RESUMEN
Two studies were conducted to determine the relationship of feeding behavior to a phenotypic expression of residual feed intake (RFI), a measure of efficiency. In Exp. 1, a feedlot diet containing roughage was fed (traditional). In Exp. 2, a no-roughage diet was fed. Residual feed intake, a measure of feed efficiency, was calculated for both studies. In Exp. 1, six feed-efficient (low RFI) steers and six feed-inefficient steers (high RFI) were selected from a contemporary group of 80 steers, and feeding behaviors were analyzed. In Exp. 2, nine feed-efficient and eight feed-inefficient steers were selected from a contemporary group of 40 steers. There were no differences (P > 0.13) in initial or final BW or ADG between efficient and inefficient groups in either Exp. 1 or 2. In Exp. 1, DMI and average eating bouts daily differed (P < 0.001), with efficient steers consuming less feed and eating fewer times per day. In Exp. 2, efficient steers consumed less (P < 0.001) feed, and average eating bouts daily tended (P = 0.07) to be fewer in efficient animals. Limited differences were noted in feeding behavior between groups, with inefficient steers from both studies having a more variable eating pattern throughout the day. The average daily eating rate did not differ (P > 0.20) between groups in either experiment. The average number of days comprising a feeding pattern for both efficiency groups in Exp. 1 and 2 was found to be 2 to 3 d and multiples of 2 to 3 d. In Exp. 1, the feed intake pattern of efficient and inefficient steers changed once they reached a BW of approximately 391 and 381 kg, respectively. This occurred near d 47 for the efficient steers and near d 32 for inefficient steers. In Exp. 2, the feed intake pattern of both efficient and inefficient steers changed once they reached a BW of approximately 399 kg, which occurred on d 31 for the efficient steers and on d 33 for the inefficient steers. From the measured variables, there were no differences in growth and limited differences noted in feeding behavior between efficient and inefficient groups. The results of the trials suggest increased variability of feed intake throughout the day for inefficient animals.
Asunto(s)
Alimentación Animal/análisis , Bovinos/crecimiento & desarrollo , Dieta/veterinaria , Conducta Alimentaria/fisiología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Composición Corporal , Masculino , Factores de TiempoRESUMEN
The project objective was to determine the CLA content of three muscles (Longissimus lumborum, LD; Semimembranosus, SM; Triceps brachii, TB), in both raw and cooked states, in cattle finished on pasture or with grain supplements. Cattle were randomly assigned to one of four finishing regimens; pasture (n=11), pasture with grain supplement (n=11), pasture with grain supplement containing soyoil (n=12), and feedlot (n=12). In the raw state, TB had higher (P<0.05) CLA than LD or SM on a mg/g sample basis. Total CLA was higher (P<0.05) in the soyoil diet when compared to the other three feeding regimes on a mg/g sample basis and when expressed as mg/g fat in both raw and cooked analyses. Pasture inclusion produced higher levels (P<0.05) of total CLA than the feedlot diet on a mg/g fat basis for cooked samples while maintaining acceptable eating quality.
RESUMEN
The objective of this study was to determine the relationships of uncoupling protein 2 and 3 expression, SNP of mitochondrial DNA, and residual feed intake (RFI) in Angus steers selected to have high or low RFI. Individual feed intake was measured via the GrowSafe feed intake system over a 3-mo period and used to calculate RFI, a measure of efficiency. Based on these calculations, 6 low- (average RFI = -1.57 kg) and 6 high- (average RFI = 1.66 kg) RFI steers were selected for further study. Blood was collected via jugular venipuncture 1 wk before slaughter for the isolation of mitochondrial DNA. The steers were then killed to collect LM for the measurement of uncoupling protein 2 and 3 mRNA and protein expression. Protein and mRNA expression of uncoupling protein 2 and 3 were determined by Western blotting and quantitative PCR, respectively. To determine SNP of mitochondrial DNA, total DNA was isolated from blood via standard phenol/chloroform extraction; fragments were amplified with PCR and sequenced with an automated nucleotide sequencer. Average daily gain and carcass composition were not different (P > 0.13) between the high- and low-RFI steers; however, ADFI by the high-RFI animals was 3.77 kg greater (P < 0.001) than the low-RFI animals. No difference (P > 0.55) was observed between the high- and low-RFI animals in their expression of uncoupling protein 2 or 3 mRNA or protein. On average 9.8 and 8.9 polymorphisms were found per mitochondrial genome for the low- and high-RFI steers, respectively. None of these polymorphisms were related to RFI. It seems that the expression of uncoupling protein 2 and 3 and mitochondrial DNA sequence are not related to RFI status.
Asunto(s)
Bovinos/metabolismo , ADN Mitocondrial/genética , Conducta Alimentaria/fisiología , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Polimorfismo de Nucleótido Simple , Alimentación Animal , Animales , Regulación de la Expresión Génica , Canales Iónicos/genética , Masculino , Proteínas Mitocondriales/genética , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
The objective of this study was to examine the relationship between mitochondrial function and residual feed intake in Angus steers. Individual feed intakes were recorded for a contemporary group of 40 steers via the GrowSafe feed intake system. Intakes were then used to calculate residual feed intake (RFI), a measure of efficiency. Based on these calculations, 9 low (RFI = -0.83) and 8 high (RFI = 0.78) RFI animals were selected for further study. Blood samples were collected via jugular venipuncture 1 wk before slaughter for the determination of plasma glucose and insulin concentrations. Tissue samples were taken from the LM from both the high and low RFI animals and mitochondria were isolated for measurement of oxygen consumption and hydrogen peroxide production. Average daily gain and carcass composition were not different between the high and low RFI steers; however, ADFI by the high RFI animals was 1.54 kg/d greater (P < 0.001) than for the low RFI animals. Low RFI steers exhibited a greater (P < 0.05) rate of state 2 and 3 respiration, respiratory control ratio, and hydrogen peroxide production than high RFI steers when provided with glutamate or succinate as a respiratory substrate. The acceptor control and adenosine diphosphate:oxygen ratios were not different between the 2 groups for either substrate. When hydrogen peroxide production was expressed as a ratio to respiration rate there was no difference between groups, signifying that electron leak was similar for both groups. Plasma glucose concentration was greater (P < 0.05) in the high RFI steers than in the low RFI steers; however, plasma insulin concentration was not different (P = 0.22) between the 2 groups. The ratio between plasma glucose and insulin concentration was similar (P = 0.88) between the 2 groups indicating no difference in glucose metabolism. The increased plasma glucose concentration observed in the high RFI steers was presumed to be the result of a greater feed intake by these animals. It seems that mitochondrial function is not different between the high and low RFI groups but rather the rate of mitochondrial respiration is increased in low RFI steers compared with high RFI steers.
Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales , Bovinos/metabolismo , Metabolismo Energético/fisiología , Conducta Alimentaria/fisiología , Mitocondrias/metabolismo , Animales , Peróxido de Hidrógeno/metabolismo , Masculino , Aumento de PesoRESUMEN
Three new Anabaena sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of sigD, sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD and sigE mutant strains to establish diazotrophic growth. A sigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of sigD or sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the sigE gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes. luxAB reporter fusions with sigD, sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains, Anabaena sp. strain PCC 7120, Synechococcus sp. strain PCC 7002, and Synechocystis sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains, Anabaena sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.
Asunto(s)
Anabaena/genética , Factor sigma/genética , Anabaena/crecimiento & desarrollo , Proteínas Bacterianas/genética , Clonación Molecular , ARN Polimerasas Dirigidas por ADN/genética , Eliminación de Gen , Datos de Secuencia Molecular , Filogenia , Factores de Transcripción/genéticaRESUMEN
The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms a developmental pattern of single heterocysts separated by approximately 10 vegetative cells. Heterocysts differentiate from vegetative cells and are specialized for nitrogen fixation. The patS gene, which encodes a small peptide that inhibits heterocyst differentiation, is expressed in proheterocysts and plays a critical role in establishing the heterocyst pattern. Here we present further analysis of patS expression and heterocyst pattern formation. A patS-gfp reporter strain revealed clusters of patS-expressing cells during the early stage of heterocyst differentiation. PatS signaling is likely to be involved in the resolution of these clusters. Differentiating cells were inhibited by PatS during the time period 6 to 12 h after heterocyst induction, when groups of differentiating cells were being resolved to a single proheterocyst. Increased transcription of patS during development coincided with expression from a new transcription start site. In vegetative cells grown on nitrate, the 5' end of a transcript for patS was localized 314 bases upstream from the first translation initiation codon. After heterocyst induction, a new transcript with a 5' end at -39 bases replaced the vegetative cell transcript. A patS mutant grown for several days under nitrogen-fixing conditions showed partial restoration of the normal heterocyst pattern, presumably because of a gradient of nitrogen compounds supplied by the heterocysts. The patS mutant formed heterocysts when grown in the presence of nitrate but showed no nitrogenase activity and no obvious heterocyst pattern. We conclude that PatS and products of nitrogen fixation are the main signals determining the heterocyst pattern.
Asunto(s)
Anabaena/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Fijación del Nitrógeno , Anabaena/genética , Anabaena/metabolismo , Secuencia de Bases , Medios de Cultivo , Datos de Secuencia Molecular , Mutación , Transcripción GenéticaRESUMEN
Many filamentous cyanobacteria grow as multicellular organisms that show a developmental pattern of single nitrogen-fixing heterocysts separated by approximately 10 vegetative cells. Overexpression of a 54-base-pair gene, patS, blocked heterocyst differentiation in Anabaena sp. strain PCC 7120. A patS null mutant showed an increased frequency of heterocysts and an abnormal pattern. Expression of a patS-gfp reporter was localized in developing proheterocysts. The addition of a synthetic peptide corresponding to the last five amino acids of PatS inhibited heterocyst development. PatS appears to control heterocyst pattern formation through intercellular signaling mechanisms.
Asunto(s)
Anabaena/crecimiento & desarrollo , Proteínas Bacterianas/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Anabaena/citología , Anabaena/genética , Anabaena/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cósmidos , Medios de Cultivo , Difusión , Genes Bacterianos , Genes Reporteros , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación Missense , Nitratos/metabolismo , Fijación del Nitrógeno , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Fenotipo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Transcripción GenéticaRESUMEN
Heterocystous cyanobacteria grow as multicellular organisms with a distinct one-dimensional developmental pattern of single nitrogen-fixing heterocysts separated by approximately ten vegetative cells. Several genes have been identified that are required for heterocyst development and pattern formation. A key regulator, HetR, has been recently shown to be aserine-type protease.
Asunto(s)
Anabaena/crecimiento & desarrollo , Anabaena/genética , Regulación Bacteriana de la Expresión Génica , Anabaena/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genes Bacterianos , Transducción de SeñalRESUMEN
A cosmid containing a wild-type Anabaena PCC 7120 DNA fragment was found to suppress heterocyst differentiation, creating a Het phenotype in an otherwise wild-type strain. Curing of the cosmid restored the full wild-type Het+ Nif+ phenotype. The cosmid contains at least four genes encoding proteins with significant sequence similarity to enzymes involved in the synthesis of fatty acids. Selection for Nif+ revertants of the suppressed strain yielded modified cosmids, one of which contained a 10.2-kb transposon, Tas1, inserted into the promoter region of a gene encoding a protein with acyl carrier and beta-keto reductase domains. This gene, called hetN, was shown previously by Black and Wolk (J. Bacteriol. (1994) 176, 2282-2292) to inhibit heterocyst differentiation when present alone on a plasmid. Oddly, hetN gene transcription is detected later than 6 h into heterocyst differentiation.
Asunto(s)
Anabaena/genética , Proteínas Portadoras , Ácidos Grasos/biosíntesis , Genes Bacterianos , Oxidorreductasas , Anabaena/citología , Proteínas Bacterianas/genética , Cósmidos , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Morfogénesis , Mutagénesis Insercional , Sistemas de Lectura Abierta , Transcripción GenéticaRESUMEN
The fdxN element, along with two other DNA elements, is excised from the chromosome during heterocyst differentiation in Anabaena sp. strain PCC 7120. Previous work showed that rearrangement of the fdxN element requires the xisF gene, which encodes a site-specific recombinase, and suggested that at least one other heterocyst-specific factor is involved. Here we report that the xisH and xisI genes are necessary for the heterocyst-specific excision of the fdxN element. Deletion of a 3.2 kb region downstream of the xisF gene blocked the fdxN-element rearrangement in heterocysts. The 3.2 kb deletion was complemented by the two overlapping genes xisH and xisI. Interestingly, extra copies of xisHI on a replicating plasmid resulted in the xisF-dependent excision of the fdxN element in vegetative cells. Therefore, xisHI are involved in the control of cell-type specificity of the fdxN rearrangement. The xisHI genes had no effect on the two other DNA rearrangements. The xisHI-induced excision of the fdxN element produced strains lacking the element and demonstrates that the 55 kb element contains no essential genes. xisH and xisI do not show similarity to any known genes.
Asunto(s)
Anabaena/genética , Ferredoxinas/fisiología , Reordenamiento Génico/genética , Genes Bacterianos/genética , Oxidorreductasas , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Ferredoxinas/genética , Reordenamiento Génico/fisiología , Genes Bacterianos/fisiología , Prueba de Complementación Genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Eliminación de Secuencia/genéticaRESUMEN
Mutants of Anabaena sp. strain PCC 7120 that form heterocysts when grown on nitrate-containing media were isolated following nitrosoguanidine mutagenesis. Six independent mutants were isolated, and the characterization of one mutant, strain AMC260, which forms 6 to 8% heterocysts in the presence of nitrate, is presented. A 1.8-kb chromosomal fragment that complemented the AMC260 mutant was sequenced, and a 1.2-kb open reading frame, named moeA, was identified. The deduced amino acid sequence of the predicted Anabaena sp. strain PCC 7120 MoeA polypeptide shows 37% identity to MoeA from Escherichia coli, which is required for the synthesis of molybdopterin cofactor. Molybdopterin is required by various molybdoenzymes, such as nitrate reductase. Interruption of the moeA gene in Anabaena sp. strain PCC 7120 resulted in a strain, AMC364, that showed a phenotype similar to that of AMC260. We show that AMC260 and AMC364 lack methyl viologen-supported nitrate reductase activity. We conclude that the inability of the moeA mutants to metabolize nitrate results in heterocyst formation on nitrate-containing media. Northern (RNA) analysis detected a 1.5-kb moeA transcript in wild-type cells grown in the presence or absence of a combined nitrogen source.
Asunto(s)
Alcohol Deshidrogenasa , Anabaena/genética , Proteínas Bacterianas/genética , Coenzimas , Metaloproteínas/metabolismo , Nitrato Reductasas/metabolismo , Nitratos/metabolismo , Pteridinas/metabolismo , Secuencia de Aminoácidos , Anabaena/crecimiento & desarrollo , Anabaena/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Mapeo Cromosómico , ADN Bacteriano , Genes Bacterianos , Prueba de Complementación Genética , Datos de Secuencia Molecular , Cofactores de Molibdeno , Nitrato-Reductasa , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The Anabaena sp. strain PCC 7120 ntcA gene showed multiple transcripts with different 5' ends. The relative abundance of transcripts varied in response to nitrogen availability. The ntcA product, NtcA, showed binding to the promoter region of its own gene. The binding site mapped to a region between the transcription start site used under nitrogen-replete conditions and the start sites used under nitrogen-limiting conditions, suggesting that NtcA regulates its own expression.
Asunto(s)
Anabaena/genética , Proteínas Bacterianas , Proteínas de Unión al ADN/genética , Genes Bacterianos , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
A transcriptional-interference selection was performed to identify genes of Anabaena sp. strain PCC 7120 that encode DNA-binding proteins able to bind to the rbcL promoter. Unexpectedly, the selection yielded the previously identified sigA gene, which encodes the principal sigma factor. Protein extracts from Escherichia coli containing the sigA gene bound the rbcL promoter fragment in mobility shift assays, and competition experiments indicated binding to rbcL and glnA but not xisA or nifH upstream regions.
Asunto(s)
Anabaena/genética , Proteínas Bacterianas/genética , Clonación Molecular/métodos , ARN Polimerasas Dirigidas por ADN , Factor sigma/genética , Proteínas Bacterianas/metabolismo , Unión Competitiva , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Genes Bacterianos/genética , Vectores Genéticos/genética , Glutamato-Amoníaco Ligasa/genética , Regiones Promotoras Genéticas/genética , Ribulosa-Bifosfato Carboxilasa/genética , Factor sigma/metabolismo , Transcripción GenéticaRESUMEN
Two site-specific DNA rearrangements occur during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120: the deletion of an 11 kb element from within the nifD gene and the deletion of a 55 kb element from within the fdxN gene. Three Nostoc and six Anabaena strains were screened for the presence of the nifD and fdxN elements by Southern hybridization with Anabaena PCC 7120 DNA probes. Eight of the nine strains contained DNA sequences that were similar to the nifD element. Three strains, Nostoc sp. strain Mac, Anabaena cylindrica and Anabaena sp. strain M131, also showed significant similarity to portions of the 55 kb fdxN element. Anabaena sp. strain CA lacked both the nifD and fdxN elements. Southern analysis of vegetative cell and heterocyst DNA from A. cylindrica and a Fox+ revertant of Nostoc Mac (isolate R2) showed rearrangement of the nifD and fdxN elements in heterocysts. We found no RFLPs between Anabaena M131 and Anabaena PCC 7120 suggesting that strain M131 is a Het- derivative of strain PCC 7120.
Asunto(s)
Cianobacterias/genética , Reordenamiento Génico , Genes Bacterianos , Fijación del Nitrógeno/genética , Operón/genética , Anabaena/genética , Proteínas Bacterianas/genética , Southern Blotting , Diferenciación Celular/genética , Mapeo Cromosómico , Cianobacterias/crecimiento & desarrollo , Ferredoxinas/genética , Especificidad de la EspecieRESUMEN
Programmed DNA rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms. Here, we identify the developmentally regulated rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. Heterocysts are terminally differentiated cells specialized for nitrogen fixation. Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element. The predicted HupL polypeptide is homologous to the large subunit of [NiFe] uptake hydrogenases. hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development. An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase. The predicted XisC polypeptide is homologous with the Anabaena sp. strain PCC 7120 site-specific recombinase XisA. Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.
Asunto(s)
Anabaena/genética , Proteínas Bacterianas/genética , Reordenamiento Génico , Genes Bacterianos/genética , Integrasas , Oxidorreductasas , Secuencia de Aminoácidos , Anabaena/crecimiento & desarrollo , Proteínas Bacterianas/química , Secuencia de Bases , Clonación Molecular , ADN Nucleotidiltransferasas/genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Recombinasas , Recombinación Genética/genética , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The Anabaena sp. strain PCC 7120 ntcA (bifA) gene encodes a sequence-specific DNA-binding protein, NtcA (BifA, VF1) that interacts with the upstream region of several genes, including glnA, xisA, rbcL, and nifH. We have constructed a ntcA null mutant by interrupting the gene with an omega Spr-Smr cassette. The ntcA mutant was not able to grow with nitrate or atmospheric dinitrogen as the sole nitrogen source but could be grown on medium containing ammonium. The ntcA mutant was unable to form heterocysts and did not rearrange the nifD or fdxN elements after induction on a medium lacking combined nitrogen. Northern (RNA) analysis of ntcA in the wild-type strain during nitrogen stepdown showed a peak of ntcA message at an early stage (12 h) of heterocyst induction. Complementation of the ntcA mutant with a DNA fragment containing the ntcA gene and 251 bp of upstream sequence on a shuttle vector restored a wild-type phenotype; however, a similar construction containing 87 bp of upstream sequence only partially restored the phenotype. Northern analysis of RNA samples isolated from ammonium-grown cultures of the ntcA mutant showed reduced amounts of glnA message and the absence of a 1.7-kb transcript. In the wild type, the 1.7-kb transcript represents the majority of glnA transcripts after nitrogen stepdown. The ntcA mutant showed a normal pattern of rbcLS messages under these growth conditions.
Asunto(s)
Anabaena/crecimiento & desarrollo , Proteínas de Unión al ADN/genética , Genes Bacterianos/genética , Compuestos de Nitrógeno/metabolismo , Factores de Transcripción/genética , Anabaena/genética , Proteínas Bacterianas/genética , Diferenciación Celular , Ferredoxinas/genética , Regulación Bacteriana de la Expresión Génica , Reordenamiento Génico , Glutamato-Amoníaco Ligasa/genética , Mutación , Nitratos/metabolismo , Fijación del Nitrógeno/genética , Regiones Promotoras Genéticas/genética , Compuestos de Amonio Cuaternario/metabolismo , Transcripción GenéticaRESUMEN
The DNA-binding factor BifA (previously called VF1) binds upstream of the developmentally regulated site-specific recombinase gene xisA in the cyanobacterium Anabaena sp. strain PCC 7120. Besides binding xisA, BifA also binds the glnA, rbcL, and nifH promoter regions. DNase I footprint analysis of BifA binding to glnA showed a protected region -125 to -148 bp upstream of the translation start site. The binding site is between the major glnA transcription start site used in vegetative cells (RNAII) and the major transcription start site used under nitrogen-deficient conditions (RNAI). The two BifA-binding sites on the rbcL promoter were localized to a 24-bp region from +12 to -12 nucleotides and to a 12-bp region from -43 to -54 nucleotides with respect to the transcription start site. Comparison of the BifA binding sites on the glnA, xisA, and rbcL upstream regions revealed the consensus recognition sequence TGT(N9 or 10) ACA. We have identified a second DNA-binding activity (factor 2) that interacts with rbcL and xisA upstream regions. Factor 2 can be resolved from BifA by heparin-Sepharose chromatography and was present in a bifA mutant. Analysis of partially purified vegetative cell and heterocyst extracts showed that whereas BifA was present in both cell types, factor 2 was present only in vegetative cells. DNase I footprint analysis of factor 2 binding to rbcL showed protection of a 63-bp region between positions -15 and -77 with respect to the transcription start site. The factor 2 binding site on xisA was localized to a 68-bp region that showed considerable overlap with the BifA binding sites.
Asunto(s)
Anabaena/metabolismo , Proteínas Bacterianas , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Genes Bacterianos , Genes de Plantas , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Anabaena/genética , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Two DNA elements are excised from the chromosome during Anabaena heterocyst differentiation. We have identified the gene xisF which encodes the site-specific recombinase responsible for the excision of a 55-kb element from within the fdxN gene. The cloned xisF gene is sufficient to cause site-specific rearrangement of an artificial substrate in Escherichia coli. Inactivation of xisF in the Anabaena chromosome prevents excision of the fdxN element and growth in nitrogen-deficient medium but does not alter the development of heterocysts. Forced transcription of xisF in vegetative cells did not result in excision of the fdxN element, suggesting that other factors may be involved in cell-type specificity. The predicted XisF protein shows significant similarity to the Bacillus subtilis SpoIVCA recombinase.
Asunto(s)
Anabaena/enzimología , ADN Nucleotidiltransferasas/genética , Integrasas , Factor sigma , Factores de Transcripción , Secuencia de Aminoácidos , Anabaena/genética , Anabaena/crecimiento & desarrollo , Proteínas Bacterianas/genética , Secuencia de Bases , ADN , ADN Nucleotidiltransferasas/antagonistas & inhibidores , ADN Nucleotidiltransferasas/metabolismo , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Plásmidos , Recombinasas , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
VF1 is a DNA-binding protein from the cyanobacterium Anabaena sp. strain PCC 7120. VF1 was originally identified on the basis of its binding affinity to the upstream region of xisA, which encodes a heterocyst-specific site-specific recombinase. VF1 also binds to the glnA, rbcL, and nifH promoters in vitro, suggesting that VF1 interacts with genes expressed in both vegetative cells and heterocysts. The role of VF1 in regulating gene expression in PCC 7120 is unknown. As a step towards the goal of understanding the role of VF1 in regulating gene expression, we have cloned the bifA gene by using a genetic selection strategy. bifA encodes a protein, BifA, that has chromatographic and DNA-binding properties indistinguishable from those of VF1. The cloning strategy was based on a transcriptional interference assay in which a strong synthetic promoter, conII, interferes with the expression of an aadA gene, which provides resistance to spectinomycin and streptomycin (S. J. Elledge, P. Sugiono, L. Guarente, and R. W. Davis, Proc. Natl. Acad. Sci. USA 86:3689-3693, 1989). A selection plasmid, pAM994, which has the conII promoter negatively regulated by a VF1-binding site, was used to enrich for VF1-producing clones from an expression library containing PCC 7120 DNA fragments. Mobility shift assays were used to identify a 672-bp open reading frame that encoded VF1-like binding activity. The deduced BifA amino acid sequence shows 77% identity to NtcA, which is a global regulator involved in nitrogen control in Synechococcus sp. strain PCC 7942. Both BifA and NtcA belong to the cyclic AMP receptor protein (CRP) family of prokaryotic regulatory proteins. Genes similar to envM, hisB, and ORF60-5 were found near the bifA gene.
Asunto(s)
Anabaena/genética , Proteínas Bacterianas , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteína Receptora de AMP Cíclico/genética , Proteínas de Unión al ADN/biosíntesis , Escherichia coli/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos/genética , Selección Genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The filamentous cyanobacterium Anabaena sp. strain PCC 7120 expresses the genes required for nitrogen fixation in terminally differentiated cells called heterocysts. The nifHDK operon encodes the nitrogenase polypeptides and is expressed at high levels in heterocysts. During heterocyst differentiation, an 11-kb DNA element is excised from the nifD gene by site-specific recombination. The xisA gene, located on the 11-kb element, is required for the excision of the element. Transcription and DNA rearrangement of the nifHDK operon both occur late during heterocyst differentiation, about 18 to 24 h after induction, suggesting that the regulation of these events might be coupled. We show that heterocyst-specific transcription and DNA rearrangement of the nifHDK operon are independent of one another. Northern (RNA) analysis of the xisA mutant strain DW12-2.2, which cannot excise the nifD 11-kb element or fix nitrogen, showed that the nifH and nifD genes are transcribed on unrearranged chromosomes. The nifK gene was not transcribed in DW12-2.2, indicating that its expression is dependent on the nifH promoter and excision of the 11-kb element from the operon. A 1.68-kb DNA fragment containing the nifH promoter was deleted from the chromosome to produce the mutant strain LW1. LW1 formed heterocysts but did not grow on nitrogen-free medium and showed no transcription through nifD. Southern analysis of LW1 showed normal excision of the 11-kb element from the nifHDK operon, indicating that transcription from the nifH promoter is not required for the developmentally regulated DNA rearrangement.