用于质粒DNA规模化生产的大肠杆菌发酵培养基的筛选

为降低质粒DNA的生产成本,在标准LB培养基的基础上,利用国产试剂配制成十种大肠杆菌液体培养基,以pEGFPC3、pcDNAlacZ和pcDNKLYZ质粒转化的JM109和DH5α大肠杆菌为指示菌进行小规模发酵培养,定时采样测量OD600值及质粒产量,获得一种高性价比培养基。用该培养基培养重组大肠肝菌,绘制生长曲线,并于其对数生长中期进行42℃诱导。结果表明经42℃诱导后,重组大肠肝菌JM109和DH5α的质粒产量均有提高,重组JM109的产量比重组DH5α约提高20％,为低成本、大规模生产重组质粒提供了良好的技术保障。     

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC) can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE) cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp) end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA^? strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid engineering.     

Ultrasound-mediated DNA transfer for bacteria

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5α and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl₂, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 × 10⁹ CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 × 10⁻⁶ transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5α and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 × 10⁻⁶ and 4.33 ± 0.78 × 10⁻⁶ transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.     

Biocontrol activity and root colonization by Pseudomonas corrugata strains CCR04 and CCR80 against Phytophthora blight of pepper

Previously, we selected Pseudomonas corrugata strains CCR04 and CCR80 as rhizobacteria suppressive to Phytophthora blight of pepper caused by Phytophthora capsici. In this study, we investigated soil microbial activity in pepper plants root-drenched with strains CCR04 and CCR80 in relation to their biocontrol activity, root colonization by using bacterial population counts and scanning electron microscopy, biofilm formation and cell motility as well as cell sensitivity to hydrogen peroxide (H₂O₂). As a result, strains CCR04 and CCR80 more effectively suppressed disease expression in pepper plants through root colonization than did Paenibacillus polymyxa AC-1 (positive control), Escherichia coli DH5α (negative control) or MgSO₄ solution (untreated control). Strains CCR04 and CCR80 had efficient biofilm formation and cell motility (swimming and swarming activities) abilities and responded to certain tested compounds (amino acids, organic acids and sugars), which can be found in root exudates. Strains CCR04 and CCR80 and the positive control strain AC-1 were relatively insensitive to H₂O₂, a reactive oxidative species at concentration up to 20 mM, unlike the negative control strain DH5α. Taken together, these results suggest that P. corrugata CCR04 and CCR80 can effectively inhibit P. capsici infection of pepper plants through successful colonization of plant roots. This bacterial colonization may be facilitated by the biofilm formation ability and cell motility in addition to reduced sensitivity to H₂O₂ and probably the production of antimicrobial compounds. These findings highlight the potential of strains CCR04 and CCR80 as biocontrol agents for the management of Phytophthora blight of pepper.     

微藻光密度与细胞密度及生物质的关系

以四种常见微藻,小球藻(Chlorella sp.XQ-20044)、栅藻(Scenedesmus sp.SS-200716)、绿球藻(Chlorococcum sp.)和螺旋藻(Spirulina sp.CH-164)为实验材料,用梯度稀释法测定对数生长期不同浓度藻液的光密度(OD)、细胞密度和生物质干重(DW),在光自养分批培养模式下对4种微藻进行OD-波长(350—800 nm)扫描,同时测定细胞密度和生物质干重,分析藻液OD与细胞密度、生物质干重的关系。结果表明:在任何波长下,对数生长期的4种微藻细胞密度与OD值、生物质干重与OD值的变化都不成比例,波长不同其拟合曲线偏离直线的程度不同。但是,在435 nm处这种关系最接近直线,可以用直线方程近似描述(R20.98),其它波长处细胞密度-OD、干重-OD的关系都可以用二项式方程很好地描述(R20.99)。因此,光密度法适用于连续和半连续培养,可以用435 nm处测得的OD值计算细胞密度与干重。但是在分批培养模式下,4种微藻DW/OD比值随着培养时间均逐渐上升。小球藻DW/OD540为0.19—0.44 g/L,栅藻DW/OD540为0.36—0.53 g/L,绿球藻DW/OD540为0.48—0.75 g/L,螺旋藻DW/OD560为0.46—0.74 g/L,因此分批培养模式下采用测定藻液OD值反映细胞密度和生物质的方法不适用,只有直接测定细胞密度和生物质才是准确的。研究结果为正确使用分光光度法监测微藻生长提供依据。     

Co-segregation of trichorhinophalangeal syndrome with a t(8;13)(q23.3;q21.31) familial translocation that appears to increase TRPS1 gene expression

Trichorhinophalangeal syndrome type I (TRPS I) is a rare autosomal dominant syndrome caused by haploinsufficiency of TRPS1 due to point mutations or deletions. Here, we report the first familial TRPS I due to a t(8;13)(q23.3;q21.31) translocation breakpoint <100 kb from the 5′ end of TRPS1. Based on the additional abnormalities observed exclusively in the index patient that are mainly compatible with clinical features of TRPS, her phenotype was defined as expanded TRPS I including brain malformations and intellectual disability. Initial analyses did not reveal any genetic defect affecting TRPS1 or any genomic alteration within the breakpoint regions or elsewhere in the genome. The pathogenic chromosome 8q23.3 breakpoint is at position g.116,768,309_116,768,310 within a transposon type I element, 87 kb from the TRPS1 5′ end. The 13q21.31 breakpoint is within a tandem repeat region at position g.65,101,509_65,101,510 (genome assembly GRCh37/hg19). This breakpoint is flanked by protocadherin 9 (PCDH9) and protocadherin 20 (PCDH20). As an outcome of the translocation, an evolutionarily conserved non-coding VISTA enhancer element from 13q21.31 is placed within the TRPS1 5′ region, 1,294 bp from the breakpoint. The increased expression of TRPS1 found by three independent methods is most probably translocation allele derived and driven by the translocated enhancer element. The index patient’s expanded phenotype presumably involves the epithelial-to-mesenchymal transition pathway that may be due to TRPS1 overexpression. Together, these findings support that the reported translocation-associated phenotypes are “cis-ruption” and TRPS1 overexpression related, the latter most probably caused by the novel enhancer element in the TRPS1 5′ region.     

Marine bacteria antagonistic to the harmful algal bloom species Alexandrium tamarense (Dinophyceae)

As part of efforts to enhance the strategies employed to manage and mitigate algal blooms and their adverse effects, algicidal bacteria have shown promise as potential suppressors of these events. Nine strains of bacteria algicidal against the toxic dinoflagellate, Alexandrium tamarense, were isolated from the East Sea area, China. Sequence analysis of 16S rDNA showed that all the algicidal bacteria belonged to the γ-proteobacteria subclass and the genera Pseudoalteromonas (strain SP31 and SP44), Alteromonas (strain DH12 and DH46), Idiomarina (strain SP96), Vibrio (strain DH47 and DH51) and Halomonas (strain DH74 and DH77). To assess the algicidal mode of these algicidal bacteria, bacterial cells and the filtrate from bacterial cultures were inoculated into A. tamarense cultures, and fluorescein diacetate vital stain was applied to monitor the growth of the algal cells. The results showed that all the algicidal bacteria exhibited algicidal activity through an indirect attack since algicidal activity was only detected in cell free supernatants but not the bacterial cells. This is the first report of bacteria from the genus Idiomarina showing algicidal activity to the toxic dinoflagellate A. tamarense and these findings would increase our knowledge of bacterial–algal interactions and the role of bacteria during the population dynamics of HABs.     

The amoebal cell of Physarum polycephalum: colony formation and growth.

Two stages of colony growth were observed during microscopic studies of Physarum polycephalum amoebae. During the first stage, “spreading growth,” the colony is composed of dispersed single cells. During the second stage, “aggregate growth,” most of the active cells in a colony are aggregated in a ring at the colony boundary. Measurements of cell movement as a function of bacterial concentration indicate that, during both spreading and aggregate growth, cell movements are not affected by changes in bacterial concentration but that the transition from spreading to aggregate growth occurs earlier on plates with lower bacterial concentrations. These results indicate that autonomous characteristics of the amoebae are more important for the determination of colony form than local variations in the concentrations of nutrients.The genetic determination of colony form is demonstrated by the existence of mutants that display specific alterations in colony morphology. Because the aggregate rings of these mutants move at an increased rate, mutant clones appear as variant sectors of wild-type colonies. The increased rate of mutant ring movement suggests that this selection method may be a useful technique for isolating mutant myxamoebae with defects in movement and behavior.     

Construction of pYGK,an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector

A shuttle vector that is capable of replicating in Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was constructed by modifying the Actinobacillus pleuropneumoniae (Ap) plasmid pYG53. A DNA fragment containing the Km^R gene was inserted into pYG53 to generate pYGK, which confers resistance to kanamycin in both Aa and Ec. By electroporation, Ec DH5α and 17 strains of Aa were transformed with pYGK with efficiencies ranging from 0.5 to 3 × 10⁶ colonies per μg of DNA. Plasmid pYGK exists at approx. 3–4 copies per cell in Ec. This plasmid will facilitate the genetic manipulation of Aa strains and the molecular analysis of virulence factors expressed by this organism     

Edwardsiella tarda DnaK: expression, activity, and the basis for the construction of a bivalent live vaccine against E. tarda and Streptococcus iniae

Edwardsiella tarda and Streptococcus iniae are important aquaculture pathogens that affect many species of farmed fish. In this study, we analyzed the expression, activity, and immunoprotective potential of E. tarda heat shock protein DnaK. We found that dnaK expression was upregulated under conditions of heat shock, oxidative stress, and infection of host cells. Recombinant DnaK (rDnaK) purified from Escherichia coli exhibited ATPase activity and induced protection in Japanese flounder (Paralichthys olivaceus) against lethal E. tarda challenge. On the basis of these results and our previous observation that a protective S. iniae antigen Sia10 which, when expressed heterogeneously in E. coli DH5α, is secreted into the extracellular milieu, we constructed a chimeric antigen by fusing DnaK to Sia10. The resulting fusion protein Sia10-DnaK was expressed in DH5α via the plasmid pTDK. Western blot analysis indicated that Sia10-DnaK was detected in the culture supernatant of DH5α/pTDK. When flounder were vaccinated with live DH5α/pTDK, strong protection was observed against both E. tarda and S. iniae. ELISA analysis detected specific serum antibody production in fish vaccinated with rDnaK and DH5α/pTDK. Taken together, these results indicate that rDnaK is an intrinsic ATPase with immunoprotective property and that Sia10-DnaK delivered by a live bacterial host is an effective bivalent vaccine candidate against E. tarda and S. iniae infection.     

Escherichia coli viability determination using dynamic light scattering: a comparison with standard methods

To determine the concentration of bacteria in a sample is important in the food industry, medicine and biotechnology. A disadvantage of the plate-counting method is that a microorganism colony could arise from one cell or from many cells. The other standard methodology, known as optical density determination, is based on the turbidity of a suspension and registers all bacteria, dead and alive. In this article, dynamic light scattering is proposed as a fast and reliable method to determine bacterial viability and, consequently, time evolution. Escherichia coli was selected because this microorganism is well known and easy to handle. A correlation between the data from these three techniques was obtained. We were able to calculate the growth rate, usually determined by plate counting or optical density measurement, using dynamic light scattering and to predict bacterial behavior. An analytical relationship between the colony forming units and the light scattered intensity was also deduced.     

pGreen-S: A clone vector bearing absence of enhanced green fluorescent protein for screening recombinants

The bacterial cloning vector, pGreen-S, was constructed by inserting the enhanced green fluorescent protein (EGFP) gene at the XbaI restriction site of pUC18 plasmid. When expressed in Escherichia coli DH5α produced colonies that were an absinthe green color under daylight and strongly fluorescent green under longwave ultraviolet light. The pGreen-S vector was used to select for directional insert based on the loss of green fluorescence in recombinant colonies that was caused by the absence of EGFP. The EGFP reporter system differs from the conventional complementation of lacZ, making screening recombinants simpler, less expensive, and more effective.     

Appearance of new phage types and new lysogenic strains after adaptation of lysogenic B. megatherium to ammonium sulfate culture medium

1. Lysogenic B. megatherium 899a was adapted to growth in a minimal ammonium sulfate medium (ASCM). 2. Adaptation took place slowly and the following changes in the culture occurred: (a) The growth rate increased from 0.5 to 1.5–2.0/hr. (b) The culture changed from diffuse to mucoid. (c) The total phage titer, and the gelatinase concentration decreased to 1/100 or less. (d) The types of phage produced changed from >99 per cent T (wild type) to 30 to 60 per cent miscellaneous clear types. The original T phage was replaced by a different smaller t, never observed in the original 899a culture. (e) Several new colony types also appeared, but the colony morphology was not correlated with the phage types produced. None of the colony types was stable on repeated transfer either in peptone or ASCM, but continued to disassociate into different colony types (cf. Ivánovics, 1955). 3. Control experiments showed that these changes in phage production and colony types could not be brought about by growing sensitive B. megatherium in the presence of the various new phages, in ASCM. It is therefore unlikely that the changes observed in adapted culture were due to infection of a sensitive cell with phage. 4. Continued growth of the ASCM-adapted strain in peptone resulted in increasing the total phage titer, and also the gelatinase concentration. The growth rate returned to its original value and the ability to grow rapidly in ASCM was soon lost. The phage types, however, remained the same as in the ASCM. 5. An improved cell for steady state growth is described.     

Whole-exome sequencing in Tricho-rhino-phalangeal syndrome (TRPS) type I in a Korean family

Tricho-rhino-phalangeal syndrome (TRPS) is a rare autosomal dominant and monogenic disease. Among three types of TRPS, it is known that TRPS type I and type III are caused by deletions or substitutions in the TRPS1 gene, located on chromosome 8 (8q23.3). Although the mutations in TRPS1 gene are responsible for human TRPS, some cases are not detected by the mutations of TRPS1 gene and several cases are presented with different genetic variations. The present case was a sporadic and without TRPS1 mutation. Therefore, we performed whole-exome sequencing (WES) with one patient and his family (father, mother, and brother) and validated novel mutations using PCR and Sanger sequencing. Through family-based WES, we found the two de novo mutations such as ZNF 134 and EXD 3 genes. Through functional effect prediction using disease association Ensembl database, we propose that the de novo mutation of ZNF134 (p.Ser207Arg) could be one of potential candidate genes for causing TRPS and develope the TRPS phenotype in the present case.     

Surface-functionalized gold nanoparticles mediate bacterial transformation: a nanobiotechnological approach

Transformation of bacteria is an important step in molecular biology. Viral and non-virus-based gene delivery techniques, including chemical/biological and physical approaches, have been applied to bacterial, mammalian and plant cells. E. coli is not competent to take up DNA; hence, different methods are used to incorporate plasmid DNA. A novel method has been developed using glutathione-functionalized gold nanoparticles to mediate transformation of plasmid DNA (pUC19) into E. coli DH5α that does not require the preparation of competent cells. The glutathione-functionalized gold nanoparticles acted as a vector and facilitated the entry of DNA into the host cell. The method also gave a higher transformation efficiency (4.2 × 10⁷/μg DNA) compared to 2.3 × 10⁵/μg DNA using the conventional CaCl₂-mediated method. It was also non-toxic to the bacterium making it suitable for biotechnological applications.     

An improved solid medium for isolation,enumeration and genetic investigations of autotrophic iron-and sulphur-oxidizing bacteria

An improved solid medium using Gelrite as a supporting gel for isolation and enumeration of acidophilic chemolithotrophic Thiobacillus strains was tested. Dark-brown circular colonies, which were robust and well-differentiated, developed on this medium within 72–96 h. The plating efficiency of T. ferrooxidans strains was 92.1±5.8%. Linear correlations with an optical density (OD) at 400 nm of 0.5, corresponding to 7·10⁶ and 5·10⁷ cells·ml^–1 for strains ATCC 13661 and TMB, respectively, were obtained. Linear correlations between OD, total bacterial protein, dry biomass and cell number were also determined. These data can be used for modelling and scale-up design of microbial leaching operations. With this plating technique, one prerequisite for selection of different mutants of T. ferrooxidans has been fulfilled. Correspondence to: A. M. Khalid     

Effect of Calcium Cation on Plating Efficiency of the Sulfate-Reducing Bacterium Desulfovibrio vulgaris

The presence of Ca²⁺ (concentration, ca. 0.4 mM) in the growth medium causes cells of Desulfovibrio vulgaris (Hildenborough) to aggregate, leading to a decrease in plating efficiency. When the Ca²⁺ concentration in the medium was reduced 20-fold, cell aggregation did not occur and the plating efficiency increased from an initial value of 34% to a final value of 56%.     

Sequence Analysis of a Novel Insertion Site of Transposon IS10

A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5′-CTGAGAGATCCCCTCATAATTT-3′ and 5′-AAATCATTAGGGGATTCATCAG-3′, which were the similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5′-TGCTTGGTT-3′ instead of the previously reported 5′-NGCTNAGCN-3′. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5α and that there were two copies in the DH5α genome.     

Adherence and intracellular survival within human macrophages of Enterococcus faecalis isolates from coastal marine sediment

Enterococcus faecalis is part of the human intestinal microbiota and an important nosocomial pathogen. It can be found in the marine environment, where it is also employed as a fecal indicator. To assess the pathogenic potential of marine E. faecalis, four strains isolated from marine sediment were analyzed for their ability to survive in human macrophages. Escherichia coli DH5α was used as a negative control. The number of adherent and intracellular bacteria was determined 2.5 h after the infection (T₀) and after further 24h (T₂₄) by CFU and qPCR counts. At T₂₄ adherent and intracellular enterococcal CFU counts were increased for all strains, the increment in intracellular bacteria being particularly marked. No CFU of E. coli DH5α were detected. In contrast, qPCR counts of intracellular enterococcal and E. coli bacteria were similar at both time points. These findings suggest that whereas E. coli was killed within macrophages (no CFU, positive qPCR), the E. faecalis isolates not only escaped killing, but actually multiplied, as demonstrated by the increase in the viable cell population. These findings support earlier data by our group, further documenting that marine sediment can be a reservoir of pathogenic enterococci.