Biblioteca Digital

Este trabalho visa avaliar o desenvolvimento microbiano e a capacidade de degradação de carbono orgânico total (COT) em quatro colunas adsorvedoras pósfiltração, em escala de bancada, em três ensaios em batelada: o primeiro de longa duração (84 dias) com afluente de água filtrada clorada com baixa concentração de COT (1,4 mg/L); o segundo de longa duração (84 dias) com afluente de solução de glicose e água filtrada clorada com concentração de COT de 6,0 mg/L e o terceiro de curta duração (21 dias) e com o mesmo afluente utilizado no segundo teste. Foi utilizado carvão ativado granular (CAG) de mesmas características nas colunas adsorvedoras, exceto as faixas de diâmetros dos grânulos, que variaram de 0,5 a 0,71 mm (coluna 1A), de 0,84 a 1,0 mm (coluna 1B) e de 1,0 a 1,19 mm (coluna 1C) nas três colunas que tinham o diâmetro interno de 1,2 cm. A coluna 2, com diâmetro interno de 2,1 cm, recebeu CAG com grânulos na mesma faixa de diâmetros da coluna 1B (0,84 a 1,0 mm). As eficiências de remoção de COT no primeiro ensaio foram bem menores (46 a 74%) do que aquelas dos ensaios 2 (94 a 97%) e 3 (96 a 97%). Estes últimos apresentaram grande desenvolvimento da microbiota e elevadas eficiências de remoção de COT...

Studies on microbial activity and biomass in forestry plantations often overlook the role of litter, typically focusing instead on soil nutrient contents to explain plant and microorganism development. However, since the litter is a significant source of recycled nutrients that affect nutrient dynamics in the soil, litter composition may be more strongly correlated with forest growth and development than soil nutrient contents. This study aimed to test this hypothesis by examining correlations between soil C, N, and P; litter C, N, P, lignin content, and polyphenol content; and microbial biomass and activity in pure and mixed second-rotation plantations of Eucalyptus grandis and Acacia mangium before and after senescent leaf drop. The numbers of cultivable fungi and bacteria were also estimated. All properties were correlated with litter C, N, P, lignin and polyphenols, and with soil C and N. We found higher microbial activity (CO2 evolution) in litter than in soil. In the E. grandis monoculture before senescent leaf drop, microbial biomass C was 46 % higher in litter than in soil. After leaf drop, this difference decreased to 16 %. In A. mangium plantations, however, microbial biomass C was lower in litter than in soil both before and after leaf drop. Microbial biomass N of litter was approximately 94 % greater than that of the soil in summer and winter in all plantations. The number of cultivable fungi and bacteria increased after leaf drop...

Soil structure has major influence on ecosystem sustainability and plant growth. Arbuscular mycorrhizal fungi (AMF) are an important functional group of soil microbiota, acting in the process of aggregation, especially in agroecosystems and related to the production and plant diversity. AMF are widely distributed in tropical agroecosystems and are extremely important for development of many agricultural crops. The present study evaluated the effect of inoculation with Glomus macrocarpum in different crop sequences on plant growth, microbial activity and aggregation of a Cerrado Oxisol. The study was conducted in a completely randomized design in a4 x 4 factorial scheme. Treatments consisted of four conditions of elimination and/or introduction of AMF (NS-NI: non-sterilized and non-inoculated; NS-I: non-sterilized and inoculated; S-NI: sterilized and non-inoculated; andS-I: sterilized and inoculated) and four crop sequences (Panicum maximum/Panicum maximum, Brachiaria ruziziensis/ soybean, sorghum/soybean and Stylosanthes spp./soybean). Inoculation favored growth of Stylosanthes spp. by increasing plant growth in up to 91% when inoculated. None of the grasses benefited from G. macrocarpum introduction. We observed that G. macrocarpum inoculation associated with indigenous AMF increased microbial biomass...

Van Etten, James L. (University of Illinois, Urbana), and David Gottlieb. Biochemical changes during the growth of fungi. II. Ergosterol and fatty acids in Penicillium atrovenetum. J. Bacteriol. 89:409–414. 1965.—Changes in the lipid constituents of Penicillium atrovenetum were studied during the growth and development of this fungus. The stages in development, as measured by the dry weight, were divided into four phases: lag, log, stationary, and death. The total fatty acids on a dry-weight basis increased from a minimum in the spores to a maximum near the end of the log phase of growth. The major fatty acids were palmitic, stearic, oleic, and linoleic. Myristic, pentadecanoic, palmitoleic, heptadecanoic, linolenic, arachidic, and heptadecenoic acids, together with two unidentified components, were also present in the fatty acid fraction. Compared to ungerminated spores, young mycelium contained a much lower percentage (on the basis of total fatty acids) of linoleic acid. There was a corresponding increase of oleic acid. Except for palmitic acid, which remained constant, the remaining fatty acids increased slightly. During subsequent growth of the fungus, linleic acid decreased, whereas the percentage of palmitic and stearic acids increased steadily from the lag phase to the end of the log phase. Ergosterol was the only sterol detected. The percentage of ergosterol...

Buono, F. (Syracuse University, Syracuse, N.Y.), R. Testa, and D. G. Lundgren. Physiology of growth and sporulation in Bacillus cereus. I. Effect of glutamic and other amino acids. J. Bacteriol. 91:2291–2299. 1966.—Growth and sporulation were studied in Bacillus cereus by use of an active culture technique and a synthetic medium. A high level of glutamic acid (70 mm) was required for optimal growth and glucose oxidation followed by sporulation even though relatively little glutamic acid was consumed (14 mm). Optimal growth occurred with a combination of 14 mm glutamic acid and 56 mm (NH4)2SO4, aspartic acid, or alanine. Ornithine or arginine at 70 mm could replace glutamic acid in the synthetic medium without affecting the normal growth cycle. Glutamic acid was not replaced by any other amino acid, by (NH4)2SO4, or by a combination of either α-ketoglutarate or pyruvate plus (NH4)2SO4. Enzyme assays of cell-free extracts prepared from cells harvested at different times were used to study the metabolism of glutamic acid. Glutamic-oxaloacetic and glutamic-pyruvate transaminases were completely activated (or derepressed) during early stages of sporulation (period of 6 to 8 hr). Alanine dehydrogenase responded in a similar manner, but the levels of this enzyme were much higher throughout the culture cycle. Neither glutamic dehydrogenase nor α-ketoglutarate dehydrogenase was detected. Sporulation in a replacement salts medium was studied with cells harvested at different times from the synthetic medium. Cultures 2 to 6 hr old were unable to sporulate in the replacement salts medium unless glutamic acid (7.0 mm) was present. By the 6th hr...

In many organisms, phosphatase expression and phosphate (P) uptake are coordinately regulated by the Pho regulon. In Myxococcus xanthus P limitation initiates multicellular development, a process associated with changes in phosphatase expression. We sought here to characterize the link between P acquisition and development in this bacterium, an organism capable of preying upon other microorganisms as a sole nutrient source. M. xanthus seems to possess no significant internal P stores, as reducing the P concentration to less than 10 μM retarded growth within one doubling time. Pyrophosphate, polyphosphate, and glyceraldehyde-3-phosphate could support growth as sole P sources, although many other P-containing biomolecules could not (including nucleic acids and phospholipids). Several Pho regulon promoters were found to be highly active during vegetative growth, and P limitation specifically induced pstSCAB, AcPA1, and pho3 promoter activity and repressed pit expression. Enhanced pstSCAB and pho3 promoter activities in a phoP4 mutant (in the presence of high and low concentrations of P) suggested that PhoP4 acts as a repressor of these genes. However, in a phoP4 background, the activities of pstSCAB remained P regulated, suggesting that there is additional regulation by a P-sensitive factor. Initiation of multicellular development caused immediate down-regulation of Pho regulon genes and caused pstSCAB and pho3 promoter activities to become P insensitive. Hence...

Peptidoglycan is a major cell wall constituent of gram-positive bacteria. It is a dynamic macromolecule that is actively remodeled to enable cell growth and differentiation through a tightly choreographed interplay of hydrolytic and biosynthetic enzyme activities. The filamentous bacterium Streptomyces coelicolor has a complex life cycle that likely requires considerable cell wall remodeling to enable both extension of vegetative hyphae and formation of differentiated cell types. In silico analysis of the S. coelicolor genome enabled identification of 56 candidate cell wall hydrolase genes. We found that seven of these genes shared a highly conserved 5′ untranslated region and were expressed during both vegetative growth and sporulation; four of these genes were selected for more extensive biochemical and biological characterization. The proteins encoded by these genes, termed RpfA, SwlA, SwlB, and SwlC, were confirmed to be hydrolytic enzymes, as they could efficiently cleave S. coelicolor cell walls. Phenotypic analyses revealed that these enzymes are important throughout development; deletion of each hydrolase gene resulted in a mutant strain that was heat sensitive, defective in spore formation, and either altered in vegetative growth or delayed in spore germination. Our results indicate that these enzymes play key roles at multiple stages in the growth and development of S. coelicolor...

Plant growth and development involves a tight coordination of the spatial and temporal organization of cell division, cell expansion and cell differentiation. Orchestration of these events requires the exchange of signaling molecules between the root and shoot, which can be affected by both biotic and abiotic factors. The interactions that occur between plants and their associated microorganisms have long been of interest, as knowledge of these processes could lead to the development of novel agricultural applications. Plants produce a wide range of organic compounds including sugars, organic acids and vitamins, which can be used as nutrients or signals by microbial populations. On the other hand, microorganisms release phytohormones, small molecules or volatile compounds, which may act directly or indirectly to activate plant immunity or regulate plant growth and morphogenesis. In this review, we focus on recent developments in the identification of signals from free-living bacteria and fungi that interact with plants in a beneficial way. Evidence has accumulated indicating that classic plant signals such as auxins and cytokinins can be produced by microorganisms to efficiently colonize the root and modulate root system architecture. Other classes of signals...

Controlling microbial growth in artificial diets is a key component in the rearing of laboratory insects. In this study an antimicrobial agent, Diet Antimicrobial Agent (DAA), was tested for its ability to suppress microbial growth on a range of different diets, and for its effect on larval and pupal performance of individuals from two different strains of Heliothis virescens Fabricus (Lepidoptera: Noctuidae). In the first experiment, it was found that the presence of DAA in a pinto bean-based diet was highly effective at suppressing microbial growth relative to other methods, and that survival of caterpillars on diets with DAA was superior to other treatments. Caterpillars also performed best on diets with DAA, although this may have been the result of laboratory selection pressure as these caterpillars had been reared on pinto bean-based diets with DAA for several hundred generations. A second experiment was conducted, using different diets and a different strain of H. virescens to more fully evaluate DAA. Here it was found that DAA significantly suppressed microbial growth and development, particularly in synthetic diets. There was no significant effect of DAA on pupal development time or mass gain. There was a statistically significant effect of DAA on eclosion time for two of the diets...

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is the most important pest of wheat and barley in wide areas of the world. Different aspects of the insect’s life history have been studied, but to date nothing is known about their microbial symbionts. Here, the con-tribution of symbiotic bacteria to the fitness of the bug was investigated by combining two different approaches to manipulate the host’s microbial community: the supplementation of anti-biotics into the insects’ diet and egg surface sterilization. First, bacteria cultured from gut homogenates were subjected to antibiotic screening tests using 20 different antibiotics. Norfloxa-cin was the most effective antibiotic, with the greatest inhibition zone among all antibiotics tested. Feeding norfloxacin to adult E. integriceps individuals significantly impaired growth and development of the offspring in a dose-dependent manner, i.e., higher antibiotic doses increased the negative effects on nymphal growth and development. Total developmental time from first nymphal instars to adult emergence in control animals was 30.1 days, but when adults had been offered diets with 10, 20, and 30 µg antibiotic per mg diet, the offspring’s developmental time was prolonged to 32.8...

Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor σG that acts at this stage has remained enigmatic. σG activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for σG activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require σG. These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and σG activity during spore maturation.

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein–protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall–targeting β-lactams. Furthermore...

Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA), an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1), as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein–protein interactions between enzymes with antagonistic functions.

Identifying genomic elements required for viability is central to our understanding of the basic physiology of bacterial pathogens. Recently, the combination of high-density mutagenesis and deep sequencing has allowed for the identification of required and conditionally required genes in many bacteria. Genes, however, make up only a part of the complex genomes of important bacterial pathogens. Here, we use an unbiased analysis to comprehensively identify genomic regions, including genes, domains, and intergenic elements, required for the optimal growth of Mycobacterium tuberculosis, a major global health pathogen. We found that several proteins jointly contain both domains required for optimal growth and domains that are dispensable. In addition, many non-coding regions, including regulatory elements and non-coding RNAs, are critical for mycobacterial growth. Our analysis shows that the genetic requirements for growth are more complex than can be appreciated using gene-centric analysis.

Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo—protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth...

Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES (‘MP’) medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.; Organismic and Evolutionary Biology

We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell’s buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell’s water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell’s dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density – the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein), we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells...

Soil salinity is a serious land degradation problem which reduces plant growth and microbial activity due to (1) low osmotic potential which causes plant water stress, and (2) ion toxicity and ion imbalances (nutrient deficiencies) as result of high salt concentrations in the soil solution. Therefore, salinity affects organic matter turnover by influencing the amount of organic matter input in the soil and decomposition rate. Microbial activity and biomass in saline soils have been extensively studied, but a little is known about the effect of organic carbon (OC) addition on adaptation of soil microbes to salinity. The objective of this thesis was to determine the effect of OC availability on adaptation of soil microbial activity and biomass to salinity. In most experiments described in this thesis, one non-saline and four saline soils from the field with similar texture (sandy clay loam) and electrical conductivities in a 1:5 soil: water extract (EC₁﹕₅) of 0.1, 1.1, 3.1 and 5.2 dS m⁻¹ or electrical conductivity of the saturation extract (ECₑ) of 1, 11, 24 and 43 dS m⁻¹ were used. In other experiments a non-saline loamy sand was amended with NaCl to achieve a range of EC levels. The optimum water content for respiration was determined by incubating the soils amended with glucose at different water contents and measuring the respiration for 10 days at 25ºC. Glucose...

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is the most important pest of wheat and barley in wide areas of the world. Different aspects of the insect's life history have been studied, but to date nothing is known about their microbial symbionts. Here, the contribution of symbiotic bacteria to the fitness of the bug was investigated by combining two different approaches to manipulate the host's microbial community: the supplementation of antibiotics into the insects' diet and egg surface sterilization. First, bacteria cultured from gut homogenates were subjected to antibiotic screening tests using 20 different antibiotics. Norfloxacin was the most effective antibiotic, with the greatest inhibition zone among all antibiotics tested. Feeding norfloxacin to adult E. integriceps individuals significantly impaired growth and development of the offspring in a dose-dependent manner, i.e., higher antibiotic doses increased the negative effects on nymphal growth and development. Total developmental time from first nymphal instars to adult emergence in control animals was 30.1 days, but when adults had been offered diets with 10, 20, and 30 µg antibiotic per mg diet, the offspring's developmental time was prolonged to 32.8...

Soil structure has major influence on ecosystem sustainability and plant growth. Arbuscular mycorrhizal fungi (AMF) are an important functional group of soil microbiota, acting in the process of aggregation, especially in agroecosystems and related to the production and plant diversity. AMF are widely distributed in tropical agroecosystems and are extremely important for development of many agricultural crops. The present study evaluated the effect of inoculation with Glomus macrocarpum in different crop sequences on plant growth, microbial activity and aggregation of a Cerrado Oxisol. The study was conducted in a completely randomized design in a4 x 4 factorial scheme. Treatments consisted of four conditions of elimination and/or introduction of AMF (NS-NI: non-sterilized and non-inoculated; NS-I: non-sterilized and inoculated; S-NI: sterilized and non-inoculated; andS-I: sterilized and inoculated) and four crop sequences (Panicum maximum/Panicum maximum, Brachiaria ruziziensis/ soybean, sorghum/soybean and Stylosanthes spp./soybean). Inoculation favored growth of Stylosanthes spp. by increasing plant growth in up to 91% when inoculated. None of the grasses benefited from G. macrocarpum introduction. We observed that G. macrocarpum inoculation associated with indigenous AMF increased microbial biomass...