Production of Calcite Nanocrystal by a Urease-Positive Strain of Enterobacter ludwigii and Study of Its Structure by SEM

Helicobacter pylori (H. Pylori) is an actively dividing spiral bacterium that changes to coccoid morphology under stressful environments. The infectivity of the coccoids is still controversial. The aim of this study was to determine the viability and expression of two important virulence genes (babA and cagE), in antibiotic-induced coccoid forms. Three strains of H. pylori, the standard 26695 and two clinical isolates (p1, p2) were converted to coccoid form by amoxicillin. Coccoids were identified according to Gram-staining and microscopic morphology. The viability of the cells was analyzed by flow cytometry. The expression of cagE and babA in coccoid forms were evaluated and compared to the spirals by quantitative PCR assay. The coccoid forms were developed after 72 h exposure of H. pylori to ½ MIC of amoxicillin, and the conversion form was completed (100 %) at 144 h in all of three isolates. Flow cytometry analyses showed that the majority of the induced coccoids (90–99.9 %) were viable. Expression of cagE and babA was seen in coccoids; however, in lower rate (cagE, ~3-fold and babA, ~10-fold) than these in spiral forms. Coccoid forms of two clinical isolates significantly expressed higher rate of cagE and babA than standard 26695 strain (P = 0.01). These results suggest that the induced coccoid form of H. pylori is not a passive entity but can actively infect the human by expression of the virulence genes for long time in stomach and probably play a role in chronic and severe disease.     

Overexpression of <Emphasis Type="Italic">spoT</Emphasis> gene in coccoid forms of clinical <Emphasis Type="Italic">Helicobacter pylori</Emphasis> isolates

Helicobacter pylori (H. pylori) can convert to coccoid form in unfavorable conditions or as a result of antibiotic treatment. In order to adapt to harsh environments, H. pylori requires a stringent response which, encoded by the spoT gene, has a bifunctional enzyme possessing both (p)ppGpp synthetic and degrading activity. Our goal in this study was to compare spoT gene expression in spiral and induced coccoid forms of H. pylori with use of amoxicillin. First, clinical isolate coccoid forms were induced with amoxicillin; then, the viability test was analyzed by flow cytometer. After RNA extraction, cDNA synthesis and designing a specific primer for spoT gene, evaluation of the desired gene expression in both forms were studied. Bacterial isolates exposed to amoxicillin at MIC and 1/2 MIC induced morphological conversion better and faster than other MIC concentration. The expression of spoT gene was significantly downregulated in spiral forms of H. pylori, while the gene expression was upregulated and + 30.3-fold changes was seen in coccoid forms of bacterium. To summarize, spoT gene is one of the key factors for antibiotic resistance and its enhanced expression in coccoid form can be a valuable diagnostic marker for recognition of H. pylori during morphological conversion.     

Coccoid Form of Helicobacter pylori as a Morphological Manifestation of Cell Adaptation to the Environment

After characterization of preferred conditions for Helicobacter pylori survival in the sessile state, it was observed that the bacterium transforms from spiral to coccoid under mild circumstances, whereas under extreme ones it is unable to undergo shape modification. This strongly supports the view that transformation into the coccoid form is an active, biologically led process, switched on by the bacterium as a protection mechanism.     

Ultrastructure of Calcareous Dinophytes (Thoracosphaeraceae,Peridiniales) with a Focus on Vacuolar Crystal-Like Particles

Biomineralization in calcareous dinophytes (Thoracosphaeracaea, Peridiniales) takes place in coccoid cells and is presently poorly understood. Vacuolar crystal-like particles as well as collection sites within the prospective calcareous shell may play a crucial role during this process at the ultrastructural level. Using transmission electron microscopy, we investigated the ultrastructure of coccoid cells at an early developmental stage in fourteen calcareous dinophyte strains (corresponding to at least ten species of Calciodinellum, Calcigonellum, Leonella, Pernambugia, Scrippsiella, and Thoracosphaera). The shell of the coccoid cells consisted either of one (Leonella, Thoracosphaera) or two matrices (Scrippsiella and its relatives) of unknown element composition, whereas calcite is deposited in the only or the outer layer, respectively. We observed crystal-like particles in cytoplasmic vacuoles in cells of nine of the strains investigated and assume that they are widespread among calcareous dinophytes. However, similar structures are also found outside the Thoracosphaeraceae, and we postulate an evolutionarily old physiological pathway (possibly involved in detoxification) that later was specialized for calcification. We aim to contribute to a deeper knowledge of the biomineralization process in calcareous dinophytes.     

Alpha-ketoglutarate oxidoreductase, an essential salvage enzyme of energy metabolism, in coccoid form of Helicobacter pylori

In the Krebs cycle of Helicobacter pylori, the absence of alpha-ketoglutarate dehydrogenase and succinyl CoA synthetase are shown. Instead, alpha-ketoglutarate is converted to succinyl CoA and succinate by alpha-ketoglutarate oxidoreductase (KOR) and CoA transferase (CoAT). In the present study, when H. pylori transformed to the coccoid form, a viable but non-culturable form of H. pylori with reduced metabolic activity, the KOR activity was enhanced while the CoAT activity was reduced. Direct inactivation of KOR could potently kill the bacteria without allowing conversion to the coccoid form, suggesting a novel treatment strategy for the eradication of H. pylori, especially in cases infected with multiple antibiotic-resistant strains.     

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.     

Methanosarcina mazei LYC, a New Methanogenic Isolate Which Produces a Disaggregating Enzyme

A methanogenic coccoid organism, Methanosarcina mazei LYC, was isolated from alkaline sediment obtained from an oil exploration drilling site. The isolate resembled M. mazei S-6 by exhibiting different morphophases during its normal growth cycle. It differed from M. mazei S-6 by undergoint a spontaneous shift from large, irregular aggregates of cells to small, individual, irregular, coccoid units. In batch cultures at pH 7.0, M. mazei LYC grew as aggregates during the early growth stage. As the batch culture began exponential growth, the cell aggregates spontaneously dispersed: the culture liquid became turbid, and myriads of tiny (diameter, 1 to 3 μm) coccoid units were observed under phase-contrast microscopy. Disaggregation apparently was accomplished by the production of an enzyme which hydrolyzed the heteropolysaccharide component of the cell wall; the enzyme was active on other Methanosarcina strains as well. Although the enzyme was active when tested at pH 6.0, it apparently was not produced at that pH: when strain LYC was grown at pH 6.0, only cell aggregates were present throughout batch growth. Individual coccoid cells of M. mazei LYC were sensitive to sodium dodecyl sulfate, but the large aggregates of cells were not. Strain LYC rapidly used H₂-CO₂, in addition to methanol, and mono-, di-, and trimethylamine as methanogenic substrates; acetate was used very slowly. Its optimum growth temperature was 40°C, and its optimum pH was 7.2.     

Mucocytes with micronuclei and sowing with the coccoid forms of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in a mucous membrane of human stomach

In accordance with the solution of IARC, the Helicobacter pylori (H. pylori) refers to carcinogens of the first group. As the carcinogenic factors have a mutagen effect, we have undertaken the cytogenetic testing of 62 patients with chronic nonatrophic gastritis (40 of which have H. pylori-associated gastritis) by account of the micronuclei in mucocytes of tectorial-pit epithelium of the mucous membrane of the antral region of the stomach. The detection of H. pylori cells in the mucous membrane of the stomach (SMM) was performed with the help of immunocytochemical method that permitted us to visualize both the bacillar and coccoid forms, as well as to evaluate the degree of sowing of SMM with the coccoid forms of H. pylori. In the patient group with H. pylori-associated gastritis, the frequency rate of mucocytes with micronuclei in SMM appears to be considerably higher than in the group of patients whose SMM was not infected with H. pylori (P < 0,05). A high scale of sowing with the coccoid forms of H. pylori was accompanied by a significantly heightened level of mucocytes with micronuclei in the SMM. In connection with this and on the basis of modern notions of carcinogenesis, based on mutagen modifications in somatic cells, patients that exhibit high sowing with coccoid forms of H. pylori may be placed in the group of heightened oncologic hazards.     

Isolation and Characterization of Methanogenic Bacteria from Landfills

Methanogenic bacteria were isolated from landfill sites in the United Kingdom. Strains of Methanobacterium formicicum, Methanosarcina barkeri, several different immunotypes of Methanobacterium bryantii, and a coccoid methanogen distinct from the reference immunotypes were identified.     

Effects of Calcium, Magnesium, pH, and Extent of Growth on the Morphology of Methanosarcina mazei S-6

Methanosarcina mazei S-6 grew faster and its morphology changed to individual coccoid cells in medium with elevated concentrations of divalent cations and a large amount of catabolic substrate.     

Endobacterial morphotypes in nudibranch cerata tips: a SEM analysis

The SEM investigation of nudibranch cerata material exhibits endobacterial morphotypes found in 12 out of 13 species tested: Aeolidia papillosa, Berghia caerulescens, Coryphella brownii, Coryphella lineata, Coryphella verrucosa, Cuthona amoena, Facelina coronata, Flabellina pedata, Dendronotus frondosus, Doto coronata, Tritonia plebeia and Janolus cristatus. Endobacteria could not be detected inside Tritonia hombergi. Endobacterial morphology found inside nudibranch species was compared to bacterial morphotypes detected earlier in tentacles of cnidarian species. SEM micrographs show endobacterial analogy among nudibranch species, but also similarity to cnidarian endobacteria investigated earlier. Of course, morphological data of microbes do not allow their identification. However, since most of these nudibranch species prey on cnidaria, it cannot be excluded that many of the endobacteria detected inside nudibranch species may originate from their cnidarian prey. Our previous data describing genetic affiliation of endobacteria from nudibranchian and cnidarian species support this assumption. Dominant coccoid endobacteria mostly exhibit smooth surface and are tightly packed as aggregates and/or wrapped in envelopes. Such bacterial aggregate type has been described previously in tentacles of the cnidarian species Sagartia elegans. Similar coccoid bacteria, lacking envelopes were also found in other nudibranch species. A different type of coccoid bacteria, characterized by a rough surface, was detected inside cerata of the nudibranch species Berghia caerulescens, and surprisingly, inside tentacles of the cnidarian species Tubularia indivisa. In contrast to cnidarian endobacteria, rod-shaped microorganisms are largely absent in nudibranch cerata.     

Temperature-Dependent Genome Degradation in the Coccoid Form of Campylobacter jejuni

Campylobacter jejuni undergoes a dramatic morphological transformation from a corkscrew-shaped rod to a coccoid form in response to unfavorable conditions. It has been speculated that the coccoid plays an important role in the survival and dissemination of C. jejuni but questions still remain regarding the viability of coccoid cells. Characterization of the genome of coccoid cells found that newly formed coccoid cells (i.e., 1–3 days) had a SmaI-digestion profile identical to that of spiral-shaped cells; however, there was a progressive degradation of the DNA with continued incubation at 37°C. Concomitant with genome degradation was the detection of DNA in supernatants of coccoid cells. In contrast, cells incubated at 4°C retained a spiral shape and their SmaI-digestion profile for 8 weeks and released little DNA into the medium. Thus, low temperature inhibited both coccoid formation and genome degradation. Collectively, these data support the theory that the coccoid form of C. jejuni is a manifestation of cellular degradation and spiral-shaped cells, or possibly coccoid cells formed at low temperature, are the most probable candidates for a viable but nonculturable form of this pathogen.     

Thoracosphaera heimii (Lohmann) Kamptner is a dinophyte: Observations on its morphology and life cycle

Thirty clones of Thoracosphaera heimii (Lohmann) Kamptner have been isolated and cultured from oceanic waters of the western North Atlantic and Gulf of Mexico. Observations on the life cycle and morphology of one clone isolated from the Sargasso Sea are reported herein. The calcareous cell wall is present in the coccoid, vegetative life phase. Reproduction is accomplished by the formation of transitory aplanospore or planospore stages or occasionally by binary fission of weakly calcified cells. The planospore is non-thecate and Gymnodinium-like with an undulating transverse flagellum and a whip-like longitudinal flagellum. All life stages possess chloroplasts and a nucleus with continually condensed chromosomes. The planospore morphology and the dinocaryotic nucleus demonstrate that T. heimii is a dinophyte and not a coccolithophorid. The taxonomic affinity and classification of T. heimii within the Dinophyceae is discussed and a new order Thoracosphaerales Tangen, ord. nov. is proposed for primarily coccoid marine dinoflagellates that possess a calcified cell wall in the vegetative life phase.     

Chlorophycean parasite on a marine fish,Sillago japonica (Japanese sillago)

A green spotted Japanese sillago (Sillago japonica) was caught by a fisherman and brought to the laboratory for pathological inspection. The green spots were abundant on the lateral line and more extensively so within the mouth cavity. In both sites, green spots were embedded within the fish flesh and formed 2–3 mm dome-shaped colonies. SEM revealed these colonies to harbor numerous unknown cells with small, surface warts (ornamentations). Molecular analysis showed the cells were Desmodesmus (D. komarekii), a common freshwater coccoid green alga found in ponds and rivers worldwide. It is uncertain how the host fish came to be infected with the alga which was not merely attached externally but embedded within the flesh and inside the mouth cavity. This is the first case of parasitic form of coccoid green algae in marine fish and provides new insights into the variable nature of green algae.     

The most ancient terrestrial lichen <Emphasis Type="Italic">Winfrenatia reticulata</Emphasis>: A new find and new interpretation

Silicified fossils from Rhynie cherts in Scotland are studied. A lichen belonging to the genus Winfrenatia is detected and studied. This oldest terrestrial lichen is dated to the Pragian (=Siegenian) of the Early Devonian. New characters of the lichen are described, and their new interpretation is given. The main component of the lichen thallus is a filamentous cyanobacterium (Nostocales). Structures which were interpreted as fungal hyphae are probably hollow sheaths of this cyanobacterium. Mycobiont hyphae develop at the base of the thallus and symbiose with a coccoid cyanobacterium. Thus, Winfrenatia reticulata is a three-parted organism, constituted of a mycobiont and filamentous and coccoid cyanobacteria.     

Ultrastructure of the endosymbionts of the whitefly,Bemisia tabaci andTrialeurodes vaporariorum

Summary The ultrastructure of the mycetocytes and mycetome micro-organisms of the sweetpotato whitefly,Bemisia tabaci Genn. andTrialeurodes vaporariorum West are described. InB. tabaci, two morphologically distinct types of micro-organisms were observed in mycetocytes. The predominant type lacked a distinct cell wall, was pleomorphic in shape with a surrounding vacuole. The second type was a coccoid organism, with inner and outer cell membranes. The coccoid organism was often found in groups of varying number within vacuoles, and in many cases appeared to be undergoing degradation. InT. vaporariorum mycetocytes, pleomorphic and coccoid organisms were found, although the coccoid micro-organism inT. vaporariorum, had a thicker cell wall than the coccoid micro-organism inB. tabaci.Abbreviations C coccoid micro-organism - P pleomorphic micro-organism     

Urease Activity and Urea Gene Sequencing of Coccoid Forms of H. pylori Induced by Different Factors

Helicobacter pylori exists in two morphologic forms: spiral shaped and coccoid. The nonculturable coccoid forms were believed to be the morphologic manifestations of cell death for a long time. However, recent studies indicate the viability of such forms. This form of H. pylori is now suspected to play a role in the transmission of the bacteria and is partly responsible for relapse of infection after antimicrobial treatment. Urease activity of H. pylori is an important maintenance factor. Determination of urease activity and possible mutations in the DNA sequences of coccoid bacteria will hence contribute to the understanding of pathogenesis of infections, which these forms might be responsible for. In this study, our aim was to analyze the urease activity and investigate the urease gene sequences of coccoid H. pylori forms induced by different factors with respect to the spiral form. For this purpose, the urease activities of H. pylori NCTC 11637 standard strain and two clinical isolates were examined before and after transformation of the cells to coccoid forms by different methods such as exposure to amoxicillin, aerobiosis, cold starvation, and aging. The effects of these conditions on the urease gene were examined by the amplification of 411-bp ureA gene and 115-bp ureB gene regions by PCR technique and sequencing of the ureA gene. The urease activities of coccoid cells were found to be lower than those of the spiral form. ureA and ureB gene regions were amplified in all coccoid cells by PCR. Inducing the change to coccoid form by different methods was found to have no effect on the nucleotide sequence of the ureA gene. These results show that the urease gene region of coccoid H. pylori is highly protected under various mild environmental conditions.     

The Campylobacter jejuni helical to coccoid transition involves changes to peptidoglycan and the ability to elicit an immune response

Campylobacter jejuni is a prevalent enteric pathogen that changes morphology from helical to coccoid under unfavorable conditions. Bacterial peptidoglycan maintains cell shape. As C. jejuni transformed from helical to coccoid, peptidoglycan dipeptides increased and tri‐ and tetrapeptides decreased. The DL‐carboxypeptidase Pgp1 important for C. jejuni helical morphology and putative N‐acetylmuramoyl‐L‐alanyl amidase AmiA were both involved in the coccoid transition. Mutants in pgp1 and amiA showed reduced coccoid formation, with ?pgp1?amiA producing minimal coccoids. Both ?amiA and ?amiA?pgp1 lacked flagella and formed unseparated chains of cells consistent with a role for AmiA in cell separation. All strains accumulated peptidoglycan dipeptides over time, but only strains capable of becoming coccoid displayed tripeptide changes. C. jejuni helical shape and corresponding peptidoglycan structure are important for pathogenesis‐related attributes. Concomitantly, changing to a coccoid morphology resulted in differences in pathogenic properties; coccoid C. jejuni were non‐motile and non‐infectious, with minimal adherence and invasion of epithelial cells and an inability to stimulate IL‐8. Coccoid peptidoglycan exhibited reduced activation of innate immune receptors Nod1 and Nod2 versus helical peptidoglycan. C. jejuni also transitioned to coccoid within epithelial cells, so the inability of the immune system to detect coccoid C. jejuni may be significant in its pathogenesis.     

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.     

Phylogenetic analyses of 18s rdna sequences reveal a new coccoid lineage of the prasinophyceae (Chlorophyta)

Phylogenetic analyses of 18S rDNA sequences from 25 prasinophytes, including 10 coccoid isolates, reveals that coccoid organisms are found in at least three prasinophyte lineages. The coccoid Ostreococcus tauri is included in the Mamiellales lineage and P ycnococcus provasolii is allied with the flagellate P seudoscourfieldia marina. A previously undescribed prasinophyte lineage is comprised of the coccoid Prasinococcus cf. capsulatus (CCMP 1407) and other isolates tentatively identified as Prasinococcus sp. (CCMP 1202, CCMP 1614, and CCMP 1194), as well as three unnamed coccoids (CCMP 1193, CCMP 1413, and CCMP 1220). No flagellate organisms are known from this lineage. Organisms of this new lineage share some characteristics of both the Pycnococcaceae and the Mamiellales, although relationships among these separate lineages were not supported by bootstrap analyses. An additional unnamed coccoid isolate (CCMP 1205) is separate from all major prasinophyte lineages. The analyses did not resolve the relationships among the major prasinophyte lineages, although they support previous conclusions that the Prasinophyceae are not monophyletic.