Predominant Catalase-negative Soil Bacteria. II. Occurrence and Characterization of Actinomyces humiferus, sp. N

A microorganism resembling an Actinomyces species was found to be a numerically predominant inhabitant of various organically rich soils. This organism forms a hyphal-like structure with true branching that fragments into gram-positive diphtheroid and coccoid elements. Its cells ferment carbohydrates and contain both lysine and ornithine as the major basic amino acids of the cell wall. It is catalase-negative, microaerophilic to aerobic, and sensitive to lysozyme, and it is dependent on an organic nitrogen source and incubation at 30 C for optimum growth. Based on these characteristics, a new species, Actinomyces humiferus, is proposed. The ecological and medical implications of a large soil population of this microorganism are discussed.     

Morphological changes of synchronized Campylobacter jejuni populations during growth in single phase liquid culture

Campylobacter jejuni strains demonstrate a variety of growth phase-linked distinct morphological forms when grown in liquid culture. The typical spiral form of the organism, evident during logarithmic phase, undergoes elongation during stationary phase before becoming coccoid via the formation of membrane blebs and budded forms in decline phase. Cellular elongation and coccoid formation occurred despite the inhibition of protein synthesis and without a detectable change in the protein components of the inner and outer cell membranes.     

Production and viability of coccoid forms of Campylobacter jejuni

Studies were conducted into the formation and physiological state of coccoid cells of a strain of the human and animal pathogen Campylobacter jejuni. It was found that growth phase and the presence of chloramphenicol did not affect the rate of shape transformation from spiral to coccoid, while nutrient limitation, aeration of the medium and the presence of free-radical scavengers had profound effects. Coccoid cells were found to reduce the tetrazolium salts INT (2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) to their respective formazans and this was linked to cellular respiration. However, respiring coccoid cells could not sustain their existence in prolonged adverse conditions, and it was concluded that they represent a degenerative stage rather than a dormant state of the organism.     

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     

Parietochloris incisa comb. nov. (Trebouxiophyceae, Chlorophyta)

A coccoid green alga, Myrmecia incisa Reisigl, was isolated from the soil of Mt Tateyama, Japan. Electronmicroscopy revealed that the organism has pyrenoids sparsely covered with starch segments and traversed by many parallel thylakoid membranes, and zoo-spores with counterclockwise basal body orientation. Due to the presence of these features, we have proposed a reclassification of M. incisa into the genus Parietochloris, Trebouxiophyceae.     

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.     

Production of methanethiol from methionine by Brevibacterium linens CNRZ 918

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.     

Two new species of arthrobacter requiring respectively vitamin B12 and the terregens factor

Summary The morphological, cultural, nutritional and general physiological properties of two species of bacteria, isolated from soil, are described which require for growth vitamin B₁₂ and the terregens factor respectively. Both organisms undergo the change in morphology from a rod stage in young culture to a coccoid stage in older culture which is characteristic of members of the genus Arthrobacter Conn and Dimmick and are considered to be most appropriately assigned to this genus. In view of their differences from other described species and from each other the organism for which vitamin B₁₂ is essential is named Arthrobacter duodecadis n. sp. and that requiring the terregens factor Arthrobacter flavescens n. sp.Herrn Professor Dr. Dr. h. c. A. Rippel zum 70. Geburtstag.Contribution No. 455, Bacteriology Division.     

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.     

EFFECT OF AN OXYGEN-REDUCING MEMBRANE FRACTION ON HELICOBACTER PYLORI

The emerging gastric pathogen Helicobacter pylori is an oxygen-sensitive fastidious microaerophile. Culturability of this organism is rapidly lost in oxygen levels present in the atmosphere due to its morphological transformation into a viable but not culturable state. The effect of the Oxyrase^TM system of oxygen-reducing membrane fragments on H. pylori was evaluated at levels ranging from 0.1 to 0.6 Units/mL in Brucella broth supplemented with 5% horse serum. Duplicate sets of Oxyrase^TM dilutions inoculated with H. pylori were incubated at 35C aerobically and microaerobically. At these Oxyrase^TM levels, a logarithmic loss of H. pylori viability was evident in the aerobic cultures. The inoculum remained recoverable for 24 h in the presence of Oxyrase^TM, whereas recovery of inoculum in untreated broth was greatly reduced after 8 h of aerobic incubation, and the organism was unrecoverable after 24 h. Oxyrase^TM-containing broth cultures of H. pylori incubated microaerobically showed a similar drop in viable counts for the first 48 h of incubation; however, at the lower levels of Oxyrase^TM, some cells survived, and resumed logarithmic growth at 96 h. To explore the effects of short term aerobic incubation in the presence of 0, 0.005, 0.05, and 0.5 Units Oxyrase^TM, cultures were examined microscopically after 4, 8, and 24 h. In the Oxyrase^TM-containing broths, <90% of the cells exhibited rod shape morphology after 8 h, whereas in the untreated broth, most cells appeared coccoid. After 24 h, all cells exhibited coccoid morphology.     

Dissimilatory Fe(III) reduction by an electrochemically active lactic acid bacterium phylogenetically related to Enterococcus gallinarum isolated from submerged soil

AIMS: The isolation and identification of a glucose-oxidizing Fe(III)-reducing bacteria (FRB) with electrochemical activity from an anoxic environment, and characterization of the role of Fe(III) in its metabolism. METHODS AND RESULTS: A Gram-positive (Firmicutes), nonmotile, coccoid and facultative anaerobic FRB was isolated based on its ability to reduce Fe(III). Using the Vitek Gram-positive identification card kit and 16S rRNA gene sequence analysis, the isolate was identified as Enterococcus gallinarum, designated strain MG25. On glucose this isolate produced lactate plus small amounts of acetate, formate and CO2 and its growth rates were similar in the presence and absence of Fe(O)OH. These results suggest that MG25 can couple glucose oxidation to Fe(III) reduction, but without conservation of energy to support growth. Cyclic voltammetry showed that strain MG25 was electrochemically active. CONCLUSIONS: An electrochemically active and FRB, E. gallinarum MG25, was isolated from submerged soil. Fe(III) is used in the bacterial metabolism as an electron sink. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report concerning the electrochemical activity of glucose-oxidizing FRB, E. gallinarum. This organism and others like it could be used as new biocatalysts to improve the performance of a mediator-less microbial fuel cell.     

Mycoplasma lactucae sp. nov., a sterol-requiring mollicute from a plant surface

Strain 831-C4T (T = type strain), isolated from the surface of lettuce plants (Lactuca sativa) obtained from a retail food market, was shown to be a sterol-requiring mollicute. Morphological examination of this organism by electron and dark-field microscopic techniques showed that it consists of small, nonhelical, nonmotile, pleomorphic coccoid cells, with individual cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew rapidly in all conventional culture medium formulations for mollicutes in either aerobic or anaerobic environments. The optimum temperature for growth was 30 degrees C, but multiplication occurred at 18 to 37 degrees C. Strain 831-C4T catabolized glucose, but hydrolysis of arginine or urea could not be demonstrated. The genome size of strain 831-C4T was determined to be about 569 megadaltons, while the base composition (guanine-plus-cytosine content) of the DNA was 30.0 mol%. Recent studies in which we compared the 16S rRNA sequences of strain 831-C4T with those of more than 40 other mollicutes indicated that this organism is phylogenetically related to the Spiroplasma-Mycoplasma mycoides clade. Strain 831-C4T was serologically unrelated to the type strains of previously described Mycoplasma species and to 18 other unclassified sterol-requiring isolates cultivated from various animal, plant, or insect sources. Strain 831-C4T (= ATCC 49193) is the type strain of Mycoplasma lactucae sp. nov.     

Kalinella bambusicola gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel coccoid Chlorella-like subaerial alga from Southeast Asia

The traditional green algal genus Chlorella , which comprised coccoid algae surrounded by a smooth cell wall and reproducing solely by autosporulation, has proved to be polyphyletic and extremely diverse in phylogenetic terms. We studied a new subaerial Chlorella -like strain CAUP H7901 and morphological, ultrastructural, and molecular phylogenetic investigations indicated that it represents a new lineage of the trebouxiophycean Watanabea clade, dissimilar from other members of this group. The alga has globular coccoid cells with a single parietal pyrenoid-bearing chloroplast. The pyrenoid is transected by multiple radial thylakoid bands. The alga reproduces exclusively by means of asexual autospores of unequal size. In 18S rDNA sequence phylogenies, it was nested within the Watanabea clade close to lineages containing Chlorella saccharophila , Chlorella luteoviridis , Heveochlorella hainangensis , and two uncharacterized strains, but alternative positions within the Watanabea clade could not be rejected by an approximately unbiased (AU) test. Here we describe this organism as a new genus and species Kalinella bambusicola gen. et sp. nov. Furthermore, we describe Heterochlorella gen. nov. to accommodate a species previously referred to as Chlorella luteoviridis .     

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).     

Ultrastructure and transovarial transmission of endosymbiotic microorganisms in Palaeococcus fuscipennis (Burmeister) (Insecta, Hemiptera, Coccinea: Monophlebidae)

Ovaries ofPalaeococcus fuscipennis (Burmeister) are accompanied by large organs termed bacteriomes which are composed of large cells termed bacteriocytes. Each bacteriocyte is surrounded with small epithelial cells. The bacteriocyte cytoplasm is tightly packed with pleomorphic bacteria, whereas in epithelial cells small coccoid microorganisms are present. The number of coccoid bacteria is significantly lower than pleomorphic bacteria. The ovarioles containing choriogenic oocytes are invaded both by pleomorphic as well by coccoid bacteria. Microorganisms traverse the follicular epithelium and enter the perivitelline space. During advanced choriogenesis, endosymbionts are accumulated in the deep depression of the oocyte. Bacteria do not enter the ooplasm until the end of oocyte growth.     

Control of Morphogenesis in Geodermatophilus: Ultrastructural Studies

Geodermatophilus grows in two major forms, a nonmotile irregularly shaped aggregate of coccoid cells (C-form) and a motile budding rod (R-form). Morphogenesis can be controlled by an unidentified factor in Tryptose which is required for maintenance of the organism in the C-form and for differentiation of the R-form to the C-form. Morphogenetic events occur synchronously in the described system. Ultrastructural studies show that the major difference between C- and R-forms is in cells envelope structure. R-form cell walls consist of two layers, an inner transparent membranous layer (10 to 12.5 nm thick) and an outer dense diffuse layer (7.5 to 10 nm). In addition to these layers, the C-form has a thick fibrous layer (30 nm) over the dense layer. This layer appears to be a cementing substance which holds the coccoid cells together.     

Isolation and characterization of a marine methanogenic bacterium from the biofilm of a shiphull in Los Angeles harbor

A marine mesophilic, irregular coccoid methanogen, which shows close resemblance toMethanococcus sp., was isolated from the biofilm of shiphulls docked in Los Angeles harbor. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8–7.0 and a temperature optimum of 37°C. Elevated levels of sodium chloride were required for optimum growth. Optimum levels of total sulfide and magnesium chloride for growth were 1.0mm and 10mm respectively. The isolate used ammonia as nitrogen source. The concentration of 30mm ammonium chloride supported maximum growth of the isolate.     

Helicobacter pylori: A Fickle Germ

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.     

Growth and pigment production by Arthrobacter pyridinolis n. sp.

A new bacterium capable of growing on 2-hydroxypyridine as sole source of carbon and nitrogen was isolated from soil. During its growth on solid medium, approximately 50% of this substrate was converted to a brilliant blue crystalline pigment which was deposited extracellularly in the colony mass. The pigment was identical to that produced by Arthrobacter crystallopoietes during its growth on 2-hydroxypyridine. The new isolate exhibited the typical cycle of morphogenesis characteristic of the genus Arthrobacter. The organism is different from all other reported species of Arthrobacter. It is proposed that the organism be named Arthorbacter pyridinolis n. sp.     

Isolation of a <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> strain from soil that can degrade polyurethane diol

Polyurethane diol (PUR-diol), a synthetic polymer, is widely used as a modifier for water-soluble resins and emulsions in wood appliances and auto coatings. Non-biodegradability of polyurethanes (PUR) and PUR-based materials poses a threat to environment that has led scientists to isolate microbes capable of degrading PUR. However, the bio-degradation of PUR-diol has not yet been reported. In this study, we report isolation of a soil bacterium that can survive using PUR-diol as sole carbon source. PUR-diol degradation by the organism was confirmed by thin layer chromatographic analysis of the conditioned medium obtained after the growth wherein a significant reduction of PUR-diol was observed compared to non-inoculated medium. To quantify the PUR-diol degradation, a sensitive assay based on High Performance Thin Layer Chromatography has been developed that showed 32% degradation of PUR-diol by the organism in 10 days. Degradation kinetics showed the maximal depletion of PUR-diol during logarithmic growth of the organism indicating a direct relation between the growth and PUR-diol degradation. Mutagenic study and GC-MS analysis revealed that esterase activity is involved in this degradation event. The ribotyping and metabolic fingerprinting analysis showed that this organism is a strain of Pseudomonous aeruginosa (P. aeruginosa). It has also been observed that this strain is able to degrade Impranil DLN™, a variety of commercially available PUR. Therefore this study identifies a new bacterium from soil that has the potential to reduce PUR-related waste burden and adds a new facet to diverse functional activities of P. aeruginosa.