Inactivation of dinoflagellate Scripsiella trochoidea in synthetic ballast water by advanced oxidation processes.

Ship-borne ballast water contributes significantly to the transfer of non-indigenous species across aquatic environments. To reduce the risk of bio-invasion, ballast water should be treated before discharge. In this study, the efficiencies of several conventional and advanced oxidation processes were investigated for potential ballast water treatment, using a marine dinoflagellate species, Scripsiella trochoidea, as the indicator organism. A stable and consistent culture was obtained and treated by ultraviolet (UV) light, ozone (O3), hydrogen peroxide (H2O2), and their various combinations. UV apparently inactivated the cells after only 10 s of irradiation, but subsequently photo-reactivation of the cells was observed for all methods involving UV. O3 exhibited 100% inactivation efficiency after 5 min treatment, while H2O2 only achieved maximum 80% inactivation in the same duration. Combined methods, e.g. UV/O3 and UV/H2O2, were found to inhibit photo-reactivation and improve treatment efficiency to some degree, indicating the effectiveness of using combined treatment processes. The total residual oxidant (TRO) levels of the methods were determined, and the results indicated that UV and O3 generated the lowest and highest TRO, respectively. The synergic effect of combined processes on TRO generation was found to be insignificant, and thus UV/O3 was recommended as a potentially suitable treatment process for ballast water.

Does seeding density affect in vitro mineral nodules formation in novel composite scaffolds?

This study investigated the human alveolar osteoblasts (AOs) proliferation and extracellular matrix formation at seeding density of 0.05, 0.1, 0.2, 0.4, and 0.8 million (M) per 3x4x4 mm3 on medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) scaffolds designed for bone regeneration. Over 80-90% of the initial seeded cells were retained in the scaffolds after 24 h. AOs bridged over pores at density of 0.2M/scaffold and below, but formed cell balls at density of 0.4M/scaffold and above. At seeding density of 0.2M and below, cell proliferation increased with time having DNA content peaked to 1600 ng/scaffold at day 21 and 28, respectively, whereas at 0.4 and 0.8M, the corresponding DNA content decreased to 1600 ng in 28 days. At day 7, higher alkaline phosphatase (ALP) activity and higher osteocalcin (OCN) secretion were detected at 0.2M/scaffold and below. After 28 days, multilayered cell-sheet formation and collagen fibers were observed at all densities. ALP and OCN in matrix and mineral nodules were found mainly at the border of AOs-scaffold construct. These findings demonstrated that the density of 0.2M and below per 3 x 4 x 4 mm(3) scaffold resulted in better cell proliferation and extracellular matrix synthesis, potentially resulting in better mineralized tissue formation.

Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system.

BACKGROUND: Polyethylenimines (PEIs) with high molecular weights are effective nonviral gene delivery vectors. However, the in vivo use of these PEIs can be hampered by their cellular toxicity. In the present study we developed and tested a new PEI polymer synthesized by linking less toxic, low molecular weight (MW) PEIs with a commonly used, biocompatible drug carrier, beta-cyclodextrin (CyD). METHODS AND RESULTS: The terminal CyD hydroxyl groups were activated by 1,1'-carbonyldiimidazole. Each activated CyD then linked two branched PEI molecules with MW of 600 Da to form a CyD-containing polymer with MW of 61 kDa, in which CyD served as a part of the backbone. The PEI-CyD polymer developed was soluble in water and biodegradable. In cell viability assays with sensitive neurons, the polymer performed similarly to low-MW PEIs and displayed much lower cellular cytotoxicity compared to PEI 25 kDa. The gene delivery efficiency of the polymer was comparable to, and at higher polymer/DNA ratios even higher than, that offered by PEI 25 kDa in neural cells. Attractively, intrathecal injection of plasmid DNA complexed by the polymer into the rat spinal cord provided levels of gene expression close to that offered by PEI 25 kDa. CONCLUSIONS: The polymer reported in the current study displayed improved biocompatibility over non-degradable PEI 25 kDa and mediated gene transfection in cultured neurons and in the central nervous system effectively. The new polymer would be worth exploring further as an in vivo delivery system of therapeutic genetic materials for gene therapy of neurological disorders.

Dopamine-related and caspase-independent apoptosis in dopaminergic neurons induced by overexpression of human wild type or mutant alpha-synuclein.

Human wild type (WT) and mutant alpha-synuclein (alpha-syn) genes were overexpressed using a Tet-on expression system in stably transfected dopaminergic MN9D cells. Their overexpression induced caspase-independent and dopamine-related apoptosis not rescued by general caspase inhibitor Z-VAD-FMK. While apoptosis due to overexpression of WT alpha-syn was completely abrogated by a specific tyrosine hydroxylase (TH) inhibitor, alpha-methyl-p-tyrosine (alpha-MT), the inhibitor only partially rescued apoptosis caused by overexpression of alpha-syn mutants. In addition, overexpression of mutants enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxyldopamine (6-OHDA) to MN9D cells, whereas overexpression of WT protected MN9D cells against MPP+ toxicity, but not against 6-OHDA. We conclude that WT alpha-syn is beneficial to dopaminergic neurons but its overexpression in the presence of endogenous dopamine makes it a potential threat to the cells. In contrast, mutant alpha-syn not only caused the loss of WT protective function but also the gain-of-toxicity which becomes more serious in the presence of dopamine and neurotoxins.

Improved neuronal transgene expression from an AAV-2 vector with a hybrid CMV enhancer/PDGF-beta promoter.

BACKGROUND: Adeno-associated virus type 2 (AAV-2) vectors are highly promising tools for gene therapy of neurological disorders. After accommodating a cellular promoter, AAV-2 vectors are able to drive sustained expression of transgene in the brain. This study aimed to develop AAV-2 vectors that also facilitate a high level of neuronal expression by enhancing the strength of a neuron-specific promoter, the human platelet-derived growth factor beta-chain (PDGF) promoter. METHODS AND RESULTS: A hybrid promoter approach was adopted to fuse the enhancer of human cytomegalovirus immediately early (CMV) promoter to the PDGF promoter. In cultured cortex neurons, AAV-2 vectors containing the hybrid promoter augmented transgene expression up to 20-fold over that mediated by titer-matched AAV-2 vectors with the PDGF promoter alone and 4-fold over the CMV enhancer/promoter. Injection of AAV-2 vectors with the hybrid promoter into the rat striatum resulted in neuron-specific transgene expression, the level of which was about 10-fold higher than those provided by the two control AAV-2 expression cassettes at 4 weeks post-injection and maintained for at least 12 weeks. Gene expression in the substantia nigra through possible retrograde transport of the AAV-2 vectors injected into the striatum was not obvious. After direct injection of AAV-2 vectors into the substantia nigra, transgene expression driven by the hybrid promoter was observed specifically in dopaminergic neurons and its level was about 3 and 17 times higher than that provided by the PDGF promoter alone and the CMV enhancer/promoter, respectively. CONCLUSIONS: Enhanced transgene capacity plus neuron-specificity of the AAV-2 vectors developed in this study might prove valuable for gene therapy of Parkinson's disease.

Compensatory caspase activation in MPP+-induced cell death in dopaminergic neurons.

Many have hypothesized that cell death in Parkinson's disease is via apoptosis and, specifically, by the mitochondrial-mediated apoptotic pathway. We tested this hypothesis using a mouse dopaminergic cell line of mesencephalic origin, MN9D, challenged with the Parkinsonism-causing neurotoxin MPP+ (1-methyl-4-phenylpyridinium ion). Apoptosis was the main mode of cell death when the cells were subjected to MPP+ treatment under serum-free conditions for 24 h. Caspase-3 and caspase-9, however, were not activated, thus indicating the existence of alternate or compensatory cell death pathway(s) in dopaminergic neuronal cells. Using caspase inhibitors, we demonstrated that these pathways involve caspase-2, -8, -6 and -7. A time-course study indicated that activation of caspase-2 and -8 occurred upstream of caspase-6 and caspase-7. Upon MPP+ challenge, the apoptosis-inducing factor was translocated from the mitochondria into the MN9D cytosol and nucleus. These results suggest the existence of alternative apoptotic pathways in dopaminergic neurons.

Photocatalytic degradation of toluene by platinized titanium dioxide photocatalysts.

A photoreactor has been set up to study the photodegradation of volatile organic compound (VOC) in situ. In the reactor, TiO2 and Pt/TiO2 photocatalysts were immobilized on to UV-transparent quartz support. Scanning electron microscope (SEM) studies and Brunauer-Emmett-Teller (BET) surface area measurements revealed that the quartz fiber support was mostly coated with catalyst with a total surface area of 4.0 +/- 0.3 m2/g. The photocatalytic activity of the photocatalysts was evaluated for the photodegradation of 160 ppm toluene-laden air. It was found that 50-70% of toluene was degraded within the first 5 min of UV illumination. Both TiO2 and Pt/TiO2 photocatalysts suffered from deactivation after 18 hours of continuous operation, and the photocatalysts' activity was significantly reduced. However, platinization doubled the photocatalyst life and delayed the onset of de-activation. The presence of moisture was found to shift the onset of catalyst de-activation to an earlier time. It is concluded that the de-activation of the photocatalyst was due to the accumulation of intermediates on the photocatalysts surface preventing the toluene being adsorbed on the photocatalyst surface for degradation.

An audit of upper gastrointestinal bleeding at Seremban Hospital.

We retrospectively analyzed all patients presenting with upper gastrointestinal bleeding to Seremban Hospital over a one-year period. A quarter of the oesophagogastro-duodenoscopies (OGD) performed were performed as emergency for upper gastrointestinal tract bleeding. Gastric ulcers and duodenal ulcers were the two most common findings. Our results suggest that there is a male preponderance of 2:1, the Chinese were more likely to be affected and the elderly (> 60 years) were at highest risk.

Opsonized virulent Edwardsiella tarda strains are able to adhere to and survive and replicate within fish phagocytes but fail to stimulate reactive oxygen intermediates.

Edwardsiella tarda is responsible for hemorrhagic septicemia (edwardsiellosis) in fish and also causes diseases in higher vertebrates such as birds, reptiles, and mammals, including humans. Interactions of E. tarda with blue gourami phagocytes were studied by light microscopy as well as by adherence, intracellular replication, and superoxide anion assays. Both nonopsonized virulent (PPD130/91 and AL9379) and avirulent (PPD125/87 and PPD76/87) bacteria could adhere to and survive and replicate within phagocytes, while only opsonized virulent strains replicated within the phagocytes. Furthermore, only avirulent E. tarda elicited a higher rate of production of reactive oxygen intermediates (ROIs) by phagocytes, indicating that they were unable to avoid and/or resist reactive oxygen radical-based killing by the fish phagocytes. TnphoA transposon mutagenesis was used to construct a library of 200 alkaline phosphatase (PhoA+) fusion mutants from a total of 182,000 transconjugants derived from E. tarda PPD130/91. Five of these mutants induced more ROI production in phagocytes than the wild-type strain. Two mutants had lower replication ability inside phagocytes and moderately higher 50% lethal dose values than the wild-type strain. Sequence analysis revealed that three of these mutants had insertions at sequences having homology to PhoS, dipeptidase, and a surface polymer ligase of lipid A core proteins of other pathogens. These three independent mutations might have changed the cell surface characteristics of the bacteria, which in turn induced phagocytes to produce increased ROIs. Sequences from two other mutants had no homology to known genes, indicating that they may be novel genes for antiphagocytic killing. The present study showed that there are differences in the interactions of virulent and avirulent E. tarda organisms with fish phagocytes and PhoA+ fusion mutants that could be used successfully to identify virulence genes. The information elucidated here would help in the development of suitable strategies to combat the disease caused by E. tarda.

Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity.

Edwardsiella tarda is a Gram-negative bacterium that causes a systemic infection, edwardsiellosis, in fish. The virulence factors of this pathogen and its genetic determinants have not been systematically examined. In this study, TnphoA transposon mutagenesis was used to construct a library of 440 alkaline phosphatase (PhoA(+)) fusion mutants from a total of 400000 transconjugants derived from Ed. tarda PPD130/91. This library included genes for secreted and membrane-associated proteins normally involved in virulence. The library was screened for four virulence factors: siderophore production, motility, serum resistance and catalase production. Eight mutants deficient in one or more of these phenotypes were grouped into four classes. They were further characterized for their stimulation of reactive oxygen intermediate production by fish phagocytes, for their adhesion to and internalization into EPC (epithelioma papillosum of carp) cells, and for attenuation of virulence in blue gourami. Mutants 2A and 34 were highly attenuated in fish, with LD(50) values about 10 times higher than for the wild-type. These strains had mutations in the genes encoding arylsulfate sulfotransferase (mutant 2A) and a catalase precursor protein (mutant 34). One hyperinvasive/adhesive mutant and four pst mutants that were pleiotropic and slightly attenuated in fish were also isolated.

Green fluorescent protein-tagged Edwardsiella tarda reveals portal of entry in fish.

The application of green fluorescent protein (GFP) to identify the portal(s) of entry of bacterial pathogens in animal hosts was studied using the fish pathogen Edwardsiella tarda and blue gourami, Trichogaster trichopterus. An immersion challenge model was utilized to mimic natural infection conditions in fish. Gastrointestinal tract, gills and the body surface of fish were found to be the sites of entry of virulent E. tarda (PPD130/91) by histological and infection kinetics studies. On the other hand, avirulent E. tarda (PPD125/87) was mainly found in the gastrointestinal tract, and the bacterial population in tissue declined over a period of 7 days.

Mortality in the Department of Surgery, Alor Setar Hospital.

This is a retrospective study of the annual mortality that occurred in the Department of Surgery, Alor Setar Hospital, for the years 1995 to 1997. This study looks at the number of admissions to the surgical wards and categorizes the causes of death. The annual mortality rates were 2.60, 2.89 and 3.25 per hundred admissions for the year 1995, 1996 and 1997 respectively. Head injury was the leading cause of death whilst sepsis and advanced malignancies second and third commonest causes. We hope that with the publication of these figures, we can initiate more studies to analyse similar local data.

Type I and type II cytokeratin cDNAs from the zebrafish (Danio rerio) and expression patterns during early development.

Full-length cDNAs of a type I (zfCKI), and a type II (zfCKII) cytokeratin from the adult zebrafish, Danio rerio, were characterized and their expressions studied during early development and in the adult. The 1,426 bp long zfCKI cDNA encodes a 46.7 kD protein, whereas the 2,398 bp zfCKII cDNA encodes a protein of 58.6 kD. zfCKI and zfCKII each have a central rod domain that is characteristic of intermediate filaments and which share 73%-91% and 87%-93% similarity, respectively, with those of type I and type II cytokeratins from zebrafish, goldfish, and the rainbow trout. The central rod domains of zfCKI and zfCKII also contain the IF signature motif, IA[T/E]YR[K/R]LL[D/E]. zfCKI has, in addition, a leucine-zipper motif at a.a. residues 184-205 and 191-212. Both zfCKI and zfCKII mRNAs are expressed in the epidermis of the zebrafish. zfCKII mRNA was both maternally inherited and zygotically transcribed and was detected from the one-cell embryo to adult stages. zfCKII was also strongly expressed specifically during the 20-somites, protruding-mouth, and adult stages. In the adult, it was uniformly expressed in the skin, fins and scale epidermis. In contrast, zfCKI mRNA was undetectable in the oocyte but was zygotically transcribed from the epiboly stage onwards. Its expression in the skin was strong only up to the swimming larva stage and was weak and patchy in the adult. Both zfCKI and zfCKII were expressed in the neurons and glial cells of the brain and spinal cord. In the adult eye, zfCKI and zfCKII were expressed in the ganglion cell layer and the retina, but zfCKII was also strongly expressed in the cornea as well as in chondrocytes in the skull.

Asynchronous activation of 10 muscle-specific protein (MSP) genes during zebrafish somitogenesis.

In the present study, 10 zebrafish cDNA clones coding for muscle-specific proteins (MSPs) were characterized and most of them encode fast skeletal muscle isoforms. They are skeletal muscle alpha-actin (acta1), fast skeletal muscle a-tropomyosin (tpma), fast skeletal muscle troponin C (tnnc), fast skeletal muscle troponin T (tnnt), fast skeletal muscle myosin heavy chain (myhz1), fast skeletal muscle myosin light chain 2 (mylz2), fast skeletal muscle myosin light chain 3 (mylz3), muscle creatine kinase (ckm), parvalbumin (pvalb), and desmin (desm). Using these cDNA probes, their expression patterns in developing embryos and adults were compared by Northern blot hybridization and whole-mount in situ hybridization. All of the 10 genes are expressed in both embryos and adult fish, and the expression is highly abundant in skeletal muscle. Among them, acta1, tpma, tnnc, tnnt, myhz1, mylz2, mylz3 and pvalb, are expressed specifically in fast skeletal muscle while ckm and desm are expressed in both fast and slow skeletal muscles. In addition, tpma, ckm, and desm are also expressed in the heart. Ontogenetically, the onset of expression of these MSP genes in zebrafish skeletal muscle varies and the expression occurs rostral-caudally in developing somites. Shortly after the expression of myoD, desm is the first to be activated at approximately 9 hpf, followed by tpma (approximately 10 hpf), tnnc (approximately 12 hpf), acta1 (approximately 12 hpf), ckm (approximately 14 hpf), myhz1 (approximately 14 hpf), mylz2 (approximately 16 hpf), mylz3 (approximately 16.5 hpf), tnnt (approximately 16.5 hpf), and pvalb (approximately 16.5 hpf). At later stages (after 48 hpf), these MSP genes are also expressed in fin buds and head muscles including eye, jaw, and gill muscles. Thus, our experiment demonstrated the order of expression of the 10 MSP genes, which may reflect the sequence of muscle filament assembly. In spite of the asynchrony in activation of these MSP genes, the timing of expression for each individual MSP gene appears to be synchronous to somite development as each somite has an identical timetable to express the set of MSP genes.

Characterization of a zebrafish (Danio rerio) desmin cDNA: an early molecular marker of myogenesis.

Desmin is a muscle-specific protein and a constitutive subunit of the intermediate filaments (IF) in skeletal, cardiac and smooth muscles. It is an early marker of skeletal muscle myogenesis. We have characterized a clone of desmin cDNA from an embryonic zebrafish (Danio rerio) cDNA library. The full-length cDNA comprised 1798 nucleotides, encoding a protein of 473 amino acids. The predicted amino acid sequence of the zebrafish desmin shares a high degree of similarity to other vertebrate desmins, but also contains a sequence at the carboxyl terminal of the tail domain that is unique to the zebrafish. It carries many features which are distinctive of IF subunit proteins. These include the T/SSYRRXF/Y motif in the head domain, and the intermediate filament signature consensus, [I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E], located in the carboxyl terminus of the central helical rod. Unlike other 3' UTR sequences, the 3' UTR of the zebrafish cDNA sequence has two CAYUG elements flanking a single polyadenylation site. The temporal and spatial expression patterns of desmin mRNA during early zebrafish development were studied. The onset of desmin expression occurred at the 1-3 somite stage (11 hpf). It increased throughout somitogenesis, with maximum expression at the Prim-6 stage (25 hpf), and decreasing expression towards the protruding-mouth stage (72 hpf). Desmin mRNA was initially localised exclusively to the somites, but was subsequently also detected in other musculature in the developing heart and fins. The onset of expression and the spatial localization of desmin mRNA in the zebrafish coincides with that reported for MyoD and myogenin.

Neuronal localization of sterol regulatory element binding protein-1 in the rodent and primate brain: a light and electron microscopic immunocytochemical study.

Sterol regulatory element binding proteins are membrane-bound transcription factors that activate expression of several genes controlling cellular cholesterol and fatty acid homeostasis. The present study aimed to investigate the in vivo expression of sterol regulatory element binding protein-1 in the normal rodent and primate brain, and in the brain in Niemann-Pick type C disease mice. These mutant animals have lysosomal cholesterol accumulation and progressive neurodegeneration caused by an inactivating mutation of the NPC1 gene whose protein product functions in vesicular lipid trafficking. Western blot analysis of rat hippocampal homogenates with an affinity purified rabbit polyclonal antibody directed against an internal epitope of sterol regulatory element binding protein-1 identified a major 68,000 mol. wt protein consistent with the amino-terminal, transcriptionally active fragment of sterol regulatory element binding proteins-1. Immunocytochemically, this antibody revealed dense sterol regulatory element binding protein-1 staining of nuclei and light staining of the cytoplasm of cells in the neocortex and hippocampus in the rat, mouse and monkey brain. By electron microscopy of immunogold-labeled brain sections, these densely labeled cells were found to be neurons. In contrast, normal glial cells had little or no sterol regulatory element binding protein-1 immunoreactivity even at a developmental stage (postnatal day 9) which coincides with active myelination in the rat brain. Also, in contrast to the normal mouse brain, Niemann-Pick type C mice showed reduced staining of cortical and hippocampal neuronal nuclei. Since sterol regulatory element binding protein-1 has been shown to be a transcriptional regulator of fatty acid synthesis in vivo, the current findings of a predominantly neuronal nuclear expression of the 68,000 mol. wt transcriptionally active fragment of sterol regulatory element binding protein-1 highlights the established role of phospholipid metabolites and other fatty-acid containing lipids in neuronal signal transduction and other neuronal functions. Reduced sterol regulatory element binding protein-1 expression in neurons in Niemann-Pick type C may reflect a deficiency in fatty acid synthesis that could contribute to the neuronal dysfunction in this disorder.

Kainate-induced neuronal injury leads to persistent phosphorylation of cAMP response element-binding protein in glial and endothelial cells in the hippocampus.

Intracerebroventricular kainate treatment in rats induces neuronal cell death, followed by proliferation and hypertrophy of glial cells in the lesioned area. To further understand the activated signal transduction pathways and to get insights into potential target gene activation, the present study aims to elucidate long-term effects on the phosphorylation state of cAMP response element-binding protein (CREB) in the hippocampal formation. One to four weeks after kainate injection, we found high levels of phosphorylated and hence activated CREB (pCREB) in glial cells of the degenerating CA fields. As shown by electron microscopy, pCREB immunoreactivity was present in reactive astrocytes, oligodendrocyte precursor cells and endothelial cells of blood vessels. It is postulated that pCREB could drive the expression of downstream genes in these cells to promote cell proliferation and survival.

Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models.

Edwardsiella tarda is a fish pathogen that causes systemic infections in many food and ornamental fish. E. tarda PPD130/91 and PPD125/87 were selected as representatives of the virulent and avirulent groups, respectively, from eight fish isolates, and transformed with plasmids encoding either green fluorescent protein (pGFPuv) or blue fluorescent protein (pBFP2). Two host models were used to study the invasion pathway of E. tarda in vitro and in vivo. Epithelioma papillosum of carp (EPC) was used as the first model. Virulent and avirulent E. tarda strains were found to adhere to and invade EPC cells. Interactions between E. tarda and host cells examined under confocal microscopy and intracellular growth were followed at different time points. Bacterial internalization of PPD130/91 and PPD125/87 involved microfilaments and protein tyrosine kinase since cytochalasin D (an inhibitor of microfilament polymerization) and genistein (an inhibitor of protein tyrosine kinase) prevented internalization. Confocal studies revealed co-localization of polymerized actin with bacteria. Staurosporine, a protein kinase C inhibitor, accelerated internalization of PPD125/87, whereas PD098059, a mitogen-activated protein kinase (MAPK) kinase inhibitor prevented internalization of PPD130/91. In the second model, blue gourami were infected with E. tarda intramuscularly. Mortalities were observed in PPD130/91(pGFPuv)-infected fish with high bacterial numbers detectable in all organs. PPD125/87(pBFP2)-infected fish did not die and the bacterial population decreased over time. Mixed infections comprised of both PPD130/91(pGFPuv) and PPD125/87(pBFP2), where inoculum size was similar to the single infections, caused mortalities in fish. High bacterial populations were noted only in the fish body muscle. The PPD125/87(pBFP2) population in the fish decreased after 5 d. The number of PPD130/91(pGFPuv) also decreased in the fish organs, except for continued high growth in the body muscle. Histology revealed necrosis of the tissue (body muscle and liver) and fluorescent bacteria in fish that were infected with PPD130/91(pGFPuv) but not with PPD125/87(pBFP2). This study showed that fluorescent proteins are a useful tool for investigating bacterial host cell infection, and information elucidated here sheds new light on the interactions between E. tarda and its hosts.

GFP-aided confocal laser scanning microscopy can monitor Agrobacterium tumefaciens cell morphology and gene expression associated with infection.

We tagged Agrobacterium tumefaciens cells with a mini-Tn5 transposon containing a promoterless gene encoding a green fluorescent protein (GFP). Some of the GFP-tagged individual bacterial cells exhibited strong green fluorescence, which reflected the expression levels of the GFP-tagged genes. Those cells could be readily detected with a confocal laser scanning microscope (CLSM). We observed that the fluorescence and morphology of A. tumefaciens cells grown in plant tissues resembled those grown in a minimal medium of low pH, which is required for expression of the virulence genes responsible for tumorigenesis. This suggests that GFP-aided CLSM can be used to determine which growth medium is more representative of the nutritional conditions that a pathogen encounters in plant tissues. We also observed that the fluorescence and morphology of A. tumefaciens cells changed dramatically during the course of infection. Our data suggested that A. tumefaciens cells were probably better fed upon successful colonization. We believe that GFP-aided CLSM can help study the fate of A. tumefaciens cells inside plant tissues by monitoring cell morphology and gene expression associated with the infection process in situ.