Stratified growth in Pseudomonas aeruginosa biofilms

In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp(1) promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 microm wide in colony biofilms and 30 microm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 microm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.     

基于xkdB假基因座位的瓦雷兹芽孢杆菌FZB42超折叠GFP标记研究

【目的】利用一个GFP的超折叠变体SfGFP(superfolder GFP)对瓦雷兹芽孢杆菌(Bacillus velezensis)FZB42菌株进行标记,以期确立一种瓦雷兹芽孢杆菌及近缘芽孢杆菌通用的高亮GFP标记手段,同时,为了方便后续生物膜和分子互作的相关研究,测试SfGFP基因插入位点,假基因xkdB,作为外源基因表达座位的可行性。【方法】利用基因工程技术构建了一系列质粒,然后通过同源重组的方式,分别获得xkdB敲除菌株FBS373和SfGFP标记菌株FBS374,分别测试这些菌株在生长速度、碳源利用、荧光亮度、生物膜形成、swarming运动性等方面的差异。【结果】本研究成功构建了SfGFP标记的瓦雷兹芽孢杆菌FZB42,其荧光亮度是gfp+变体标记菌株的5倍以上;xkdB基因敲除对瓦雷兹芽孢杆菌FZB42生长速度、不同碳源利用、生物膜形成和运动性等方面无明显影响。【结论】通过本研究我们确认了xkdB基因位点作为瓦雷兹芽孢杆菌FZB42基因组上外源基因表达的中性位点的可行性,同时,通过在xkdB基因座位表达了SfGFP基因,成功对FZB42进行了高亮标记,对同类菌株的标记...     

Lentivirus‐mediated bifunctional cell labeling for in vivo melanoma study

Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long‐term culture and colony formation of several LV‐labeled mouse melanoma cells showed that promoters derived from mammalian house‐keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase–green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP‐labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP‐positive cells can be isolated from the tumors by fluorescence‐activated cell sorter. Pol2‐Luc/GFP labeling, while lower in activity, was more sustainable than FerH‐Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol‐2‐Luc/GFP labeling allows long‐term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models.     

GFP质控芯片的初步研究

目的：建立一种质量控制芯片来监测样品标记、杂交和检测过程中的失误。方法：针对GFP基因设计的4条60mer寡核苷酸探针和1条阳性对照探针polv（U）与流感寡核苷酸探针一起打印在DAKO玻片上,并构建了GFP基因的克隆载体和体外表达载体,将从这两种重组载体上获得的绿色荧光蛋白（Green Fluorescent Protein,GFP）基因的ILNA、DNA片段和人的全血样品中的DNA用限制性显示技术（Restriction Display technology,RD）扩增标记,将标记的样品和荧光标记的通用引物U分别与芯片杂交、检测,并对扫描的结果进行统计分析。结果：GFP探针与相应的样品杂交时出现阳性信号,阳性对照探针在所有的杂交中均出现阳性信号,而空白对照则未检测荧光信号。结论：建立的质控芯片具有较好的敏感性和特异性,可以用于基因芯片中的质量监控。     

Expression of green fluorescent protein in Streptococcus gordonii DL1 and its use as a species-specific marker in coadhesion with Streptococcus oralis 34 in saliva-conditioned biofilms in vitro

Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we isolated and characterized an endogenous S. gordonii promoter, PhppA, which is situated upstream of the chromosomal hppA gene encoding an oligopeptide-binding lipoprotein. A chromosomal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was constructed and used to demonstrate that PhppA was highly active throughout the growth of bacteria in batch culture. A promoterless 0.8-kb gfp ('gfp) cassette was PCR amplified from pBJ169 and subcloned to replace the cat cassette downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating pMA1. Subsequently, the PhppA-'gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S. gordonii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S. gordonii DL1 aerobically to ensure GFP expression. Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 from PhppA and that S. gordonii DL1 transformed with the PhppA-'gfp fusion plasmid stably maintained the fluorescent phenotype. Fluorescent S. gordonii DL1 transformants were used to elucidate the spatial arrangement of S. gordonii DL1 alone in biofilms or with the coadhesion partner Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned in vitro flowcell system. These results show for the first time that GFP expression in oral streptococci can be used as a species-specific marker in model oral biofilms.     

Gene expression analysis of monospecies Salmonella typhimurium biofilms using differential fluorescence induction

Bacterial biofilm formation is an important cause of environmental persistence of food-borne pathogens, such as Salmonella Typhimurium. As the ensemble of bacterial cells within a biofilm represents different physiological states, even for monospecies biofilms, gene expression patterns in these multicellular assemblages show a high degree of heterogeneity. This heterogeneity might mask differential gene expression that occurs only in subpopulations of the entire biofilm population when using methods that average expression output. In an attempt to address this problem and to refine expression analysis in biofilm studies, we used the Differential Fluorescence Induction (DFI) technique to gain more insight in S. Typhimurium biofilm gene expression. Using this single cell approach, we were able to identify 26 genetic loci showing biofilm specific increased expression. For a selected number of identified genes, we confirmed the DFI results by the construction of defined promoter fusions, measurement of relative gene expression levels and construction of mutants. Overall, we have shown for the first time that the DFI technique can be used in biofilm research. The fact that this analysis revealed genes that have not been linked with Salmonella biofilm formation in previous studies using different approaches illustrates that no single technique, in casu biofilm formation, is able to identify all genes related to a given phenotype.     

Rapid,long‐term labeling of cells in the developing and adult rodent visual cortex using double‐stranded adeno‐associated viral vectors

Chronic in vivo imaging studies of the brain require a labeling method that is fast, long‐lasting, efficient, nontoxic, and cell‐type specific. Over the last decade, adeno‐associated virus (AAV) has been used to stably express fluorescent proteins in neurons invivo. However, AAV's main limitation for many studies (such as those of neuronal development) is the necessity of second‐strand DNA synthesis, which delays peak transgene expression. The development of double‐stranded AAV (dsAAV) vectors has overcome this limitation, allowing rapid transgene expression. Here, we have injected different serotypes (1, 2, 6, 7, 8, and 9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype‐specific tropism. dsAAV‐GFP labeling showed high levels of neuronal GFP expression as early as 2 days postinjection and as long as a month, surpassing conventional AAV's onset of expression and matching its longevity. Neurons labeled with dsAAV‐GFP appeared structurally and electrophysiologically identical to nonlabeled neurons, suggesting that dsAAV‐GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV‐labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long‐term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Effect of biofilm model, mode of growth, and strain on streptococcusmutans protein expression as determined by two-dimensional difference gel electrophoresis

The effect of biofilm model, strain and mode of growth (biofilm or planktonic) on protein expression in Streptococcus mutans, a dental pathogen, was determined by two-dimensional difference gel electrophoresis. The bacterial strain (21-28% differentially expressed proteins) and the biofilm model (0.3-7.8% differential expression) used have a much larger effect on protein expression than the mode of growth (0.2-0.7% differential expression), something that has been ignored in biofilm studies up to now.     

Rapid in vivo labeling of identified zebrafish neurons

We report a simple and rapid method to label individual neurons in live zebrafish embryos and to examine their gene expression profiles. Injection of plasmid DNA encoding an alpha-tubulin promotor driving GFP expression results in mosaic embryos containing a limited number of GFP-positive neurons. Labeled neurons express GFP in their soma and axon, providing the opportunity to analyze pathfinding behaviors of identified neurons in vivo. Moreover, the presence of only a small subset of GFP tagged neurons permits the rapid anatomical identification of these neurons based on soma position and axonal trajectory. Analysis of injected embryos reveals that most, if not all, spinal cord cell types and many other neuronal cell types elsewhere in the nervous system can be GFP tagged. Finally, by combining GFP labeling of individual neurons with fluorescent in situ hybridization, we demonstrate the potential of this method to elucidate gene expression patterns at single cell resolution.     

Red fluorescent protein DsRed from Discosoma sp. as a reporter protein in higher plants

GFP from Aequorea victoria is a standard genetic marker widely used to visualize cellular events in a noninvasive manner. For simultaneous imaging of different processes, in vivo mutants of GFP with shifted wavelength spectra (e.g., blue fluorescent protein) are conventionally used. The recently reported red fluorescent protein from Discosoma sp., DsRed, represents a new marker that can be used together with GFP variants for multicolor imaging. DsRed is an interesting marker protein for use in plants because of its red-shifted wavelength spectrum that will avoid damaging cells and tissues by excitation light. In this report, we show that DsRed is an excellent marker in higher plants in spite of the interfering red autofluorescence of chlorophyll, which can be eliminated by using the appropriate filter sets. Transient expression of DsRed1-C1 and a soluble-modified, red-shifted GFP variant has been carried out both individually and jointly in the epidermal cells of three different Nicotiana species and Chenopodium quinoa, which gives rise to dual labeling in plants. For this purpose, a human codon-optimized variant of DsRed has been adopted for expression in plants. Moreover, the DsRed reporter gene was expressed by using a labeled plant viral vector derived from an infectious full-length clone of potato virus X.     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .     

Timing and localization of rhamnolipid synthesis gene expression in Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoyl-homoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 mum have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture.     

慢病毒介导的绿色荧光蛋白报告基因对人多能干细胞的生物风险评估研究

摘要 目的：探讨慢病毒介导的绿色荧光蛋白（GFP）标记人诱导多能干细胞（hiPSC）是否影响其细胞生物学特性,为多角度评价生物学风险提供实验基础。方法：慢病毒感染hiPSC后24小时,通过抗性基因表达筛选成功标记GFP的hiPSC。利用流式细胞分析法（FACS）检测GFP阳性的细胞比例。通过碱性磷酸酶染色检验干细胞的多能性,并通过免疫荧光染色检测多能性标记基因OCT4,NANOG,SOX2,SSEA4的表达情况。体外拟胚体分化实验检测GFP标记的hiPSC分化为不同胚层细胞的能力。结果：慢病毒感染不仅可以成功hiPSC标记上GFP,而且抗性基因表达筛选使GFP阳性细胞比例从37.5%提高到97.4%。AP染色和多能性标记基因的免疫染色证明标记后的细胞能维持多能性。体外分化实验显示感染后hiPSC可以形成拟胚体并实现三个胚层细胞共存。结论：慢病毒能够高效的标记hiPSC,并且不影响其多能性和拟胚体形成能力,可以用于后续的分化和细胞示踪研究。     

In situ monitoring of biofilm formations ofEscherichia coli andPseudomonas putida by use of lux and GFP reporters

A plasmid vector containing two reporter genes,mer-lux andlac-GFP, was transformed to bothEscherichia coli andPseudomonas putida. Their cellular activities and biofilm characteristics were investigated in flow-cell units by measuring bioluminescent lights and fluorescent levels of GFP. Bioluminescence was effective to monitor temporal cell activities, whereas fluorescent level of GFP was useful to indicate the overall cell activities during biofilm development. The light production rates ofE. coli andP. putida cultures were dependent upon concentrations of HgCl₂. Mercury molecules entrapped inP. putida biofilms were hardly washed out in comparison with those inE. coli biofilms, indicating thatP. putida biofilms may have higher affinity to mercury molecules thanE. coli biofilms. It was observed thatP. putida expressed GFP cDNA in biofilms but not in liquid cultures. This may indicate that the genetic mechanisms ofP. putida were favorably altered in biofilm conditions to make a foreign gene expression possible.     

Differential rates of gene expression monitored by green fluorescent protein

The use of green fluorescent protein (GFP) as a reporter gene has made a broad impact in several areas, especially in studies of protein trafficking, localization, and expression analysis. GFP's many advantages are that it is small, autocatalytic, and does not require fixation, cell disruption, or the addition of cofactors or substrates. Two characteristics of GFP, extreme stability and chromophore cyclization lag time, pose a hindrance to the application of GFP as a real-time gene expression reporter in bioprocess applications. In this report, we present analytical methods that overcome these problems and enable the temporal visualization of discrete gene regulatory events. The approach we present measures the rate of change in GFP fluorescence, which in turn reflects the rate of gene expression. We conducted fermentation and microplate experiments using a protein synthesis inhibitor to illustrate the feasibility of this system. Additional experiments using the classic gene regulation of the araBAD operon show the utility of GFP as a near real-time indicator of gene regulation. With repetitive induction and repression of the arabinose promoter, the differential rate of GFP fluorescence emission shows corresponding cyclical changes during the culture.     

Finding gene-expression patterns in bacterial biofilms

The production of biofilms by bacteria is a lifestyle that is thought to require or involve a differential gene expression compared with that of planktonic bacteria. Recently, we have witnessed a change of focus from the simple hunt for hypothetical essential biofilm genes to the identification of late and more complex biofilm functions. However, finding common bacterial biofilm gene-expression patterns through global expression analysis remains difficult. Owing to the apparently minimal overlap between functions involved in biofilm formation by different bacteria, exploring the biofilm lifestyle could prove to be a case-by-case task for which global approaches show their limits.