Nitrospirillum viridazoti sp. nov., an Efficient Nitrogen-Fixing Species Isolated from Grasses.

A group of Gram-negative plant-associated diazotrophic bacteria belonging to the genus Nitrospirillum was investigated, including both previously characterized and newly isolated strains from diverse regions and biomes, predominantly in Brazil. Phylogenetic analysis of 16S rRNA and recA genes revealed the formation of a distinct clade consisting of thirteen strains, separate from the formally recognized species N. amazonense (the closest species) and N. iridis. Comprehensive taxonomic analyses using the whole genomes of four strains (BR 11140T = AM 18T = Y-2T = DSM 2788T = ATCC 35120T, BR 11142T = AM 14T = Y-1T = DSM 2787T = ATCC 35119T, BR 11145 = CBAmC, and BR 12005) supported the division of these strains into two species: N. amazonense (BR 11142 T and BR 12005) and a newly proposed species (BR 11140 T and BR 11145), distinct from N. iridis. The phylogenomic analysis further confirmed the presence of the new Nitrospirillum species. Additionally, MALDI-TOF MS analysis of whole-cell mass spectra provided further evidence for the differentiation of the proposed Nitrospirillum species, separate from N. amazonense. Analysis of chemotaxonomy markers (i.e., genes involved in fatty acid synthesis, metabolism and elongation, phospholipid synthesis, and quinone synthesis) revealed that the new species highlights high similarity and evolutionary convergence with other Nitrospirillum species. This new species exhibited nitrogen fixation ability in vitro, it has similar NifHDK protein phylogeny position with the closest species, lacked denitrification capability, but possessed the nosZ gene, enabling N2O reduction, distinguishing it from the closest species. Despite being isolated from diverse geographic regions, soil types, and ecological niches, no significant phenotypic or physiological differences were observed between the proposed new species and N. amazonense. Based on these findings, a new species, Nitrospirillum viridazoti sp. nov., was classified, with the strain BR 11140T (DSM 2788T, ATCC 35120T) designated as the type strain.

Tips and tools to obtain and assess mosquito viromes.

Due to their vectorial capacity, mosquitoes (Diptera: Culicidae) receive special attention from health authorities and entomologists. These cosmopolitan insects are responsible for the transmission of many viral diseases, such as dengue and yellow fever, causing huge impacts on human health and justifying the intensification of research focused on mosquito-borne diseases. In this context, the study of the virome of mosquitoes can contribute to anticipate the emergence and/or the reemergence of infectious diseases. The assessment of mosquito viromes also contributes to the surveillance of a wide variety of viruses found in these insects, allowing the early detection of pathogens with public health importance. However, the study of mosquito viromes can be challenging due to the number and complexities of steps involved in this type of research. Therefore, this article aims to describe, in a straightforward and simplified way, the steps necessary for obtention and assessment of mosquito viromes. In brief, this article explores: the capture and preservation of specimens; sampling strategies; treatment of samples before DNA/RNA extraction; extraction methodologies; enrichment and purification processes; sequencing choices; and bioinformatics analysis.

Genomic analysis reveals genes that encode the synthesis of volatile compounds by a Bacillus velezensis-based biofungicide used in the treatment of grapes to control Aspergillus carbonarius.

Fungal control strategies based on the use of Bacillus have emerged in agriculture as eco-friendly alternatives to replace/reduce the use of synthetic pesticides. Bacillus sp. P1 was reported as a new promising strain for control of Aspergillus carbonarius, a known producer of ochratoxin A, categorized as possible human carcinogen with high nephrotoxic potential. Grape quality can be influenced by vineyard management practices, including the use of fungal control agents. The aim of this study was to evaluate, for the first time, the quality parameters of Chardonnay grapes exposed to an antifungal Bacillus-based strategy for control of A. carbonarius, supporting findings by genomic investigations. Furthermore, genomic tools were used to confirm that the strain P1 belongs to the non-pathogenic species Bacillus velezensis and also to certify its biosafety. The genome of B. velezensis P1 harbors genes that are putatively involved in the production of volatiles and hydrolytic enzymes, which are responsible for releasing the free form of aroma compounds. In addition to promote biocontrol of phytopathogenic fungi and ochratoxins, the treatment with B. velezensis P1 did not change the texture (hardness and firmness), color and pH of the grapes. Heat map and hierarchical clustering analysis (HCA) of volatiles evaluated by GC/MS revealed that Bacillus-treated grapes showed higher levels of compounds with a pleasant odor descriptions such as 3-hydroxy-2-butanone, 2,3-butanediol, 3-methyl-1-butanol, 3,4-dihydro-ß-ionone, ß-ionone, dihydroactinidiolide, linalool oxide, and ß-terpineol. The results of this study indicate that B. velezensis P1 presents desirable properties to be used as a biocontrol agent.

Genome-guided purification of high amounts of the siderophore ornibactin and detection of potentially novel burkholdine derivatives produced by Burkholderia catarinensis 89T.

AIM: The increased availability of genome sequences has enabled the development of valuable tools for the prediction and identification of bacterial natural products. Burkholderia catarinensis 89T produces siderophores and an unknown potent antifungal metabolite. The aim of this work was to identify and purify natural products of B. catarinensis 89T through a genome-guided approach. MATERIALS AND METHODS: The analysis of B. catarinensis 89T genome revealed 16 clusters putatively related to secondary metabolism and antibiotics production. Of particular note was the identification of a nonribosomal peptide synthetase (NRPS) cluster related to the production of the siderophore ornibactin, a hybrid NRPS-polyketide synthase Type 1 cluster for the production of the antifungal glycolipopeptide burkholdine, and a gene cluster encoding homoserine lactones (HSL), probably involved in the regulation of both metabolites. We were able to purify high amounts of the ornibactin derivatives D/C6 and F/C8, while also detecting the derivative B/C4 in mass spectrometry investigations. A group of metabolites with molecular masses ranging from 1188 to 1272 Da could be detected in MS experiments, which we postulate to be new burkholdine analogs produced by B. catarinensis. The comparison of B. catarinensis BGCs with other Bcc members corroborates the hypothesis that this bacterium could produce new derivatives of these metabolites. Moreover, the quorum sensing metabolites C6-HSL, C8-HSL, and 3OH-C8-HSL were observed in LC-MS/MS analysis. CONCLUSION: The new species B. catarinensis is a potential source of new bioactive secondary metabolites. Our results highlight the importance of genome-guided purification and identification of metabolites of biotechnological importance.

Genome-based taxonomy of Burkholderia sensu lato: Distinguishing closely related species.

The taxonomy of Burkholderia sensu lato (s.l.) has been revisited using genome-based tools, which have helped differentiate closely related species. Many species from this group are indistinguishable through phenotypic traits and 16S rRNA gene sequence analysis. Furthermore, they also exhibit whole-genome Average Nucleotide Identity (ANI) values in the twilight zone for species circumscription (95-96%), which may impair their correct classification. In this work, we provided an updated Burkholderia s.l. taxonomy focusing on closely related species and give other recommendations for those developing genome-based taxonomy studies. We showed that a combination of ANI and digital DNA-DNA hybridization (dDDH) applying the universal cutoff values of 95% and 70%, respectively, successfully discriminates Burkholderia s.l. species. Using genome metrics with this pragmatic criterion, we demonstrated that i) Paraburkholderia insulsa should be considered a later heterotypic synonym of Paraburkholderia fungorum; ii) Paraburkholderia steynii differs from P. terrae by harboring symbiotic genes; iii) some Paraburkholderia are indeed different species based on dDDH values, albeit sharing ANI values close to 95%; iv) some Burkholderia s.l. indeed represent new species from the genomic viewpoint; iv) some genome sequences should be evaluated with care due to quality concerns.

Synthesizing the connections between environmental disturbances and zoonotic spillover.

Zoonotic spillover is a phenomenon characterized by the transfer of pathogens between different animal species. Most human emerging infectious diseases originate from non-human animals, and human-related environmental disturbances are the driving forces of the emergence of new human pathogens. Synthesizing the sequence of basic events involved in the emergence of new human pathogens is important for guiding the understanding, identification, and description of key aspects of human activities that can be changed to prevent new outbreaks, epidemics, and pandemics. This review synthesizes the connections between environmental disturbances and increased risk of spillover events based on the One Health perspective. Anthropogenic disturbances in the environment (e.g., deforestation, habitat fragmentation, biodiversity loss, wildlife exploitation) lead to changes in ecological niches, reduction of the dilution effect, increased contact between humans and other animals, changes in the incidence and load of pathogens in animal populations, and alterations in the abiotic factors of landscapes. These phenomena can increase the risk of spillover events and, potentially, facilitate new infectious disease outbreaks. Using Brazil as a study model, this review brings a discussion concerning anthropogenic activities in the Amazon region and their potential impacts on spillover risk and spread of emerging diseases in this region.

Alternative nitrogenase of Paenibacillus sonchi genomovar Riograndensis: An insight in the origin of Fe-nitrogenase in the Paenibacillaceae family.

Paenibacillus sonchi genomovar Riograndensis is a nitrogen-fixing bacteria isolated from wheat that displays diverse plant growth-promoting abilities. Beyond conventional Mo-nitrogenase, this organism also harbors an alternative Fe-nitrogenase, whose many aspects related to regulation, physiology, and evolution remain to be elucidated. In this work, the origins of this alternative system were investigated, exploring the distribution and diversification of nitrogenases in the Panibacillaceae family. Our analysis showed that diazotrophs represent 17% of Paenibacillaceae genomes, of these, only 14.4% (2.5% of all Paenibacillaceae genomes) also contained Fe or V- nitrogenases. Diverse nif-like sequences were also described, occurring mainly in genomes that also harbor the alternative systems. The analysis of genomes containing Fe-nitrogenase showed a conserved cluster of nifEN anfHDGK across three genera: Gorillibacterium, Fontibacillus, and Paenibacillus. A phylogeny of anfHDGK separated the Fe-nitrogenases into three main groups. Our analysis suggested that Fe-nitrogenase was acquired by the ancestral lineage of Fontibacillus, Gorillibacterium, and Paenibacillus genera via horizontal gene transfer (HGT), and further events of transfer and gene loss marked the evolution of this alternative nitrogenase in these groups. The species phylogeny of N-fixing Paenibacillaceae separated the diazotrophs into five clades, one of these containing all occurrences of strains harboring alternative nitrogenases in the Paenibacillus genus. The pangenome of this clade is open and composed of more than 96% of accessory genes. Diverse functional categories were enriched in the flexible genome, including functions related to replication and repair. The latter involved diverse genes related to HGT, suggesting that such events may have an important role in the evolution of diazotrophic Paenibacillus. This study provided an insight into the organization, distribution, and evolution of alternative nitrogenase genes in Paenibacillaceae, considering different genomic aspects.

Pangenome analyses of Bacillus pumilus, Bacillus safensis, and Priestia megaterium exploring the plant-associated features of bacilli strains isolated from canola.

Previous genome mining of the strains Bacillus pumilus 7PB, Bacillus safensis 1TAz, 8Taz, and 32PB, and Priestia megaterium 16PB isolated from canola revealed differences in the profile of antimicrobial biosynthetic genes when compared to the species type strains. To evaluate not only the similarities among B. pumilus, B. safensis, and P. megaterium genomes but also the specificities found in the canola bacilli, we performed comparative genomic analyses through the pangenome evaluation of each species. Besides that, other genome features were explored, especially focusing on plant-associated and biotechnological characteristics. The combination of the genome metrics Average Nucleotide Identity and digital DNA-DNA hybridization formulas 1 and 3 adopting the universal thresholds of 95 and 70%, respectively, was suitable to verify the identification of strains from these groups. On average, core genes corresponded to 45%, 52%, and 34% of B. pumilus, B. safensis, and P. megaterium open pangenomes, respectively. Many genes related to adaptations to plant-associated lifestyles were predicted, especially in the Bacillus genomes. These included genes for acetoin production, polyamines utilization, root exudate chemoreceptors, biofilm formation, and plant cell-wall degrading enzymes. Overall, we could observe that strains of these species exhibit many features in common, whereas most of their variable genome portions have features yet to be uncovered. The observed antifungal activity of canola bacilli might be a result of the synergistic action of secondary metabolites, siderophores, and chitinases. Genome analysis confirmed that these species and strains have biotechnological potential to be used both as agricultural inoculants or hydrolases producers. Up to our knowledge, this is the first work that evaluates the pangenome features of P. megaterium.

Pangenome inventory of Burkholderia sensu lato, Burkholderia sensu stricto, and the Burkholderia cepacia complex reveals the uniqueness of Burkholderia catarinensis.

Here the pangenome analysis of Burkholderia sensu lato (s.l.) was performed for the first time, together with an updated analysis of the pangenome of Burkholderia sensu stricto, and Burkholderia cepacia complex (Bcc) focusing on the Bcc B. catarinensis specific features of its re-sequenced genome. The pangenome of Burkholderia s.l., Burkholderia s.s., and of the Bcc was open, composed of more than 96% of accessory genes, and more than 62% of unknown genes. Functional annotations showed that secondary metabolism genes belonged to the variable portion of genomes, which might explain their production of several compounds with varied bioactivities. Taken together, this work showed the great variability and uniqueness of these genomes and revealed an underexplored unknown potential in poorly characterized genes. Regarding B. catarinensis 89T, its genome harbors genes related to hydrolases production and plant growth promotion. This draft genome will be valuable for further investigation of its biotechnological potentials.

Aspergillus carbonarius-derived ochratoxins are inhibited by Amazonian Bacillus spp. used as a biocontrol agent in grapes.

Bacillus spp. have been used as a biocontrol strategy to eliminate/reduce toxic fungicides in viticulture. Furthermore, the presence of fungi that are resistant to commonly used products is frequent, highlighting the need for new biocontrol strains. Aspergillus carbonarius can produce ochratoxins, including ochratoxin A (OTA), which has a regulatory maximum allowable limit for grape products. The purpose of this study was to assess the ability of four Amazonian strains of Bacillus (P1, P7, P11, and P45) to biocontrol A. carbonarius and various forms of ochratoxins in grapes. Berries treated with strain P1 presented no fungal colonies (100% reduction), while P7, P11 and P45 strains caused a reduction of 95, 95 and 61% on fungal counts, respectively. Six forms of ochratoxin were found in the grapes inoculated with A. carbonarius, including ochratoxin α, ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide, and OTA. Four of these ochratoxin forms (ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide) are reported for the first time in grapes. These ochratoxins were identified using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS). All Bacillus strains inhibited the synthesis of OTA, which is the most toxic form of ochratoxin. No ochratoxin form was found when P1 and P7 were used. Although some forms of ochratoxin were detected in grapes treated with Bacillus spp. P11 and P45, the levels decreased by 97%. To our knowledge, this is the first report on the inhibition of Aspergillus carbonarius-derived ochratoxin by Bacillus species. P1 strain, identified as Bacillus velezensis, was found to be the most promising for completely inhibiting fungal growth and production of all ochratoxins.

Burkholderia in the genomic era: from taxonomy to the discovery of new antimicrobial secondary metabolites.

Species of Burkholderia are highly versatile being found not only abundantly in soil, but also as plants and animals' commensals or pathogens. Their complex multireplicon genomes harbour an impressive number of polyketide synthase (PKS) and nonribosomal peptide-synthetase (NRPS) genes coding for the production of antimicrobial secondary metabolites (SMs), which have been successfully deciphered by genome-guided tools. Moreover, genome metrics supported the split of this genus into Burkholderia sensu stricto (s.s.) and five new other genera. Here, we show that the successful antimicrobial SMs producers belong to Burkholderia s.s. Additionally, we reviewed the occurrence, bioactivities, modes of action, structural, and biosynthetic information of thirty-eight Burkholderia antimicrobial SMs shedding light on their diversity, complexity, and uniqueness as well as the importance of genome-guided strategies to facilitate their discovery. Several Burkholderia NRPS and PKS display unusual features, which are reflected in their structural diversity, important bioactivities, and varied modes of action. Up to now, it is possible to observe a general tendency of Burkholderia SMs being more active against fungi. Although the modes of action and biosynthetic gene clusters of many SMs remain unknown, we highlight the potential of Burkholderia SMs as alternatives to fight against new diseases and antibiotic resistance.

Antifungal potential against Sclerotinia sclerotiorum (Lib.) de Bary and plant growth promoting abilities of Bacillus isolates from canola (Brassica napus L.) roots.

Endophytic bacteria show important abilities in promoting plant growth and suppressing phytopathogens, being largely explored in agriculture as biofertilizers or biocontrol agents. Bacteria from canola roots were isolated and screened for different plant growth promotion (PGP) traits and biocontrol of Sclerotinia sclerotiorum. Thirty isolates belonging to Bacillus, Paenibacillus, Lysinibacillus, and Microbacterium genera were obtained. Several isolates produced auxin, siderophores, hydrolytic enzymes, fixed nitrogen and solubilized phosphate. Five isolates presented antifungal activity against S. sclerotiorum by the dual culture assay and four of them also inhibited fungal growth by volatile organic compounds production. All antagonistic isolates belonged to the Bacillus genus, and had their genomes sequenced for the search of biosynthetic gene clusters (BGC) related to antimicrobial metabolites. These isolates were identified as Bacillus safensis (3), Bacillus pumilus (1), and Bacillus megaterium (1), using the genomic metrics ANI and dDDH. Most strains showed several common BGCs, including bacteriocin, polyketide synthase (PKS), and non-ribosomal peptide synthetase (NRPS), related to pumilacidin, bacillibactin, bacilysin, and other antimicrobial compounds. Pumilacidin-related mass peaks were detected in acid precipitation extracts through MALDI-TOF analysis. The genomic features demonstrated the potential of these isolates in the suppression of plant pathogens; however, some aspects of plant-bacterial interactions remain to be elucidated.

Genomic metrics made easy: what to do and where to go in the new era of bacterial taxonomy.

With the advent of high-throughput DNA sequencing technologies, traditional methodologies for taxonomic classification of bacteria as DNA-DNA hybridization and 16S rRNA identity analyses are being challenged by the development of a fast-growing number of genomic metrics. The large amount of portable and digitized genome sequences available in public repositories constitutes an invaluable data for bacterial classification. Consequently, several genomic metrics and tools were developed to aid the interpretation of these massive data. Genomic metrics are based on the assumption that higher genome similarities would reflect closer phylogenetic relationships. Different metrics would vary in their methodology of analysis, resolution power, limitations and easiness of use. The aim of this review is to highlight the differences among available genome-based methods and tools to provide a guide for in silico bacterial identification and classification.

The combined analysis as the best strategy for Dual RNA-Seq mapping.

In Dual RNA-Seq experiments the simultaneous extraction of RNA and analysis of gene expression data from both interacting organisms could be a challenge. One alternative is separating the reads during in silico data analysis. There are two main mapping methods used: sequential and combined. Here we present a combined approach in which the libraries were aligned to a concatenated genome to sort the reads before mapping them to the respective annotated genomes. A comparison of this method with the sequential analysis was performed. Two RNA-Seq libraries available in public databases consisting of a eukaryotic (Zea mays) and a prokaryotic (Herbaspirillum seropediceae) organisms were mixed to simulate a Dual RNA-Seq experiment. Libraries from real Dual RNA-Seq experiments were also used. The sequential analysis consistently attributed more reads to the first reference genome used in the analysis (due to cross-mapping) than the combined approach. More importantly, the combined analysis resulted in lower numbers of cross-mapped reads. Our results highlight the necessity of combining the reference genomes to sort reads previously to the counting step to avoid losing information in Dual RNA-Seq experiments. Since most studies first map the RNA-Seq libraries to the eukaryotic genome, much prokaryotic information has probably been lost.

Paenibacillus helianthi sp. nov., a nitrogen fixing species isolated from the rhizosphere of Helianthus annuus L.

Three facultatively anaerobic endospore-forming bacteria were isolated from the rhizosphere of sunflowers grown in fields of Rio Grande do Sul State, Brazil. The designated type strain P26ET was previously identified as a sunflower growth promoting bacterium and is able to fix nitrogen and to excrete ammonia. According to analyses of 16S rRNA gene sequences, P26ET presented similarity values above 98.8% in relation to Paenibacillus azotifigens NF2-4-5T, Paenibacillus graminis RSA19T, Paenibacillus jilunlii Be17T, Paenibacillus salinicaeni LAM0A28T, and Paenibacillus sonchi X19-5T. Phylogenetic reconstructions based on 16S rRNA gene and core proteome data showed that the strains P26ET, P3E and P32E form a distinct clade, which did not include any type strain of the currently described Paenibacillus species. Also, genomic comparisons using average nucleotide identity (ANI), Orthologous ANI and in silico DNA-DNA hybridization revealed similarity ranges below the recommended thresholds when the three isolates from sunflower were compared to their close relatives. The DNA G + C content of strain P26ET was determined to be 49.4 mol%. The major cellular fatty acids are anteiso-C15:0 and iso-C15:0, representing about 58 and 14% of the total fatty acids in P26ET, respectively. Based on different taxonomic genomic metrics, phylogeny, and phenotypic data, we propose that strain P26ET (= DSM 102269 = BR10509) represents a novel species within the genus Paenibacillus, for which the name Paenibacillus helianthi sp. nov. is proposed.

Reclassification of Paenibacillus riograndensis as a Genomovar of Paenibacillus sonchi: Genome-Based Metrics Improve Bacterial Taxonomic Classification.

Species from the genus Paenibacillus are widely studied due to their biotechnological relevance. Dozens of novel species descriptions of this genus were published in the last couple of years, but few utilized genomic data as classification criteria. Here, we demonstrate the importance of using genome-based metrics and phylogenetic analyses to identify and classify Paenibacillus strains. For this purpose, Paenibacillus riograndensis SBR5T, Paenibacillus sonchi X19-5T, and their close relatives were compared through phenotypic, genotypic, and genomic approaches. With respect to P. sonchi X19-5T, P. riograndensis SBR5T, Paenibacillus sp. CAR114, and Paenibacillus sp. CAS34 presented ANI (average nucleotide identity) values ranging from 95.61 to 96.32%, gANI (whole-genome average nucleotide identity) values ranging from 96.78 to 97.31%, and dDDH (digital DNA-DNA hybridization) values ranging from 68.2 to 73.2%. Phylogenetic analyses of 16S rRNA, gyrB, recA, recN, and rpoB genes and concatenated proteins supported the monophyletic origin of these Paenibacillus strains. Therefore, we propose to assign Paenibacillus sp. CAR114 and Paenibacillus sp. CAS34 to P. sonchi species, and reclassify P. riograndensis SBR5T as a later heterotypic synonym of P. sonchi (type strain X19-5T), with the creation of three novel genomovars, P. sonchi genomovar Sonchi (type strain X19-5T), P. sonchi genomovar Riograndensis (type strain SBR5T), P. sonchi genomovar Oryzarum (type strain CAS34T = DSM 102041T; = BR10511T).

Detection of misidentifications of species from the Burkholderia cepacia complex and description of a new member, the soil bacterium Burkholderia catarinensis sp. nov.

The correct identification of bacteria from the Burkholderia cepacia complex (Bcc) is crucial for epidemiological studies and treatment of cystic fibrosis infections. However, genome-based identification tools are revealing many controversial Bcc species assignments. The aim of this work is to re-examine the taxonomic position of the soil bacterium B. cepacia 89 through polyphasic and genomic approaches. recA and 16S rRNA gene sequence analysis positioned strain 89 inside the Bcc group. However, based on the divergence score of seven concatenated allele sequences, and values of average nucleotide identity, and digital DNA:DNA hybridization, our results suggest that strain 89 is different from other Bcc species formerly described. Thus, we propose to classify Burkholderia sp. 89 as the novel species Burkholderia catarinensis sp. nov. with strain 89T (=DSM 103188T = BR 10601T) as the type strain. Moreover, our results call the attention to some probable misidentifications of Bcc genomes at the National Center for Biotechnology Information database.

How to transform a recalcitrant Paenibacillus strain: From culture medium to restriction barrier.

Paenibacillus riograndensis SBR5T is a plant growth-promoting bacterium isolated from the wheat rhizosphere. Its recalcitrance to genetic manipulation is a major bottleneck for molecular studies, as has been reported for other Paenibacillus environmental isolates. An efficient electroporation protocol was established by evaluating diverse parameters and optimizing the culture medium, culture growth phase, electroporation solution, recovery medium, DNA input, and electric field strength. Efficiencies of approximately 2.8×104transformantsµg-1 of plasmid DNA were obtained. The optimized protocol was tested with other Paenibacillus species, and the relevance of bypassing the restriction DNA defense system to transform Paenibacillus was highlighted. This protocol is the tool needed to deepen molecular studies with this strain and will aid in the manipulation of other new environmental isolates that also exhibit recalcitrant transformation difficulties.

Complete genome sequence of Paenibacillus riograndensis SBR5(T), a Gram-positive diazotrophic rhizobacterium.

Paenibacillus riograndensis is a Gram-positive rhizobacterium which exhibits plant growth promoting activities. It was isolated from the rhizosphere of wheat grown in the state of Rio Grande do Sul, Brazil. Here we announce the complete genome sequence of P. riograndensis strain SBR5(T). The genome of P. riograndensis SBR5(T) consists of a circular chromosome of 7,893,056bps. The genome was finished and fully annotated, containing 6705 protein coding genes, 87 tRNAs and 27 rRNAs. The knowledge of the complete genome helped to explain why P. riograndensis SBR5(T) can grow with the carbon sources arabinose and mannitol, but not myo-inositol, and to explain physiological features such as biotin auxotrophy and antibiotic resistances. The genome sequence will be valuable for functional genomics and ecological studies as well as for application of P. riograndensis SBR5(T) as plant growth-promoting rhizobacterium.

Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils.

The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45-55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.