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 stxA 2 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 stxA 2 gene. Higher-contrast images were obtained with this method than with in situ PCR.

An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Ha wobacterium-Cytophaga; Burkholderia~seudomonas (rRNA Ill)-authentic Alcaligenes; VibriwAeromonas; Pseudomonas (rRNA I); the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. A t the eutrophic site, 54-68% of all cells visualized by staining with DAPl (4',6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 3 9 4 5 % of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five group-specific probes; the bacterial community structure was dominated by the Flawobacteriu~Cytophaga group and BurkholderiaPseudomonas (rRNA Ill)-authentic Alcaligenes group. A t the oligotrophic site, only 2638% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.

Lateral gene transfer by phages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency and range of phage-mediated gene transfer, it is important to understand the movement of DNA among microbes. Using an in situ DNA amplification technique (cycling primed in situ amplification-fluorescent in situ hybridization; CPRINS-FISH), we examined the propensity for phage-mediated gene transfer in freshwater environments at the single-cell level. Phage P1, T4 and isolated Escherichia coli phage EC10 were used as vectors. All E. coli phages mediated gene transfer from E. coli to both plaque-forming and non-plaque-forming Enterobacteriaceae strains at frequencies of 0.3-8 Â 10À3 per plaque-forming unit (PFU), whereas culture methods using selective agar media could not detect transductants in non-plaque-forming strains. The DNA transfer frequencies through phage EC10 ranged from undetectable to 9 Â 10 À2 per PFU (undetectable to 2 Â 10 À3 per total direct count) when natural bacterial communities were recipients. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viability in most cases. These results indicate that the exchange of DNA sequences among bacteria occurs frequently and in a wide range of bacteria, and may promote rapid evolution of the prokaryotic genome in freshwater environments.

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.

The phylogenetic composition and physiological activity of bacterioplankton communities in two different rivers in Southeast Asian countries, the Kelang River basin in Malaysia and Chao Phraya River in Thailand, which are polluted by untreated or incompletely treated sewage, were analyzed by fluorescent in situ hybridization (FISH) and FISH combined with the direct viable count technique (DVC-FISH). The results were also compared with those from temperate zone habitats in eutrophic rivers in Osaka, Japan. FISH detected 56% to 78% of total cells with the probe EUB338 targeted for the domain Bacteria in samples from the Kelang River basin, compared with 14% to 33% in samples from the Chao Phraya River. DVC-FISH with an antibiotic cocktail increased the fraction of bacteria detectable with EUB338 in the Chao Phraya River (72% to 75% of total bacteria), while no appreciable change was found in samples from the Kelang River basin. These results show that in situ physiologic activity of resident bacteria was generally high in the Kelang River basin and low but present in the Chao Phraya River. Bacterial community structures in both rivers were dominated by the beta (5% to 39%) and gamma (4% to 41%) subclasses of Proteobacteria. In river water samples from Osaka, bacterial community structures determined by FISH were dominated by the beta subclass, but those determined by DVC-FISH were dominated by both beta (26% to 39%) and gamma (17% to 47%) subclasses. This result implies that in situ physiological activity of the gamma subclass is low in the eutrophic river in Osaka, but those bacteria have the potential for cell division.

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10 6 -fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.

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 3 ؋ 10 ؊8 to 2 ؋ 10 ؊6 , 1 ؋ 10 ؊8 to 4 ؋ 10 ؊8 , and <4 ؋ 10 ؊9 to 4 ؋ 10 ؊8 per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 ؋ 10 ؊4 to 1 ؋ 10 ؊3 , 9 ؋ 10 ؊4 to 3 ؋ 10 ؊3 , and 5 ؋ 10 ؊4 to 4 ؋ 10 ؊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.Recent nucleotide and whole-genome analyses have revealed that most bacterial genomes contain large amounts of bacteriophage DNA (18). This finding suggests that lateral gene transfer by bacteriophages has contributed significantly to the acquisition of new genetic traits, the ability of bacteria to exploit new environments, and the genetic diversity of bacteria (27). Since bacteriophage-mediated gene transfer was first recognized (32), transduction has been found to occur in many phage-host systems, and various aspects of transduction, including molecular mechanisms, physiologic and genetic characterization of transductants, and ideal environments for transduction, have been investigated (29).For nearly a half-century, culture methods using selective agar media have played a leading role in the study of gene transfer (5). Genetic characteristics such as amino acid deficiency repair and antibiotic resistance have been used for selection of transductants (9). Transduction frequencies were shown to differ over orders of magnitude from 10 Ϫ11 to 10 Ϫ5 per bacteriophage, which are lower than those for conjugation and transformation (14, 27). However, current knowledge of horizontal gene transfer via bacteriophages in the environment is rather limited because of methodological constraints. Conventional methods for the detection of gene transfer depend on high levels of gene expression and culturability on selective media. Although these methods have led to an understanding of the genetic and physiologic characteristics of transductants and the molecular mechanism of transduction, they have limited abilities to quantify the genetic material introduced into individua...

In situ enumeration methods utilizing fluorescent probes were used to estimate the number of physiologically active bacteria in river water. Two fluorogenic compounds, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 6-carboxy fluorescein diacetate (6CFDA), were chosen for direct epifluorescent microscopic detection of active bacteria. CTC is a soluble redox indicator which is reduced by respiring bacteria to fluorescent CTC-formazan crystals. 6CFDA is hydrolyzed by nonspecific esterases to produce the fluorescent compound, 6-carboxy fluorescein. Estimates of the number of active bacteria identified by these fluorescent probes were compared with those obtained with the plate count method. Bacteria with respiratory activity, as determined by CTC reduction, accounted for approximately 10% of the total bacteria at oligotrophic sites and 15 to 20% at eutrophic sites. These values exceeded those obtained with the plate count method. Estimates of enzymatically active bacteria, as determined by 6CFDA hydrolysis, were also higher than those obtained with the plate count method, constituting 40 to 50% of the total bacteria at both oligotrophic and eutrophic sites. These results indicate that some non-culturable bacteria retain physiologic activity and may play an important role in the ecosystem.