High-frequency phage-mediated gene transfer among Escherichia coli cells, determined at the single-cell level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3x10(-8) to 2x10(-6), 1x10(-8) to 4x10(-8), and <4x10(-9) to 4x10(-8) per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7x10(-4) to 1x10(-3), 9x10(-4) to 3x10(-3), and 5x10(-4) to 4x10(-3) for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Human sialidase as a cancer marker

Altered sialylation of cell surface glycoproteins and glycolipids is closely related to the malignant phenotype of cancer cells, including the metastatic potential and invasiveness. Many cancer-related antigens in clinical use contain sialic acids at the terminal position of sugar chains in the molecules. To elucidate the molecular mechanism, we focused our investigation on sialidase, which catalyzes the removal of sialic acid residues from the glycoconjugates. Four types of human sialidases identified to date behave in different manners during carcinogenesis. One of the sialidases, found in the lysosomes, showed downregulation in cancers, promoting anchorage-independent growth, and metastatic ability, while another, found in the plasma membrane, showed marked upregulation, causing apoptosis suppression. It was found that estimation of the mRNA levels of sialidases by real-time PCR allowed discrimination of cancerous from noncancerous tissues and even determination of the pathological stage in some cancers. Immunohistochemistry of cancer tissues using the antibody against the plasma membrane sialidase was useful for clinical diagnosis. This paper briefly summarizes our findings of the altered sialidase expression in cancers and the possibility of their clinical application as cancer markers. Human sialidases are indeed related to malignancy and may be potential targets for cancer diagnosis and therapy.     

Further evaluation of an in vivo micronucleus test on rat and mouse skin: results with five skin carcinogens.

In a previous paper, we presented a practical in vivo micronucleus (MN) test that used rat skin as the target organ. To evaluate the test, as well as to determine the reproducibility and applicability of the method to mice, we used it to test the effect of five skin carcinogens (N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P)) on rat and mouse skin. All five compounds significantly and dose-dependently increased the MN frequencies in the basal cells of the chemical-treated skin. These results indicated the reproducibility of the test results and also the applicability of the test to mice as well as rats.     

Detection of Bacteria Carrying the stx2 Gene by In Situ Loop-Mediated Isothermal Amplification

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA₂ in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA₂ gene. Higher-contrast images were obtained with this method than with in situ PCR.     

Prostaglandin production in cultured gastric mucosal cells: Role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE₂, PGI₂ and thromboxaneA₂ (TXA₂) in amounts of 316±18, 100±7 and 30±5 pg per 10⁵ cells in 10 min, respectively, in response to 10μM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1–5mM) did not alter the amount of subsequent AA-induced PGE₂ production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE₂ synthesis, while it increased intracellular cAMP accumulation. Our studies suggest (1) AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, (2) AA-induced PG production is limited in these cells, and (3) it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Genetic and Physiological Characterization of the Intestinal Bacterial Microbiota of Bluegill (Lepomis macrochirus) with Three Different Feeding Habits

Bluegill (Lepomis macrochirus) in Lake Biwa, Japan, feed on benthic invertebrates (benthivorous type), aquatic plants (herbivorous type), and zooplankton (planktivorous type). To evaluate the effect of food on intestinal bacterial microbiota, we characterized and compared the intestinal microbiota of these three types of bluegill in terms of community-level physiological profile (CLPP) and genetic structure. The CLPP was analyzed using Biolog MicroPlates (Biolog, Inc., Hayward, CA, USA), and multivariate analysis of variance revealed that the CLPP of intestinal microbiota differed significantly between any pairs of the three types of bluegill. The genetic profiles were analyzed by temperature gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments, and multidimensional scaling indicated the existence of specific intestinal bacterial structures for both the benthivorous and the planktivorous types. These results suggest that the host's feeding habit can be one factor controlling the intestinal microbiota of fish in the natural environment.     

Are minichromosomes valid model systems for DNA replication control? Lessons learned from Escherichia coli

Initiation of chromosome replication is a key event in the life cycle of any organism. Little is known, however, about the regulatory mechanisms of this vital process. Conventionally, the initiation mechanism of chromosome replication in microorganisms has been studied using plasmids in which an origin of chromosome replication has been cloned, rather than using the chromosome itself. The reason for this is that even bacterial chromosomes are so large that biochemical and genetic manipulations become difficult and cumbersome. Recently, the combination of flow cytometry and genetic methods, in which modifications of the replication origin are systematically introduced onto the chromosome, has made possible detailed studies of the molecular events involved in the control of replication initiation in Escherichia coli . The results indicate that requirements for initiation at the chromosomal origin, oriC , are drastically different from those for initiation at cloned oriC .     

Simplified sample preparation using frame spotting method for direct counting of total bacteria by fluorescence microscopy

A new preparation method for direct counting of bacteria in liquid samples with fluorescence microscope was developed using a glass slide coated with 3-aminopropyltriethoxy silane and ring-shaped polyester seal as a retainer. The experimental steps of this method were spotting samples onto the coated slides with the seal, drying under vacuum, staining with SYBR Green II, drying and covering with immersion oil and coverslip to allow counting. This simplified method provided consistent results when compared with the conventional filtration method for fluorescence microscopy, and is rapid, inexpensive and reproducible.