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.     

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Inducible Gene Expression by Nonculturable Bacteria in Milk after Pasteurization

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5°C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.     

Sequence analysis and insertional inactivation of a gene encoding Moraxella sp. CK-1 cell wall hydrolase

Sequencing of the Moraxella sp. CK-1 autolysin (cell wall hydrolases) gene showed the presence of an open reading frame which encodes a polypeptide of 273 amino acids with a molecular mass of 33,316 Da. A presumed ribosomal binding site, a possible –10 and –35 region, and rho-dependent terminators were found. The C-terminal region of the mature protein showed considerable homology with the Thermus sp. serine proteinase. Enzyme assay suggests that the recombinant autolysin has amidase or endopeptidase activity. Analysis of the peptidoglycan fragments, following the treatment with the autolysin, indicates that this protein is an N-acetylmuramyl-L-alanine amidase. Insertional inactivation of the autolysin of Moraxella sp. CK-1 chromosome led to a decrease in cell wall hydrolytic activity, clumping of the cells, and color change. No lytic band present in inactivated magA mutant by renaturing SDS-PAGE.     

Response of Tetraselmis suecica to nutrient and grazer manipulation

Three methods of algal quantification (direct cell counts, chlorophyll a extraction, in vivo fluorescence) were used to evaluate the response of the unicellular green flagellate Tetraselmis suecica to nutrients and grazers. Nutrient enrichment enhanced total cell counts, chlorophyll a concentration and in vivo and DCMU-fluorescence. Photosynthetic efficiency was reduced in the complete F2 medium as indicated by the high level of in vivo fluorescence, whereas photosynthetic efficiency was increased by the introduction of mussels to the F2 medium. The addition of mussels significantly increased the proportion of non-motile cells, but did not reduce the total cell count. The effect of mussel grazing on algae could be underestimated if only total cells were counted or only the chlorophyll a concentration was measured. The results indicate that these three methods measure different properties of an algal culture and are complementary to each other in assessing the quality and quantity of an algal population. Direct algal counting offers a reliable numerical assessment for cell population abundance. Chlorophyll a concentration was closely correlated to the total cell count. In the presence of mussels, in vivo fluorescence did not correlate with either algal cell counts or chlorophyll a concentration, indicating that the measurement of in vivo fluorescence may be misleading for estimating algal abundance under different culture conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth and Starvation of a Strain of Klebsiella pneumoniae Isolated from a Brazilian Oil Formation

Summary The changes that occur in cell size and shape during experimental starvation and resuscitation of bacteria may find practical application in the biotechnological processes used in microbial enhanced oil-recovery (MEOR), in in situ bioremediation and in the creation of biobarriers. In the work described here, the aim was to observe certain aspects of the response of a strain of Klebsiella pneumoniae, isolated from oil producer wells belonging to PETROBRAS (National Oil Company of Brazil), to starvation and growth under various culture conditions. Cells of K. pneumoniae were taken from an exponentially-growing culture, washed and suspended in phosphate-buffered saline solution, without nutrients, where they remained for 91 days. Aliquots were withdrawn periodically, to perform viable and total cell counts and measure the optical density and cell dimensions. The starved cells were resuspended in sodium citrate medium, where they recuperated and exhibited a typical growth curve. The results indicate that this strain is a viable option for future trials on the transport and growth of microorganisms inside porous media, aimed at possible applications in MEOR.     

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing,cell death and T-DNA loss

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ~10⁸ bacteria/ml (equivalent to ~10⁷ CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Characterization and resuscitation of viable but nonculturable <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> VIB283

The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4°C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10¹⁰ to 10⁹ CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.     

Metabolism of 2-hydroxyphenylglyoxylate by Moraxella sp. strain VS1

A bacterium, designated as Moraxella sp., was enriched with 2-hydroxyphenylglyoxylate (2HPGA) as sole source of carbon and energy. Identified metabolites and enzyme activities determined with whole cells and extracts indicated that 2HPGA was degraded by an inducible sequence of enzymes via salicylaldehyde, salicylate, and gentisate; only minute amounts of salicylate were converted to catechol. Further evidence was obtained that permeases were necessary for the uptake of most aromatic compounds utilized for growth. For the direct determination of 2HPGA decarboxylase activity, an enzyme assay involving high-performance liquid chromatography for quantitation of the substrate was developped to study the initial step of the degradative pathway.     

Survival and viability of nonculturableEscherichia coli andVibrio cholerae in the estuarine and marine environment

Plating methods for estimating survival of indicator organisms, such asEscherichia coli, and water-borne pathogens includingVibrio cholerae, have severe limitations when used to estimate viable populations of these organisms in the aquatic environment. By combining the methods of immunofluorescent microscopy, acridine orange direct counting, and direct viable counting, with culture methods such as indirect enumeration by most probable number (MPN) estimation and direct plating, it was shown that bothE. coli andV. cholerae undergo a nonrecoverable stage of existence, but remain viable. Following 2-week incubations in saltwater (5–25%o NaCl) microcosms, total counts, measured by direct microscopic examination of fluorescent antibody and acridine orange stained cells, remained unchanged, whereas MPN estimates and plate counts exhibited rapid decline. Results of direct viable counting, a procedure permitting estimate of substrate-responsive viable cells by microscopic examination, revealed that a significant proportion of the nonculturable cells were, indeed, viable. Thus, survival of pathogens in the aquatic environment must be re-assessed. The die-off or decay concept may not be completely valid. Furthermore, the usefulness of the coliform and fecal coliform indices for evaluating water quality for public health purposes may be seriously compromised, in the light of the finding reported here.     

Maize Ac/Ds transposon system leads to highly efficient germline transmission of transgenes in medaka (Oryzias latipes)

As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.     

The usefulness of the <Emphasis Type="Italic">gfp</Emphasis> reporter gene for monitoring <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of potato dihaploid and tetraploid genotypes

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. ‘Baltica’, respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.     

Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-Tagged Pseudomonas fluorescens A506

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5°C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30°C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30°C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50°C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.     

Construction of a Tightly Regulated Plasmid Vector for Streptococcus pneumoniae: Controlled Expression of the Green Fluorescent Protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P_M promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P_M promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P_M-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

Visualizing the infection process of Xanthomonas campestris in cabbage using green fluorescent protein

The plant pathogen, Xanthomonas campestris NRRL B-1459 was chromosomally tagged with gfp, and the transformant, which was subjected to Southern hybridization showed the presence of gfp in the chromosome. The virulence-related gene of the transformant was not affected by the insertion of gfp. After inoculation into cabbage plants, the infection process was visually studied in planta. Using a fluorescence microscope, the migration and distribution of gfp-labelled bacteria was visualized in real time. As the gfp-labelled cells were easily visualized from the beginning of infection, we observed a time delay of 2 days between distribution of the Xanthomonas cells in cabbage plant and the appearance of visible necrosis.     

Use of a simple semiquantitative method for appraisal of green fluorescent protein gene expression in transgenic tobacco plants

We have applied a simple method for evaluation of gfp gene expression in plants using a CCD camera and computerized processing of images. Transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transfer of plasmid T-DNA bearing a m-gfp5-ER sequence governed by the 35S promoter together with the nptII selectable marker gene. Presence of the gfp gene in plants was confirmed by a polymerase chain reaction method. Mean brightness values measured using image analysis software showed differences between transgenic and control plants and suggest the possibility of rapid selection of transgenic individuals among regenerants and their progenies.     

Starvation survival of Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT7137, exopolysaccharide-producing bacterium, was isolated from groundwater contaminated with leachate in a former municipal landfill site (São Paulo, Brazil). The strain was submitted to starvation in phosphate-buffered saline solution for 56 days so as to evaluate its behavior regarding cuturability and cell morphology. As a response to starvation, G. polyisoprenivorans CCT7137 presented reduction in viable cell count, cell size and cell shape alteration. The initial number of viable cells was 1.51 × 10⁷ c.f.u. ml. After 7 days of starvation culturability dropped to 13.70% (2.07 × 10⁶ c.f.u/ml) and, after 56 days, to 3.25% (4.93 × 10⁵ c.f.u/ml). It was also observed that after 7 days of starvation the cell size presented an average reduction in the values of length, length/width ratio, volume and area of 50, 58, 40 and 42%, respectively. The length/width ratio showed a change of shape from rod to coccobacillus. Changes in cell dimensions and distribution of cell into classes were not significant after day 7 of starvation. The results obtained show that it is not necessary for the strain to starve for more than a week to obtain G. polyisoprenivorans CCT7137 size- reduced cells. The results also indicate the potential for its starved forms to be used in future tests in porous medium to study the production of exopolysaccharide in situ.