Study of ectoparasitism of ultramicrobacteria of the genus <Emphasis Type="Italic">Kaistia</Emphasis>, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gramnegative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated [1], but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.     

Ultrastructural organization and development cycle of soil ultramicrobacteria belonging to the class Alphaproteobacteria

Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development cycle, the strains formed small coccoid cells of 400-800 nm and ultrasmall cells of 200-300 nm. Phylogenetically, the strains NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated. The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm. The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB: these include the following: (a) piles of rod-like subunits, ca. 30 A in diameter and 150-250 angstroms in length: (b) long bunches (up to 300-400 angstroms) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200-300 angstroms in diameter) localized in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial (CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB eytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2-3) located directly on the mother cell, without formation of intennediate hyphae.     

Antibacterial activity of some unsymmetrical diorganyltellurium(IV) dichlorides

Six unsymmetrical diorganyltellurium(IV) dichlorides RR'TeCl2 (where R= phenacyl-, 1-naphthacyl-, and styrylacyl- and R' = p-methoxyphenyl, p-hydroxyphenyl-, and 3-methyl-4-hydoxyphenyl-) were tested for their antibacterial activity against gram-positive (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 25923) and gram-negative (Escherichia coli ATCC 25922. Pseudomonas aeruginosa ATCC 27853 and Salmonella sp.) bacteria. Antibacterial activity was measured by disk diffusion method. Inhibition zones demonstrated that all the compounds showed good activity against gram-negative strains. Phenacyl (3-methyl-4-hydroxyphenyl) tellurium(IV) dichloride and naphthacyl (3-methyl-4-hydroxyphenyl) tellurium(IV) dichloride showed significant activity against both gram-positive and gram-negative strains. Among the tested compounds, the former exhibited maximum activity against gram-positive bacteria, while the latter against all the bacteria under study and styrylacyl (p-methoxyphenyl) tellurium(IV) dichloride against all the three gram-negative bacteria.     

Ultrastructural organization and development cycle of soil ultramicrobacteria belonging to the class <Emphasis Type="Italic">Alphaproteobacteria</Emphasis>

Gram-negative chemoorganotrophic soil ultramicrobacteria (UMB), strains NF1 and NF3, have been isolated. In their development cycle, the strains formed small coccoid cells of 400–800 nm and ultrasmall cells of 200–300 nm. Phylogenetically, the strains NF1 and NF3 belong to Alphaproteobacteria and are close to the type strain of the recently described species Kaistia adipata. The ultrastructure of UMB cells has been studied using ultrathin sections and freeze-fracturing. It has been shown that the structure of UMB cell walls is of the gram-negative type; the outer membrane and peptidoglycan layer are well differentiated. The cell surface has numerous protrusions (prosthecae) of conical or spherical shape filled with the contents of the periplasm. The formation of unusual cellular structures (not occurring in known free-living bacteria) is a feature of UMB; these include the following: (a) piles of rod-like subunits, ca. 30 Å in diameter and 150–250 Å in length; (b) long bunches (up to 300–400 Å) comprised of filamentous subunits; and (c) large electron-dense spherical bodies (up to 200–300 Å in diameter) localized in the periplasm. A distinctive feature of UMB is their ability to grow as facultative parasites on living cyanobacterial (CB) cells. In this case, three types of interaction between UMB and CB have been revealed: (1) adsorption of UMB cells on the surface of CB cells; (2) penetration of UMB into polysaccharide sheathes; and (3) penetration of UMB into CB cytoplasm. UMB cells have been shown to reproduce by budding, with buds (up to 2–3) located directly on the mother cell, without formation of intermediate hyphae.     

Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Ultramicrobacteria: Formation of the concept and contribution of ultramicrobacteria to biology

Ultramicrobacteria (UMB) are species of the domain Bacteria characterized by very small sizes of proliferating cells (less than 0.1 μm³ in volume) and small genomes (3.2 to 0.58 Mb). Some authors use the term nanobacteria as a synonym of UMB. Several tens of UMB species have been isolated from various natural habitats: sea water, soil, silt, Greenland ice sheet, permafrost soils, and intestines of humans and insects. Under laboratory conditions, they are cultivated on different nutrient media. In the second prokaryotic domain, the Archaea, ultrasmall forms (ultramicroarchaea) have also been described, including nanoarchaea (members of the genus Nanoarchaeum) with a cell volume of less than 0.1 μm³. The term nanobacteria is used in the literature also to denote ultrasmall bacterium-like particles occurring in rocks, sands, soils, deep sub-surface layers, meteorites, and clinical samples. The systematic position and the capacity for self-reproduction of these particles are still unclear. The cultured UMB forms are characterized by highly diverse morphology, ultrastructural organization, physiology, biochemistry, and ecology. UMB form three groups according to the type of cell wall structure and the reaction to Gram staining: (1) gram-negative, (2) gram-positive, and (3) cell wall-lacking. Their cells divide by constriction, septation, or budding. The unique processes performed by UMB are dehalorespiration and obligate or facultative epibiotic parasitism. The UMB that synthesize organic compounds in ocean waters with the involvement of proteorhodopsin play a great role in the biosphere. UMB have been found in seven large phylogenetic groups of prokaryotes, where their closest relatives are organisms with larger cells typical of bacteria, which is evidence of the polyphyletic origin of the currently known UMB species and the reductive mode of their evolution.     

Bactericidal effect of gentamicin-induced membrane vesicles derived from Pseudomonas aeruginosa PAO1 on gram-positive bacteria

Previous studies have shown that gentamicin-induced membrane vesicles (g-MVs) from Pseudomonas aeruginosa PAO1 possess both the antibiotic (gentamicin) and a potent peptidoglycan hydrolase (PGase; autolysin) that is effective in killing gram-negative pathogens. This present study evaluated the therapeutic potential of g-MVs against four gram-positive bacteria. Bactericidal assays and electron microscopy of thin sections revealed that Bacillus subtilis 168 and Staphylococcus aureus D2C were susceptible to killing mediated by g-MVs, Listeria monocytogenes ATCC 19113 was slightly susceptible, whereas Enterococcus hirae ATCC 9790 was unaffected. g-MVs were generally more effective against the bacteria than was soluble gentamicin, suggesting they could have more killing power than natural membrane vesicles containing no antibiotic. Electron microscopy and hydrophobic interaction chromatography showed that more membrane vesicles (MVs) initially attached to B. subtilis (hydrophilic) than to predominantly hydrophobic E. hirae, L. monocytogenes, and S. aureus. Zymograms containing murein sacculi as an enzyme substrate illustrated that all organisms except E. hirae were sensitive to the 26-kDa autolysin to varying degrees. Peptidoglycan O-acetylation did not influence susceptibility to MV-mediated lysis. Though not universally effective, the g-MV delivery system remains a promising therapeutic alternative for specific gram-positive infections.     

Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride

In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions (α to θ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

Actinomycetes in Karstic caves of northern Spain (Altamira and Tito Bustillo).

A variety of isolation procedures were carried out to study the involvement of bacteria in the colonisation and biodeterioration of Spanish caves with paleolithic rock art (Altamira and Tito Bustillo). The applied techniques mainly aimed to isolate heterotrophic bacteria such as streptomycetes, nocardioform and coryneform actinomycetes, and other gram-positive and gram-negative bacteria. The results demonstrated that actinomycetes were the most abundant gram-positive bacteria in the caves. Actinomycetes revealed a great taxonomic diversity with the predominant isolates belonging to the genus Streptomyces. Members of the genera Nocardia, Rhodococcus, Nocardioides, Amycolatopsis, Saccharothrix, Brevibacterium, Microbacterium, and coccoid actinomycetes (family Micrococcaceae) were also found.     

Accumulation in gram-postive and gram-negative bacteria as a mechanism of resistance to erythromycin

Erythromycin was recovered in high yield after incubation with gram-negative bacteria. The cell-free protein-synthesizing preparation from gram-negative bacteria is equally as susceptible to the antibiotic as is that from gram-positive bacteria. Thus, neither destruction of erythromycin nor the absence of the step susceptible to the antibiotic plays an important role in the resistance mechanism of gram-negative bacteria. A 100-fold difference in accumulation of erythromycin between gram-positive and gram-negative bacteria was observed. This alone explains the resistance of gram-negative bacteria to erythromycin. Furthermore, data showed that the inhibition of growth is closely related to the accumulation of erythromycin. The concentration of intracellular erythromycin in gram-positive bacteria was found to be 44- to 90-fold greater than that of the extracellular medium. However, the antibiotic did not accumulate on the cell walls, nor was the accumulation energy-dependent. It is proposed that it takes place by the binding of erythromycin to the bacterial ribosomes, forming a very stable complex. The dissociation constants of erythromycin-Staphylococcus aureus complex and erythromycin-Bacillus subtilis complex were determined to be 1.1 x 10(-7) and 3.4 x 11(-7)m, respectively.     

Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168.

The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells. Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX. In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis. It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B. subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E. coli proteins. The B. subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection. The B. subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B. subtilis LH45, which is a subtilin-producing mutant of B. subtilis 168. B. subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B. subtilis ATCC 6633 (W. Liu and J. N. Hansen, J. Bacteriol. 173:7387-7390, 1991). Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633. Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium. Conversely, a mutant (LH45 deltaproU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine. We conclude that the proU-like locus in B. subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.     

A cytological characterization of the parasitic action of ultramicrobacteria NF1 and NF3 of the genus Kaistia on chemoorganotrophic and phototrophic bacteria

Two strains (NF1 and NF3) of free-living chemoorganotrophic bacteria have been isolated from multiyear oil slime and Pedilanthus tithymaloides rhizosphere and ascribed to the genus Kaistia of the class Alphaproteobacteria on the basis of the nucleotide sequences of 16S rRNA gene and phenotypic characteristics. These strains can be assigned to ultramicrobacteria as their populations are represented by two subpopulations: (1) ultrasmall cells, on average 200–300 nm in diameter and <0.1 μm³ in volume, of up to 60% of the total number of cells in a population, and (2) cells 400–800 nm in diameter and 0.15–0.5 μm³ in volume, of up to 40% of the total number of cells in a population. The interaction of the isolated ultramicrobacteria strains (IUMB) with different bacterial species has been studied in cocultures grown under starvation and in complete nutrient media. It has been found that IUMB can be facultative parasites on certain species of chemoorganotrophic and phototrophic bacteria. The interaction of IUMB with prey bacteria exhibits the extracellular type of parasitism and involves establishing stable cell–cell contacts between the parasites and their prey to cause destruction of host cells.     

A flow-cytometric gram-staining technique for milk-associated bacteria

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50 degrees C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18 degrees C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5 degrees C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.     

The Influence of Preculture Conditions and Food Quality on the Ingestion and Digestion Process of Three Species of Heterotrophic Nanoflagellates

The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated. Three species of gram-negative bacteria, one species of gram-positive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria). Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles. In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis. Digestion was found to be particle-specific. Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles. The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content. "Selective digestion" could be shown to be an important selection mechanism concerning natural food particles. The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field. In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.     

Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.     

Characterization of the bacterial microflora of the tympanic cavity of eastern box turtles with and without aural abscesses

Aerobic bacterial cultures of the tympanic cavity of the middle ear were performed in eight eastern box turtles (Terrapene carolina carolina) with aural abscesses and 15 eastern box turtles without aural abscesses (controls) that were admitted to The Wildlife Center of Virginia, Virginia, USA during 2003. Twenty-two bacterial isolates were identified from 17 turtles including 10 gram-negative and 12 gram-positive bacteria. Ten of 15 control animals had bacterial growth, resulting in identification of 13 bacteria, including six gram-negative and seven gram-positive agents. Seven of eight turtles with aural abscesses had bacterial growth, and 10 isolates were identified, including four gram-negative and six gram-positive organisms. The most frequently isolated bacteria from control animals were Micrococcus luteus (n = 3) and Pantoea agglomerans (n = 2). Morganella morganii (n = 2) was the only species isolated from the tympanic cavity of more than one turtle with aural abscesses. Staphylococcus epidermidis (n = 2) was the only species isolated from both groups. A trend toward greater bacterial growth in tympanic cavities of affected turtles compared with turtles without aural abscesses was noted. No single bacterial agent was responsible for aural abscesses in free-ranging eastern box turtles in this study, an observation consistent with the hypothesis that aerobic bacteria are not primary pathogens, but secondary opportunistic invaders of environmental origin.     

Occurrence and ultrastructural characterization of bacteria in association with and isolated from Azolla caroliniana.

The occurrence and ultrastructure of bacteria in leaf cavities of symbiotic Azolla caroliniana were examined by transmission electron microscopy. Bacteria were observed in all leaf cavities of Azolla cultures. Five ultrastructurally distinct types of bacteria were observed in each individual leaf cavity. Features used to characterize the bacteria included morphology, cell wall structure, and cytoplasmic organization. At least one gram-positive and as many as four gram-negative types of bacteria reside in leaf cavities of A. caroliniana. The morphological and ultrastructural characteristics of the gram-positive bacterium suggest that it is an Arthrobacter sp. The gram-negative bacteria could not be cultured; therefore, they have not been classified further. Bacterial cell shape and cell wall structure were similar in leaf cavities of different ages, but cell size and cytoplasmic composition varied. The relative contributions of each bacterial type to the total community within individual leaves was determined. Ultrastructural characteristics of bacterial isolates cultured from A. caroliniana in a free-living state were also examined.     

Sporulation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in binary cultures with ultramicrobacteria

The effect of ultramicrobacterial epibionts of the genera Kaistia (strain NF1), Chryseobacterium (strain NF4), and Stenotrophomonas (strain FM3) on the process of sporulation of Bacillus subtilis ATCC 6633 was studied. The investigated strains of ultramicrobacteria (UMB) were found to inhibit the sporulation process of B. subtilis ATCC 6633 in binary mixed cultures, exhibiting a 3-day delay of the onset of sporulation compared to the control one, an extended period of the prospore maturation, formation of the fraction of immature spores, and development of ultrastructural defects in many endospores. Thus, investigation of binary mixed cultures of B. subtilis and UMB revealed that, apart from suppression of reproduction and lysis of host vegetative cells, inhibition of spore formation and destruction of endospores was yet another feature of intermicrobial parasitism. The UMB parasites of the studied genera are assumed to participate in the regulation of development and reproduction of B. subtilis in natural habitats of this spore-forming bacterium.     

Characterization of the chemotaxis fli Y and che A genes in Bacillus cereus

This paper describes the first identification of chemotaxis genes in Bacillus cereus. We sequenced and studied the genomic organization and the expression of the cheA and fliY genes in two different B. cereus strains, ATCC 14579 and ATCC 10987. While cheA encodes a highly conserved protein acting as the main regulator of the chemotactic response in flagellated eubacteria, fliY, which has been previously described only in B. subtilis, is one of the three genes encoding proteins of the flagellar switch complex. Although the sequences and relative position of cheA and fliY were found to be identical in the two B. cereus strains analyzed, the restriction fragment containing both genes was located differently on the physical maps of B. cereus ATCC 14579 and ATCC 10987. Evidence is shown that the genomic organization and the expression of fliY and cheA in B. cereus differ significantly from that described for B. subtilis, which is considered a model microorganism for chemotaxis in gram-positive bacteria.