RESUMO
Repair of uracils in DNA is initiated by uracil DNA glycosylases (UDGs). Family 1 UDGs (Ung) are the most efficient and ubiquitous proteins having an exquisite specificity for uracils in DNA. Ung are characterized by motifs A (GQDPY) and B (HPSPLS) sequences. We report a novel dimeric UDG, Blr0248 (BdiUng) from Bradyrhizobium diazoefficiens. Although BdiUng contains the motif A (GQDPA), it has low sequence identity to known UDGs. BdiUng prefers single stranded DNA and excises uracil, 5-hydroxymethyl-uracil or xanthine from it. BdiUng is impervious to inhibition by AP DNA, and Ugi protein that specifically inhibits family 1 UDGs. Crystal structure of BdiUng shows similarity with the family 4 UDGs in its overall fold but with family 1 UDGs in key active site residues. However, instead of a classical motif B, BdiUng has a uniquely extended protrusion explaining the lack of Ugi inhibition. Structural and mutational analyses of BdiUng have revealed the basis for the accommodation of diverse substrates into its substrate binding pocket. Phylogenetically, BdiUng belongs to a new UDG family. Bradyrhizobium diazoefficiens presents a unique scenario where the presence of at least four families of UDGs may compensate for the absence of an efficient family 1 homologue.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/enzimologia , Reparo do DNA , DNA Bacteriano/metabolismo , Uracila-DNA Glicosidase/metabolismo , Uracila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Bradyrhizobium/genética , Clonagem Molecular , Cristalografia por Raios X , Dano ao DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/genéticaRESUMO
The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C(1) compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/Methylobacterium symbiosis.
Assuntos
Fabaceae/microbiologia , Metanol/metabolismo , Methylobacterium/isolamento & purificação , Nódulos Radiculares de Plantas/microbiologia , Oxirredutases do Álcool/genética , Meios de Cultura , Genes Bacterianos , Genes de RNAr , Genoma Bacteriano , Methylobacterium/genética , Methylobacterium/crescimento & desenvolvimento , Methylobacterium/metabolismo , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , África do Sul , Especificidade da Espécie , SimbioseRESUMO
Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N(2) was evaluated. N(2) fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed. Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells. The Sm1021-M. truncatula symbiosis is poorly matched for N(2) fixation and the strain could possess broader N(2) fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N(2) fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.
Assuntos
Medicago truncatula/microbiologia , Fixação de Nitrogênio/fisiologia , Sinorhizobium meliloti/metabolismo , Simbiose , Medicago truncatula/anatomia & histologia , Medicago truncatula/crescimento & desenvolvimento , Modelos Biológicos , Dados de Sequência Molecular , Brotos de Planta/anatomia & histologia , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/microbiologia , Nódulos Radiculares de Plantas/anatomia & histologia , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Sinorhizobium meliloti/genéticaRESUMO
ActS-ActR proteins belong to a highly conserved family of two-component signal transduction systems involved in global regulation in the alpha-proteobacteria; they were first identified in Sinorhizobium medicae (previously Sinorhizobium meliloti) as essential for acid-tolerance. This paper reports on the identification of genes regulated by ActS and/or ActR in S. medicae. To do this, random gusA fusions were created in S. medicae to follow gene transcription in an actS chromosomal knockout mutant containing plasmid-borne actS. Plasmid borne actS was cured from the mutants and beta-glucuronidase (GUS) activity compared between the different genetic backgrounds. We detected actS-dependent regulation of the genes gst1 (detoxification), hyuA (hydantoin utilization) and fixN2 (microaerobic respiration). We show that ActR is involved in regulating cbbS (CO2 fixation), narB (nitrate assimilation) and required for low pH and microaerobic induction of the nitrogen fixation regulators fixK and nifA. In particular, we demonstrate that the transcriptional activation of fixN2 is regulated by ActR through FixK.
Assuntos
Ácidos/metabolismo , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais/genética , Sinorhizobium/genética , Sinorhizobium/metabolismo , Proteínas de Bactérias/genética , Dióxido de Carbono/metabolismo , Genes Reporter , Testes Genéticos , Concentração de Íons de Hidrogênio , Mutagênese Insercional , Fixação de Nitrogênio/genética , Oxigênio/metabolismo , Sinorhizobium/crescimento & desenvolvimento , Ativação Transcricional/fisiologiaRESUMO
Biserrula pelecinus L. is a pasture legume that was introduced to Australia from the Mediterranean basin in 1993. Although the native rhizobial population could not nodulate B. pelecinus at the time of its introduction, recent research has shown the emergence of a diversity of strains (novel isolates) that are able to do so. Three novel isolates, WSM2073T, WSM2074 and WSM2076, had nearly identical 16S rRNA gene sequences, and clustered separately with all recognized species of the genus Mesorhizobium. Conversely, the novel isolate WSM2075T had >23 nt mismatches with the above three isolates. All four novel isolates shared 97-99% 16S rRNA gene sequence similarity with the type strains of all recognized Mesorhizobium species. However, strains WSM2073T, WSM2074 and WSM2076 showed <95.2% dnaK gene sequence similarity to the type strains of recognized Mesorhizobium species, and <92.9% to WSM2075T (which also shared <95.5% dnaK gene sequence similarity to the type strains of recognized Mesorhizobium species). Results for GSII gene sequencing were consistent with those for the dnaK gene. The fatty acid profiles of the novel isolates were diagnostic of root-nodule bacteria, but did not match those of recognized bacterial species. Strain WSM2075T had a significantly different fatty acid profile from the other three isolates. The above results indicated that strains WSM2073T, WSM2074 and WSM2076 represent the same species. Strain WSM2073T showed <45% DNA-DNA relatedness and WSM2075T<5% DNA-DNA relatedness with the type strains of recognized Mesorhizobium species; these two novel isolates shared 59% DNA-DNA relatedness. Collectively, these data indicate that strains WSM2073T, WSM2074 and WSM2076, and strain WSM2075T belong to two novel species of the genus Mesorhizobium, for which the names Mesorhizobium australicum sp. nov. and Mesorhizobium opportunistum sp. nov. are proposed, respectively. The type strain of Mesorhizobium australicum sp. nov. is WSM2073T (=LMG 24608T=HAMBI 3006T) and the type strain of Mesorhizobium opportunistum sp. nov. is WSM2075T (=LMG 24607T=HAMBI 3007T).
Assuntos
Alphaproteobacteria/classificação , Alphaproteobacteria/isolamento & purificação , Fabaceae/microbiologia , Alphaproteobacteria/genética , Austrália , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Biserrula pelecinus L. is a pasture legume species that forms a highly specific nitrogen-fixing symbiotic interaction with a group of bacteria that belong to Mesorhizobium. These mesorhizobia have >98.8 % sequence similarity to Mesorhizobium ciceri and Mesorhizobium loti for the 16S rRNA gene (1440 bp) and >99.3 % sequence similarity to M. ciceri for the dnaK gene (300 bp), and strain WSM1271 has 100 % sequence similarity to M. ciceri for GSII (600 bp). Strain WSM1271 had 85 % relatedness to M. ciceri LMG 14989(T) and 50 % relatedness to M. loti LMG 6125(T) when DNA-DNA hybridization was performed. WSM1271 also had a similar cellular fatty acid profile to M. ciceri. These results are strong evidence that the Biserrula mesorhizobia and M. ciceri belong to the same group of bacteria. Significant differences were revealed between the Biserrula mesorhizobia and M. ciceri in growth conditions, antibiotic resistance and carbon source utilization. The G+C content of the DNA of WSM1271 was 62.7 mol%, compared to 63-64 mol% for M. ciceri. The Biserrula mesorhizobia contained a plasmid ( approximately 500 bp), but the symbiotic genes were detected on a mobile symbiosis island and considerable variation was present in the symbiotic genes of Biserrula mesorhizobia and M. ciceri. There was <78.6 % sequence similarity for nodA and <66.9 % for nifH between Biserrula mesorhizobia and M. ciceri. Moreover, the Biserrula mesorhizobia did not nodulate the legume host of M. ciceri, Cicer arietinum, and M. ciceri did not nodulate B. pelecinus. These significant differences observed between Biserrula mesorhizobia and M. ciceri warrant the proposal of a novel biovar for Biserrula mesorhizobia within M. ciceri. The name Mesorhizobium ciceri biovar biserrulae is proposed, with strain WSM1271 (=LMG 23838=HAMBI 2942) as the reference strain.
Assuntos
Alphaproteobacteria/classificação , Fabaceae/microbiologia , Aciltransferases/genética , Alphaproteobacteria/genética , Alphaproteobacteria/fisiologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Metabolismo dos Carboidratos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Farmacorresistência Bacteriana , Fabaceae/fisiologia , Ácidos Graxos/análise , Genes de RNAr , Ilhas Genômicas/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oxirredutases/genética , Filogenia , Plasmídeos/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Simbiose/genéticaRESUMO
The multi-billion dollar asset attributed to symbiotic nitrogen fixation is often threatened by the nodulation of legumes by rhizobia that are ineffective or poorly effective in N(2) fixation. This study investigated the development of rhizobial diversity for the pasture legume Biserrula pelecinus L., 6 years after its introduction, and inoculation with Mesorhizobium ciceri bv. biserrulae strain WSM1271, to Western Australia. Molecular fingerprinting of 88 nodule isolates indicated seven were distinctive. Two of these were ineffective while five were poorly effective in N(2) fixation on B. pelecinus. Three novel isolates had wider host ranges for nodulation than WSM1271, and four had distinct carbon utilization patterns. Novel isolates were identified as Mesorhizobium sp. using 16S rRNA, dnaK and GSII phylogenies. In a second study, a large number of nodules were collected from commercially grown B. pelecinus from a broader geographical area. These plants were originally inoculated with M. c bv. biserrulae WSM1497 5-6 years prior to isolation of strains for this study. Nearly 50% of isolates from these nodules had distinct molecular fingerprints. At two sites diverse strains dominated nodule occupancy indicating recently evolved strains are highly competitive. All isolates tested were less effective and six were ineffective in N(2) fixation. Twelve randomly selected diverse isolates clustered together, based on dnaK sequences, within Mesorhizobium and distantly to M. c bv. biserrulae. All 12 had identical sequences for the symbiosis island insertion region with WSM1497. This study shows the rapid evolution of competitive, yet suboptimal strains for N(2) fixation on B. pelecinus following the lateral transfer of a symbiosis island from inoculants to other soil bacteria.
Assuntos
Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Fabaceae/microbiologia , Transferência Genética Horizontal , Ilhas Genômicas/genética , Fixação de Nitrogênio/genética , Sequência de Bases , Evolução Biológica , Fabaceae/metabolismo , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Microbiologia do SoloRESUMO
Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA and nifH from an inoculant to soil bacteria. Transfer of these chromosomal genes and the presence of an identical integrase gene adjacent to a Phe tRNA gene in both the inoculant and recipients indicate that there was lateral transfer of a symbiosis island.
Assuntos
Evolução Molecular , Fabaceae/microbiologia , Transferência Genética Horizontal , Ilhas Genômicas/genética , Rhizobium/genética , Aciltransferases/genética , Proteínas de Bactérias/genética , Integrases/genética , Oxirredutases/genética , Aminoacil-RNA de Transferência/genética , Fatores de TempoRESUMO
Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Sinorhizobium/genética , Sinorhizobium/fisiologia , Transativadores/fisiologia , Adaptação Fisiológica/genética , Antibacterianos/farmacologia , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Deleção de Genes , Glucuronidase/análise , Glucuronidase/genética , Concentração de Íons de Hidrogênio , Medicago/microbiologia , Lipídeos de Membrana/análise , Viabilidade Microbiana , Dados de Sequência Molecular , Fixação de Nitrogênio , Regiões Promotoras Genéticas , Nódulos Radiculares de Plantas/microbiologia , Análise de Sequência de DNARESUMO
The actA gene, which is disrupted by Tn5 in the acid-sensitive mutant of Rhizobium meliloti TG2-6, was cloned and sequenced. It encodes a protein of 541 amino acids with a calculated molecular mass of 57,963 Da and an estimated pl of 9.0. The ActA protein sequence has 30% identity, and much higher similarity (69%), with the CutE protein of Escherichia coli. Like the cutE mutant of E. coli, TG2-6 is sensitive to copper. The reconstructed wild-type actA gene complemented the low pH- and copper-sensitive phenotype of TG2-6. Studies with an actA-lacZ gene fusion showed that actA is constitutively expressed at pH 5.8 and 7.0. The actA gene appears to be chromosomal and is present in all seven strains of R. meliloti tested.
Assuntos
Acetiltransferases , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Sinorhizobium meliloti/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Análise de Sequência , Sinorhizobium meliloti/metabolismoRESUMO
A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by Tn5 in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71.3% identity over 892 bp). At the protein level there was 70% identity and 93.3% similarity over 267 amino acids with the ExoR protein of R. meliloti Rm1021. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM710. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/fisiologia , Polissacarídeos Bacterianos/biossíntese , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Genes Bacterianos/genética , Teste de Complementação Genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese/genética , Fenótipo , Polissacarídeos Bacterianos/química , Rhizobium leguminosarum/isolamento & purificação , Análise de Sequência de DNA , Simbiose/genética , Simbiose/fisiologiaRESUMO
The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivity of Medicago species (such as lucerne) sown in acidic soils. To investigate the pH response of an acid-tolerant Sinorhizobium medicae strain, a pool of random promoter fusions to gusA was created using minitransposon insertional mutagenesis. Acid-activated expression was identified in 11 mutants; rhizobial DNA flanking insertions in 10 mutants could be cloned and the DNA sequences obtained were used to interrogate the genome database of Sinorhizobium meliloti strain 1021. Acid activated expression was detected for fixNO, kdpC, lpiA, and phrR and for genes encoding a putative lipoprotein, two ABC-transporter components, a putative DNA ligase and a MPA1-family protein. These findings implicate cytochrome synthesis, potassium ion cycling, lipid biosynthesis and transport processes as key components of pH response in S. medicae.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Sinorhizobium/genética , DNA Bacteriano/genética , Marcadores Genéticos , Medicago/microbiologia , Mutagênese , Doenças das Plantas/microbiologia , Transcrição Gênica/genéticaRESUMO
The phrR gene in Sinorhizobium meliloti (previously known as Rhizobium meliloti) WSM419, directly downstream from actA, is induced by low pH or certain stresses (e.g. high concentrations of Zn2+, Cu2+, H2O2 or ethanol), but not in stationary phase or by other stresses (e.g. phosphate limitation, elevated temperature, high concentrations of sucrose or iron). A DNA fragment containing the wild-type phrR gene could not be cloned and inverse PCR was therefore used to amplify a 3.5 kb BamHI fragment containing phrR from the mutant S. meliloti TG2-6 (actA::Tn5). DNA fragments from a BamHI/SalI digest of the amplified product were cloned into pUK21 and sequenced. The phrR open reading frame contiguous to actA appears to code for a 15.2 kDa protein showing significant identity with the proteins encoded by y4wC and y4aM in Rhizobium sp. NGR234. All three proteins resemble transcriptional regulators in containing a DNA-binding helix-turn-helix motif similar to that reported for URF4 in Rhodospirillum rubrum and repressors in coliphage.
Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Sinorhizobium meliloti/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Concentração de Íons de Hidrogênio , Proteínas de Membrana/genética , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Two 'calcium-irreparable' acid-sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti with Tn5. Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P-type ATPase that belongs to the CPx heavy metal-transporting subfamily. Here, we show that both actP-knockout mutants show sensitivity to copper; omission of this heavy metal from low pH-buffered media restores acid tolerance to these strains. Furthermore, complementation of the mutant phenotype requires only the actPgene. An actP-gusA fusion in R. leguminosarum was transcriptionally regulated by copper in a pH-dependent manner.Downstream to actP in both organisms is the hmrR gene that encodes a heavy metal-responsive regulator (HmrR) that belongs to the merR class of regulatory genes. Insertional Inactivation of hmrR abolished transcriptional activation of actP by copper ions and increased the basal level of its expression in their absence. These observations suggest that HmrR can regulate actP transcription positively and negatively. We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Cobre/metabolismo , Rhizobium leguminosarum/enzimologia , Sinorhizobium meliloti/enzimologia , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Cálcio , Proteínas de Transporte/genética , Elementos de DNA Transponíveis , DNA Bacteriano , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Homeostase , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta , Fenótipo , Rhizobium leguminosarum/genética , Análise de Sequência de DNA , Sinorhizobium meliloti/genética , Simbiose , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.
Assuntos
Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Mutagênese Insercional , Rhizobiaceae/genética , Sequência de Bases , Clonagem Molecular/métodos , Elementos de DNA Transponíveis/genética , Resistência Microbiana a Medicamentos/genética , Genes Reporter/genética , Engenharia Genética , Vetores Genéticos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mapeamento por Restrição , Transcrição GênicaRESUMO
To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we have identified acid-activated gene transcription and now complement this approach by using a proteomic analysis to identify the changes that occur following exposure to acidity. Protein profiles of persistently or transiently acid-stressed S. medicae cells were compared to those grown in pH neutral, buffered media. Fifty pH-regulated proteins were identified; N-terminal sequences for 15 of these were obtained using the Edman degradation. Transient acid exposure downregulated GlnA and GlnK and upregulated a hypothetical protein. Continuing acid exposure downregulated ClpP, an ABC transporter, a hypothetical protein, a lipoprotein, the Trp-like repressor WrbA1 and upregulated DegP, fructose bisphosphate aldolase, GroES, malate dehydrogenase and two hypothetical proteins. These findings implicate proteolytic, chaperone and transport processes as key components of pH response in S. medicae.