Genome rearrangement and nitrogen fixation in Anabaena blocked by inactivation of xisA gene.

Two genome rearrangements involving 11- and 55-kilobase DNA elements occur during the terminal differentiation of an Anabaena photosynthetic vegetative cell into a nitrogen-fixing heterocyst. The xisA gene, located on the nifD 11-kilobase DNA element, was inactivated by recombination between the chromosome and a copy of the xisA gene that was mutated by inserting an antibiotic gene cassette. Site-directed inactivation of the Anabaena xisA gene blocked rearrangement of the 11-kilobase element and nitrogen fixation, but did not affect rearrangement of the 55-kilobase element, heterocyst differentiation, or heterocyst pattern formation.

Heterocyst pattern formation controlled by a diffusible peptide.

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.

A pheromone influences larval development in the nematode Caenorhabditis elegans.

A Caenorhabditis-specific pheromone and the food supply influence both entry into and exit from a developmentally arrested juvenile stage called the dauer larva. The pheromone increases the frequency of dauer larva formation and inhibits recovery but does not affect adult behavior such as chemotaxis and egg laying. The fatty acid--like pheromone has been partially purified and characterized by a new bioassay. If similar developmental control mechanisms are used by parasitic nematodes, such mechanisms might be exploited to develop highly selective anthelmintic agents.

Conjugated linoleic acid content of beef differs by feeding regime and muscle.

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.

Heterocyst formation in Anabaena.

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.

A new filamentous phage cloning vector: fd-tet.

We have constructed a hybrid chromosome composed of the genome of wild-type fd (a filamentous, male-specific bacteriophage) and a segment of transposon Tn10 coding for tetracycline resistance but not including the Tn10 insertion sequences. The hybrid phage infects male E. coli, thereby transducing the infected cells to tetracycline resistance. The phage DNA can also be propagated in F- cells after transfection. This new phage, fd-tet, may be used as a cloning vector to produce large quantities of cloned DNA in single-stranded form. Its usefulness has been demonstrated by cloning of a fragment from bacteriophage lambda. Some unexpected sequence alterations have been identified in lambda cloning experiments.

Suppression of heterocyst differentiation in Anabaena PCC 7120 by a cosmid carrying wild-type genes encoding enzymes for fatty acid synthesis.

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.

The relationship of feeding behavior to residual feed intake in crossbred Angus steers fed traditional and no-roughage diets.

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.

The relationship between mitochondrial function and residual feed intake in Angus steers.

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.

The relationships among mitochondrial uncoupling protein 2 and 3 expression, mitochondrial deoxyribonucleic acid single nucleotide polymorphisms, and residual feed intake in Angus steers.

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.

Two heterocyst-specific DNA rearrangements of nif operons in Anabaena cylindrica and Nostoc sp. strain Mac.

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.

A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process.

Formation of a developmentally arrested dispersal stage called the dauer larva is enhanced by a Caenorhabditis-specific pheromone and is inhibited by increasing amounts of food. Pheromone-induced dauer larva formation of three tested wild-type strains is temperature-dependent, so that an increased percentage of the population forms dauer larvae at 25 degrees C compared to lower temperatures. Dauer-defective mutants fail to respond to added pheromone, and some behavioral mutants affected in thermotaxis or egg-laying also exhibit abnormal responses. Temperature-sensitive (ts) dauer-constitutive mutants form dauer larvae at a restrictive temperature regardless of environmental stimuli. At the permissive temperature (17.5 degrees C), alleles of six out of seven dauer-constitutive genes tested overrespond to the dauer-inducing pheromone. All known mutations in daf-4 (eight alleles) and daf-7 (five alleles) produce a ts dauer-constitutive phenotype. One daf-4 and one daf-7 allele are suppressed by the amber nonsense suppressor, sup-7(st5). At least these two dauer-constitutive mutations are likely to cause production of nonfunctional rather than ts gene products. These mutations appear to indirectly result in a ts phenotype by enhancing the expression of a wild-type ts developmental process.

PatS and products of nitrogen fixation control heterocyst pattern.

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.

Identification and inactivation of three group 2 sigma factor genes in Anabaena sp. strain PCC 7120.

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.

ACaenorhabditis elegans dauer-inducing pheromone and an antagonistic component of the food supply.

The free-living soil nematodeCaenorhabditis elegans forms a nonfeeding dispersal stage at the second molt called the dauer larva when exposed to environmental cues indicating crowding and limited food. An improved bioassay, tenfold more sensitive than that used previously, has been used in the characterization of the two chemical cues which act competitively in controlling this developmental process. The pheromone concentration provides a measure of the population density; it enhances dauer larva formation, and inhibits recovery (exit) from the dauer stage. The pheromone is a family of related molecules which are nonvolatile, very stable, and possess physical and Chromatographie properties similar to those of hydroxylated fatty acids and bile acids. A food signal, with effects on development opposite those of the pheromone, is produced by bacteria, and is also present in yeast extract. In contrast to the pheromone, the food signal is a labile substance which is neutral and hydrophilic.

The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature.

Three environmental cues influence both the entry into and exit from the developmentally arrested dispersal stage called the dauer larva: a dauer-inducing pheromone, food, and temperature. The pheromone, which is a measure of population density, induces dauer larva formation at the second (L2) molt and inhibits recovery in a dose-dependent manner. Food acts competitively to reduce the frequency of dauer larva formation and to enhance recovery. The pheromone causes a specific extension of the second larval stage, coupled with a transient decrease in the growth rate of the L2. Second-stage larvae grown in the presence of added pheromone are morphologically distinguishable from L2 larvae grown without pheromone. We have named the pre-dauer L2 larva the L2d. Commitment to dauer larva formation can occur at the L2d molt. When L2d larvae are shifted out of pheromone to a lawn of E. coli just before the L2d molt, a few worms complete development into dauer larvae. In contrast, worms are essentially committed to the non-dauer life cycle by the first larval molt if the L1 larvae are not grown in appropriately high levels of pheromone. In the presence of pheromone, the percentage of dauer larva formation is enhanced at higher temperatures within the normal growth range. Temperature down-shifts induce dauer larva recovery. Temperature-shift experiments show that the enhancement of dauer larva formation requires exposure to the higher temperature around the L1 molt. Two sensory mutants defective in thermotaxis are altered in their sensitivity to the dauer-inducing pheromone, but their pheromone response retains temperature dependence. Response of dauer larvae to environmental cues is highly age dependent, with older dauer larvae exhibiting an increased tendency to recover.

A gene affecting production of the Caenorhabditis elegans dauer-inducing pheromone.

A nematode mutant lacking pheromone activity does not enter the developmentally arrested dispersal stage called the dauer larva unless exogenous pheromone is added to the growth medium, indicating that the pheromone is required for wild-type dauer larva formation. In contrast, a class of temperature-sensitive mutant forms dauer larvae even in the absence of detectable pheromone, indicating that such mutants bypass the normal pheromone requirement. A rapid bioassay of pheromone produced by individual nematodes has been developed for genetic analysis of pheromone production.

Nitrate reductase activity and heterocyst suppression on nitrate in Anabaena sp. strain PCC 7120 require moeA.

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.

Two Anabaena sp. strain PCC 7120 DNA-binding factors interact with vegetative cell- and heterocyst-specific genes.

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.

Independent regulation of nifHDK operon transcription and DNA rearrangement during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

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.