejuni influenced the proportions of coccoid forms in cultures. It was deduced from electron microscopy observations during prolonged incubation of cultures that the process of conversion to coccoid forms involves a loss of spiral morphology, a shortening of the cell and retraction of the cytoplasm towards a cell terminal region. Coccoid forms and some intermediate forms in thin sections were found to lack cell integrity. It is concluded that coccoid form production in cultures is a degenerate response to toxic oxygen derivatives in cultures.     

A comparative study of the rod and coccoid forms of Campylobacter jejuni ATCC 29428

Coccoid forms in cultures of a strain of the enteric pathogen Campylobacter jejuni were investigated. A culture containing 100% coccoid forms was non-viable. Coccoid forms had a lesser content of cytoplasmic components and nucleic acids than rods of C. jejuni. During the conversion to coccoid forms nucleotides leaked from the cells. The results of treatments with ionic and non-ionic detergents, and lysozyme and ethylenediaminetetraacetic acid indicated a changed cell wall in coccoid forms compared with rods. Using rate-zonal centrifugation coccoid forms were found to be less dense than rods. The results of this study indicate that the coccoid form of C. jejuni ATCC 29428 is a degenerate cell form which is undergoing cellular degradation.     

Effect of medium supplements, illumination and superoxide dismutase on the production of coccoid forms of Campylobacter jejuni ATCC 29428

Factors influencing the production of coccoid forms in cultures and suspensions of a strain of the enteric pathogen Campylobacter jejuni during storage in air were investigated. Addition of blood or a supplement containing ferrous sulphate, sodium metabisulphite and sodium pyruvate minimized conversion of rods to coccoid forms in cultures. Exposure of cultures to light during storage in air increased the rate of production of coccoid forms. Ultraviolet radiation was shown to effect the viability of cells in suspensions but the increase in production of coccoid forms was low after irradiation. The presence of hydrogen peroxide and its dissociation products in bacterial suspensions increased conversion to coccoid forms. Addition of active superoxide dismutase, a superoxide anion scavenging enzyme, minimized production of coccoid forms in suspensions stored in air. Coccoid forms contained a lower level of superoxide dismutase than rods. It is deduced that a decreased level of the enzyme in cells is linked with production of coccoid forms.     

Effect of medium supplements, illumination and superoxide dismutase on the production of coccoid forms of Campylobacter jejuni ATCC 29428

Factors influencing the production of coccoid forms in cultures and suspensions of a strain of the enteric pathogen Campylobacter jejuni during storage in air were investigated. Addition of blood or a supplement containing ferrous sulphate, sodium metabisulphite and sodium pyruvate minimized conversion of rods to coccoid forms in cultures. Exposure of cultures to light during storage in air increased the rate of production of coccoid forms. Ultraviolet radiation was shown to effect the viability of cells in suspensions but the increase in production of coccoid forms was low after irradiation. The presence of hydrogen peroxide and its dissociation products in bacterial suspensions increased conversion to coccoid forms. Addition of active superoxide dismutase, a superoxide anion scavenging enzyme, minimized production of coccoid forms in suspensions stored in air. Coccoid forms contained a lower level of superoxide dismutase than rods. It is deduced that a decreased level of the enzyme in cells is linked with production of coccoid forms.     

Factors affecting production of coccoid forms by Campylobacter jejuni on solid media during incubation

Conditions influencing the conversion of oxygen into toxic derivatives in media were investigated for their effects on production of coccoid forms in cultures of the enteric pathogen Campylobacter jejuni. Compared with stored media, production of coccoid forms was less on freshly prepared media. Whether freshly prepared or stored before use, brucella agar media produced the fewest coccoid forms under the test conditions. Addition of supplements used as detoxifying agents minimized production of these forms on media but antibiotic formulations used in selective media did not influence production of coccoid forms. Furthermore, the type of incubation atmosphere and the strain of C. jejuni influenced the proportions of coccoid forms in cultures. It was deduced from electron microscopy observations during prolonged incubation of cultures that the process of conversion to coccoid forms involves a loss of spiral morphology, a shortening of the cell and retraction of the cytoplasm towards a cell terminal region. Coccoid forms and some intermediate forms in thin sections were found to lack cell integrity. It is concluded that coccoid form production in cultures is a degenerate response to toxic oxygen derivatives in cultures.     

A comparative study of the rod and coccoid forms of Campylobacter jejuni ATCC 29428

Coccoid forms in cultures of a strain of the enteric pathogen Campylobacter jejuni were investigated. A culture containing 100% coccoid forms was non-viable. Coccoid forms had a lesser content of cytoplasmic components and nucleic acids than rods of C. jejuni. During the conversion to coccoid forms nucleotides leaked from the cells. The results of treatments with ionic and non-ionic detergents, and lysozyme and ethylenediaminetetraacetic acid indicated a changed cell wall in coccoid forms compared with rods. Using rate-zonal centrifugation coccoid forms were found to be less dense than rods. The results of this study indicate that the coccoid form of C. jejuni ATCC 29428 is a degenerate cell form which is undergoing cellular degradation.     

斑马鱼肠道中一株酵母菌菌株的鉴定与系统发育分析

从斑马鱼肠道中分离到一株酵母菌,编号为ZF-5,进行了形态学观察、生理特征测定和26S rDNA D1/D2序列分析,并构建系统发育树。结果表明ZF-5菌株细胞呈卵圆形或杆状,为芽殖,有假菌丝;除乳糖外,能够发酵葡萄糖、蔗糖、麦芽糖等多种碳源;26S rDNA D1/D2区序列分析表明与季也蒙毕赤酵母Pichia guilliermondii的序列相似性最高,构建的系统发育进化树显示菌株ZF-5与Pichia guilliermondii模式菌株CBS 2030（= NRRL Y-2075）亲缘关系最近,     

Identification of two Saccharomyces cerevisiae cell wall mannan chemotypes

We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 --> 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 --> 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. alpha-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties.     

Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.     

SAME BUT DIFFERENT: TWO NOVEL BICARINATE SPECIES OF EXTANT CALCAREOUS DINOPHYTES (THORACOSPHAERACEAE,PERIDINIALES) FROM THE MEDITERRANEAN SEA1

The diversity of extant calcareous dinophytes (Thoracosphaeraceae, Dinophyceae) is currently not sufficiently recorded. The majority of their coccoid stages are cryptotabulate or entirely atabulate, whereas relatively few forms exhibit at least some degree of tabulation more than the archeopyle. A survey of coastal surface sediment samples from the Mediterranean Sea resulted in the isolation and cultivation of several strains of calcareous dinophytes showing a prominent tabulation. We investigated the morphologies of the thecate and the coccoid cells and conducted phylogenetic analyses using Maximum Likelihood and Bayesian approaches. The coccoid cells showed a distinct reflection of the cingulum (and were thus cingulotabulate), whereas thecal morphology corresponded to the widely distributed and species‐rich Scrippsiella. As inferred from molecular sequence data (including 81 new GenBank entries), the strains belonged to the Scrippsiella sensu lato clade of the Thoracosphaeraceae and represented two distinct species. Morphological details likewise indicated two distinct species with previously unknown coccoid cells that we describe here as new, namely S. bicarinata spec. nov. and S. kirschiae spec. nov. Cingulotabulation results from the fusion of processes representing the pre‐ and postcingular plate series in S. bicarinata, whereas the ridges represent sutures between the cingulum and the pre‐ and postcingular series in S. kirschiae, respectively. Bicarinate cingulotabulation appears homoplasious among calcareous dinophytes, which is further supported by a comparison to similar, but only distantly related fossil forms.     

Micronuclei in mucose cells and colonization of human stomach epithelium with coccoid forms of Helicobacter pylori

International Agency for Research on Cancer recognized as sufficient the evidence of Helicobacter pylori (HP) infection carcinogenicity and placed it into the 1 st group of carcinogens. Micronucleus level in gastric epithelial cells of antral stomach region of patients with chronic non-atrophy gastritis (n = 62) was studied. 40 patients of 62 had HP-associated gastritis. The HP-bacterium exists in a spiral and coccoid form. Both morphological forms were examined using immunocytochemistry. Significantly increased micronucleus number was observed in the cells of HP-infected patients compared with non-infected person (P < 0.05). The frequency of stomach epithelium cells with micronuclei was enhanced considerably in the patients infected with the coccoid HP form. Therefore the patients with HP-associated chronic gastritis caused by the coccoid form with high degree of colonization must be considered as a group of enhanced risk of gastric carcinogenesis.     

Morphological forms and viability of Campylobacter species studied by electron microscopy.

Electron microscopic studies of Campylobacter revealed that different morphological forms predominate at different parts of a colony. At the periphery, cells were almost all spirals, while in the center of the colony cells were mainly coccus shaped. Unusual ring-shaped cells, "donuts", were observed in the raised, peripheral region of the colony. Donut or ring forms have not previously been reported for Campylobacter organisms. Our data indicate that young or actively growing cells are mainly spiral shaped. Older cells undergo a degenerative change to coccoid forms. The donut shape appears to be an intermediate stage between spirals and cocci. Comparisons of plate counts of actively growing and inactive cells confirmed that coccoid cells are probably nonviable.     

Searching the point of no return in Helicobacter pylori life: necrosis and/or programmed death?

AIMS: Ultrastructural and molecular studies to support the hypothesis of programmed cell death in Helicobacter pylori were conducted. METHODS AND RESULTS: Evidence of programmed death in H. pylori is provided through electron microscopic detection and cytochemical labelling of electrondense bodies (EDB), containing packaged DNA in coccoid cells, resembling micronuclei of apoptotic eukaryotic cells. This morphological evidence is also supported by DNA cleavage in homogeneous fragments of about 100 base pairs. Programmed cell death was observed in H. pylori cultures at 37 degrees C, with a maximum of 37.5% of EDB coccoid cells after 7 days. The non-permissive temperature of 4 degrees C anticipated this process, with 40% of EDB coccoid forms within 3 days, and it remained substantially unaffected during the observation time of 14 days. CONCLUSION: In these experiments, deprivation of nutrients and a non-permissive temperature acted as a powerful trigger for programmed cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Helicobacter pylori bacterial populations, under stressing stimuli, can respond with programmed cell suicide as a means of species preservation.     

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.