Applied and Environmental Microbiology

Direct isolation of nucleic acids from the environment may be useful in several respects, including the estimation of total biomass, detection of specific organisms and genes, estimations of species diversity, and cloning applications. We have developed a method that facilitates the concentration of microorganisms from aquatic samples and the extraction of their nucleic acids. Natural water samples of 350 to greater than 1,000 ml are concentrated on a single cylindrical filter membrane (type SVGS01015; Millipore Corp., Bedford, Mass.), and cell lysis and proteolysis are carried out within the filter housing. Crude, high-molecular-weight nucleic acid solutions are then drawn off the filter. These solutions can be immediately analyzed, concentrated, or purified, depending on the intended application. The method is simple, rapid, and economical and provides high-molecular-weight chromosomal DNA, plasmid DNA, and speciated RNAs which comigrate with 5S, 16S, and 23S rRNAs. The methods presented here should prove useful in studying both the ecology and the phylogeny of microbes that resist classical culture methods.

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p -nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg ⁻¹ protein for Kre1/EstA/Cwp2p and 72 mU mg ⁻¹ protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg ⁻¹ protein for Kre1/EstA/Cwp2p and 1.27 U mg ⁻¹ protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli , can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

Pseudomonas fluorescens Pf-5, a rhizosphere bacterium, produces a suite of secondary metabolites that are toxic to seed- and root-rotting plant pathogens. Among these are the polyketide compounds pyoluteorin and 2,4-diacetylphloroglucinol. We provide evidence that pyoluteorin production is influenced by positive autoregulation. Addition of pyoluteorin to liquid cultures of Pf-5 enhanced pyoluteorin production. In addition, pyoluteorin and 2,4-diacetylphloroglucinol mutually inhibit one another's production in Pf-5. For pyoluteorin, both positive autoregulation and negative influences on production by 2,4-diacetylphloroglucinol were demonstrated at the transcriptional level by measuring activity from transcriptional fusions of an ice nucleation reporter gene ( inaZ ) to three separate pyoluteorin biosynthetic genes. The occurrence of pyoluteorin autoregulation in the rhizosphere was assessed on cucumber seedlings in pasteurized soil with cross-feeding experiments. In the rhizosphere, expression of a pyoluteorin biosynthesis gene by a pyoluteorin-deficient mutant of Pf-5 was enhanced by pyoluteorin produced by coinoculated cells of Pf-5. These data establish that the polyketide pyoluteorin is an autoregulatory compound and functions as a signal molecule influencing the spectrum of secondary metabolites produced by the bacterial cell.

We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

Propionibacterium freudenreichii được sử dụng như một chất khởi đầu trong quá trình ủ phô mai và như một loại probiotic. Các tác động sinh lý đã được báo cáo của nó ở cấp độ ruột, bao gồm việc điều chỉnh bifidobacteria, sự tăng sinh và apoptosis của tế bào biểu mô ruột, và viêm ruột, phụ thuộc vào sự chuyển hóa hoạt động in situ . Khả năng sống sót và hoạt động vì vậy là những yếu tố then chốt quyết định hiệu quả của nó, tạo ra thách thức về thích ứng với căng thẳng và dung nạp cho các ứng dụng probiotic. Môi trường nuôi cấy và điều kiện tăng trưởng xác định việc tiếp thu dung nạp. Chúng tôi đã nghiên cứu khả năng sử dụng whey ngọt, một sản phẩm phụ từ sữa, để duy trì P. freudenreichii trong quá trình tăng trưởng. Nó được sử dụng ở các nồng độ khác nhau (chất rắn khô) như một môi trường nuôi cấy. Sử dụng whey ngọt siêu đậm đặc dẫn đến việc gia tăng khả năng chịu đựng đa căng thẳng, sự biểu hiện quá mức của các protein chịu stress chính, và sự tích lũy các phân tử lưu trữ nội bào và các solute tương thích, cũng như tăng cường khả năng sống sót trong quá trình phun khô. Một quy trình đơn giản hóa từ sự phát triển đến việc phun khô propionibacteria đã được phát triển sử dụng whey ngọt như một môi trường 2-trong-1 vừa nuôi cấy P. freudenreichii vừa bảo vệ nó khỏi bị tổn thương nhiệt và thẩm thấu mà không cần các bước thu hoạch và rửa. Do phun khô rẻ hơn và tiết kiệm năng lượng hơn so với đông khô, công trình này mở ra những triển vọng mới cho sự phát triển bền vững của các chế phẩm khởi đầu và probiotic mới với độ bền cao hơn.

Ý NGHĨA Trong nghiên cứu này, chúng tôi chứng minh rằng whey ngọt, một sản phẩm phụ của ngành công nghiệp sữa, không chỉ cho phép sự phát triển của các propionibacteria dairy probiotic, mà còn kích hoạt một phản ứng đa dung nạp thông qua thích ứng thẩm thấu và phản ứng căng thẳng tổng quát. Chúng tôi cũng chỉ ra rằng các propionibacteria tích lũy solute tương thích trong các điều kiện nuôi cấy này, có thể giải thích cho sự mất mát độ sống sót hạn chế sau quá trình phun khô. Công trình này mở ra những triển vọng mới cho việc sản xuất các chế phẩm khởi đầu và probiotic từ sữa với hiệu quả năng lượng cao hơn.

Biofilms are complex and dynamic communities of microorganisms that are studied in many fields due to their abundance and economic impact. Biofilm thickness is an important parameter in biofilm characterization. Current methods of measuring biofilm thicknesses have several limitations, including application, availability, and costs. Here, we present low-load compression testing (LLCT) as a new method for measuring biofilm thickness. With LLCT, biofilm thicknesses are measured during compression by inducing small loads, up to 5 Pa, corresponding to 0.1% deformation, making LLCT essentially a nondestructive technique. Comparison of the thicknesses of various bacterial and yeasts biofilms obtained by LLCT and by using confocal laser scanning microscopy (CLSM) resulted in the conclusion that CLSM underestimates the biofilm thickness due to poor penetration of different fluorescent dyes, especially through the thicker biofilms, whereas LLCT does not suffer from this thickness limitation.

Phosphorus was added as a nutrient to bench-scale and pilot-scale biologically active carbon (BAC) reactors operated for perchlorate and nitrate removal from contaminated groundwater. The two bioreactors responded similarly to phosphorus addition in terms of microbial community function (i.e., reactor performance), while drastically different responses in microbial community structure were detected. Improvement in reactor performance with respect to perchlorate and nitrate removal started within a few days after phosphorus addition for both reactors. Microbial community structures were evaluated using molecular techniques targeting 16S rRNA genes. Clone library results showed that the relative abundance of perchlorate-reducing bacteria (PRB) Dechloromonas and Azospira in the bench-scale reactor increased from 15.2% and 0.6% to 54.2% and 11.7% after phosphorus addition, respectively. Real-time quantitative PCR (qPCR) experiments revealed that these increases started within a few days after phosphorus addition. In contrast, after phosphorus addition, the relative abundance of Dechloromonas in the pilot-scale reactor decreased from 7.1 to 0.6%, while Zoogloea increased from 17.9 to 52.0%. The results of this study demonstrated that similar operating conditions for bench-scale and pilot-scale reactors resulted in similar contaminant removal performances, despite dramatically different responses from microbial communities. These findings suggest that it is important to evaluate the microbial community compositions inside bioreactors used for drinking water treatment, as they determine the microbial composition in the effluent and impact downstream treatment requirements for drinking water production. This information could be particularly relevant to drinking water safety, if pathogens or disinfectant-resistant bacteria are detected in the bioreactors.

The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli . Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has K _m and V _max values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B . pumilus (strain 8A3) was cloned and expressed. This suggests that cynD , the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B . pumilus and is not a reflection of its origin at a mining waste site.

Four commercial composts were added to soil to study their effect on plant growth, total rhizosphere microflora, and incidence of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of tomato plants. Three of the compost treatments significantly improved plant growth, while one compost treatment significantly depressed it. Compost amendments caused only small variations in the total numbers of bacteria, actinomycetes, and fungi in the rhizosphere of tomato plants. A total of 709 bacteria were isolated from the four compost treatments and the soil control to determine the percentage of PGPR in each treatment. The PGPR tests measured antagonism to soilborne root pathogens, production of indoleacetic acid, cyanide, and siderophores, phosphate solubilization, and intrinsic resistance to antibiotics. Our results show that the addition of some composts to soil increased the incidence in the tomato rhizosphere of bacteria exhibiting antagonism towards Fusarium oxysporum f. sp. radicis-lycopersici, Pyrenochaeta lycopersici, Pythium ultimum, and Rhizoctonia solani. The antagonistic effects observed were associated with marked increases in the percentage of siderophore producers. No significant differences were observed in the percentage of cyanogens, whereas the percentages of phosphate solubilizers and indoleacetic acid producers were affected, respectively, by one and two compost treatments. Intrinsic resistance to antibiotics was only marginally different among the rhizobacterial populations. Our results suggest that compost may stimulate the proliferation of antagonists in the rhizosphere and confirm previous reports indicating that the use of composts in container media has the potential to protect plants from soilborne root pathogens.

The biodegradation of trichloroethylene (TCE) and toluene, incubated separately and in combination, by indigenous microbial populations was measured in three unsaturated soils incubated under aerobic conditions. Sorption and desorption of TCE (0.1 to 10 micrograms ml-1) and toluene (1.0 to 20 micrograms ml-1) were measured in two soils and followed a reversible linear isotherm. At a concentration of 1 micrograms ml-1, TCE was not degraded in the absence of toluene in any of the soils. In combination, both 1 microgram of TCE ml-1 and 20 micrograms of toluene ml-1 were degraded simultaneously after a lag period of approximately 60 to 80 h, and the period of degradation lasted from 70 to 90 h. Usually 60 to 75% of the initial 1 microgram of TCE ml-1 was degraded, whereas 100% of the toluene disappeared. A second addition of 20 micrograms of toluene ml-1 to a flask with residual TCE resulted in another 10 to 20% removal of the chemical. Initial rates of degradation of toluene and TCE were similar at 32, 25, and 18 degrees C; however, the lag period increased with decreasing temperature. There was little difference in degradation of toluene and TCE at soil moisture contents of 16, 25, and 30%, whereas there was no detectable degradation at 5 and 2.5% moisture. The addition of phenol, but not benzoate, stimulated the degradation of TCE in Rindge and Yolo silt loam soils, methanol and ethylene slightly stimulated TCE degradation in Rindge soil, glucose had no effect in either soil, and dissolved organic carbon extracted from soil strongly sorbed TCE but did not affect its rate of biodegradation.