Analysis of eryBI , eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A␣mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis. Received: 13 August 1997 / Accepted: 27 November 1997     

Genetic organization and transposition properties of IS 511

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobactercrescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISRI, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5′ end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family. Received: 18 August 1996 / Accepted: 17 September 1996     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway. Received: 15 August 1996 / Accepted: 23 October 1996     

Oxygen consumption in the foraging honeybee depends on the reward rate at the food source

Oxygen consumption of the honeybee Apis mellifera ligustica was measured as a function of the flow rate supply of sucrose solution at an automatic feeder located inside a respirometric chamber. Trained bees freely entered the respirometric chamber and collected the sucrose solution supplied. The mean value of the O₂ consumption rate per visit increased with the sucrose flow rate, and for a given flow rate, with increasing locomotor activity. However, when no locomotor activity was displayed, O₂ consumption also increased with increasing nectar flow rate. Crop load attained at the end of the visit showed a positive relationship with the nectar flow rate; however, for a given flow rate, O₂ consumption showed either no correlation or a negative one with the final crop load attained. It is concluded that the energy expenditure of the foraging bee is controlled by a motivational drive whose intensity depends on the reward rate at the food source. Accepted: 30 July 1996     

c-fos expression in the putative avian suprachiasmatic nucleus

c-fos induction was investigated as a potential component in the avian photic entrainment pathway and as a possible means of locating the central pacemaker in birds. In both quail (Coturnix coturnix japonica) and starlings (Sturnus vulgaris) exposure to 1 h of light induced Fos-lir in the visual suprachiasmatic nucleus but not in the medial suprachiasmatic nucleus. However, the degree of c-fos induction in the visual suprachiasmatic nucleus was similar at different circadian times despite the fact that the light pulses caused differential phase shifts in the locomotor rhythm. For golden hamsters the same experiment resulted in significantly different levels of Fos-lir in the suprachiasmatic nucleus, as well as different phase shifts. Starlings and hamsters were also entrained to T-cycles that caused a large daily phase shift (T = 21.5 h in starlings, T = 22.67 hours in hamsters), or no daily phase shift (T = free running period). No difference in the induced levels of Fos-lir in the visual suprachiasmatic nucleus region was observed between the two groups of starlings, but in hamsters there were significantly different levels of Fos-lir in the suprachiasmatic nucleus between the two groups. Accepted: 15 November 1996     

The Cdk-like protein PCTAIRE-1 from mouse brain associates with p11 and 14-3-3 proteins

PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk)-like class of proteins, and is localized mainly in the mammalian brain. Using the yeast two-hybrid system we screened a mouse brain cDNA library with PCTAIRE-1 as bait, and isolated several clones coding for the mouse homologs of the following proteins: p11 (also known as calpactin I light chain) and the η, θ (also known asτ) and ζ isoforms of 14-3-3 proteins. We confirmed that these four proteins interact with PCTAIRE-1 by demonstrating the biochemical interactions using the pure recombinant proteins. The fact that 14-3-3 proteins are known to interact with many other intracellular proteins (such as C-kinase, Raf, Bcr, PI3-kinase) and p11 with annexin II (a major pp60^v-src and C-kinase substrate) suggests that PCTAIRE-1 might be part of multiple signal transduction cascades and cellular protein networks. Received: 23 September 1996 / Accepted: 10 January 1997     

Second messenger and cAMP-dependent protein kinase responses to dehydration and anoxia stresses in frogs

The effects of whole body dehydration (up to 40% of total body water lost) or anoxia exposure (up to 2 days under N₂ gas) at 5 °C on tissue levels of adenosine 3′–5′ cyclic monophosphate (cAMP) and the percentage of cAMP-dependent protein kinase present as the free catalytic subunit (PKAc), as well as the levels of the protein kinase C (PKC) second messenger, inositol 1,4,5-trisphosphate (IP₃), were assessed in two anurans, the freeze-tolerant wood frog, Rana sylvatica, and the freeze-intolerant leopard frog, Rana pipiens. Dehydration of wood frogs resulted in a rapid elevation of liver cAMP and PKAc; cAMP was 3.4-fold greater than control values in animals that had lost 5% of total body water, whereas PKAc was elevated threefold in 20% dehydrated frogs. These results indicate protein kinase A mediation of the liver glycogenolysis and hyperglycemia that is induced by dehydration in this species. Skeletal muscle PKAc content also rose with dehydration but neither cAMP nor PKAc was affected by dehydration in leopard frog tissues. Anoxia exposure had different effects on signal transduction systems. PKAc was elevated after 1 h anoxia in R. sylvatica brain and was sustained over time but the enzyme was unaffected in other organs; by contrast, R. pipiens showed variable responses by PKAc to anoxia in three organs. Both species showed rapid (within 30 min) and large (3 to 7.8-fold) increases in IP₃ in liver of anoxic frogs that decreased slowly with continued anoxia. IP₃ also increased quickly in heart of anoxia-exposed wood frogs. This suggests that PKC may mediate various metabolic adjustments that promote hypoxia/anoxia resistance such as coordinating metabolic rate depression. A progressive rise in liver IP₃ during dehydration in wood frogs (reaching fourfold higher than controls in 40% dehydrated animals) may also mediate similar hypoxia resistance adaptations under this stress since anurans experience progressive hypoxia due to increased blood viscosity when water loss reaches high values. The patterns of second messenger and PKAc changes in wood frog liver during dehydration closely parallel the changes seen in these same parameters during natural freezing suggesting that the freeze tolerance of selected terrestrially hibernating anurans may have evolved out of various anuran mechanisms of dehydration resistance. Accepted: 2 January 1997     

The effect of a thiamin derivative on exercise performance

The purpose of this study was to investigate the effect of a thiamin derivative, thiamin tetrahydrofurfuryl disulfide (TTFD), on oxygen uptake (˙VO₂), lactate accumulation and cycling performance during exercise to exhaustion. Using a randomized, double-blind, cross-over design with a 10-day washout between trials, 14 subjects ingested either 1 g · day⁻¹ of TTFD or a placebo (PL) for 4 days. On day 3, subjects performed a progressive exercise test to exhaustion on a cycle ergometer for the determination of ˙VO_2submax, ˙VO_2peak, lactate concentration ([La⁻ ]), lactate threshold (Th_La) and heart rate ( f _c). On day 4, subjects performed a maximal 2000-m time trial on a cycle ergometer. A one-way analysis of variance (ANOVA) with repeated measures was used to determine significant differences between trials. There were no significant differences detected between trials for serial measures of ˙VO_2submax, [La⁻] or f _c. Likewise, ˙VO_2peak [PL 4.06 (0.19) TTFD 4.12 (0.19) l · min⁻¹, P = 0.83], Th_La [PL 2.47 (0.17), TTFD 2.43 (0.16) l · min⁻¹, P = 0.86] and 2000-m performance time [PL 204.5 (5.5), TTFD 200.9 (4.3) s, P = 0.61] were not significantly different between trials. The results of this study suggest that thiamin derivative supplementation does not influence high-intensity exercise performance. Accepted: 19 December 1996     

Rhythmicity of torpor in a marsupial hibernator, the mountain pygmy-possum (Burramysparvus), under natural and laboratory conditions

Circadian rhythms have been observed in most mammals, but their importance and function remain controversial with respect to daily cycles during hibernation. We investigated the timing of arousals from and entries into hibernation for both free-living and captive mountain pygmy-possums (Burramys parvus). Under both natural and laboratory conditions most arousals and entries were entrained with the light-dark cycle. Entries occurred mainly during the night and arousals preferably around dusk, which coincides with the onset of the normal activity phase for the nocturnal pygmy-possums. This entrainment prevailed throughout the hibernation season although only the laboratory animals were constantly subjected to photoperiodic stimuli, whereas under natural conditions hibernacula are shielded from photic cues and diurnal temperature fluctuations. Nevertheless, possums left their hibernacula frequently throughout winter and were occasionally trapped close to the snow surface suggesting that during the periods of post-arousal normothermia they can be exposed to environmental stimuli. It thus appears that the synchronisation with the photocycle was governed by a temperature-compensated circadian clock which was reset periodically during short activity periods. For the mountain pygmy-possum, entrainment with the photocycle probably has two functions: 1. Entrainment ensures that foraging bouts during the hibernation season remain synchronised with the dark phase. 2. Information about the prevailing climatic conditions sampled during short activity periods enables them to time final spring emergence from hibernation when snow melt begins and ensures that the breeding season can commence as early as possible. Accepted: 26 August 1998