枯草芽孢杆菌中高效响应N-乙酰神经氨酸生物传感器的构建

枯草芽孢杆菌(Bacillus subtilis)是公认的食品安全菌株,目前已被用于多种高附加值产品的生物合成,包括被广泛用作营养化学品和药物中间体的N-乙酰神经氨酸(N-acetylneuraminic acid, NeuAc)。响应目标产物的生物传感器被广泛用于代谢工程中的动态调控和高通量筛选等方面,以提高生物合成效率。但是,枯草芽孢杆菌中缺乏可高效响应NeuAc的生物传感器。因此,本文首先测试和优化了能将胞外NeuAc转运进胞内的转运蛋白,获得了一系列具有不同转运能力的菌株,以用于后续响应NeuAc的生物传感器的验证;随后将响应NeuAc的转录因子Bbr_NanR的结合位点插入枯草芽孢杆菌组成型启动子的不同位置,筛选具有活性的杂合启动子;接下来,通过在具有NeuAc转运能力的枯草芽孢杆菌中表达Bbr_NanR,选择能响应NeuAc的杂合启动子,并进一步通过优化Bbr_NanR表达量获得了一系列动态范围广、激活倍数高的生物传感器,其中生物传感器P535-N2能灵敏地响应胞内NeuAc浓度的变化,具有最大的动态范围,为(180–20 245) AU/OD;P566-N2则具有最高的激活倍数,为122倍,是已报道的枯草芽孢杆菌中响应N-乙酰神经氨酸的生物传感器的2倍。本文构建的响应NeuAc的生物传感器可用于高产NeuAc的酶突变体和枯草芽孢杆菌菌株的筛选,为枯草芽孢杆菌生物合成NeuAc提供了高效、灵敏的分析和调控工具。     

Interrelationships between <Emphasis Type="Italic">Dunaliella</Emphasis> and halophilic prokaryotes in saltern crystallizer ponds

Thanks to their often very high population densities and their simple community structure, saltern crystallizer ponds form ideal sites to study the behavior of halophilic microorganisms in their natural environment at saturating salt concentrations. The microbial community is dominated by square red halophilic Archaea, recently isolated and described as Haloquadratum walsbyi, extremely halophilic red rod-shaped Bacteria of the genus Salinibacter, and the unicellular green alga Dunaliella as the primary producer. We review here, the information available on the microbial community structure of the saltern crystallizer brines and the interrelationships between the main components of their biota. As Dunaliella produces massive amounts of glycerol to provide osmotic stabilization, glycerol is often postulated to be the most important source of organic carbon for the heterotrophic prokaryotes in hypersaline ecosystems. We assess here, the current evidence for the possible importance of glycerol and other carbon sources in the nutrition of the Archaea and the Bacteria, the relative contribution of halophilic Bacteria and Archaea to the heterotrophic activity in the brines, and other factors that determine the nature of the microbial communities that thrive in the salt-saturated brines of saltern crystallizer ponds. Three-letter abbreviations for names of genera of Halobacteriaceae conform the recommendations of the ICSP Subcommittee on the Taxonomy of Halobacteriaceae.     

Nitrate reductase activity of free-living and symbiotic uptake hydrogenase-positive and uptake hydrogenase-negative strains of Bradyrhizobium japonicum

The nitrate reductase activity (NR) of selected uptake hydrogenase-positive (hup ⁺) and uptake hydrogenase-negative (hup ^-) strains of Bradyrhizobium japonicum were examined both in free-living cells and in symbioses with Glycine max L. (Marr.) cv. Williams. Bacteria were cultured in a defined medium containing either 10 mM glutamate or nitrate as the sole nitrogen source. Nodules and bacteriods were isolated from plants that were only N₂-dependent or grown in the presence of 2 mM KNO₃. Rates of activity in nodules were determined by an in vivo assay, and those of cultured cells and bacteriods were assayed after permeabilization of the cells with alkyltrimethyl ammonium bromide. All seven strains examined expressed NR activity as free-living cells and as symbiotic forms, regardless of the hup genotype of the strain used for inoculation. Although the presence of nitrate increased nitrate reduction by cultures cells and nodules, no differences in NR activity were observed between bacteroids isolated from nodules of plants fed with nitrate or grown on N₂-fixation exclusively. Cultured cells, nodules and bacteriods of strains with hup ^- genotype (USDA 138, L-236, 3. 15B3 and PJ17) had higher rates of NR activity than those with hup ⁺ genotype (USDA 110, USDA 122 DES and CB1003). These results suggest that NR activity is reduced in the presence of a genetic determinant associated with the hup region of B. japonicum.Abbreviations EDTA ethylene-diamine tetraacetic acid - Hup hydrogen uptake - MOPS 3-(N-morpholino)-propane sulfonic acid - NR nitrate reductase - PVP polyvinyl-polypyrrolidone - Tris Tris(hydroxymethyl)-aminomethane     

All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes

Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Q_i site of the bacterial and bovine bc₁ complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q_i pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Q_i pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc₁ complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc₁ complex and only rarely in the bovine bc₁ complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc₁ complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc₁ complex.     

The development and multiple uses of a standardised bioassay method to select hypocrealean fungi for biological control of aphids

A technically standardised bioassay method was designed, evaluated and used to assess virulence and host range of hypocrealean fungi against aphids. A track mounted sprayer was used to apply conidia because hand held versions of the same sprayer can be used for field applications, thereby allowing the outcome from laboratory experiments to predict activity in the field accurately. Eighteen fungal isolates were assessed in single concentration bioassays against the black bean aphid Aphis fabae Scopoli. Isolates comprised commercially available mycoinsecticides (based on Beauveria bassiana and Lecanicillium longisporum) and isolates of B. bassiana, Lecanicillium spp., Paecilomyces fumosoroseus and Metarhizium anisopliae. Aphid mortality was in excess of 80% for 15 isolates, and HRI 1.72 (L. longipsorum), Z11 (P. fumosoroseus), Mycotech strain GHA (B. bassiana) and ARSEF 2879 (B. bassiana) were studied further. Multiple concentration bioassays identified HRI 1.72 as the most virulent isolate against A. fabae with significantly smaller LC₅₀ and LT₅₀ values compared to other isolates. A precise LC₅₀ value (2.95 × 10² conidia ml⁻¹) was calculated for HRI 1.72 using a second multiple concentration assay with smaller concentrations of conidia. The four isolates were applied at a single concentration (1 × 10⁸ conidia ml⁻¹) against Myzus persicae, A. fabae, Acyrthosiphon pisum, Metopolophium dirhodum, Sitobion avenae and Rhopalosiphum padi. A ranking of aphid susceptibility was obtained, such that S. avenae > M. persicae, A. pisum, A. fabae > R. padi. Results indicate the importance of standardising bioassay methods to reduce bioassay variability without compromising the ability to use the bioassay to investigate fungus–host interactions under varying abiotic and biotic conditions.     

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N₂ fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.     

气候变暖下水圈甲烷排放及其微生物学机制

大气温室气体浓度升高导致的气候变暖已对人类社会可持续发展带来了严重影响。水圈生态系统既是全球最为重要的碳汇之一,也是全球最为重要的甲烷自然排放源。因此,阐明气候变暖背景下水圈甲烷排放格局及其相关微生物调控机制,是认识未来地球气候系统演变机理、预测未来全球变化潜在情景的关键命题,也将为如何高效发挥水圈碳汇潜力提供基础理论支撑,更好应对全球气候变化问题。本文主要综述了气候变暖背景下主要水圈生态系统中微生物介导的甲烷排放研究的现状与趋势,介绍了水圈甲烷排放格局及其气候变暖背景下的演变趋势,回顾了气候变暖对甲烷代谢相关微生物群落与功能的复杂调控作用。基于目前的研究现状,未来亟需通过微观机制与宏观过程相结合的途径,并基于生态系统复杂性和气候变暖长期性开展相关研究。同时,建议应加强对海洋等相对薄弱区域的研究。     

Chlamydial endocytobionts of free-living amoebae differentially affect the growth rate of their hosts

Bacteria closely related to chlamydiae live and multiply as endocytobionts within free-living amoebae, making these amoebae potential vehicles of new emerging bacterial pathogens of humans. Hartmannella vermiformis containing endobiotic Neochlamydia hartmannellae grew more rapidly than those without endobionts, whilst Acanthamoeba sp. harbouring the Parachlamydia-related endocytobiont UWE25 multiplied more slowly than those without endobionts. The cause for the opposite effect of chlamydial endocytobionts on the growth of their host cells remains unknown.     

Enzymatic synthesis of 2-keto- -gluconate and 2-keto- -galactonate from -glucose and -galactose with cell culture of Pseudomonas fluorescens and 2-keto-galactonate from -galactono 1,4-lactone with partially purified 2-ketogalactonate reductase

2-Keto- -gluconate and 2-keto- -galactonate were prepared from -glucose (with a yield of 40%) and -galactose (with a yield of 25%), respectively, with cell culture of Pseudomonas fluorescens. However, 2-keto- -mannoate was not prepared in this method. The time courses of the reactions showed that 2-keto- -gluconic acid and 2-keto- -galactonic acid were produced from -glucose and -galactose through -gluconate and -galactonate, respectively. When using -galactono 1,4-lactone as a starting material, 2-keto- -galactonate was produced with partially purified NADP-dependent 2-ketogalactonate reductase from P. fluorescens. Some fundamental properties of the 2-ketogalactonate reductase were compared with those of 2-ketogluconate reductase from Acetobacter and Gluconobacter.     

The Genus Gluconobacter Oxydans: Comprehensive Overview of Biochemistry and Biotechnological Applications

ABSTRACT

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in “historical” production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.     

What Does the Microsporidian <Emphasis Type="Italic">E. cuniculi</Emphasis> Tell Us About the Origin of the Eukaryotic Cell?

The relationship among the three cellular domains Archaea, Bacteria, and Eukarya has become a central problem in unraveling the tree of life. This relationship can now be studied as the completely sequenced genomes of representatives of these cellular domains become available. We performed a bioinformatic investigation of the Encephalitozoon cuniculi proteome. E. cuniculi has the smallest sequenced eukaryotic genome, 2.9 megabases coding for 1997 proteins. The proteins of E. cuniculi were compared with a previously characterized set of eukaryotic signature proteins (ESPs). ESPs are found in a eukaryotic cell, whether from an animal, a plant, a fungus, or a protozoan, but are not found in the Archaea and the Bacteria. We demonstrated that 85% of the ESPs have significant sequence similarity to proteins in E. cuniculi. Hence, E. cuniculi, a minimal eukaryotic cell that has removed all inessential proteins, still preserves most of the ESPs that make it a member of the Eukarya. The locations and functions of these ESPs point to the earliest history of eukaryotes.Reviewing Editor: Dr. Manyuan Long     

Serial Selection for Resistance to a Wild-Type and to a Genetically Modified Nucleopolyhedrovirus in Trichoplusia ni

In previous work, cabbage loopers (Trichoplusia ni) evolved 22-fold resistance to the single nucleocapsid nucleopolyhedrovirus of T. ni (TnSNPV) after 26 generations of selection with the virus. The goal of the present study was to determine if T. ni could evolve resistance to the recombinant Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV-AaIT) that expresses an insect specific neurotoxin and to determine if it was influenced by prior development of resistance to TnSNPV. To answer these questions, the T. ni line that had been exposed to TnSNPV was divided into two sublines at generation 27. One of them was serially selected for resistance to AcMNPV-AaIT (subline TnSNPV/AcMNPV-AaIT), while the other one was mock infected with distilled water (subline TnSNPV/H₂O). The same was done with the line that was used as a control from generations 1 to 26 (subline H₂O/AcMNPV-AaIT and subline H₂O/H₂O). After 17 generations of selection with AcMNPV-AaIT, T. ni that had not been previously exposed to TnSNPV evolved only twofold resistance to AcMNPV-AaIT. However, those that had been selected with TnSNPV evolved fourfold resistance to AcMNPV-AaIT. Exposure to AcMNPV-AaIT conferred cross-resistance to TnSNPV in only one subline, subline H₂O/AcMNPV-AaIT. Resistance to AcMNPV-AaIT did not affect the developmental time, pupal weight, egg production, or percentage of egg hatch of T. ni.     

Terminal Oxidases in Representatives of Different Genera of the Family Microbacteriaceae

The nature of terminal oxidases in representatives of four different genera of the family Microbacteriaceae was studied. It was found that the late-logarithmic and early-stationary cells of all of the investigated strains of the genera Plantibacter and Okibacterium contain the aa ₃-type cytochrome oxidase. Bacteria of the genera Rathayibacter and Agreia synthesize three oxidases, the bb ₃- and aa ₃-type cytochrome oxidases and nonheme cyanide-resistant oxidase, in proportions dependent on the cultivation conditions and the growth phase. Oxygen deficiency in the cultivation medium induces the synthesis of the bd-type oxidase in all of the microorganisms studied. The data obtained provide evidence that the type of terminal oxidases, along with the known chemotaxonomic characteristics, may serve to differentiate the genera of the family Microbacteriaceae at the phenotypic level.     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

The Phylogeny of Acetic Acid Bacteria Based on the Partial Sequences of 16S Ribosomal RNA: The Elevation of the Subgenus Gluconoacetobacter to the Generic Level

Thirty-six strains of acetic acid bacteria classified in the genera Acetobacter, Gluconobacter, and Acidomonas were examined for their partial base sequences in positions 1220 through 1375, 156 bases, of 16S rRNA. The strains of the Q₁₀-equipped Gluconobacter species examined were divided into two subgroups, which included the type strains of Gluconobacter oxydans, the type species of the genus Gluconobacter, and of a second species, Gluconobacter cerinus, respectively. The base differences numbered four between the two type strains. The strains of the Q₉-equipped species examined classified in the type subgenus Acetobacter of the genus Acetobacter were not very distant phylogenetically from those of the genus Gluconobacter. The calculated number of base differences was 9–6 between the type strains of G. oxydans and G. cerinus and the type strains of Acetobacter aceti and Acetobacter pasteurianus. In contrast, the strains of the Q₁₀-equipped species examined classified in the subgenus Gluconoacetobacter of the genus Acetobacter were very distant phylogenetically from those of the Acetobacter and Gluconobacter species mentioned above. The number of base differences was calculated to be 14-8. Furthermore, the strains of the methanol-assimilating, Q₁₀-equipped species of the genus Acidomonas examined were located in phylogenetically isolated positions. The type strain of Acidomonas methanolica (≡ Acetobacter methanolicus), the type species of the genus Acidomonas, had 16–9 base differences. The data obtained here indicated that the members of the subgenus Gluconoacetobacter of the genus Acetobacter can be distinguished at the generic level. The new genus Gluconoacetobacter was proposed with the type species, Gluconoacetobacter liquefaciens, in recognition of the genus Acidomonas along with the genera Acetobacter and Gluconobacter in the classification of the acetic acid bacteria.     

Isolation and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> and <Emphasis Type="Italic">Azospirillum</Emphasis> strains from the sugarcane rhizosphere

Bacteria with the ability to grow on nitrogen-free media and with nitrogenase activity under aerobic or microaerobic conditions were isolated from sugarcane roots collected from four different agricultural locations in Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter chroococcum and Azospirillum brasilense. Our results suggest that Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and that, on the contrary, Azospirillum isolates show specific association and perhaps endophytic colonization of sugarcane. However, obligate endophytes (Gluconacetobacter diazotrophicus) were not found in the apoplastic fluid of the stems and macerates extracts of sugarcane tissues with the procedure applied. Population of this microorganism might be in low number in the Spanish sugarcane varieties studied which is also discussed.     

Development of a system for integrative and stable transformation of the zygomycete<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis> by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated DNA transfer

Two transformation systems, based on the use of CaCl₂/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS⁺ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl₂/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl₂/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at Communicated by C. P. Hollenberg