Conventional confocal laser scanning microscopy uses continuous wave visible or UV lasers for one-photon excitation and imaging. These instruments are now enhanced with pulsed infrared lasers allowing two-photon excitation. With the appropriate technical setup two-photon excitation may be used for intensity imaging or for lifetime imaging. Both of these techniques have specific advantages if compared with one-photon excitation. Despite this fact, to date mainly one-photon excitation has been employed in microbial ecology. In this review the potential of two-photon intensity and lifetime imaging is discussed, using appropriate examples from cell biology as well as the first reports in microbiology.
We identified phycoerythrin-pigment phenotypes of Synechococcus strains in samples obtained from the Uwa Sea, and investigated the seasonal changes in the pigment-type composition of the Synechococcus community in this sea from January 2001 to March 2002. Two Synechococcus strains (UT01 and UT02) with different flow cytometric signatures were isolated by sorting and subsequent culturing. Excitation spectra showed that these two strains possessed different ratios of phycourobilin (PUB) to phycoerythrobilin (PEB). Flow cytometric signatures of the two types were also different; UT01 showed a higher green to red fluorescence ratio than UT02. The ranges of green to red fluorescence intensity of the strains did not overlap, though the strains were cultured under various light intensities (7-160 μE m-2 s-1). In seawater samples, we could divide Synechococcus into lower-and higher-PUB types based on the ratio. There were two peaks of total Synechococcus abundance in June (2.8×105 cells ml-1) and August (1.6×105 cells ml-1) in the upper layer at a depth of 0 to 15 m. Major types during these two peaks were different; the higher-PUB type contributed 75.6% to the total Synechococcus community in June, and the lower-PUB type, 58.6% in August. This is the first report which describes the seasonal succession of two phycoerythrin-pigment types of Synechococcus in a coastal sea.
DNA extraction has been difficult from some types of soil. Seven soil samples from agricultural fields and a forest, which were mainly volcanic ash soils, were used. Soil DNA could be extracted from only two of them using a commercially available kit exploiting bead-beating. When skim milk was added to the extraction buffer at 40 mg g-1 soil, DNA could be detected by electrophoresis from all the samples, indicating that the DNA from lysed cells was adsorbed by soil colloids. The addition of skim milk did not affect PCR-DGGE profiles. The improved method is applicable to the analysis of molecular communities in soils which strongly adsorb DNA.
Virus-like particles (VLPs) were collected from geothermal vent water samples in the drift-way at Toyoha Mine, Hokkaido, Japan (−500 m level, 63.5°C) whose VLP and bacterial abundance was (No/ml±SD, n: 500), VLP: 9.60±0.29×108 and bacteria: 3.61±0.14×106. VLPs ranged in diameter from 30 to 320 nm, and the major size distribution (ca 62%) was 83.33.3 nm (n: 843). Ultrafiltration followed by CsCl density equilibrium ultracentrifugation gave purified TY-VLPs: 6.64×1013. Regardless of UV treatment, TY-VLP reduced the efficiency of plating to 68.6-83.4% at a multiplicity of infection of ca 0.3 on Escherichia coli AB1157. Generalised transduction was observed on E. coli AB1157 with a frequency between 10-4 and 10-5 cells/particle using TY-VLPs without UV-treatment. The growth of generated E. coli transductants (TY-E-trans) was compared to that of an E. coli transductant (ST-E-trans) generated by Aquificales originating VLP (Chiura, 2002). The extent of the maximum growth of both transductants was ca 40% of the parental E. coli used as a recipient. TY-E-trans acquired "budding-like" particle productivity, which has been demonstrated for ST-E-trans. ST-E-trans produced five different size particles, whose DNA content ranged between 291.6 and 382.0 kb, and TY-E-trans produced ten different size particles between 68.5 and 190.2 kb, respectively.
Low concentrations of L-lysine and L-lysine-containing-peptides strongly inhibited the growth of axenic strains of 4 species belonging to the genus Microcystis (cyanobacteria). Inhibitory activities decreased in the order of L-lysine>tri-L-lysine>di-L-lysine>α-poly-L-lysine>L-lysyl-L-histidine>L-lysyl-L-alanine. The addition of 50 μM of L-lysine to growing cells of axenic strains of M. novacekii TAC20-1 and M. viridis NIES102 resulted in a loss of buoyancy, a decrease in chlorophyll a content and consequent cell lysis. The cells took up most of the L-lysine added to the culture medium of M. viridis NIES-102 in one day. However, the amino acid was released back into the medium with cell lysis 3 days after the addition. Laboratory results were confirmed in experimental ponds outdoors by spraying L-lysine onto natural Microcystis blooms in the summer of 1999 and 2000. The spraying of 50 μM L-lysine caused Microcystis colonies to vanish from the surface water within two days; there was a dramatic change in the color and transparency of the pond surface water. After the immediate disappearance of Microcystis sp., both Euglena sp. and/or Phormidium tenue appeared, the latter becoming the dominant species in the phytoplankton community of the pond. In the experiment in summer 2000, the L-lysine provided to the pond was assayed. The level decreased gradually and L-lysine was not detected in the pond water after 6 days. The mechanisms of growth inhibition by L-lysine and a possible mitigation of Microcystis blooms by the amino acid are discussed.
Stem samples of three sweetpotato cultivars were taken from an experimental field two and four months after planting (MAP). The occurrence of diazotrophic endophytes in different stem portions of sweetpotato was examined by stem-pieces-incubation (SPI) and the acetylene reduction activity (ARA) method using a semi-solid modified Rennie medium. Positive responses of the stem-pieces to ARA were detected, confirming the occurrence of diazotropohic endophytes in the stems of Japanese sweetpotato cultivars. The frequency of ARA-positive portions was higher at 4 MAP than at 2 MAP. However, the site of ARA-positive portions varied between replicated stems in each cultivar. Both positive and negative responses to ARA were detected in tubes inoculated with five neighboring thinly sliced pieces from the same internode portion. These results suggest that the diazotrophic endophytes that are culturable on semi-solid modified Rennie medium were discretely localized in sweetpotato stems.
Enterobacter cloacae IFO 3320 exhibits chemotactic responses to inorganic phosphate (Pi) when starved of Pi. One Tn1737KH-induced mutant failed to exhibit Pi taxis even under conditions of Pi limitation. The mutant showed normal chemotactic responses to amino acids and citrate, suggesting that it is specifically defective in Pi taxis. Southern blot analysis revealed that the insertion Tn1737KH was located in the indigenous plasmid pEC01. The pEC01-cured strain of IFO 3320 did not show Pi taxis even under conditions of Pi limitation, indicating that pEC01 is required for Pi taxis in E. cloacae IFO 3320. This plasmid is 5002 bp long, consisting of four mobilization genes, mbeCABD, and an origin of replication homologous to ColE1-type plasmids. pEC01 contains no open reading frame (ORF) encoding a protein with a highly conserved motif of methyl-accepting chemotaxis proteins (MCPs), suggesting that a gene encoding a MCP responsible for Pi taxis is located on the chromosome. An ORF encoding a putative protein of 102 amino acids was responsible for the complementation of the pEC01-cured strain. The putative protein had a helix-turn-helix motif, suggesting that it is involved in the gene regulation of the Pi taxis MCP gene.
Different composts were added to a conducive soil to study their effects on the bacterial wilt of tomato caused by Ralstonia solanacearum. The soils to which bark compost, coffee manure and pork manure were added showed higher wilt incidence (more than 5 plants out of 6 wilted) after 30 days of cultivation compared to those in the non-treated soil, while the soils with poultry and farmyard manure (FYM) showed consistently lower wilt incidence (less than one plant out of 6 wilted). Survival of the pathogen was better in the control and in the bark, coffee and pork manure added soils than in the poultry and FYM added soils. A lower C/N ratio and higher amounts of water soluble organic carbon and nitrogen were found in the poultry and FYM than the other composts, suggesting that the contents of easily decomposable organic matters might be higher in the added soils. This was supported by higher numbers of culturable bacteria and fungi and higher microbial activity (measured by CO2 emission and dehydrogenase activity) in the poultry and farmyard manure added soils than in the other compost added soils. This study demonstrated that the bacterial wilt of tomato was suppressed in the poultry and FYM added soils, and higher microbial activity was likely responsible.
A fluorochrome-staining method using 5-cyano-2,3-ditoryl tetrazolium chloride (CTC) was improved to enhance the sensitivity of detection of metabolically active bacteria in activated sludge. The highest efficiency of CTC staining was obtained with 6 mM CTC in MOPS buffer at pH 6.5. CTC staining efficiency was also increased by adding a substrate mixture (0.05% peptone, 0.05% yeast extract and 1 mM glucose), Meldola's Blue as an electron transfer mediator and KCN. The bacterial counts detected by the CTC staining method thus improved accounted for 41-76% of the total counts in activated sludge with the average value being 54%. This value was approximately two-fold higher than those obtained by the traditional CTC staining assay which has been performed in phosphate buffer or phosphate-buffered saline without any addition of external substrate or electron transfer mediator. There was a high positive correlation between CTC-stained direct counts and the plate counts of aerobic chemoorganotrophic bacteria or viable cell counts measured using a LIVE/DEAD BacLight Bacterial Viability kit. The viable population in the activated sludges as measured by BacLight staining and plate counting accounted for 73±7.6% and 8.7±4.7% of the total population, respectively. These data suggested that most of the "viable but nonculturable" bacteria (i.e., BacLight-positive but unculturable population) potentially existing in the sludge system were so metabolically active as to be detectable by the improved CTC staining method.
Two-dimensional gel electrophoresis was used to identify differentially displayed proteins of Bradyrhizobium japonicum USDA110 in the symbiotic and non-symbiotic state. When proteome maps were compared and the characterization of bacteroid-specific proteins was performed by N-terminal amino acid sequencing and matrix-assisted desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, putative identity was assigned to 61 bacteroid protein spots, including many metabolic proteins and ABC transporters as well as nitrogenase proteins like NifH. This study shows that proteome analysis will be a useful tool for surveying genes contributing to rhizobial functions in symbiosis.
In the cellular slime mold Dictyostelium discoideum, Countin2 and Countin3 proteins are thought to limit the minimum size of the multicellular structure since countin2- and countin3- strains form small fruiting bodies. Expression levels of the cell-cell adhesion proteins, gp24 and gp80, in wild type and null mutants were compared. During the process of aggregation, countin2- cells expressed lower levels of gp24 and gp80 than those expressed by the wild type. The expression levels of gp24 and gp80 in countin3- cells were almost the same as those in wild-type cells in the early stages of aggregation but were lower than wild-type levels in the late stages. These results indicate that Countin2 and Countin3 proteins enhance the expression of gp24 and gp80 in D. discoideum in different ways.