The ability of soil-borne fungi to degrade organophosphonate carbon-to-phosphorus bonds

The ability of a wide variety of soil-borne fungal strains to degrade four structurally different com pounds containing PC bonds, namely the naturally occurring amino acid ciliatine, the popular herbicide glyphosate, phosphonoacetic acid and 2-amino-3-phosphonopropionic acid, was studied in order to show that soil fungi may play an important role in the biodegradation of organophosphonates. Most of the strains appeared to utilize ciliatine as the sole source of phosphorus for growth. Only a limited number of strains were able to grow on the other phosphonates used in this work. The strains of Trichoderma harzianum, Scopulariopsis sp. and Aspergillus niger chosen for more detailed study show the ability to degrade ciliatine, glyphosate and also amino(3-methoxyphenyl)mehtylphosphonic acid effectively. Received: 14 May 1997 / Received revision: 10 June 1997 / Accepted: 14 June 1997     

Physiological aspects of glyphosate degradation in Alcaligenes spec. strain GL

Alcaligenes spec. strain GL (IMET 11314) is able to grow on glyphosate (N-[phosphonomethyl]glycine) and other phosphonates as sole source of phosphorus. Degradation of glyphosate to inorganic phosphate and sarcosine by this strain is subject to several regulatory principles. While uptake and dephosphonation of glyphosate are regulated by P_i starvation, the intensity of glyphosate degradation is also controlled by the cellular ability to utilize the C-skeleton derived from glyphosate. Depending on the external concentration of glyphosate, the liberated sarcosine is differentially metabolised. Utilization of the sarcosine moiety and complete incorporation of 3-[¹⁴C]-label of glyphosate into cellular material occur only in cultures adapted to higher concentrations (5 mM) of the herbicide. At low concentrations of glyphosate (1 mM) only the P_i required by the growing cultures is utilized but not the sarcosine. Initially high rates of glyphosate uptake obtained after P_i-starvation decrease in the presence of low glyphosate concentrations. It is suggested that uptake and metabolism of glyphosate are connected with the expression of the sarcosine metabolizing capacity of the Alcaligenes cells.Abbreviation AMPA aminomethylphosphonic acid     

Response of Biofilm Bacteria to Dissolved Organic Matter from Decomposing Maple Leaves

Stream bacteria play an important role in the utilization of dissolved organic matter (DOM) leached from leaves, and in transfer of this DOM to other trophic levels. Leaf leachate is a mixture of labile, recalcitrant, and inhibitory compounds, and bacterial communities vary in their ability to utilize leachate. The purpose of this study was to determine the effects of DOM from sugar maple leaves on bacterial populations in biofilms on decomposing leaf surfaces. Populations of Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida were enumerated on decomposing maple leaves in a northeast Ohio stream using fluorescence in situ hybridization. Additionally, artificial substrata consisting of PVC-end caps filled with agar supplemented with leaf leachate and covered with cellulose filters were used to determine bacterial response to leachate from leaves at different stages of decomposition. Population sizes of bacterial species exhibited different responses. Leachate did not affect A. calcoaceticus. B. cepacia was tolerant of phenolic compounds released from leaves and the population size increased when DOM concentrations were greatest. In contrast, P. putida was inhibited by phenolic components of leachate when total DOM concentrations were greatest. Differences in response of the bacterial species to components of leaf leachate indicate the complexity of microbial population dynamics and interactions with DOM. Differences among species in response to DOM have the potential to influence transport and retention of organic matter in stream ecosystems.     

Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide

Endophytic bacteria are ubiquitous in most plant species influencing the host fitness by disease suppression, contaminant degradation, and plant growth promotion. This endophytic bacterial community may be affected by crop management such as the use of chemical compounds. For instance, application of glyphosate herbicide is common mainly due to the use of glyphosate-resistant transgenic plants. In this case, the bacterial equilibrium in plant–endophyte interaction could be shifted because some microbial groups are able to use glyphosate as a source of energy and nutrients, whereas this herbicide may be toxic to other groups. Therefore, the aim of this work was to study cultivable and noncultivable endophytic bacterial populations from soybean (Glycine max) plants cultivated in soil with and without glyphosate application (pre-planting). The cultivable endophytic bacterial community recovered from soybean leaves, stems, and roots included Acinetobacter calcoaceticus, A. junii, Burkholderiasp., B. gladioli, Enterobacter sakazaki, Klebsiella pneumoniae, Pseudomonas oryzihabitans, P. straminea, Ralstonia pickettii,and Sphingomonassp. The DGGE (Denaturing Gradient Gel Electrophoresis) analysis from soybean roots revealed some groups not observed by isolation that were exclusive for plants cultivated in soil with pre-planting glyphosate application, such as Herbaspirillum sp., and other groups in plants that were cultivated in soil without glyphosate, such as Xanthomonas sp. and Stenotrophomonas maltophilia. Furthermore, only two bacterial species were recovered from soybean plants by glyphosate enrichment isolation. They were Pseudomonas oryzihabitans and Burkholderia gladioliwhich showed different sensibility profiles to the glyphosate. These results suggest that the application at pre-planting of the glyphosate herbicide may interfere with the endophytic bacterial communitys equilibrium. This community is composed of different species with the capacity for plant growth promotion and biological control that may be affected. However, the evaluation of this treatment in plant production should be carried out by long-term experiments in field conditions.     

Root nodule bacteria isolated from South African <Emphasis Type="Italic">Lotononis bainesii,L. listii</Emphasis> and <Emphasis Type="Italic">L. solitudinis</Emphasis> are species of <Emphasis Type="Italic">Methylobacterium</Emphasis> that are unable to utilize methanol

The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C₁ compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/Methylobacterium symbiosis.     

Common Reed Phragmites australis: Control and Effects Upon Biodiversity in Freshwater Nontidal Wetlands

Phragmites australis (common reed) has expanded in many wetland habitats. Its ability to exclude other plant species has led to both control and eradication programs. This study examined two control methods—herbicide application or a herbicide‐burning combination—for their efficacy and ability to restore plant biodiversity in non‐tidal wetlands. Two Phragmites‐dominated sites received the herbicide glyphosate. One of these sites was burned following herbicide application. Plant and soil macroinvertebrate abundance and diversity were evaluated pre‐treatment and every year for four years post‐treatment using belt transects. The growth of Phragmites propagules—seeds, rhizomes, and rooted shoots—was examined in the greenhouse and under bare, burned, or vegetated soil conditions. Both control programs greatly reduced Phragmites abundance and increased plant biodiversity. Plant re‐growth was quicker on the herbicide‐burn site, with presumably a more rapid return to wetland function. Re‐growth at both sites depended upon a pre‐existing, diverse soil seed bank. There were no directed changes in soil macroinvertebrate abundance or diversity and they appeared unaffected by changes in the plant community. Phragmites seeds survived only on bare soils, while buried rhizomes survived under all soil conditions. This suggests natural seeding of disturbed soils and inadvertent human planting of rhizomes as likely avenues for Phragmites colonization. Herbicide control, with or without burning, can reduce Phragmites abundance and increase plant biodiversity temporarily. These changes do not necessarily lead to a more diverse animal community. Moreover, unless Phragmites is eradicated and further human disturbance is prohibited, it will likely eventually re‐establish dominance.     

The Response of Three Bacterial Populations to Pollution in a Stream

Abstract Practical methods for biomonitoring of natural systems are still under development. Bacteria are potentially useful indicators of water quality because of their species diversity and ability to rapidly respond to changing environmental conditions. In this study, bacterial populations from unpolluted and polluted stream sites in two watersheds were compared to determine their suitability for use as environmental indicators. Upper Three Runs Creek and Four Mile Creek headwaters have had little anthropogenic disturbance, as opposed to lower Four Mile Creek which received thermal, radioactive, and chemical perturbations. Chemical and physical measurements provided evidence that seepage from holding ponds polluted Four Mile Creek. Polluted sites did not have altered total bacterial numbers but had decreased numbers of colony-forming units. Abundances of three bacterial species, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, were determined by colony hybridization with species-specific rDNA probes. Contribution of A. calcoaceticus to the assemblage was higher at polluted sites, which indicated either tolerance of polluted conditions or the ability to utilize compounds existing at these sites to reach larger populations. No differences in B. cepacia populations were detected, and differences in P. putida populations could not be attributed solely to disturbance. The pollution of Four Mile Creek induced differences in bacterial populations that could be monitored using the described approach. Received: 24 September 1996; Accepted: 20 December 1996     

异裂菊属根际微生物群落结构及功能多样性

异裂菊属是广西喀斯特石山区典型的特有属,根际微生物是其能否有效吸收、有效利用土壤养分和适应石山恶劣土壤环境的最直接表征之一。该研究采用DGGE和Biolog两种方法对异裂菊属植物根际和非根际微生物多样性进行了研究。结果表明:(1)异裂菊属5个种根际pH、碱解氮等9个养分含量都高于非根际。(2)5个种的根际、非根际存在2个共有细菌类群,但在数量上存在差异,3个种的根际条带小于非根际;5个种的根际、非根际微生物群落较为相似,较易聚在一起。(3)绢叶异裂菊根际微生物对碳源利用能力最强,凹脉异裂菊非根际最弱,其他对碳源的利用能力较接近;异裂菊属种根际微生物利用碳源的能力都高于非根际,根际微生物多样性指数均高于非根际,优势度指数与非根际基本相同或略高于非根际,丰富度和均匀度指数与优势度指数规律相似;异裂菊属根际、非根际微生物利用的碳源主要是糖类、羧酸类和氨基酸类化合物,4个种根际微生物利用碳源的能力高于非根际。(4)阳离子交换量、黏粒含量百分率和碱解氮是影响异裂菊属根际微生物碳源利用模式的最重要因子。总体来说,土壤理化性质对异裂菊属植物根际微生物群落多样性具有重要影响,异裂菊属通过分泌羧酸、糖等多类化合物提高了根际微生物的活性,进而有效地提高了根际肥力水平。     

Pleiotropic mutants affecting the control of nitrogen metabolism in Aspergillus nidulans

Summary Mutants of Aspergillus nidulans with lesions in gene amdT are pleiotropically affected in their ability to utilize a wide variety of nitrogen sources in the presence of glucose. Ability to utilize a number of these compounds as sole sources of carbon and nitrogen is not altered. One of these mutants, amdT102, has properties consistent with it being derepressed for glucose repression of the utilization of most (but not all) nitrogen sources. The amdT102 mutant can grow strongly on histidine, lysine and cystine as sole nitrogen sources while the wild type strain grows extremely poorly on these amino acids. Similar but less extreme effects apply to many other nitrogen sources. The amdT19 mutant is unable to utilize most nitrogen sources in the presence of glucose, suggesting that it is subject to greatly increased repression of nitrogen source utilization. The amdT mutants are not affected in their ability to use many compounds as sole carbon sources. Carbon sources other than glucose also affect utilization of nitrogen sources in the amdT mutants.     

Substrate Utilization Profiles of Bacterial Strains in Plankton from the River Warnow, a Humic and Eutrophic River in North Germany

Bacteria are very important degraders of organic substances in aquatic environments. Despite their influential role in the carbon (and many other element) cycle(s), the specific genetic identity of active bacteria is mostly unknown, although contributing phylogenetic groups had been investigated. Moreover, the degree to which phenotypic potential (i.e., utilization of environmentally relevant carbon substrates) is related to the genomic identity of bacteria or bacterial groups is unclear. The present study compared the genomic fingerprints of 27 bacterial isolates from the humic River Warnow with their ability to utilize 14 environmentally relevant substrates. Acetate was the only substrate utilized by all bacterial strains. Only 60% of the strains respired glucose, but this substrate always stimulated the highest bacterial activity (respiration and growth). Two isolates, both closely related to the same Pseudomonas sp., also had very similar substrate utilization patterns. However, similar substrate utilization profiles commonly belonged to genetically different strains (e.g., the substrate profile of Janthinobacterium lividum OW6/RT-3 and Flavobacterium sp. OW3/15-5 differed by only three substrates). Substrate consumption was sometimes totally different for genetically related isolates. Thus, the genomic profiles of bacterial strains were not congruent with their different substrate utilization profiles. Additionally, changes in pre-incubation conditions strongly influenced substrate utilization. Therefore, it is problematic to infer substrate utilization and especially microbial dissolved organic matter transformation in aquatic systems from bacterial molecular taxonomy.     

The use of genetic markers selected in vitro for the isolation and genetic verification of intraspecific somatic hybrids of Nicotiana tabacum L.

Summary A scheme employing genetic markers obtained by in vitro selection was developed for the stringent isolation of hybrid somatic cells of Nicotiana tabacum. Mesophyll protoplasts that carried two dominant alleles of nuclear genes conferring resistance to the herbicide picloram (pmR1) and the ability to utilize glycerol as the sole source of carbon (Gut) were fused with suspension-culture protoplasts that were marked with the dominant nuclear allele (HuR9) conferring resistance to hydroxyurea. Putative somatic hybrid cell lines were identified by selecting for the Gut and HuR9 markers, followed by an assay for the unselected marker PmR1. Plants regenerated from six of these cell lines were proved to be true somatic hybrids by demonstrating the segregation of each of the three parental markers in the progeny of crosses of those plants with normal seed-derived plants.     

Phosphonate utilization by bacterial cultures and enrichments from environmental samples.

A selection of axenic microbial strains and a variety of environmental samples were investigated with respect to the utilization of a series of natural and xenobiotic phosphonates as the sole phosphorus source for growth. Phosphonate degradation was observed only with bacteria and not with eucaryotic microorganisms. All representatives of the phosphonates examined supported bacterial growth, with the exception of methylphosphonate diethylester. Yet, distinctly different phosphonate utilization patterns were noted between phosphonate-positive strains. C-P bond cleavage by a photosynthetic bacterium is reported for the first time; growing photoheterotrophically, Rhodobacter capsulatus ATCC 23782 was able to utilize 2-aminoethylphosphonate and alkylphosphonates. Bacteria with the potential to utilize at least one of the phosphonate moieties from the xenobiotic phosphonates Dequest 2010, Dequest 2041, and Dequest 2060 were detected in all environments, with only two exceptions for Dequest 2010. Phosphonate P utilization to an extent of 94 and 97%, for Dequest 2010 and Dequest 2041, respectively, provided evidence that a complete breakdown of these compounds with respect to the C-P bond cleavage can be achieved by some bacteria. The results suggest that phosphonate-utilizing bacteria are ubiquitous, and that selected strains can degrade phosphonates that are more complex than those described previously.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Background  

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.     

Phosphonate utilization by bacterial cultures and enrichments from environmental samples

A selection of axenic microbial strains and a variety of environmental samples were investigated with respect to the utilization of a series of natural and xenobiotic phosphonates as the sole phosphorus source for growth. Phosphonate degradation was observed only with bacteria and not with eucaryotic microorganisms. All representatives of the phosphonates examined supported bacterial growth, with the exception of methylphosphonate diethylester. Yet, distinctly different phosphonate utilization patterns were noted between phosphonate-positive strains. C-P bond cleavage by a photosynthetic bacterium is reported for the first time; growing photoheterotrophically, Rhodobacter capsulatus ATCC 23782 was able to utilize 2-aminoethylphosphonate and alkylphosphonates. Bacteria with the potential to utilize at least one of the phosphonate moieties from the xenobiotic phosphonates Dequest 2010, Dequest 2041, and Dequest 2060 were detected in all environments, with only two exceptions for Dequest 2010. Phosphonate P utilization to an extent of 94 and 97%, for Dequest 2010 and Dequest 2041, respectively, provided evidence that a complete breakdown of these compounds with respect to the C-P bond cleavage can be achieved by some bacteria. The results suggest that phosphonate-utilizing bacteria are ubiquitous, and that selected strains can degrade phosphonates that are more complex than those described previously.     

Novel α‐ketoglutarate dioxygenase tfdA‐related genes are found in soil DNA after exposure to phenoxyalkanoic herbicides

Phenoxyalkanoic herbicides such as 2,4‐dichlorophenoxyacetate (2,4‐D), 2,4‐dichlorophenoxybutyrate (2,4‐DB) or mecoprop are widely used to control broad‐leaf weeds. Several bacteria have been reported to degrade these herbicides using the α‐ketoglutarate‐dependent, 2,4‐dichlorophenoxyacetate dioxygenase encoded by the tfdA gene, as the enzyme catalysing the first step in the catabolic pathway. The effects of exposure to different phenoxyalkanoic herbicides in the soil bacterial community and in the tfdA genes diversity were assessed using an agricultural soil exposed to these anthropogenic compounds. Total community bacterial DNA was analysed by terminal restriction fragment length polymorphism of the 16S rRNA and the tfdA gene markers, and detection and cloning of tfdA gene related sequences, using PCR primer pairs. After up to 4 months of herbicide exposure, significant changes in the bacterial community structure were detected in soil microcosms treated with mecoprop, 2,4‐DB and a mixture of both plus 2,4‐D. An impressive variety of novel tfdA gene related sequences were found in these soil microcosms, which cluster in new tfdA gene related sequence groups, unequally abundant depending on the specific herbicide used in soil treatment. Structural analysis of the putative protein products showed small but significant amino acid differences. These tfdA gene sequence variants are, probably, required for degradation of natural substrate(s) structurally related to these herbicides and their presence explains self‐remediation of soils exposed to phenoxyalkanoic herbicides.     

Ultrasound-mediated stable transformation of potato tuber discs

Summary Plasmid DNA (pBARGUS), containing the selectable bar gene for resistance to the herbicide Basta, was delivered into potato tuber discs via ultrasonication. Transformed plants were identified by their ability to grow on a medium containing 1mg phosphinothricin/l. Southern hybridization and plant resistance to the application of Basta indicated that a functional bar gene had integrated into potato chromosomal DNA.     

Denitrifying capacity of rhizobial strains of Argentine soils and herbicide sensitivity

Agrochemical application in soils is a matter of environmental concern, and among soil microorganisms, rhizobia and their action before different pesticides are interesting to study, due to their taxonomic and functional diversity. The objectives of the present work were to assess the capacity of rhizobial populations to use herbicides as source of nutrients, as well as their ability to reduce nitrates and / or denitrify. Eighty-one strains belonging to four populations of different genera of rhizobia (Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium) were assessed. The effect of glyphosate, 2,4-dichlorophenoxyacetic acid, and atrazine on growth of the strains, as well as the ability of the strains to act on herbicide transformation to reduce nitrate and denitrify, were evaluated. The genera studied showed different responses to pesticides. Bradyrhizobium had the greater capacity to utilize the herbicides and among the compounds evaluated, atrazine was the most used as a source of energy. To conclude, some Bradyrhizobium strains were able both to denitrify and to use the atrazine herbicide. The results obtained in this study increase expectations of the use of rhizobia as inoculants, causing changes at the agricultural and environmental level and allowing an appropriate management of agricultural soil fertilization, efficiency in nitrogen fixation and a faster biodegradation of pesticides in soil.     

Plasmid Involvement in Linalool Metabolism by Pseudomonas fluorescens

A bacterial strain was isolated from a wastewater lagoon and identified as Pseudomonas fluorescens. This isolate was able to utilize linalool as a sole carbon and energy source. The ability was found to be encoded on a 60-megadalton transmissible plasmid, pSRQ60. The plasmid was also mated into a commercial waste treatment strain, which expanded its ability to utilize other isoprenoid compounds.     

Organophosphonate Utilization by the Wild-Type Strain of Penicillium notatum

We studied the biodegradation of compounds containing phosphorus-to-carbon bonds by using a wild-type strain of Penicillium notatum. The substrate specificity of this strain was studied, and we found that it is able to utilize structurally diverse organophosphonates as sole sources of phosphorus. This ability seems to be inducible, as indicated by the presence of a lag phase during growth. A popular herbicide, glyphosate, inhibited fungal growth, but it was also degraded by the fungus if it was applied in sublethal doses. This indicates that P. notatum may play an important role in biodegradation of organophosphonates. The strain which we used did not metabolize any of the phosphonates which we tested when they were used as sole carbon or nitrogen sources.     

Phosphate-limited ocean regions select for bacterial populations enriched in the carbon–phosphorus lyase pathway for phosphonate degradation

In tropical and subtropical oceanic surface waters phosphate scarcity can limit microbial productivity. However, these environments also have bioavailable forms of phosphorus incorporated into dissolved organic matter (DOM) that microbes with the necessary transport and hydrolysis metabolic pathways can access to supplement their phosphorus requirements. In this study we evaluated how the environment shapes the abundance and taxonomic distribution of the bacterial carbon–phosphorus (C–P) lyase pathway, an enzyme complex evolved to extract phosphate from phosphonates. Phosphonates are organophosphorus compounds characterized by a highly stable C–P bond and are enriched in marine DOM. Similar to other known bacterial adaptions to low phosphate environments, C–P lyase was found to become more prevalent as phosphate concentrations decreased. C–P lyase was particularly enriched in the Mediterranean Sea and North Atlantic Ocean, two regions that feature sustained periods of phosphate depletion. In these regions, C–P lyase was prevalent in several lineages of Alphaproteobacteria (Pelagibacter, SAR116, Roseobacter and Rhodospirillales), Gammaproteobacteria, and Actinobacteria. The global scope of this analysis supports previous studies that infer phosphonate catabolism via C–P lyase is an important adaptive strategy implemented by bacteria to alleviate phosphate limitation and expands the known geographic extent and taxonomic affiliation of this metabolic pathway in the ocean.