Isolation and characterization of megaplasmid DNA from lithoautotrophic bacteria

A method is described for the preparative isolation of megaplasmids ranging in size from 340 to 700 kb. These plamids were isolated from chemolithoautotrophic bacteria including the species Alcaligenes, Pseudomonas, and Paracoccus. The procedure was based on alkaline sodium dodecyl sulfate lysis of the cells, followed by heat treatment, salt precipitation, several phenol extractions, dialysis steps, and proteinase and RNase treatment. The various parameters were evaluated and controlled. Hydrogen-oxidizing-ability (Hox) encoding plasmids were compared by EcoRI restriction enzyme analysis. pHG plasmids from Alcaligenes eutrophus wild-type strains appeared to be closely related; plasmids derived from the type strain TF93 and from A. hydrogenophilus exhibited major differences in restriction sites. Two cryptic plasmids harbored by Pseudomonas facilis and Paracoccus denitrificans showed scarcely detectable similarity to the plasmid species of Alcaligenes.     

A generally applicable cryopreservation method for nitrite-oxidizing bacteria

Nitrite-oxidizing bacteria are key members of the global nitrogen cycle but their study is hampered by their limited availability in culture, mostly due to laborious cultivation procedures and the lack of stable preservation methods. In this study, it was demonstrated that long-term cryopreservation of nitrite-oxidizing bacteria assigned to the genera Nitrobacter, Nitrospina, Nitrococcus, Nitrotoga and Nitrospira was possible using a simple and rapid protocol. Their survival was tested with different cryoprotecting agents, DMSO and Hatefi, and in various carbon-rich preservation media, ten-fold diluted TSB, and ten-fold diluted TSB supplemented with 1% trehalose, and 1% sucrose. Optimal preservation conditions were strain-dependent and marine strains appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide with or without ten-fold diluted trypticase soy broth as a preservation medium allowed successful preservation of all tested strains.     

Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.

Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia- and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor's photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.     

Fatty acid profiles of nitrite-oxidizing bacteria reflect their phylogenetic heterogeneity

The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter, Nitrococcus, Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis-9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis-9-hcxadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis-7 and cis-11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

Numbers of nitrite-oxidizing bacteria in the root zone of grassland plants

Abstract The results of Most Probable Number determinations applying low and high concentrations of nitrite reveal the presence of at least two different communities of potential nitrite-oxidizing bacteria in a number of soil types. The effect of plant roots on these two communities was studied in pot experiments with soil from natural grassland in the presence or absence of either Festuca rubra or Plantago lanceolata . Both plant species are dominant on the grassland soil used in this study. Plant roots had a stimulating effect on the numbers of nitrite-oxidizing bacteria determined with 0.05 mM nitrite in the enumeration medium as well as on the potential nitrite-oxidizing activity. On the other hand, plants roots, especially in younger plants, repressed the numbers of nitrite-oxidizing bacteria enumerated with 5.0 mM nitrite in the counting medium. Pure culture studies with organotrophically grown Nitrobacter species clearly showed that this type of potential nitrite-oxidizing bacteria could not have been responsible for the relatively high Most Probable Numbers observed in the root zones when applying 0.05 mM nitrite in the enumeration medium.     

复合载体固定亚硝酸盐氧化菌处理养殖废水

利用新型复合载体固定亚硝酸盐氧化菌,确定了挂膜条件,考察了水力停留时间对NO2--N去除率的影响,并进行对虾养殖水处理.结果表明,新型载体可以较好地固定亚硝酸盐氧化菌,在水力停留时间为12 h、6 h、3 h、1 h的条件下,NO2--N的最大去除率分别为100%、98.77%、90.59%、59.02%.在HRT为1 h时,固定化的亚硝酸盐氧化菌可以高效去除养殖水体中的NO2--N,去除率达76.94%.     

Identification of nitrite-oxidizing bacteria with monoclonal antibodies recognizing the nitrite oxidoreductase.

Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the genera Nitrospira, Nitrococcus, and Nitrospina. The MAb designated Hyb 153-2, which recognized the alpha subunit of the NOR (alpha-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the beta-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the beta-NOR, bound to members of the genera Nitrobacter and Nitrococcus. The molecular masses of the beta-NOR of the genus Nitrobacter and the beta subunit of the NOS (beta-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the beta-NOS of the genera Nitrospina and Nitrospira were different (48 and 46 kDa). When the genus-specific reactions of the MAbs were correlated with 16S rRNA sequences, they reflected the phylogenetic relationships among the nitrite oxidizers. The specific reactions of the MAbs allowed us to classify novel isolates and nitrite oxidizers in enrichment cultures at the genus level. In ecological studies the immunoblot analyses demonstrated that Nitrobacter or Nitrospira cells could be enriched from activated sludge by using various substrate concentrations. Fluorescence in situ hybridization and electron microscopic analyses confirmed these results. Permeated cells of pure cultures of members of the four genera were suitable for immunofluorescence labeling; these cells exhibited fluorescence signals that were consistent with the location of the NOS.     

Effects of roxithromycin on ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the rhizosphere of wheat

In a pot-cultural experiment, the impact of the antibiotic roxithromycin (ROX) addition was assessed on the diversities of microbial structure and function communities, especially involved in ammonia and nitrite oxidation in wheat rhizosphere soil with and without the addition of earthworms. The abundances of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and total bacteria were surveyed by the quantitative PCR. The quantities of total bacteria, AOB, and NOB with earthworms were higher than those without earthworms because of the synergistic effect. ROX inhibited the growth of AOB in all treatments, although the quantities of AOB were in a light increase in medium and heavy polluted treatments compared with that in the light polluted treatments. Different from AOB, the quantities of NOB were lowest in light polluted treatments, but the quantities of NOB were rapidly increased in medium and heavy polluted treatments compared with that in the control. These results indicated that the application of ROX principally had a negative effect on nitrification performance by affecting the abundances and relative ratios of both AOB and NOB in soil communities, which affected the N cycle in an agricultural ecosystem. According to the metabolic diversities evaluated by the biologic assay, the tendency of metabolic diversities was quite contrary to the quantities of NOB in all treatments and showed the contrast growing relation of autotrophic and heterotrophic bacteria under ROX pollution pressure in agricultural ecosystems.     

污水短程硝化体系氨氧化菌与亚硝酸盐氧化菌的生态学研究进展

短程硝化(partial nitrification, PN)是一种绿色低碳的生物脱氮创新技术,伴随厌氧氨氧化(anaerobic ammonia oxidation, Anammox)污水脱氮技术的进一步推广,短程硝化作为提供其电子受体的重要环节,已成为了污水脱氮领域的研究热点。氨氧化菌(ammonia-oxidizing bacteria,AOB)和亚硝酸盐氧化菌(nitrite-oxidizing bacteria, NOB)是该技术的核心竞争微生物,掌握这两类微生物的生态学特征,借助生态学理论和手段调控AOB淘汰NOB,提高种群的可预测性,对于实现稳定高效的短程硝化具有重要意义。本文基于生态学角度介绍了AOB和NOB基础分类、生理性能及生态位分离,重点综述了短程硝化系统中AOB和NOB的生长动力学、群落构建、环境因素和相互作用,最后对这两类微生物的未来研究重点和研究方法进行了展望,为短程硝化工艺的快速启动和稳定运行提供理论指导。     

Regulation of citrate synthase in facultative methylotrophic bacteria

Commonly the TCA cycle fulfils an anabolic and a catabolic function in case of aerobic chemoorganoheterotrophic nutrition. In methylotrophic growth the TCA cycle is dispensable as a bioenergetic pathway. This is reflected by properties of citrate synthase in facultative methylotrophic bacteria. Two citrate synthases, a "chemoorganoheterotrophic" one, which is inhibited by NADH (or ATP in Acetobacter MB 58), and a "methylotrophic" one, which is not or less affected by energy indicators, were found in Pseudomonas oleovorans, Pseudomonas MS, Pseudomonas MA, and Acetobacter MB 58. The concentration of these citrate synthases depends on the manner of nutrition. Bacteria with ICL-negative-variant of the serine pathway and with ribulosebisphosphate pathway seem to possess only a "chemoorganoheterotrophic" citrate synthase. Possibly the anabolic function of this citrate synthase can be realized by metabolites.     

Biodegradation of estrogens by facultative anaerobic iron-reducing bacteria

Natural estrogens such as estrone, 17β-estradiol, estriol, and the synthetic component of contraceptive pills, 17α-ethinylestradiol, enter the municipal wastewater treatment plant via human excretions. A significant portion of these substances is found to remain in reject water produced after anaerobic digestion of activated sludge. In this study, the effect of the oxidant, Fe(III), and facultative anaerobic strain of iron-reducing bacteria on the anaerobic degradation of estrogens in reject water was investigated. Synthetic 17α-ethinylestradiol remained resistant to anaerobic biodegradation by iron-reducing bacteria, while natural estrogens such as 17β-estradiol, estriol, and estrone were removed by 92%, 60% and 27%, respectively, after 15 days of batch cultivation of iron-reducing bacteria in reject water with the addition of all estrogens to concentrations 100 μg l⁻¹ each. The ability of facultative anaerobic iron-reducing bacteria to degrade estrogens can be used for the anaerobic removal of trace organics from reject water in municipal wastewater treatment plant.     

亚硝酸盐氧化细菌的生态位以及对海洋环境变化的响应机制

硝酸盐是海洋微生物可利用氮的主要形式,也是限制表层海洋生物生产力的主要营养物质,海洋中的硝酸盐主要由氨和亚硝酸盐的氧化产生。探索亚硝酸盐氧化细菌在海洋生态系统中的生态位以及对环境变化的响应机制,对认识微生物参与的氮循环具有十分重要的意义。本文综述了海洋亚硝酸盐氧化细菌的研究进程及其主要种类,并总结了其主要的生理生态学特征,指出微生物在海洋生态系统变迁中所衍生出的适应对策。基于当前的研究现状,展望亚硝酸盐氧化细菌未来的研究方向,以期更好地了解海洋中亚硝酸盐的氧化过程,为进一步认识氮在生物地球化学中的循环奠定基础。     

Isolation of thermotolerant,halotolerant, facultative biosurfactant-producing bacteria

Several facultative bacterial strains tolerant to high temperature and salinity were isolated from the oil reservoir brines of an Iranian oil field (Masjed-I Soleyman). Some of these isolates were able to grow up to 60°C and at high concentration of NaCl (15% w/v). One of the isolates grew at 40°C, while it was able to grow at 15% w/v NaCl. Tolerances to NaCl levels decreased as the growth temperatures were increased. Surfactant production ability was detected in some of these isolates. The use of biosurfactant is considered as an effective mechanism in microbial-enhanced oil recovery processes detected in some of these isolates. The surfactant producers were able to grow at high temperatures and salinities to about 55°C and 10% w/v, respectively. These isolates exhibited morphological and physiological characteristics of the Bacillus genus. The partial sequencing of the 16S ribosomal deoxyribonucleic acid gene of the selected isolates was assigned them to Bacillus subtilis group. The biosurfactant produced by these isolates caused a substantial decrease in the surface tension of the culture media to 26.7 mN/m. By the use of thin-layer chromatography technique, the presence of the three compounds was detected in the tested biosurfactant. Infrared spectroscopy and ¹H nuclear magnetic resonance analysis were used, and the partial structural characterization of the biosurfactant mixture of the three compounds was found to be lipopeptidic in nature. The possibility of use of the selected bacterial strains reported, in the present study, in different sectors of the petroleum industry has been addressed.     

Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization

Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP—BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l^−l), MDW 80 (301 mg l^−l), M. komagatae (279 mg l^−l), and MSF 34 (202 mg l^−l), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.     

Regulation of C3-enzymes in facultative phototrophic bacteria

Pyruvate kinase (EC2.7.1.40) from Rhodopseudomonas sphaeroides was purified 40-fold by precipitation with protamine sulfate and ammonium sulfate followed by gelfiltration. The preparations obtained from cells grown with different carbon sources or cultural conditions differ with respect to specific activity but not with respect to molecular weight (250000 dalton) or regulatory properties. The phosphoenolpyruvate (PEP)-saturation curve of the enzyme is sigmoidal with Hill coefficients varying from n _H =1.8 (pH 9.2) to 2.7 (pH 6.0). The enzyme is activated by adenosinemonophosphate (AMP) and the sugarmonophosphates ribose-5-phosphate (R-5-P), glucose-6-phosphate (G-6-P), and-to a lesser extent-fructose-6-phosphate (F-6-P). Fructose-1.6-bisphosphate (FDP) has no measurable effect. Inhibitors of the enzyme are adenosintriphosphate (ATP), inorganic phosphate (P _i ) and the dicarboxylic acids succinate and fumarate. Kinetic analysis reveals that the sugar-phosphates and the dicarboxylic acids act as true allosteric ligands, wheras the effects of AMP, ATP, and P _i cannot be interpreted solely in terms of allosteric interactions. Cold-treatment of the enzyme leads to a rapid loss of activity, but does not change the regulatory properties of the enzyme. Analysis of the kinetics of cold-inactivation and its reversal at 30°C, together with studies on the gelfiltration behaviour of the native and the cold-treated enzyme make it likely that the cold-induced loss of activity is due to a dissociation of the enzyme.     

Viability of facultative anaerobic bacteria in commercial transport systems

The possibilities of the transport systems manufactured by Copan (Italy) and Deltalab (Spain) were studied on 15 microbial strains: representatives of the family Enterobacteriaceae (enterobacterial swabs with Cary-Blair medium) and the genus Streptococcus, as well as the species Neisseria meningitidis, Haemophilus influenzae (universal swabs with charcoal-enriched Amies medium). Microorganisms were shown to retain their viability (in colony-forming units, %) for 48 hours in the systems of both firms. H. influenzae exhibited greater viability in the system manufactured by Copan than in that manufactured by Deltalab (respectively, 62% and 28% in 24 hours, 19% and 6% in 48 hours).     

Haemoproteins and haem synthesis in facultative photosynthetic and denitrifying bacteria

1. A simple spectrophotometric method is described for the measurement of various haemoproteins in extracts of photosynthetic and non-photosynthetic bacteria. The method is based on measurements of difference spectra at the Soret maxima. 2. In photosynthetic bacteria of the Athiorhodaceae group the concentration of carbon monoxide-binding haemoprotein and of cytochromes of the b and c types is two to three times as high in anaerobically grown cells as in those grown aerobically. 3. During the adaptation of Rhodopseudomonas spheroides 8253 to form photosynthetic pigments the concentration of each of these haemoproteins increases in parallel with that of the bacteriochlorophyll. 4. The carbon monoxide-binding haemoprotein in aerobically grown Rps. spheroides 8253, in contrast with anaerobically grown cells, is predominantly in the particulate fraction of extracts prepared by ultrasonic vibration. The b- and c-type cytochromes are approximately equally distributed between each fraction in extracts from both types of cell. 5. Extracts of Micrococcus denitrificans grown anaerobically on nitrate contain more cytochromes of the b and c types, as well as of the carbon monoxide-binding pigment, than do those from aerobically grown cells. 6. The activity of ferrochelatase in both Rps. spheroides 8253 and M. denitrificans was similar in extracts from cells grown aerobically and anaerobically, though the haemoprotein content was higher under the latter conditions. Coproporphyrinogen oxidative decarboxylase could not be demonstrated in cell-free extracts of either organism.     

In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants.

Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO(3)(-) or as CO(2)) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.