Plasmid gene AZOBR_p60126 impacts biosynthesis of lipopolysaccharide II and swarming motility in Azospirillum brasilense Sp245

The facultative plant endophyte Azospirillum brasilense Sp245 synthesizes two high-molecular-weight lipopolysaccharides, LPSI and LPSII, which comprise identical d -rhamnan O-polysaccharides and, presumably different core oligosaccharides. Previously, using random insertion mutagenesis, we constructed the LpsII⁻ mutant KM139 of strain Sp245 that possessed an Omegon-Km insertion in plasmid AZOBR_p6. Here, we found that in KM139, Omegon-Km disrupted the coding sequence AZOBR_p60126 for a putative glycosyltransferase related to mannosyltransferases and rhamnosyltransferases. To verify its function, we cloned the AZOBR_p60126 gene of strain Sp245 in the expression vector plasmid pRK415 and transferred the construct pRK415–p60126 into KM139. In the complemented mutant KM139 (pRK415–p60126), the wild-type LPSI⁺ LPSII⁺ profile was recovered. We also compared the swimming and swarming motilities of strains Sp245, Sp245 (pRK415), KM139, KM139 (pRK415), and KM139 (pRK415–p60126). All these strains had the same flagellar-dependent swimming speeds, but on soft media, the LpsI⁺ LpsII⁻ strains KM139 and KM139 (pRK415) swarmed significantly faster than the other LpsI⁺ LpsII⁺ strains. Such interstrain differences in swarming motility were more pronounced on 0.4% than on 0.5% soft agar plates. These data show that the AZOBR_p60126-encoded putative glycosyltransferase significantly affects the lipopolysaccharide profile and, as a consequence, the social motility of azospirilla.     

Colonization of the cecal mucosa by Helicobacter hepaticus impacts the diversity of the indigenous microbiota

Establishment of mucosal and/or luminal colonization is the first step in the pathogenesis of many gastrointestinal bacterial pathogens. The pathogen must be able to establish itself in the face of competition from the complex microbial community that is already in place. We used culture-independent methods to monitor the colonization of the cecal mucosa of Helicobacter-free mice following experimental infection with the pathogen Helicobacter hepaticus. Two days after infection, H. hepaticus comprised a minor component of the mucosa-associated microbiota, but within 14 days, it became the dominant member of the community. Colonization of the mucosa by H. hepaticus was associated with a decrease in the overall diversity of the microbial community, in large part due to changes in evenness resulting from the relative dominance of H. hepaticus as a member of the community. Our results demonstrate that invasion of the complex gastrointestinal microbial community by a pathogenic microorganism causes reproducible and significant disturbances in the community structure. The use of non-culture-based methods to monitor these changes should lead to a greater understanding of the ecological principles that govern pathogen invasion and may lead to novel methods for the prevention and control of gastrointestinal pathogens.     

The biomedical potential of genetically modified flax seeds overexpressing the glucosyltransferase gene

Background

Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance.

Methods

The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated.

Results

The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated that GT seedcake preparations have no harmful effect on human skin. Moreover, GT seedcake preparations exhibited inhibitory properties toward two bacterial strains: Staphylococcus aureus and Escherichia coli.

Conclusions

We suggest that preparations derived from the new GT flax are an effective source of phenylopropanoids and that their glycoside derivatives and might be promising natural products with both healing and bacteriostatic effects. This flax-derived product is a good candidate for application in the repair and regeneration of human skin and might also be an alternative to antibiotic therapy for infected wounds.

     

Inactivation of auxin in tobacco transformed with the indoleacetic acid-lysine synthetase gene of Pseudomonas savastanoi

The iaaL gene of Pseudomonas syringae, subspecies savastanoi, encodes an indoleacetic acid (IAA)-lysine synthetase. To determine the effects of converting IAA to IAA-lysine in whole plants, the iaaL gene was fused to a constitutive plant promoter and introduced into tobacco plants. Biochemical analyses show that endogenous IAA is reduced by up to 19-fold in iaaL plants. Tobacco plants expressing the iaaL gene display reduced apical dominance, reduced rooting, and inhibition of vascular differentiation. The phenotypic effects of iaaL gene expression are reverted by crossing iaaL plants with plants that overproduce IAA. These data indicate that iaaL can act as an anti-auxin gene in vivo and confirm the role of auxin in the control of apical dominance, root growth, and vascular differentiation in whole plants.     

Effects of probiotic administration upon the composition and enzymatic activity of human fecal microbiota in patients with irritable bowel syndrome or functional diarrhea.

In a clinical trial, 10 patients suffering from irritable bowel syndrome or functional diarrhea were administered the probiotic preparation VSL-3. Preliminary results indicated that administration of VSL-3 improved the clinical picture and changed the composition and biochemistry of fecal microbiota. Titer variations of intestinal bacterial groups were evaluated by culture and PCR techniques. A significant increase in lactobacilli, bifidobacteria and Streptococcus thermophilus was observed as a consequence of probiotic treatment, while enterococci, coliforms, Bacteroides and Clostridium perfringens did not change significantly. The strains Bifidobacterium infantis Y1 and Bifidobacterium breve Y8, included in VSL-3, were specifically detected in feces of patients treated with the probiotic by using strain-specific PCR primers. In addition, fecal beta-galactosidase increased and urease activities decreased as a result of changes in the intestinal microbiota induced by VSL-3 administration.     

Nicotinic modulation of network and synaptic transmission in the immature hippocampus investigated with genetically modified mice

The hippocampus, a key structure in learning and memory processes, receives a powerful cholinergic innervation from the septum and contains nicotinic acetylcholine receptors (nAChRs). Early in postnatal development, activation of nAChRs by nicotine or endogenous acetylcholine contributes to enhance synaptic signalling. Here, the patch-clamp technique was used to assess the contribution of α7 and β2-containing (α7* and β2*) nAChRs to nicotine-elicited modulation of GABAergic and glutamatergic activity at the network and single-cell level in the immature hippocampus of wild-type (WT), α7−/− and β2−/− mice. We found that α7* and β2* nAChRs were sufficient to modulate nicotine-induced increase in frequency of spontaneously occurring giant depolarizing potentials (GDPs), which are generated at the network level by the synergistic action of glutamate and depolarizing GABA, and thought to play a crucial role in neuronal wiring. However, α7* but not β2* receptors were essential in nicotine-induced increase of interictal discharge frequency recorded after postnatal day 3 in the presence of bicuculline, when GABA shifted from the depolarizing to the hyperpolarizing direction. To correlate these observations with nicotine-elicited changes in synaptic transmission, we recorded spontaneous GABAergic and glutamatergic postsynaptic currents in pyramidal cells and interneurons localized in stratum oriens, stratum pyramidale and stratum radiatum, in slices obtained from WT and knock-out animals. We found that early in postnatal life α7* and β2* nAChRs exert a fine regional modulation of GABAergic and glutamatergic transmission that underlies nicotine-elicited changes in network synchronization.     

Physiological consequences of the P2328S mutation in the ryanodine receptor (RyR2) gene in genetically modified murine hearts

Aim: To explore the physiological consequences of the ryanodine receptor (RyR2)‐P2328S mutation associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). Methods: We generated heterozygotic (RyR2^p/s) and homozygotic (RyR2^s/s) transgenic mice and studied Ca²⁺ signals from regularly stimulated, Fluo‐3‐loaded, cardiac myocytes. Results were compared with monophasic action potentials (MAPs) in Langendorff‐perfused hearts under both regular and programmed electrical stimulation (PES). Results: Evoked Ca²⁺ transients from wild‐type (WT), heterozygote (RyR2^p/s) and homozygote (RyR2^s/s) myocytes had indistinguishable peak amplitudes with RyR2^s/s showing subsidiary events. Adding 100 nm isoproterenol produced both ectopic peaks and subsidiary events in WT but not RyR2^p/s and ectopic peaks and reduced amplitudes of evoked peaks in RyR2^s/s. Regularly stimulated WT, RyR2^p/s and RyR2^s/s hearts showed indistinguishable MAP durations and refractory periods. RyR2^p/s hearts showed non‐sustained ventricular tachycardias (nsVTs) only with PES. Both nsVTs and sustained VTs (sVTs) occurred with regular stimuli and PES with isoproterenol treatment. RyR2^s/s hearts showed higher incidences of nsVTs before but mainly sVTs after introduction of isoproterenol with both regular stimuli and PES, particularly at higher pacing frequencies. Additionally, intrinsically beating RyR2^s/s showed extrasystolic events often followed by spontaneous sVT. Conclusion: The RyR2‐P2328S mutation results in marked alterations in cellular Ca²⁺ homeostasis and arrhythmogenic properties resembling CPVT with greater effects in the homozygote than the heterozygote demonstrating an important gene dosage effect.     

Colonization with Helicobacter is concomitant with modified gut microbiota and drastic failure of the immune control of Mycobacterium tuberculosis

Epidemiological and experimental observations suggest that chronic microbial colonization can impact the immune control of other unrelated pathogens contracted in a concomitant or sequential manner. Possible interactions between Mycobacterium tuberculosis infection and persistence of other bacteria have scarcely been investigated. Here we demonstrated that natural colonization of the digestive tract with Helicobacter hepaticus in mice is concomitant with modification of the gut microbiota, subclinical inflammation, and drastic impairment of immune control of the growth of subsequently administered M. tuberculosis, which results in severe lung tissue injury. Our results provided insights upon the fact that this prior H. hepaticus colonization leads to failures in the mechanisms that could prevent the otherwise balanced cross-talk between M. tuberculosis and the immune system. Such disequilibrium ultimately leads to the inhibition of control of mycobacterial growth, outbreak of inflammation, and lung pathology. Among the dysregulated immune signatures, we noticed a correlation between the detrimental lung injury and the accumulation of activated T-lymphocytes. Our findings suggest that the impact of prior Helicobacter spp. colonization and subsequent M. tuberculosis parasitism might be greater than previously thought, which is a key point given that both species are among the most frequent invasive bacteria in human populations.     

IDO修饰的成纤维细胞培养上清对小鼠T淋巴细胞增殖的影响

目的：检测吲哚胺2—3双加氧酶（IDO）基因修饰的细胞对T淋巴细胞的免疫抑制作用。方法：将pEGFP-NI-mIDO真核表达载体转染至小鼠NIH 3T3成纤维细胞系。体外检测mIDO修饰的成纤维细胞培养液中色氨酸水平的变化；使用CFSE标记技术，检测mIDO修饰的成纤维细胞培养液对ConA刺激的小鼠T淋巴细胞增殖的影响。结果：将pEGFP-NI-mIDO转入NIH 3T3成纤维细胞，流式细胞术（FCM）检测到EGFP的表达；体外实验表明，成纤维细胞培养上清和转染pEGFP—NI的成纤维细胞培养上清可检测到一定量的色氨酸水平，而转染pEGFP-NI-mIDO的成纤维细胞培养上清未检测到色氨酸，提示pEGFP-NI-mIDO转染的成纤维细胞表达了具有活性的mIDO；使用CFSE标记技术，检测mIDO修饰的成纤维细胞培养上清对小鼠T淋巴细胞增殖的影响，发现ConA刺激的IDO组，72h后亲代细胞占各代细胞的百分率明显高于对照组。结论：IDO基因修饰的成纤维细胞可抑制T淋巴细胞的增殖。     

Evaluation of the effectiveness and safety of a genetically modified live vaccine in broilers challenged with Salmonella Heidelberg

Salmonellosis ranks among the major diseases of commercial poultry, and its presence in poultry flocks is responsible for economic losses and risks related to public health. Vaccines are an important tool within integrated programmes to control salmonellosis. The purpose of this study was to assess cross-protection provided by the Poulvac® ST vaccine in the control of Salmonella Heidelberg in experimentally challenged 3- and 21-day-old birds. Eighty birds were identified and separated into four treatments (T1: vaccinated and challenged at 3 days of age, T2: unvaccinated and challenged at 3 days of age, T3: vaccinated and challenged at 21 days of age, and T4: unvaccinated and challenged at 21 days of age). The inoculum was produced from a Brazilian field strain of SH. At the end of the experiment, caecum and liver/spleen samples were collected for quantitative and qualitative analysis of SH, respectively. Analysis of the liver/spleen showed that Poulvac® ST significantly (P?≤?0.05) reduced the percentage of SH positivity in the group challenged at 3 days of age, while in the group challenged at 21 days this difference was almost considered significant (P?=?0.1818). On the other hand, there was no statistically significant difference in SH count in the caecum (CFU/g) in the group challenged at 3 days, but for the group challenged at 21 days the SH counts were significantly (P?≤?0.05) lower in the vaccinated group when compared to the positive control.     

Effects of amoxicillin treatment on the salivary microbiota in children with acute otitis media

Amoxicillin is a first-line antibiotic treatment for acute otitis media in children and one of the most commonly used antibiotics for human bacterial infections. We investigated changes in salivary bacterial communities among children treated with amoxicillin for acute otitis media (n = 18), using a culture-independent approach based on pyrosequencing of the V3 region of the bacterial 16S rRNA gene. The control group consisted of children with acute otitis media who were not given antibiotics (n = 15). One species-level phylotype assigned to the genus Streptococcus was identified across all (n = 99) saliva samples. Two additional species-level phylotypes from the genera Gemella and Granulicatella were shared by all (n = 45) samples of control subjects. Amoxicillin treatment resulted in reduced species richness and diversity, and a significant shift in the relative abundance of 35 taxa at different ranks from phylum to species-level phylotype. At the phylum level, prevalence of TM7 and Actinobacteria decreased at the end of treatment, whereas Proteobacteria had a higher relative abundance post-treatment. Multivariate analysis showed that samples from the same control subject taken over time intervals tended to cluster together. Among antibiotic-treated subjects, samples taken before and at the end of amoxicillin treatment formed two relatively well-separated clusters both of which greatly overlapped with samples taken about 3 weeks post-treatment. Our results point to a substantial but incomplete recovery of the salivary bacterial community from the antibiotic about 3 weeks after the end of treatment.     

脑源性神经营养因子转基因成纤维细胞的构建与检测

目的:构建大鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因修饰的成纤维细胞(fibroblasts FBs).方法:真核表达载体pcDNA3/BDNF由昆明医学院神经科学研究所提供.FBs取自胎鼠.pcDNA3/BDNF重组质粒载体以阳离子脂质体Lipofect...     

Effects of long-term fertilization on the diversity of bacterial mercuric reductase gene in a Chinese upland soil

Soil mercury (Hg) pollution has received considerable attention due to its neurotoxin effects and its potential risk to food safety. The microbial transformation of Hg plays a key role in reducing Hg toxicity by the mercuric reductase (MerA) conferred by genes arranged in the mer operon. This study investigated the effects of long‐term fertilization on the diversity of bacterial mercuric reductase gene (merA), which specify the reduction of ionic Hg²⁺ to the volatile elemental form Hg⁰, in an agricultural soil with relatively high Hg content. The soil samples were collected from different treatments, including control without fertilizer (CK), fertilizer nitrogen (N), combined fertilizers (NPK) of N, phosphorus (P) and potassium (K), and NPK plus organic manure (NPK + OM). The merA gene diversity patterns were analyzed based on the merA clone libraries and sequencing measurements. Results showed that the merA gene diversity was influenced by soil variables depending on the fertilization practices. In particular, NH₄⁺ and NO₃^– contents had strong effect on the merA gene diversity pattern both in the N and NPK treatments, whereas the merA gene diversity pattern in NPK + OM treatment was distinctly influenced by the contents of organic matter, available P and K. These results suggested that long‐term fertilization had significant influences on merA gene diversity, which could be helpful to understand the Hg reduction process and potentially serve microbial remediation of Hg contaminated soil. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A gene involved in the biogenesis of cytochromes is co-transcribed with a ribosomal protein gene in wheat mitochondria

Sequence analysis of a transcribed region of the wheat mitochondrial (mt) genome revealed two open reading frames (orfs) coding for proteins of 589 and 174 amino acids. Both genes are co-transcribed in a 2.6-kb RNA. The largest orf codes for a hydrophobic protein which bears similarity to a bacterial protein involved in the biogenesis of c-type cytochromes. Its corresponding RNA sequence is fully edited at 34 positions. The second orf encodes a protein homologous to the amino-terminal third of E. coli ribosomal protein S1, corresponding to the ribosome-binding domain of this protein. Its RNA sequence is edited at four positions, one of the edits creating a stop codon. The presence of both proteins in wheat mitochondria was demonstrated using specific antibodies raised against fusion proteins obtained in E. coli from the corresponding cDNAs.