Applied and Environmental Microbiology

Một vi sinh vật khử Fe(III) và Mn(IV) đã được tách ra từ trầm tích nước ngọt của sông Potomac, Maryland. Chế phẩm này được ký hiệu là GS-15, phát triển trong môi trường kị khí xác định với axetate làm chất cho electron duy nhất và Fe(III), Mn(IV) hoặc nitrat làm chất nhận electron duy nhất. GS-15 đã oxi hóa axetate thành carbon dioxide với sự khử đồng thời oxit sắt Fe(III) vô định hình thành magnetit (Fe ₃ O ₄ ). Khi Fe(III) citrate thay thế oxit sắt Fe(III) vô định hình như chất nhận electron, GS-15 phát triển nhanh hơn và đã khử toàn bộ Fe(III) thêm vào thành Fe(II). GS-15 đã khử oxit sắt Fe(III) vô định hình tự nhiên nhưng không khử đáng kể các dạng Fe(III) có tinh thể cao. Fe(III) được khử một cách tối ưu ở pH từ 6.7 đến 7 và ở nhiệt độ từ 30 đến 35°C. Ethanol, butyrate, và propionate cũng có thể đóng vai trò như chất cho electron trong quá trình khử Fe(III). Nhiều hợp chất hữu cơ khác và hydro không thể làm điều này. MnO ₂ đã bị khử hoàn toàn thành Mn(II), gây kết tủa dưới dạng rhodochrosite (MnCO ₃ ). Nitrat đã được khử thành amoniac. Oxy không thể làm chất nhận electron, và nó đã ức chế sự phát triển với các chất nhận electron khác. Đây là lần đầu tiên cho thấy rằng vi sinh vật có thể hoàn toàn oxi hóa các hợp chất hữu cơ với Fe(III) hoặc Mn(IV) làm chất nhận electron duy nhất và rằng sự oxi hóa chất hữu cơ kết hợp với sự khử Fe(III) hoặc Mn(IV) dissimilatory có thể tạo ra năng lượng cho sự phát triển của vi sinh vật. GS-15 cung cấp một mô hình cho cách mà các phản ứng xúc tác bằng enzym có thể là những cơ chế có ý nghĩa định lượng cho sự khử sắt và mangan trong môi trường kị khí.

We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere soil from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.

A stable variant of Methylomonas methanolica , with a higher temperature optimum for growth, was obtained after mutagenic treatment and selection. The mutant strain M13V has an optimum growth temperature of 35 to 37°C and a maximum at 43°C, as compared with 30 and 40°C for the wild strain. Strain M13V and M. methanolica have similar basic characteristics and cell composition. An extracellular polysaccharide is produced by both strains, but this property is more pronounced in strain M13V. In strain M13V the production is favored by high temperature, low growth rate, and oxygen limitation. In continuous culture of strain M13V, the polysaccharide production was partly growth associated and partly independent of the growth rate. The extracellular polysaccharide acted as a flocculating agent. A relationship between polysaccharide concentration and sedimentation rate was found. Biomass production from strain M13V is most effective at 35°C with respect to both growth rate and substrate utilization. It was found that the yield coefficient for methanol was independent of the dilution rate, whereas the yield coefficient for oxygen increased and the production coefficient for carbon dioxide decreased at increasing dilution rates. These results are discussed in connection with the polysaccharide production.

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda ⁵ ]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda ⁵ ]microcystin-LR and [ d -Asp ³ ,ADMAdda ⁵ ]microcystin-LR and a partial structure of three new [ADMAdda ⁵ ]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.

A strain of the filamentous cyanobacterium Nostoc sp. isolated from a lake in Finland was found to produce at least nine hepatotoxic peptides with chemical and toxicological properties similar to those of the hepatotoxic hepta- and pentapeptides produced by other cyanobacteria. Toxins were isolated and purified by high-performance liquid chromatography. Amounts available for five of the purified toxins (P6, P14, P15, P16, and P18) were adequate for high-performance liquid chromatography amino acid analysis and determination of molecular weight by fast-atom bombardment-mass spectrometry (FAB-MS). Quantities of three toxins (P14, P15, and P16) were adequate for further analysis by high-resolution FAB-MS, FAB-MS/MS, and 1H-nuclear magnetic resonance. Analysis showed that the toxins are new types of microcystin-LR homologs. Microcystin-LR contains equimolar amounts of D-alanine, L-leucine, D-erythro-beta-methylaspartic acid, L-arginine, ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), D-glutamic acid, and N-methyldehydroalanine (molecular weight [MW], 994). Nostoc sp. strain 152 was found to produce the following microcystin-LR homologs: (i) P6 contains an extra methylene group most probably due to the presence of N-methyldehydrobutyrine instead of N-methyldehydroalanine (MW, 1,008); (ii) P14 is O-acetyl-O-demethyl ADDA-microcystin-LR (MW, 1,022); (iii) P15 is 3-demethyl-O-acetylADDA-homoarginine-microcystin-LR (MW, 1,036); (iv) P16 is 3-demethyl-O-acetyl-O-demethylADDA-microcystin-LR (MW, 1,008); (v) P18 is 3-demethyl-O-acetyl-O-demethylADDA-homoarginine-microcystin- LR (MW, 1,022). The toxicities of the new microcystin homologs were not significantly different from those of microcystin-LR or demethylmicrocystin-LR.

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli . A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, ( S )-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.

A bacterial strain which could be grown in a medium containing organic solvents and which could secrete lipolytic enzyme was isolated. The stability of the lipolytic activity of the supernatant of the culture increased significantly in the presence of organic solvents such as toluene, cyclohexane, ethanol, and acetone.

A yellow-pigmented marine bacterium, designated strain SD-21, was isolated from surface sediments of San Diego Bay, San Diego, Calif., based on its ability to oxidize soluble Mn(II) to insoluble Mn(III, IV) oxides. 16S rRNA analysis revealed that this organism was most closely related to members of the genus Erythrobacter , aerobic anoxygenic phototrophic bacteria within the α-4 subgroup of the Proteobacteria (α-4 Proteobacteria ). SD-21, however, has a number of distinguishing phenotypic features relative to Erythrobacter species, including the ability to oxidize Mn(II). During the logarithmic phase of growth, this organism produces Mn(II)-oxidizing factors of ≈250 and 150 kDa that are heat labile and inhibited by both azide and o -phenanthroline, suggesting the involvement of a metalloenzyme. Although the expression of the Mn(II) oxidase was not dependent on the presence of Mn(II), higher overall growth yields were reached in cultures incubated with Mn(II) in the culture medium. In addition, the rate of Mn(II) oxidation appeared to be slower in cultures grown in the light. This is the first report of Mn(II) oxidation within the α-4 Proteobacteria as well as the first Mn(II)-oxidizing proteins identified in a marine gram-negative bacterium.

Nhiều serovar của tác nhân gây bệnh qua thực phẩm Vibrio parahaemolyticus thường gây ra các trường hợp nhiễm trùng. Kể từ năm 1996, các chủng O3:K6 của tác nhân này đã gây ra các đại dịch ở nhiều quốc gia châu Á, bao gồm Đài Loan. Để hiểu rõ hơn về các chủng gây dịch này, các chủng O3:K6 lâm sàng gần đây được phân lập từ Ấn Độ, Nhật Bản, Hàn Quốc và Đài Loan đã được nghiên cứu dựa trên cách phân loại điện di gel xung (PFGE) và các đặc điểm sinh học khác. Sau khi thực hiện PFGE và phân tích cụm, tất cả các chủng O3:K6 đã được nhóm thành hai nhóm không liên quan. Các chủng O3:K6 gần đây được phân lập đều nằm trong một nhóm, bao gồm tám mẫu liên quan chặt chẽ, với mẫu I1 (81%) và I5 (13%) là những mẫu thường gặp nhất. Mẫu I1 là mẫu chính cho các chủng từ Nhật Bản, Hàn Quốc và Đài Loan. Tất cả các chủng O3:K6 gần đây được phân lập đều mang gen hemolysin trực tiếp bền nhiệt ( tdh ). Không có sự khác biệt đáng kể nào được quan sát giữa các chủng O3:K6 mới được phân lập và các chủng tham chiếu không phải O3:K6 hoặc các chủng O3:K6 cũ được phân lập trước năm 1996 liên quan đến độ nhạy với kháng sinh, mức độ hemolysin trực tiếp bền nhiệt và độ nhạy với các căng thẳng môi trường. Kết quả trong nghiên cứu này đã xác nhận rằng các chủng O3:K6 mới được phân lập của V. parahaemolyticus có mối quan hệ di truyền gần gũi với nhau, trong khi các đặc điểm sinh học khác được nghiên cứu thường phụ thuộc vào từng chủng, và không có đặc điểm độc nhất nào được tìm thấy trong các chủng O3:K6 vừa được phân lập.