Influence of Sugarcane Variety on Rhizosphere Microbiota Under Irrigated and Water-Limiting Conditions.

Drought is one of the main problems linked to climate change that is faced by agriculture, affecting various globally important crops, including sugarcane. Environmentally sustainable strategies have been sought to mitigate the effects of climate change on crops. Among them, the use of beneficial microorganisms offers a promising approach. However, it is still necessary to understand the mechanisms that regulate plant-microorganism interactions, in normal situations and under stress. In this work, the rhizosphere metagenomes of two sugarcane varieties, one resistant and the other susceptible to drought, were compared under normal conditions and under water-limiting conditions. The results showed that for the drought-resistant sugarcane variety, bacteria belonging to the order Sphingomonadales and the family Xanthomonadaceae presented increased activities in terms of mobility, colonization, and cell growth. In contrast, the rhizosphere associated with the drought-sensitive variety exhibited increases of bacteria belonging to the family Polyangiaceae, and the genus Streptomyces, with modifications in DNA metabolism and ribosome binding proteins. The results pointed to variation in the rhizosphere microbiota that was modulated by the host plant genotype, revealing potential bacterial candidates that could be recruited to assist plants during water-limiting conditions.

Prospecting Plant Growth-Promoting Bacteria Isolated from the Rhizosphere of Sugarcane Under Drought Stress.

In the rhizosphere, the soil bacteria and the plants are closely related, with the plant-associated microbiota playing an important role in promoting plant growth under both normal and stress conditions. In this study, the cultivable bacteria in the sugarcane rhizosphere under different levels of drought stress were characterized and screened for plant growth activities. The results suggested that the microbial community associated with the sugarcane rhizosphere was strongly affected by drought, but some important genera of bacteria such as Arthrobacter, Pseudomonas, Microbacterium, and Bacillus remained present during the entire experiment, indicating the adaptability of these organisms and their importance in the rhizosphere community. Many isolates exhibited positive results for one or more plant growth activity, and they were also capable of growing under simulated drought stress, suggesting that the microorganisms isolated from the sugarcane rhizosphere could be explored for uses such as biofertilizers or biocontrol agents in agriculture.

Diversity and antimicrobial activity of bacteria isolated from different Brazilian coral species.

Corals harbor a wide diversity of bacteria associated with their mucus. These bacteria can play an important role in nutrient cycling, degradation of xenobiotics and defense against pathogens by producing antimicrobial compounds. However, the diversity of the cultivable heterotrophic bacteria, especially in the Brazilian coral species, remains poorly understood. The present work compares the diversity of cultivable bacteria isolated from the mucus and surrounding environments of four coral species present along the Brazilian coast, and explores the antibacterial activity of these bacteria. Bacteria belonging to the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were isolated. The mucus environment presented a significantly different bacteria composition, compared to the water and sediment environments, with high abundance of Alcanivorax, Acinetobacter, Aurantimonas and Erythrobacter. No difference in the inhibition activity was found between the isolates from mucus and from the surrounding environment. Eighty-three per cent of the bacteria isolated from the mucus presented antimicrobial activity against Serratia marcescens, an opportunistic coral pathogen, suggesting that they might play a role in maintaining the health of the host. Most of the bacteria isolates that presented positive antimicrobial activity belonged to the genus Bacillus.

Worldwide Phylogenetic Group Patterns of Escherichia coli from Commensal Human and Wastewater Treatment Plant Isolates.

Escherichia coli is an important microorganism in the gastrointestinal tract of warm-blooded animals. Commensal populations of E. coli consist of stable genetic isolates, which means that each individual has only one phylogenetic group (phylogroup). We evaluated the frequency of human commensal E. coli phylogroups from 116 people and observed that the majority of isolates belonged to group A. We also evaluated the frequency of phylogroups in wastewater samples and found a strong positive correlation between the phylogroup distribution in wastewater and human hosts. In order to find out if some factors, such as geographical location, and climate could influence the worldwide phylogroup distribution, we performed a meta-analysis of 39 different studies and 24 countries, including different climates, living areas, and feeding habits. Unexpectedly, our results showed no substructuring patterns of phylogroups; indicating there was no correlation between phylogroup distribution and geographic location, climate, living area, feeding habits, or date of collection.

Draft genome sequence of Kocuria sp. SM24M-10 isolated from coral mucus.

Here, we describe the genomic features of the Actinobacteria Kocuria sp. SM24M-10 isolated from mucus of the Brazilian endemic coral Mussismilia hispida. The sequences are available under accession number LDNX01000000 (http://www.ncbi.nlm.nih.gov/nuccore/LDNX00000000). The genomic analysis revealed interesting information about the adaptation of bacteria to the marine environment (such as genes involved in osmotic and oxidative stress) and to the nutrient-rich environment provided by the coral mucus.

Characterization of the core microbiota of the drainage and surrounding soil of a Brazilian copper mine.

The core microbiota of a neutral mine drainage and the surrounding high heavy metal content soil at a Brazilian copper mine were characterized by 16S rDNA pyrosequencing. The core microbiota of the drainage was dominated by the generalist genus Meiothermus. The soil samples contained a more heterogeneous bacterial community, with the presence of both generalist and specialist bacteria. Both environments supported mainly heterotrophic bacteria, including organisms resistant to heavy metals, although many of the bacterial groups identified remain poorly characterized. The results contribute to the understanding of bacterial communities in soils impacted by neutral mine drainage, for which information is scarce, and demonstrate that heavy metals can play an important role in shaping the microbial communities in mine environments.

High-quality draft genome sequence of Kocuria marina SO9-6, an actinobacterium isolated from a copper mine.

An actinobacterial strain, designated SO9-6, was isolated from a copper iron sulfide mineral. The organism is Gram-positive, facultatively anaerobic, and coccoid. Chemotaxonomic and phylogenetic properties were consistent with its classification in the genus Kocuria. Here, we report the first draft genome sequence of Kocuria marina SO9-6 under accession JROM00000000 (http://www.ncbi.nlm.nih.gov/nuccore/725823918), which provides insights for heavy metal bioremediation and production of compounds of biotechnological interest.

Comparative metagenomic analysis of coral microbial communities using a reference-independent approach.

By comparing the SEED and Pfam functional profiles of metagenomes of two Brazilian coral species with 29 datasets that are publicly available, we were able to identify some functions, such as protein secretion systems, that are overrepresented in the metagenomes of corals and may play a role in the establishment and maintenance of bacteria-coral associations. However, only a small percentage of the reads of these metagenomes could be annotated by these reference databases, which may lead to a strong bias in the comparative studies. For this reason, we have searched for identical sequences (99% of nucleotide identity) among these metagenomes in order to perform a reference-independent comparative analysis, and we were able to identify groups of microbial communities that may be under similar selective pressures. The identification of sequences shared among the metagenomes was found to be even better for the identification of groups of communities with similar niche requirements than the traditional analysis of functional profiles. This approach is not only helpful for the investigation of similarities between microbial communities with high proportion of unknown reads, but also enables an indirect overview of gene exchange between communities.

Bacterial diversity assessment in soil of an active Brazilian copper mine using high-throughput sequencing of 16S rDNA amplicons.

Mining activities pose severe environmental risks worldwide, generating extreme pH conditions and high concentrations of heavy metals, which can have major impacts on the survival of organisms. In this work, pyrosequencing of the V3 region of the 16S rDNA was used to analyze the bacterial communities in soil samples from a Brazilian copper mine. For the analysis, soil samples were collected from the slopes (geotechnical structures) and the surrounding drainage of the Sossego mine (comprising the Sossego and Sequeirinho deposits). The results revealed complex bacterial diversity, and there was no influence of deposit geographic location on the composition of the communities. However, the environment type played an important role in bacterial community divergence; the composition and frequency of OTUs in the slope samples were different from those of the surrounding drainage samples, and Acidobacteria, Chloroflexi, Firmicutes, and Gammaproteobacteria were responsible for the observed difference. Chemical analysis indicated that both types of sample presented a high metal content, while the amounts of organic matter and water were higher in the surrounding drainage samples. Non-metric multidimensional scaling (N-MDS) analysis identified organic matter and water as important distinguishing factors between the bacterial communities from the two types of mine environment. Although habitat-specific OTUs were found in both environments, they were more abundant in the surrounding drainage samples (around 50 %), and contributed to the higher bacterial diversity found in this habitat. The slope samples were dominated by a smaller number of phyla, especially Firmicutes. The bacterial communities from the slope and surrounding drainage samples were different in structure and composition, and the organic matter and water present in these environments contributed to the observed differences.

Changes in the bacterial community of soil from a neutral mine drainage channel.

Mine drainage is an important environmental disturbance that affects the chemical and biological components in natural resources. However, little is known about the effects of neutral mine drainage on the soil bacteria community. Here, a high-throughput 16S rDNA pyrosequencing approach was used to evaluate differences in composition, structure, and diversity of bacteria communities in samples from a neutral drainage channel, and soil next to the channel, at the Sossego copper mine in Brazil. Advanced statistical analyses were used to explore the relationships between the biological and chemical data. The results showed that the neutral mine drainage caused changes in the composition and structure of the microbial community, but not in its diversity. The Deinococcus/Thermus phylum, especially the Meiothermus genus, was in large part responsible for the differences between the communities, and was positively associated with the presence of copper and other heavy metals in the environmental samples. Other important parameters that influenced the bacterial diversity and composition were the elements potassium, sodium, nickel, and zinc, as well as pH. The findings contribute to the understanding of bacterial diversity in soils impacted by neutral mine drainage, and demonstrate that heavy metals play an important role in shaping the microbial population in mine environments.

Bacterial communities and species-specific associations with the mucus of Brazilian coral species.

We investigated the existence of species-specific associations between Brazilian coral species and bacteria. Pyrosequencing of the V3 region of the 16S rDNA was used to analyze the taxonomic composition of bacterial communities associated with the mucus of four coral species (Madracis decactis, Mussismilia hispida, Palythoa caribaeorum, and Tubastraea coccinea) in two seasons (winter and summer), which were compared with the surrounding water and sediment. The microbial communities found in samples of mucus, water, and sediment differed according to the composition and relative frequency of OTUs. The coral mucus community seemed to be more stable and resistant to seasonal variations, compared to the water and sediment communities. There was no influence of geographic location on the composition of the communities. The sediment community was extremely diverse and might act as a "seed bank" for the entire environment. Species-specific OTUs were found in P. caribaeorum, T. coccinea, and M. hispida.

Use of Escherichia coli BOX-PCR fingerprints to identify sources of fecal contamination of water bodies in the State of São Paulo, Brazil.

Repetitive element sequence-based polymerase chain reaction (rep-PCR) is one of the commonest methods used to identify sources of fecal contamination of water systems. In this work, BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) was used to discriminate Escherichia coli strains originating from different animals and water sources, and the suitability of the technique for bacterial source tracking (BST) was evaluated. A total of 214 strains from humans, 150 strains from animals, 55 strains from sewage and 77 strains from water bodies were analyzed by the BOX-PCR technique. When maximum similarity between the fingerprints was used, a correct classification rate of 84% was achieved for strains from human and animal sources. Furthermore, 95% of the strains found in sewage were classified as being from human sources by at least one of the four classification tools used. Classification of the strains found in water bodies in the State of São Paulo was based on the fingerprints obtained for human and animal sources. Most of the sampling sites appeared to be affected by mixed sources of fecal contamination. The use of BOX-PCR for BST could be especially valuable in developing countries, where simplicity and cost are important considerations.

Gene expression modulation by heat stress in Acidithiobacillus ferrooxidans LR.

During bioleaching, Acidithiobacillus ferrooxidans is subjected to different types of stress, including heat stress, which affect bacterial growth. In this work, real time quantitative PCR was used to analyze the expression of heat shock genes, as well as genes that encode proteins related to several functional categories in A. ferrooxidans. Cells were submitted to long-term growth and heat shock, both at 40°C. The results showed that heat shock affected the expression levels of most genes investigated, whilst long-term growth at 40°C resulted in minor changes in gene expression, except for certain genes related to iron transport, which were strongly down-regulated, suggesting that the iron processing capability of A. ferrooxidans was affected by long-term growth at 40°C. A bioinformatic analysis of the genes' promoter regions indicated a putative transcriptional regulation by the σ(32) factor in 12 of the 31 genes investigated, suggesting the involvement of other regulatory mechanisms in the response of A. ferrooxidans to heat stress.

The small heat shock proteins from Acidithiobacillus ferrooxidans: gene expression, phylogenetic analysis, and structural modeling.

BACKGROUND: Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that has been successfully used in metal bioleaching. In this study, an analysis of the A. ferrooxidans ATCC 23270 genome revealed the presence of three sHSP genes, Afe_1009, Afe_1437 and Afe_2172, that encode proteins from the HSP20 family, a class of intracellular multimers that is especially important in extremophile microorganisms. RESULTS: The expression of the sHSP genes was investigated in A. ferrooxidans cells submitted to a heat shock at 40°C for 15, 30 and 60 minutes. After 60 minutes, the gene on locus Afe_1437 was about 20-fold more highly expressed than the gene on locus Afe_2172. Bioinformatic and phylogenetic analyses showed that the sHSPs from A. ferrooxidans are possible non-paralogous proteins, and are regulated by the σ32 factor, a common transcription factor of heat shock proteins. Structural studies using homology molecular modeling indicated that the proteins encoded by Afe_1009 and Afe_1437 have a conserved α-crystallin domain and share similar structural features with the sHSP from Methanococcus jannaschii, suggesting that their biological assembly involves 24 molecules and resembles a hollow spherical shell. CONCLUSION: We conclude that the sHSPs encoded by the Afe_1437 and Afe_1009 genes are more likely to act as molecular chaperones in the A. ferrooxidans heat shock response. In addition, the three sHSPs from A. ferrooxidans are not recent paralogs, and the Afe_1437 and Afe_1009 genes could be inherited horizontally by A. ferrooxidans.

Prevalence of virulence factors in Escherichia coli isolated from healthy animals and water sources in Brazil.

The aim of this work was to verify the presence of seven virulence factors (ST, LT, eae, stx(1), stx(2), INV and EAEC) among Escherichia coli strains isolated from healthy humans, bovines, chickens, sheep, pigs and goats, from two sewage treatment plants and from the Tietê River. We have found a high prevalence of eae, stx(1) and stx(2) in ruminants. The EAEC gene was only found in humans and sewage. No strains presented ST, LT or INV. BOX-PCR fingerprints revealed a high diversity among the strains analysed and a non-clonal origin of strains that presented the same virulence factors. Therefore, we concluded that ruminants may constitute an important reservoir of most diarrheagenic E. coli in Brazil, except for EAEC strains. These results emphasize the importance of the identification of the animal source of fecal contamination for the correct water risk assessment.

Ferric iron uptake genes are differentially expressed in the presence of copper sulfides in Acidithiobacillus ferrooxidans strain LR.

Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide ores. This work aimed to investigate the relative expression of genes related to the iron uptake system when A. ferrooxidans LR was maintained in contact with chalcopyrite or bornite as the sole energy source. Real-time quantitative PCR analysis revealed that the presence of bornite had no effect on the expression of seven genes related to the siderophore-mediated Fe(III) uptake system, while in the presence of chalcopyrite the expression of the genes was up-regulated. Bioinformatic analysis of the genomic region where these genes were found revealed the existence of three new putative DNA-binding sequences for the ferric iron uptake transcriptional regulator (Fur). Electrophoretic mobility shift assays demonstrated that a purified A. ferrooxidans His-tagged Fur protein was able to bind in vitro to each of these putative Fur boxes, suggesting that Fur regulated the expression of these genes. The expression of fur and two known Fur-regulated genes, mntH and dsrK, was also investigated in the presence of chalcopyrite. While the expression of fur and mntH was up-regulated, the expression of dsrK was down-regulated. The low amount of ferrous iron in the medium was probably responsible for the up-regulation of fur and the genes related to the siderophore-mediated Fe(III) uptake system when A. ferrooxidans LR was kept in the presence of chalcopyrite. A homology model of the A. ferrooxidans Fur was constructed and revealed that the putative DNA-binding surface presents conserved positively charged residues, supporting a previously suggested mode of interaction with DNA. The up-regulation of fur and the siderophore-mediated Fe(III) uptake genes, and the down-regulation of dsrK suggest that in the presence of chalcopyrite Fur acts as a transcription inducer and repressor.

Heat and phosphate starvation effects on the proteome, morphology and chemical composition of the biomining bacteria Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a Gram negative, acidophilic, chemolithoautotrophic bacterium that plays an important role in metal bioleaching. During bioleaching, the cells are subjected to changes in the growth temperature and nutrients starvation. The aim of this study was to gather information about the response of the A.ferrooxidans Brazilian strain LR to K2HPO4 starvation and heat stress through investigation of cellular morphology, chemical composition and differential proteome. The scanning electron microscopic results showed that under the tested stress conditions, A. ferrooxidans cells became elongated while the Fourier transform infrared spectroscopy (FT-IR) analysis showed alterations in the wavenumbers between 850 and 1,275 cm(-1), which are related to carbohydrates, phospholipids and phosphoproteins. These findings indicate that the bacterial cell surface is affected by the tested stress conditions. A proteomic analysis, using 2-DE and tandem mass spectrometry, enabled the identification of 44 differentially expressed protein spots, being 30 due to heat stress (40°C) and 14 due to K2HPO4 starvation. The identified proteins belonged to 11 different functional categories, including protein fate, energy metabolism and cellular processes. The upregulated proteins were mainly from protein fate and energy metabolism categories. The obtained results provide evidences that A. ferrooxidans LR responds to heat stress and K2HPO4 starvation by inducing alterations in cellular morphology and chemical composition of the cell surface. Also, the identification of several proteins involved in protein fate suggests that the bacteria cellular homesostasis was affected. In addition, the identification of proteins from different functional categories indicates that the A. ferrooxidans response to higher than optimal temperatures and phosphate starvation involves global changes in its physiology.

Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination.

BACKGROUND: Escherichia coli strains are commonly found in the gut microflora of warm-blooded animals. These strains can be assigned to one of the four main phylogenetic groups, A, B1, B2 and D, which can be divided into seven subgroups (A0, A1, B1, B22, B23, D1 and D2), according to the combination of the three genetic markers chuA, yjaA and DNA fragment TspE4.C2. Distinct studies have demonstrated that these phylo-groups differ in the presence of virulence factors, ecological niches and life-history. Therefore, the aim of this work was to analyze the distribution of these E. coli phylo-groups in 94 human strains, 13 chicken strains, 50 cow strains, 16 goat strains, 39 pig strains and 29 sheep strains and to verify the potential of this analysis to investigate the source of fecal contamination. RESULTS: The results indicated that the distribution of phylogenetic groups, subgroups and genetic markers is non-random in the hosts analyzed. Strains from group B1 were present in all hosts analyzed but were more prevalent in cow, goat and sheep samples. Subgroup B23 was only found in human samples. The diversity and the similarity indexes have indicated a similarity between the E. coli population structure of human and pig samples and among cow, goat and sheep samples. Correspondence analysis using contingence tables of subgroups, groups and genetic markers frequencies allowed the visualization of the differences among animal samples and the identification of the animal source of an external validation set. The classifier tools Binary logistic regression and Partial least square--discriminant analysis, using the genetic markers profile of the strains, differentiated the herbivorous from the omnivorous strains, with an average error rate of 17%. CONCLUSIONS: This is the first work, as far as we are aware, that identifies the major source of fecal contamination of a pool of strains instead of a unique strain. We concluded that the analysis of the E. coli population structure can be useful as a supplementary bacterial source tracking tool.

Gene expression modulation by chalcopyrite and bornite in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a mesophilic, acidophilic, chemolithoautotrophic bacterium that obtains energy from the oxidation of ferrous iron (Fe2+), elemental sulfur and reduced sulfur compounds. The industrial interest in A. ferrooxidans resides in its capacity to oxidize insoluble metal sulfides into soluble metal sulfates, thus allowing the recovery of the desired metals from low-grade sulfide ores. In the present work, RNA arbitrarily primed PCR (RAP-PCR) was performed to identify cDNAs differentially expressed in A. ferrooxidans cells grown in the presence of Fe2+ and cells maintained for 24 h in the presence of the copper sulfides bornite and chalcopyrite. Eighteen cDNAs corresponding to genes with known function were identified, and their relative expression was further characterized by real-time quantitative PCR. Bornite had a mild effect on the expression of the 18 genes analyzed. None of these genes was down-regulated and among the few genes up-regulated, it is worth mentioning lepA and def-2 that are involved in protein synthesis. Chalcopyrite presented the most significant changes. Five genes related to protein processing were down-regulated, and another 5 genes related to the transport system were up-regulated. The up- and down-regulation of these genes in the presence of bornite and chalcopyrite could be due to alterations in the ideal pH, presence of copper ions in solution and nutrient limitation. The results suggest that gene expression modulation might be important for the A. ferrooxidans early response to copper sulfides.

The rus operon genes are differentially regulated when Acidithiobacillus ferrooxidans LR is kept in contact with metal sulfides.

Acidithiobacillus ferrooxidans is a gram-negative bacterium that obtains energy from the oxidation of ferrous iron or reduced sulfur compounds. In this bacterium, the proteins encoded by the rus operon are involved in electron transfer from Fe(II) to O(2), and the first two proteins in this pathway also participate in the electron transfer pathway from Fe(II) to NAD(P). In this work we analyzed the expression, by real-time PCR, of the eight genes from the rus operon when A. ferrooxidans LR was grown in the presence of iron (control) and then kept in contact with chalcopyrite (CuFeS(2)) and covellite (CuS). A small decrease in rus operon gene expression was observed in the presence of chalcopyrite, while in the presence of covellite the expression of these genes showed a remarkable decrease. These results can be explained by the absence of ferrous iron in covellite. To explain the expression difference observed between the gene cyc1 and the gene rus, we investigated the information content presented at the Translation Initiation Site (TIS) of both genes. cyc1 showed a highly information content (8.4 bits) that can maximize translation, and rus showed a less favorable context (5.5 bits). Our hypothesis is that the energetic metabolism in A. ferrooxidans may be controlled at the transcriptional and posttranscriptional level by different mechanisms.