RESUMO
Molecular mechanisms of plant recognition and colonization by diazotrophic bacteria are barely understood. Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, to promote plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicaeâ SmR1 recovered 1 and 3 days after inoculation. The results indicated that nitrogen metabolism was strongly activated in the rhizosphere and polyhydroxybutyrate storage was mobilized in order to assist the survival of H. seropedicae during the early stages of colonization. Epiphytic cells showed altered transcription levels of several genes associated with polysaccharide biosynthesis, peptidoglycan turnover and outer membrane protein biosynthesis, suggesting reorganization of cell wall envelope components. Specific methyl-accepting chemotaxis proteins and two-component systems were differentially expressed between populations over time, suggesting deployment of an extensive bacterial sensory system for adaptation to the plant environment. An insertion mutation inactivating a methyl-accepting chemosensor induced in planktonic bacteria, decreased chemotaxis towards the plant and attachment to roots. In summary, analysis of mutant strains combined with transcript profiling revealed several molecular adaptations that enable H. seropedicae to sense the plant environment, attach to the root surface and survive during the early stages of maize colonization.
Assuntos
Herbaspirillum/crescimento & desenvolvimento , Zea mays/microbiologia , Aclimatação , Adaptação Fisiológica , Herbaspirillum/genética , Herbaspirillum/isolamento & purificação , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Rizosfera , Microbiologia do Solo , Zea mays/crescimento & desenvolvimentoRESUMO
The ammonium transport family Amt/Rh comprises ubiquitous integral membrane proteins that facilitate ammonium movement across biological membranes. Besides their role in transport, Amt proteins also play a role in sensing the levels of ammonium in the environment, a process that depends on complex formation with cytosolic proteins of the P(II) family. Trimeric P(II) proteins from a variety of organisms undergo a cycle of reversible posttranslational modification according to the prevailing nitrogen supply. In proteobacteria, P(II) proteins are subjected to reversible uridylylation of each monomer. In this study we used the purified proteins from Azospirillum brasilense to analyze the effect of P(II) uridylylation on the protein's ability to engage complex formation with AmtB in vitro. Our results show that partially uridylylated P(II) trimers can interact with AmtB in vitro, the implication of this finding in the regulation of nitrogen metabolism is discussed. We also report an improved expression and purification protocol for the A. brasilense AmtB protein that might be applicable to AmtB proteins from other organisms.
Assuntos
Azospirillum brasilense/química , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Nitrogênio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Uridina Monofosfato/químicaRESUMO
The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energy- expensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the P(II) signal transduction proteins act as key players.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Nitrogenase/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Transdução de Sinais , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Nitrogenase/genética , Proteínas PII Reguladoras de Nitrogênio/genética , Processamento de Proteína Pós-TraducionalRESUMO
Proteins belonging to the P(II) family coordinate cellular nitrogen metabolism by direct interaction with a variety of enzymes, transcriptional regulators and transporters. The sensing function of P(II) relies on its ability to bind the nitrogen/carbon signalling molecule 2-oxoglutarate (2-OG). In Proteobacteria, P(II) is further subject to reversible uridylylation according to the intracellular levels of glutamine, which reflect the cellular nitrogen status. A number of P(II) proteins have been shown to bind ADP and ATP in a competitive manner, suggesting that P(II) might act as an energy sensor. Here, we analyse the influence of the ADP/ATP ratio, 2-OG levels and divalent metal ions on in vitro uridylylation of the Azospirillum brasilense P(II) proteins GlnB and GlnZ, and on interaction with their targets AmtB, DraG and DraT. The results support the notion that the cellular concentration of 2-OG is a key factor governing occupation of the GlnB and GlnZ nucleotide binding sites by ATP or ADP, with high 2-OG levels favouring the occupation of P(II) by ATP. Both P(II) uridylylation and interaction with target proteins responded to the ADP/ATP ratio within the expected physiological range, supporting the concept that P(II) proteins might act as cellular energy sensors.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Cátions Bivalentes/metabolismo , Ácidos Cetoglutáricos/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas PII Reguladoras de Nitrogênio/genética , Transdução de SinaisRESUMO
BACKGROUND: Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. RESULTS: A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. CONCLUSIONS: Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice.
Assuntos
Sistemas de Secreção Bacterianos/genética , Endófitos/patogenicidade , Herbaspirillum/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas de Membrana Transportadoras/genética , Doenças das Plantas/microbiologia , Poaceae/microbiologia , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Endófitos/genética , Deleção de Genes , Herbaspirillum/genética , Dados de Sequência Molecular , Família Multigênica , Mutagênese Insercional , Análise de Sequência de DNA , Fatores de Virulência/genéticaRESUMO
PII are signal-transducing proteins that integrate metabolic signals and transmit this information to a large number of proteins. In proteobacteria, PII are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. The uridylylation/deuridylylation cycle of PII is also regulated by carbon and energy signals such as ATP, ADP and 2-oxoglutarate (2-OG). These molecules bind to PII proteins and alter their tridimensional structure/conformation and activity. In this work, we determined the effects of ATP, ADP and 2-OG levels on the in vitro uridylylation of Herbaspirillum seropedicae PII proteins, GlnB and GlnK. Both proteins were uridylylated by GlnD in the presence of ATP or ADP, although the uridylylation levels were higher in the presence of ATP and under high 2-OG levels. Under excess of 2-OG, the GlnB uridylylation level was higher in the presence of ATP than with ADP, while GlnK uridylylation was similar with ATP or ADP. Moreover, in the presence of ADP/ATP molar ratios varying from 10/1 to 1/10, GlnB uridylylation level decreased as ADP concentration increased, whereas GlnK uridylylation remained constant. The results suggest that uridylylation of both GlnB and GlnK responds to 2-OG levels, but only GlnB responds effectively to variation on ADP/ATP ratio.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Herbaspirillum/enzimologia , Ácidos Cetoglutáricos/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Herbaspirillum/metabolismo , Nitrogênio/metabolismo , Nucleotidiltransferases/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
BACKGROUND: The PII protein family comprises homotrimeric proteins which act as transducers of the cellular nitrogen and carbon status in prokaryotes and plants. In Herbaspirillum seropedicae, two PII-like proteins (GlnB and GlnK), encoded by the genes glnB and glnK, were identified. The glnB gene is monocistronic and its expression is constitutive, while glnK is located in the nlmAglnKamtB operon and is expressed under nitrogen-limiting conditions. RESULTS: In order to determine the involvement of the H. seropedicae glnB and glnK gene products in nitrogen fixation, a series of mutant strains were constructed and characterized. The glnK- mutants were deficient in nitrogen fixation and they were complemented by plasmids expressing the GlnK protein or an N-truncated form of NifA. The nitrogenase post-translational control by ammonium was studied and the results showed that the glnK mutant is partially defective in nitrogenase inactivation upon addition of ammonium while the glnB mutant has a wild-type phenotype. CONCLUSIONS: Our results indicate that GlnK is mainly responsible for NifA activity regulation and ammonium-dependent post-translational regulation of nitrogenase in H. seropedicae.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum/genética , Herbaspirillum/metabolismo , Fixação de Nitrogênio , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Mutagênese , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/genética , Regiões Promotoras Genéticas , Compostos de Amônio Quaternário/metabolismoRESUMO
BACKGROUND: Herbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism. RESULTS: In this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes. CONCLUSIONS: Our results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival in planta.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Herbaspirillum/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Proteínas de Ligação a DNA/genética , Herbaspirillum/genética , Dados de Sequência Molecular , Ligação ProteicaRESUMO
BACKGROUND: Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant. RESULTS: In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v). CONCLUSIONS: The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.
Assuntos
Proteínas de Bactérias/química , Lipase/química , Metagenômica , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Lipase/classificação , Lipase/genética , Dados de Sequência Molecular , Filogenia , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Estereoisomerismo , Especificidade por Substrato , Temperatura , Yersinia enterocolitica/enzimologiaRESUMO
The Amazon rainforest is the world's largest tropical forest, and this biome may be a significant contributor to primary biological aerosol (PBA) emissions on a global scale. These aerosols also play a pivotal role in modulating ecosystem dynamics, dispersing biological material over geographic barriers and influencing climate through radiation absorption, light scattering, or acting as cloud condensation nuclei. Despite their importance, there are limited studies investigating the effect of environmental variables on the bioaerosol composition in the Amazon rainforest. Here we present a 16S rRNA gene-based amplicon sequencing approach to investigate the bacterial microbiome in aerosols of the Amazon rainforest during distinct seasons and at different heights above the ground. Our data revealed that seasonal changes in temperature, relative humidity, and precipitation are the primary drivers of compositional changes in the Amazon rainforest aerosol microbiome. Interestingly, no significant differences were observed in the bacterial community composition of aerosols collected at ground and canopy levels. The core airborne bacterial families present in Amazon aerosol were Enterobacteriaceae, Beijerinckiaceae, Polyangiaceae, Bacillaceae and Ktedonobacteraceae. By correlating the bacterial taxa identified in the aerosol with literature data, we speculate that the phyllosphere may be one possible source of airborne bacteria in the Amazon rainforest. Results of this study indicate that the aerosol microbiota of the Amazon Rainforest are fairly diverse and principally impacted by seasonal changes in temperature and humidity.
Assuntos
Microbiota , Floresta Úmida , Aerossóis , Florestas , Humanos , RNA Ribossômico 16S/genéticaRESUMO
In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues.
Assuntos
Genes Bacterianos , Herbaspirillum/genética , Ramnose/biossíntese , Zea mays/microbiologia , Aderência Bacteriana , Biofilmes , DNA Forma A/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Glucosamina/farmacologia , Herbaspirillum/fisiologia , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/fisiologia , Mutação , Fenótipo , Raízes de Plantas/microbiologia , Polimixina B/farmacologia , Ramnose/fisiologia , Sensibilidade e EspecificidadeRESUMO
Biological aerosols (bioaerosol) are atmospheric particles that act as a dispersion unit of living organisms across the globe thereby affecting the biogeographic distribution of organisms. Despite their importance, there is virtually no knowledge about bioaerosols emitted by pristine forests. Here we provide the very first survey of the prokaryotic community of a bioaerosol collected inside pristine Amazon forest at 2â¯m above ground. Total atmospheric particles were collected at the Amazon Tall Tower Observatory, subjected to metagenomic DNA extraction and the prokaryotic diversity was determined by 16S rRNA gene amplicon sequencing. A total of 271,577 reads of 250â¯bp of the 16S rRNA gene amplicon were obtained. Only 27% of the reads could be classified using the 16S SILVA database. Most belonged to Proteobacteria, Actinobacteria and Firmicutes which is in good agreement with other bioaerosol studies. Further inspection of the reads using Blast searches and the 18S SILVA database revealed that most of the dataset was composed of Fungi sequences. The identified microbes suggest that the atmosphere may act as an important gateway to interchange bacteria between plants, soil and water ecosystems.
Assuntos
Aerossóis/análise , Microbiologia do Ar , Florestas , Biodiversidade , Brasil , Monitoramento AmbientalRESUMO
Mangroves are highly productive ecosystems located at the transition between the terrestrial and marine environments. Mangroves play an important role in carbon storage, nutrient cycling and support for the marine food web. Mangrove soils are formed by fine particles rich in organic carbon and are subject to constant fluctuations in oxygen, salinity and nutrient availability due to fresh water flux and tidal variations. Microbes play an important role in nutrient cycling in mangrove soils; however, studies on the mangrove soil microbiome are scarce. Here we compare the microbiome of pristine mangrove soil located in an environmentally protected area in Guaratuba, Southern Brazil, with the microbiome of mangrove soil affected by the presence of carbonaceaous debris eroding from an archeological site known as Sambaqui. We show that although the Sambaqui site has a major effect on soil chemistry, increasing the soil pH by 2.6â¯units, only minor changes in the soil microbiome were detected indicating resilience of the microbial community to pH variations. The high alpha diversity indexes and predicted metabolic potential suggest that the mangrove soil microbiome not only provides important ecological services but also may host a broad range of microbes and genes of biotechnological interest.
Assuntos
Monitoramento Ambiental , Microbiologia do Solo , Áreas Alagadas , Brasil , Carbono , Microbiota , SoloRESUMO
Herbaspirillum seropedicae is a diazotrophic bacterium which associates endophytically with economically important gramineae. Flavonoids such as naringenin have been shown to have an effect on the interaction between H. seropedicae and its host plants. We used a high-throughput sequencing based method (RNA-Seq) to access the influence of naringenin on the whole transcriptome profile of H. seropedicae. Three hundred and four genes were downregulated and seventy seven were upregulated by naringenin. Data analysis revealed that genes related to bacterial flagella biosynthesis, chemotaxis and biosynthesis of peptidoglycan were repressed by naringenin. Moreover, genes involved in aromatic metabolism and multidrug transport efllux were actived.
RESUMO
We report the complete genome sequence of Herbaspirillum hiltneri N3 (DSM 17495), a member of the genus Herbaspirillum of the Betaproteobacteria. The genome is contained in a single chromosome, and analysis revealed that N3 lacks the whole nitrogen fixation (nif) gene cluster, confirming its inability to fix nitrogen.
RESUMO
The Azospirillum brasilense transcription regulator NifA and the nitrogen-status signaling proteins GlnB, GlnZ and GlnK were expressed in Escherichia coli and analyzed for their ability to activate nif gene expression. When expressed separately, none of the proteins were able to activate nifH promoter expression in any tested conditions; in contrast, nifH expression was observed in cells grown in the absence of ammonium and oxygen and when expressing simultaneously NifA and GlnB proteins, but not when expressing NifA and GlnZ or GlnK. Our results show that the GlnB protein is required for transcription activation by Azospirillum brasilense NifA and it cannot be replaced by GlnZ or GlnK.
Assuntos
Azospirillum brasilense/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Genes Bacterianos , Fixação de Nitrogênio/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fixação de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio , Fatores de Transcrição/genéticaRESUMO
The transcriptional regulatory protein Fnr, acts as an intracellular redox sensor regulating a wide range of genes in response to changes in oxygen levels. Genome sequencing of Herbaspirillum seropedicae SmR1 revealed the presence of three fnr-like genes. In this study we have constructed single, double and triple fnr deletion mutant strains of H. seropedicae. Transcriptional profiling in combination with expression data from reporter fusions, together with spectroscopic analysis, demonstrates that the Fnr1 and Fnr3 proteins not only regulate expression of the cbb3-type respiratory oxidase, but also control the cytochrome content and other component complexes required for the cytochrome c-based electron transport pathway. Accordingly, in the absence of the three Fnr paralogs, growth is restricted at low oxygen tensions and nitrogenase activity is impaired. Our results suggest that the H. seropedicae Fnr proteins are major players in regulating the composition of the electron transport chain in response to prevailing oxygen concentrations.
Assuntos
Proteínas de Bactérias/metabolismo , Citocromos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Herbaspirillum/fisiologia , Oxigênio/metabolismo , Proteínas de Bactérias/genética , Citocromos/genética , Proteínas de Ligação a DNA/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mutagênese Sítio-DirigidaRESUMO
Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum/metabolismo , Hidroxibutiratos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Herbaspirillum/genética , Espectrometria de MassasRESUMO
Here we report the one-scaffold draft genome of Herbaspirillum huttiense subsp. putei strain 7-2T (IAM 15032), which was isolated from well water.
RESUMO
Lipid-A was isolated by mild acid hydrolysis from lipopolysaccharides extracted from cells of Herbaspirillum seropedicae, strain SMR1, and from two mutants deficient in the biosynthesis of rhamnose (rmlBâ» and rmlCâ»). Structural analyzes were carried out using MALDI-TOF and derivatization by per-O-trimethylsilylation followed by GC-MS in order to determine monosaccharide and fatty acid composition. De-O-acylation was also performed to determine the presence of N-linked fatty acids. Lipid-A from H. seropedicae SMR1 showed a major structure comprising 2-amino-2-deoxy-glucopyranose-(1â6)-2-amino-2-deoxy-glucopyranose phosphorylated at C4' and C1 positions, each carrying a unit of 4-amino-4-deoxy-arabinose. C2 and C2' positions were substituted by amide-linked 3-hydroxy-dodecanoic acids. Both rhamnose-defective mutants showed similar structure for their lipid-A moieties, except for the lack of 4-amino-4-deoxy-arabinose units attached to phosphoryl groups.