Root Colonization by Agrobacterium tumefaciens Is Reduced in cel, attB, attD, and attR Mutants

Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 10³ bacteria/cm of root length at the time of inoculation to more than 10⁷ bacteria/cm after 10 days. The numbers of cellulose-minus and nonattaching attB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thaliana ecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel and att mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

High Abundances of Aerobic Anoxygenic Photosynthetic Bacteria in the South Pacific Ocean

Little is known about the abundance, distribution, and ecology of aerobic anoxygenic phototrophic (AAP) bacteria, particularly in oligotrophic environments, which represent 60% of the ocean. We investigated the abundance of AAP bacteria across the South Pacific Ocean, including the center of the gyre, the most oligotrophic water body of the world ocean. AAP bacteria, Prochlorococcus, and total prokaryotic abundances, as well as bacteriochlorophyll a (BChl a) and divinyl-chlorophyll a concentrations, were measured at several depths in the photic zone along a gradient of oligotrophic conditions. The abundances of AAP bacteria and Prochlorococcus were high, together accounting for up to 58% of the total prokaryotic community. The abundance of AAP bacteria alone was up to 1.94 × 10⁵ cells ml⁻¹ and as high as 24% of the overall community. These measurements were consistent with the high BChl a concentrations (up to 3.32 × 10⁻³ μg liter⁻¹) found at all stations. However, the BChl a content per AAP bacterial cell was low, suggesting that AAP bacteria are mostly heterotrophic organisms. Interestingly, the biovolume and therefore biomass of AAP bacteria was on average twofold higher than that of other prokaryotic cells. This study demonstrates that AAP bacteria can be abundant in various oligotrophic conditions, including the most oligotrophic regime of the world ocean, and can account for a large part of the bacterioplanktonic carbon stock.     

Quantitative and Qualitative Analysis of Bacteria in Er(III) Solution by Thin-Film Magnetopheresis

Magnetic deposition, quantitation, and identification of bacteria reacting with the paramagnetic trivalent lanthanide ion, Er³⁺, was evaluated. The magnetic deposition method was dubbed thin-film magnetopheresis. The optimization of the magnetic deposition protocol was accomplished with Escherichia coli as a model organism in 150 mM NaCl and 5 mM ErCl₃ solution. Three gram-positive bacteria, Staphylococcus epidermidis, Staphylococcus saprophyticus, and Enterococcus faecalis, and four gram-negative bacteria, E. coli, Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, were subsequently investigated. Quantitative analysis consisted of the microscopic cell count and a scattered-light scanning of the magnetically deposited material aided by the computer data acquisition system. Qualitative analysis consisted of Gram stain differentiation and fluorescein isothiocyanate staining in combination with selected antisera against specific types of bacteria on the solid substrate. The magnetic deposition protocol allowed quantitative detection of E. coli down to the concentration of 10⁵ CFU ml^-1, significant in clinical diagnosis applications such as urinary tract infections. Er³⁺ did not interfere with the typical appearance of the Gram-stained bacteria nor with the antigen recognition by the antibody in the immunohistological evaluations. Indirect antiserum-fluorescein isothiocyanate labelling correctly revealed the presence of E. faecalis and P. aeruginosa in the magnetically deposited material obtained from the mixture of these two bacterial species. On average, the reaction of gram-positive organisms was significantly stronger to the magnetic field in the presence of Er³⁺ than the reaction of gram-negative organisms. The thin-film magnetophoresis offers promise as a rapid method for quantitative and qualitative analysis of bacteria in solutions such as urine or environmental water.     

The competition between Rhizobium leguminosarum bv. viciae strains progresses until late stages of symbiosis

The competition potential of 14 Rhizobium leguminosarum bv. viciae isolates originating from nodules of Pisum sativum was estimated. Genotypic analyses of the isolates revealed a high level of chromosomal and plasmid content diversity. The isolates tagged with a plasmid-bearing constitutively expressed gusA gene were used to inoculate vetch (Vicia villosa) in competition experiments carried out under laboratory conditions. Soil extract containing autochthonous rhizobial population was used as competitor for gus-tagged strains, and the competition was studied by: (i) estimation of Gus⁺ root nodules on whole root systems, (ii) the pattern of individual nodule colonization by Gus⁺/Gus^? rhizobia, and (iii) the number of Gus⁺/Gus^? bacteria recovered from individual nodules. Several patterns of nodule colonization by Gus⁺/Gus^? bacteria were found. Some nodules identified as Gus⁺ contained gus-tagged bacteria only in the young and saprophytic zones, while the symbiotic zone was occupied by unmarked soil rhizobia. In other Gus⁺ nodules, despite the visible colonization of the entire nodule by gus-marked bacteria, a high number of Gus^? soil-derived rhizobia were recovered. The results suggest that rhizobial strains compete with each other also in the late stage of nodule development. Therefore, they may use different strategies to reach the late saprophytic zone of the nodule, which serves as an optimal environment for massive proliferation.     

Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates

Tetracycline-resistant (Tet^r) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet^r gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet^r genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet^r strains transferred Tet^r genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet^r strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects

Lambda duplication phages grown for several rounds on Escherichia coli strains containing arl mutations were recombined at elevated frequencies (3 to 6-fold higher) in subsequent test infections. Enhanced recombination of Arl^? phages (grown on arl bacteria) was demonstrable by assays for altered genetic linkages as well as by the standard assay, which measures the conversion of duplication phages (EDTA-sensitive) to single-copy phages (EDTA-resistant). The accumulated potential for enhanced recombination was lost during subsequent growth of the phages on arl⁺ bacteria. Arl^? phages had the same mutation frequencies, at a variety of loci, as control phages; arl bacteria themselves exhibited normal mutation rates. Arl^? phages had normal plating efficiencies and buoyant densities. DNA extracted from Arl^? phages exhibited the same frequency of strand interruption, the same superhelical density (when circularized in vivo), and the same thermal denaturation profile as DNA from phages grown on arl⁺ bacteria. Recombination of Arl^? phages in the presence of λ repressor was very low, as is the case for normal phages. The recombination frequency of ultraviolet light irradiated (80 J/m²) Arl^? phages was more than twice the sum of the frequencies for unirradiated Arl^? phages and irradiated control phages. Substantially increased recombination of Arl^? phages was observed when either the E. coli RecBC, or RecE (but not RecF) pathway was active.     

Bacterial Population Adherent to the Epithelium on the Roo of the Dorsal Rumen of Sheep

By anaerobic procedures, the total number of adherent bacteria was determined on tissue samples obtained from the roof of the dorsal rumen of three sheep. After four washings, 1.91 × 10⁷, 0.34 × 10⁷, and 1.23 × 10⁷ bacteria per cm² were still attached to the rumen epithelium in sheep 1, 2, and 3, respectively. A total of 95 strains of bacteria were isolated from these three samples. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were presumptively identified as follows: Butyrivibrio fibrisolvens, 30 strains; atypical Butyrivibrio, 5 strains; Bacteroides ruminicola, 22 strains; Lactobacillus, 1 strain; and unknown Bacteroides species, 37 strains. For sheep 3, washing the rumen epithelium a total of 10 times reduced the adherent bacterial population by 93% (8.4 × 10⁵ bacteria per cm²). Of 30 strains isolated from this sample, 22 were presumptively identified as Butyrivibrio and Bacteroides types. These results suggest that the epithelium on the roof of the dorsal rumen is primarily colonized by two genera of bacteria, Butyrivibrio and Bacteroides. Most Butyrivibrio and Bacteroides ruminicola strains appeared to be similar to previously isolated rumen strains. However, the unknown Bacteroides species differed considerably from the three species of this genus which are commonly isolated from rumen contents.     

Heterotrophic bacteria attached to seaweeds

Viable counts of heterotrophic bacteria attached to the green algae, Monostroma nitidum Wittrock and Enteromorpha linza (Linné) J. Agardh, ranged from 10⁴ to 10⁶/cm², and those attached to the red alga Porphyra suborbiculata Kjellman from 10³ to 10⁴/cm². These bacterial populations were larger than those attached to the brown alga Eisenia bicyclis (Kjellman) setchell ranging from 10¹ to 10⁴/cm². The bacterial populations in the environmental sea water. Nabem Inlet and Otsuchi Bay (Japan), were 10³/ml. Orange and yellow pigmented bacteria were predominant on the green and red algae, but not in the bacterial populations of the brown alga and the sea water. Most of the pigmented bacteria were identified as belonging to the Flavobacterium-Cytophaga group. A beneficial relationship was suggested between the green algae and the pigmented bacteria. Proportions of Vibrionaceae were small on the green algae.     

Anoxygenic phototrophic bacteria from gypsum karst lakes of Lithuania

The spatial distribution and composition of anoxygenic phototrophic bacteria in the enriched bacterial communities from different depths of karst lakes Kirkilai and Ramunelis was studied using spectrophotometric analysis, as well as microbiological and molecular methods. In Lake Kirkilai, the highest bacterial abundance was measured in the metalimnion and near the bottom (up to 10.7 × 10⁶ cell/mL); in Lake Ramunelis it was in the anoxic hypolimnion (up to 22.4 × 10⁶ cell/mL). Increased water mineralization (0.5–1.2 g/L) with the domination of SO ₄ ^2? and Ca²⁺ ions created favorable conditions for the development of sulfate-reducing bacteria; hydrogen sulfide produced as a result of their life activity facilitated the development of sulfur-oxidizing bacteria. The pigment analysis of phototrophic green and purple sulfur bacteria showed the domination of green sulfur bacteria in the enrichment culture. The results of phylogenetic analysis showed that Chlorobium limicola dominated in the enrichment culture for the green sulfur bacteria, whereas purple nonsulfur bacteria of the genus Rhodopseudomonas dominated in the enrichment culture for the purple sulfur bacteria.     

Communities of anoxygenic phototrophic bacteria in the saline soda lakes of the Kulunda steppe (Altai Krai)

The saline soda lakes of the Kulunda steppe (Altai krai) are small and shallow; they are characterized by a wide range of salinity and alkalinity, as well as by the extreme instability of their water and chemical regimes. Accumulations of anoxygenic phototrophic bacteria (APB) visible to the unaided eye were noted only in several lakes with high rates of sulfate reduction in their bottom sediments. However, enumeration of APB cells by inoculation revealed their presence in all 17 lakes. APB cell numbers varied from 10³ to 10⁹ CFU cm^?3. In the APB communities of all lakes, purple sulfur bacteria of the family Ectothiorhodospi- raceae were predominant. In 14 out of the 17 lakes, purple nonsulfur bacteria of the family Rhodobacteraceae were also detected (10³–10⁷ CFU cm^?3). Purple sulfur bacteria of the family Chromatiaceae were less abundant: Halochromatium sp. (10⁴–10⁷ CFU cm^?3) were found in six lakes, while Thiocapsa sp. (10⁴ CFU cm³) were detected in one lake. On the whole, the APB communities of the soda lakes of the Kulunda steppe were characterized by the low diversity and evenness of their species compositions, as well as by the pronounced dominance of the members of the family Ectothiorhodospiraceae. There was no correlation between the structures of the APB communities and alkalinity. However, the dependence of the species composition of APB (mainly ectothiorhodospiras) on water mineralization was revealed. High mineralization (above 200 g l^?1) was a limiting factor that affected the APB communities on the whole, restricting the APB species diversity to extremely halophilic bacteria of the genus Halorhodospira.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.     

F'' Plasmids from Hfrh and Hfrc in recA- ESCHERICHIA COLI

We have isolated and characterized a number of clones resulting from matings of HfrH and HfrC cultures of Escherichia coli with auxotrophic recA^- E. coli. As in Low's (1968) experiments, the recA^- marker prevented integration of F' episomes into the vegetative chromosomes of the host. Both F'H F'C plasmids contained a great variety of non-selected nutritional markers. However, more F'H plasmids seemed to have expressed F⁺ characteristics than did F'C plasmids. These characteristics include (i) the presence of F-pili as determined by susceptibility to male-specific phages; (ii) fertility as determined by the merozygote's ability to transfer nutritional markers to an auxotrophic F^- strain of E. coli; and (iii) a high degree of inheritability as estimated by the proportion of F' bacteria to F^- bacteria in clones grown in a non-selective medium like broth. This proportion is seen to be affected by both factors that determine the probabilities that daughters of F' bacteria inherit the episome and from physiological factors that determine the rates of growth of F' and F^- bacteria.     

A Novel Balanced-Lethal Host-Vector System Based on glmS

During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS⁻ mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS⁺p) complemented the phenotype of the GlmS⁻ mutant, and that GlmS⁺p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS ⁺pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.     

Action of genotypical surrounding of host Drosophila melanogaster on biological effects of endosymbiont Wolbachia (strain wMelPop)

In this work, a comparative study of the structure of symbiotic bacteria Wolbachia (strain wMelPop decreasing the fly lifespan) in genotypically different Drosophila melanogaster, as well as the effect of the bacteria on the host cell ultrastructure was investigated out. As a result of special crossings, the Drosophila melanogaster [w]Trl ³⁶² and [w]Trl ^en82 lines, which are carried of mutations for the gene Trithorax-like, are synthesized (lines infected with Wolbachia are designated as [w]). The Drosophila melanogaster line free of Wolbachia was obtained by treatment with antibiotics of the initially infected [w]w ¹¹¹⁸ line. The complex of the used methods and approaches has allowed us to perform a comparative study of the morphology of cell structures for the first time before and after the infestation of insects with bacteria and to evaluate effect of the bacteria on viability and fertility of flies of these lines. Electron microscopy analysis has shown that the embryos of the analyzed lines contain typical Wolbachia in contact with various host cell compartments; the ultrastructural organization of the bacteria indicates the preservation of their functional activity. In the cytoplasm of embryos that are mutant for the gene Trithorax-like, morphologically atypical mitochondria were revealed, as well as Wolbachia (wMelPop) of unusual morphology with a modified form of membtane envelopes. The presence of Wolbachia in ovarian cells of the female mutant fly lines has been found to produce no effect on the amount of the female-ovipositioned eggs. It has been established for the first time that lifespans of the infected and Wolbachia-free Drosophila melanogaster mutant lines TM3 containing chromosome 3 as a balancer are equal. However, it is significantly shorter in the imago of the [w]w ¹¹¹⁸ line than in flies of the mutant lines. This has allowed us to suggest that either the chromosome-balancer TM3 or mutation of the gene Trl play an important role in the host-symbiont interactions. On checking this suggestion, it was found that the lifespan of homozygotes [w]Trl ³⁶² and [w]Trl ^en82 after the infection of flies with bacteria decreased markedly and was close to the lifespan of [w]w ¹¹¹⁸ line. The obtained data indicate that the chromosome-balancer TM3 can have a significant effect on the symbiont-host interaction.     

Identification of Intracellular Bacteria in Adenoid and Tonsil Tissue Specimens: The Efficiency of Culture Versus Fluorescent In Situ Hybridization (FISH)

Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14⁺ cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14⁺ cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14⁺ cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14⁺ cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.     

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica,a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d⁻¹. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13 ng C predator⁻¹d⁻¹ (0.06 cells predator⁻¹d⁻¹). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019 ng C predator⁻¹d⁻¹ (266 bacteria predator⁻¹d⁻¹), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 10⁷ cells ml⁻¹ was 0.01 ng C predator⁻¹d⁻¹ (48 Synechococcus predator⁻¹d⁻¹). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.     

Regulation of nitrogen fixation by Rhizobia export of fixed N2 as NH4+

The metabolic fate of gaseous nitrogen (¹⁵N₂) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N₂-fixation system was followed. A majority of the fixed ¹⁵N₂ was found to be exported into the cell supernatant. For example, as much as 94% of the ¹⁵N₂ fixed by Rhizobium japonicum (soybean symbiont) was recovered as ¹⁵NH₄⁺ from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH₄⁺ from l-histidine. Evidence is presented that overproduction and export of NH₄⁺ by free-living Rhizobia may be closely linked to the control of several key enzymes of NH₄⁺ assimilation. For instance, NH₄⁺ was found to repress glutamine synthetase whereas l-glutamate repressed glutamate synthase. Assimilation of NH₄⁺ as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH₄⁺ production by root nodule bacteria is discussed.     

A Pathogenic Bacterium,Enterococcus faecalis,to the Beet Armyworm,Spodoptera exigua

A bacterial disease was found in the beet armyworm, Spodoptera exigua (Hübner). Blackened body of the infected larvae was a typical symptom of the epizootic disease especially at the intersegmental areas. We isolated the bacteria from the hemolymph of the infected 5th instar larvae and identified the isolate as a gram-positive bacterium, Enterococcus faecalis. When the 4th instar larvae were injected with the bacteria, half lethal dose of the bacteria was estimated as 22,593 colony-forming units (cfu) per larva and half lethal time of the bacteria was estimated as 2 days at 10⁷ cfu injection and 6 days at 10⁸ cfu injection. The bacteria were strongly resistant to each 1,000 ppm of ampicillin, kanamycin, and streptomycin. They were, however, relatively susceptible to mixture (1,000 ppm) of different combinations of the three antibiotics.     

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity¹ and vibration². In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus³. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.EIW assays involve counting the number of eggs retained in the uterus of C. elegans⁴. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine^5-9.