Living on the edge: Daily, seasonal and annual body temperature patterns of Arabian oryx in Saudi Arabia.

Heterothermy, the ability to allow body temperature (Tb) to fluctuate, has been proposed as an adaptive mechanism that enables large ungulates to cope with the high environmental temperatures and lack of free water experienced in arid environments. By storing heat during the daytime and dissipating it during the night, arid-adapted ungulates may reduce evaporative water loss and conserve water. Adaptive heterothermy in large ungulates should be particularly pronounced in hot environments with severely limited access to free water. In the current study we investigated the effects of environmental temperature (ambient, Ta and soil, Ts) and water stress on the Tb of wild, free-ranging Arabian oryx (Oryx leucoryx) in two different sites in Saudi Arabia, Mahazat as-Sayd (MS) and Uruq Bani Ma'arid (UBM). Using implanted data loggers wet took continuous Tb readings every 10 minutes for an entire calendar year and determined the Tb amplitude as well as the heterothermy index (HI). Both differed significantly between sites but contrary to our expectations they were greater in MS despite its lower environmental temperatures and higher rainfall. This may be partially attributable to a higher activity in an unfamiliar environment for translocated animals in UBM. As expected Tb amplitude and HI were greatest during summer. Only minor sex differences were apparent that may be attributable to sex-specific investment into reproduction (e.g. male-male competition) during rut. Our results suggest that the degree of heterothermy is not only driven by extrinsic factors (e.g. environmental temperatures and water availability), but may also be affected by intrinsic factors (e.g. sex and/or behaviour).

Genetic and lifestyle causal beliefs about obesity and associated diseases among ethnically diverse patients: a structured interview study.

BACKGROUND: New genetic associations with obesity are rapidly being discovered. People's causal beliefs about obesity may influence their obesity-related behaviors. Little is known about genetic compared to lifestyle causal beliefs regarding obesity, and obesity-related diseases, among minority populations. This study examined genetic and lifestyle causal beliefs about obesity and 3 obesity-related diseases among a low-income, ethnically diverse patient sample. METHODS: Structured interviews were conducted with patients attending an inner-city hospital outpatient clinic. Participants (n=205) were asked how much they agreed that genetics influence the risk of obesity, type 2 diabetes, heart disease, and cancer. Similar questions were asked regarding lifestyle causal beliefs (overeating, eating certain types of food, chemicals in food, not exercising, smoking). In this study, 48% of participants were non-Hispanic Black, 29% Hispanic and 10% non-Hispanic White. RESULTS: Over two-thirds (69%) of participants believed genetics cause obesity 'some' or 'a lot', compared to 82% for type 2 diabetes, 79% for heart disease and 75% for cancer. Participants who held genetic causal beliefs about obesity held more lifestyle causal beliefs in total than those who did not hold genetic causal beliefs about obesity (4.0 vs. 3.7 lifestyle causal beliefs, respectively, possible range 0-5, p=0.025). There were few associations between causal beliefs and sociodemographic characteristics. CONCLUSIONS: Higher beliefs in genetic causation of obesity and related diseases are not automatically associated with decreased lifestyle beliefs. Future research efforts are needed to determine whether public health messages aimed at reducing obesity and its consequences in racially and ethnically diverse urban communities may benefit from incorporating an acknowledgement of the role of genetics in these conditions.

Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520.

FK506 and FK520 are 23-membered macrocyclic polyketides with potent immunosuppressive and antifungal activities. The gene encoding 31-O-demethyl-FK506 methyltransferase, fkbM, was isolated from Streptomyces sp. strains MA6858 and MA6548, two FK506 producers, and Streptomyces hygroscopicus subsp. ascomyceticus, an FK520 producer. The nucleotide sequence of the fkbM gene revealed an open reading frame encoding a polypeptide of 260 amino acids. Disruption of fkbM in Streptomyces sp. strain MA6548 yielded a mutant that produced 31-O-demethyl-FK506, confirming the involvement of the isolated genes in the biosynthesis of FK506 and FK520. Heterologous expression of fkbM in Streptomyces lividans established that fkbM encodes an O-methyltransferase catalyzing the methylation of the C-31 hydroxyl group of 31-O-demethyl-FK506 and FK520. A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.

The genetic and biochemical basis of polyketide metabolism in microorganisms and its role in drug discovery and development.

The possibilities for the design of new drug screening and development strategies directed to a specific objective on the basis of genetic engineering of microorganisms is discussed from two points of view. Firstly, results of work on genetic hybrids of Streptomyces species for the production of new metabolites such as mederrhodin (1) and aloespanoarin II (4) are described. Secondly, the enhanced production of known metabolites such as tetracenomycin A2 (11) and tetracenomycin C (9) by recombinant Streptomyces species is considered. Mechanistic aspects of polyketide metabolism are included.

The genetic and biochemical basis of polyketide metabolism in microorganisms and its role in drug discovery and development.

The possibilities for the design of new drug screening and development strategies directed to a specific objective on the basis of genetic engineering of microorganisms is discussed from two points of view. Firstly, results of work on genetic hybrids of STREPTOMYCES species for the production of new metabolites such as mederrhodin (1) and aloespanoarin II (4) are described. Secondly, the enhanced production of known metabolites such as tetracenomycin A (2) (11) and tetracenomycin C (9) by recombinant STREPTOMYCES species is considered. Mechanistic aspects of polyketide metabolism are included.

"Streptomyces avermitilis" mutants defective in methylation of avermectins.

"Streptomyces avermitilis" mutants defective in the methylation of the avermectins have been isolated and characterized. Four mutant strains, CR-1, CR-2, CR-3, and CR-4, were unable to methylate the oxygen at C5 of the macrolide moiety and produced essentially only the avermectin B components. These four strains lack avermectin B2 O-methyltransferase (B2OMT) activity. Two mutant strains were unable to methylate the oleandrose moiety at the oxygens at C3' and C3'' and produced essentially only demethylavermectin components. One of these mutants, strain CR-5 (derived from wild-type "S. avermitilis"), produced demethylavermectin A and B components and possessed normal B2OMT levels. The other mutant, strain CR-6 (derived from strain CR-1, which lacks B2OMT activity), produced only demethylavermectin B components. Reaction of 3"-O-demethylavermectin B2a and S-adenosylmethionine with either cell extracts or purified B2OMT resulted in the methylation of the oxygen at C5 of the macrolide moiety and yielded only 3''-O-demethylavermectin A2a as the product. These experiments indicate that different enzymes are required for methylation of the macrolide (the oxygen at C5) and the oleandrose (oxygen at C3) and that methylation of the oleandrose occurs before attachment to the macrolide ring.

Characterization of suppressible mutations in the viomycin phosphotransferase gene of the Streptomyces enteric plasmid pVE138.

The viomycin phosphotransferase gene (vph) is expressed and confers resistance to viomycin in both Streptomyces spp. and members of the family Enterobacteriaceae. We report the isolation of UGA (opal) and UAG (amber) mutations in the vph gene of shuttle plasmid pVE138. We found that the five UGA mutations in vph resulted in a temperature-sensitive phenotype in Salmonella typhimurium. Su- strains are Vior at 28 degrees C and Vios at 37 degrees C, whereas Su+UGA strains are Vior at both 28 and 37 degrees C. The single amber mutation isolated was not temperature sensitive and resulted in the expected Vios phenotype in Su- strains and Vior in Su+UAG strains.

Glutamine synthetase of Streptomyces cattleya: purification and regulation of synthesis.

Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.

Regulation of glutamine synthetase activity by adenylylation in the Gram-positive bacterium Streptomyces cattleya.

The enzymatic activity of glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from the Gram-positive bacterium Streptomyces cattleya is regulated by covalent modification. In whole cells containing high levels of GS the addition of ammonium chloride leads to a rapid decline in GS activity. Crude extracts prepared from such ammonia-shocked cells had very low levels of GS activity as measured by biosynthetic and gamma-glutamyltransferase assays. Incubation of the crude extracts with snake venom phosphodiesterase restored GS activity. In cell extracts, GS was also inactivated by an ATP- and glutamine-dependent reaction. Radioactive labeling studies demonstrated the incorporation of an AmP moiety into GS protein upon modification. Our results suggest a covalent modification of GS in a Gram-positive bacterium. This modification appears to be adenylylation of the GS subunit similar to that found in the Gram-negative bacteria.

Purification of glutamine synthetase from a variety of bacteria.

We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.

Glutamine synthetase regulation, adenylylation state, and strain specificity analyzed by polyacrylamide gel electrophoresis.

We used polyacrylamide gel electrophoresis to examine the regulation and adenylylation states of glutamine synthetases (GSs) from Escherichia coli (GS(E)) and Klebsiella aerogenes (GS(K)). In gels containing sodium dodecyl sulfate (SDS), we found that GS(K) had a mobility which differed significantly from that of GS(E). In addition, for both GS(K) and GS(E), adenylylated subunits (GS(K)-adenosine 5'-monophosphate [AMP] and GS(E)-AMP) had lesser mobilities in SDS gels than did the corresponding non-adenylylated subunits. The order of mobilities was GS(K)-AMP < GS(K) < GS(E)-AMP < GS(E). We were able to detect these mobility differences with purified and partially purified preparations of GS, crude cell extracts, and whole cell lysates. SDS gel electrophoresis thus provided a means of estimating the adenylylation state and the quantity of GS present independent of enzymatic activity measurements and of determining the strain origin. Using SDS gels, we showed that: (i) the constitutively produced GS in strains carrying the glnA4 allele was mostly adenylylated, (ii) the GS-like polypeptide produced by strains carrying the glnA51 allele was indistinguishable from wild-type GS(K), and (iii) strains carrying the glnA10 allele contained no polypeptide having the mobility of GS(K) or GS(K)-AMP. Using native polyacrylamide gels, we detected the increased amount of dodecameric GS present in cells grown under nitrogen limitation compared with cells grown under conditions of nitrogen excess. In native gels there was neither a significant difference in the mobilities of adenylylated and non-adenylylated GSs nor a GS-like protein in cells carrying the glnA10 allele.

Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase.

The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA.

Regulation of enzyme synthesis by the glutamine synthetase of Salmonella typhimurium: a factor in addition to glutamine synthetase is required for activation of enzyme formation.

In Klebsiella aerogenes but not in Salmonella typhimurium glutamine synthetase can function during nitrogen-limited growth to increase the rate of synthesis of histidase from the hut genes of S. typhimurium 15-59 (hutS. 15-59). Formation of proline oxidase is also not increased in nitrogen-limited cultures of S. typhimurium. However, in hybrid strains of Escherichia coli or K. aerogenes, the glutamine synthetase of S. typhimurium activates synthesis of histidase from the hutS. 15-59 genes. Apparently, glutamine synthetase is necessary but not sufficient for activation of transcription of the hut genes; another factor must also be present. This factor is active in both K. aerogenes and E. coli but is missing or altered in S. typhimurium.

Regulation of enzyme formation in Klebsiella aerogenes by episomal glutamine synthetase of Escherichia coli.

We studied the physiology of cells of Klebsiella aerogenes containing the structural gene for glutamine synthetase (glnA) of Escherichia coli on an episome. The E. coli glutamine synthetase functioned in cells of K. aerogenes in a manner similar to that of the K. aerogenes enzyme: it allowed the level of histidase to increase and that of glutamate dehydrogenase to decrease during nitrogen-limited growth. The phenotype of mutations in the glnA site was restored to normal by the introduction of the episomal glnA+ gene. These results are consistent with the hypothesis that glutamine synthetase regulates the function of its own structural gene.

Genetic control of glutamine synthetase in Klebiella aerogenes.

Mutations at two sites, glnA and glnB, of the Klebsiella aerogenes chromosome result in the loss of glutamine synthetase. The locations of these sites on the chromosome were established by complementation by episomes of Escherichia coli and by determination of their linkage to other genetic sites by transduction with phage P1. The glnB gene is located at a position corresponding to 48 min on the Taylor map of the E. coli chromosome; it is linked to tryA, nadB, and GUA. The glnA gene is at a position corresponding to 77 min on the Taylor map and is linked to rha and metB; it is also closely linked to rbs, located in E. coli at 74 min, indicating a difference in this chromosomal region between E. coli and K. aerogenes. Mutations in the glnA site can also lead to nonrepressible synthesis of active glutamine synthetase. The examination of the fine genetic structure of glnA revealed that one such mutation is located between two mutations leading to the loss of enzymatic activity. This result, together with evidence that the structural gene for glutamine synthetase is at glnA, suggests that glutamine synthetase controls expression of its own structural gene by repression.

Regulation of nitrogen fixation in Klebsiella pneumoniae: evidence for a role of glutamine synthetase as a regulator of nitrogenase synthesis.

Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.

Direct selection for P1-sensitive mutants of enteric bacteria.

A method has been developed to isolate mutants sensitive to coliphage P1 from bacterial genera normally not sensitive to this phage. P1clr100KM was used. This phage is heat inducible and confers kanamycin resistance when present as a prophage (in lysogens). P1-sensitive mutants of Klebsiella, Enterobacter, Citrobacter, and Erwinia have been found. This technique provides a well-known genetic system for the study of many bacterial genera that previously had either no such system or only a marginally useful means of genetic manipulation. It also extends the range of possible intergeneric hybrids that may be constructed and studied.