Faster-acting insulin aspart: earlier onset of appearance and greater early pharmacokinetic and pharmacodynamic effects than insulin aspart.

AIMS: To evaluate the pharmacokinetics and pharmacodynamics of faster-acting insulin aspart and insulin aspart in a randomized, single-centre, double-blind study. METHODS: Fifty-two patients with type 1 diabetes (mean age 40.3 years) received faster-acting insulin aspart, insulin aspart, or another faster aspart formulation (not selected for further development), each as a single 0.2 U/kg subcutaneous dose, under glucose-clamp conditions, in a three-way crossover design (3-12 days washout between dosing). RESULTS: Faster-acting insulin aspart had a faster onset of exposure compared with insulin aspart, shown by a 57% earlier onset of appearance [4.9 vs 11.2 min; ratio 0.43, 95% confidence interval (CI) 0.36; 0.51], a 35% earlier time to reach 50% maximum concentration (20.7 vs 31.6 min; ratio 0.65, 95% CI 0.59; 0.72) and a greater early exposure within 90 min after dosing. The greatest difference occurred during the first 15 min, when area under the serum insulin aspart curve was 4.5-fold greater with faster-acting insulin aspart than with insulin aspart. Both treatments had a similar time to maximum concentration, total exposure and maximum concentration. Faster-acting insulin aspart had a significantly greater glucose-lowering effect within 90 min after dosing [largest difference: area under the curve for the glucose infusion rate (AUC(GIR), 0-30 min) ratio 1.48, 95% CI 1.13; 2.02] and 17% earlier time to reach 50% maximum glucose infusion rate (38.3 vs 46.1 min; ratio 0.83, 95% CI 0.73; 0.94). The primary endpoint (AUC(GIR, 0-2 h)) was 10% greater for faster-acting insulin aspart, but did not reach statistical significance (ratio 1.10, 95% CI 1.00; 1.22). Both treatments had similar total and maximum glucose-lowering effects, indicating similar overall potency. CONCLUSIONS: Faster-acting insulin aspart was found to have earlier onset and higher early exposure than insulin aspart, and a greater early glucose-lowering effect, with similar potency.

Obesity does not influence the unique pharmacological properties of different biphasic insulin aspart preparations in patients with type 2 diabetes.

AIM: To investigate the influence of obesity in type 2 diabetic patients upon pharmacological properties of different biphasic preparations of insulin aspart. METHODS: A total of 75 type 2 diabetic patients were stratified according to their body mass index (BMI) into 40 non-obese (BMI 23-28 kg/m(2)) and 35 obese (BMI 30-35 kg/m(2)) subjects. The trial was a double-blinded crossover study. In two periods of 4 weeks each the patients received subcutaneous injections of biphasic insulin aspart 50 (BIAsp 50) or biphasic insulin aspart 70 (BIAsp 70) thrice daily in random order. Insulin doses were titrated individually. At the end of each period 24-h serum profiles of insulin aspart, C-peptide and glucose were recorded. The primary endpoint was the area under the curve of serum glucose concentration during 24 h (AUC(Glu)(0-24 h)). RESULTS: The insulin concentration profiles of BIAsp 50 and 70 were as expected according to the content of protamine-bound insulin aspart (50 and 30% respectively). AUC(Glu(0-24 h)) BIAsp 50/BIAsp 70 ratios were 0.97 (95% CI: 0.90-1.05, p = 0.49) for non-obese and 0.98 (95% CI: 0.92-1.05, p = 0.55) for obese. Fasting serum glucose (FSG) BIAsp 50/BIAsp 70 ratios were 0.90 (95% CI: 0.84-0.96, p = 0.002) for non-obese and 0.90 (95% CI: 0.84-0.97, p = 0.006) for obese. During both treatment regimens the frequency of minor hypoglycaemic episodes was highest for the non-obese group. CONCLUSIONS: The pharmacokinetic and pharmacodynamic characteristics of the two preparations of biphasic insulin aspart were different; however, they were not influenced by the degree of obesity in type 2 diabetic patients.

Chitin hydrolysis by Listeria spp., including L. monocytogenes.

Listeria spp., including the food-borne pathogen Listeria monocytogenes, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear beta-1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that L. monocytogenes and other Listeria spp. are able to hydrolyze alpha-chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30 degrees C than at 37 degrees C. In L. monocytogenes EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (chiB) and lmo1883 (chiA), but not on a gene encoding a putative chitin binding protein (lmo2467). The chiB and chiA genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of Listeria are discussed.

Chicken juice, a food-based model system suitable to study survival of Campylobacter jejuni.

AIMS: The purpose of this study was to develop a food-based model system that resembles the environment that Campylobacter jejuni experiences on raw poultry products and use this model system to investigate growth and survival of the bacterium. METHODS AND RESULTS: Chicken juice was collected from frozen chickens and subsequently cleared by centrifugation and subjected to sterile filtration. At low temperatures (5 and 10 degrees C) C. jejuni NCTC11168 remained viable in chicken juice for a remarkably longer period of time than in the reference medium BHI. When exposed to heat stress (48 degrees C) C. jejuni NCTC11168 also showed increased viability in chicken juice compared with the reference medium. Furthermore, agar plates made with chicken juice supported growth of four clinical isolates of C. jejuni and a C. jejuni strain obtained from chicken at both 37 and 42 degrees C. CONCLUSIONS: Our work shows that minimal processed and sterilized chicken juice is an ideal environment for survival of C. jejuni and that it is useful as a food-based model system. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed model system may contribute to the understanding of C. jejuni viability on poultry products and can be instrumental in the development of alternative preservation strategies.

An activator of transcription regulates phage TP901-1 late gene expression.

A promoter active in the late phase of the lytic cycle of lactococcal bacteriophage TP901-1 has been identified. The promoter is tightly regulated and requires the product of the phage TP901-1 orf29 for activity. A deletion analysis of the late promoter region showed that a fragment as small as 99 bp contains both the promoter and the region necessary for activation by ORF29. The transcriptional start site of the promoter was identified by primer extension to position 13073 on the TP901-1 genome, thus located 87 bp downstream of orf29 in a 580-bp intergenic region between orf29 and orf30. Furthermore, the region located -85 to -61 bp upstream of the start site was shown to be necessary for promoter activity. During infection, the transcript arising from the late promoter is fully induced at 40 min postinfection, and our results suggest that a certain level of ORF29 must be reached in order to activate transcription of the promoter. Several lactococcal bacteriophages encode ORF29 homologous proteins, indicating that late transcription may be controlled by a similar mechanism in these phages. With the identification of this novel regulator, our results suggest that within the P335 group of lactococcal phages at least two regulatory systems controlling transcription in the late stage of infection exist.

Social determinants of dietary habits in Denmark.

OBJECTIVE: To investigate whether there is an association between socioeconomic status and a healthy diet, taking the possible influence of other social variables such as age, gender, income and household composition into consideration. DESIGN: Cross-sectional study. SETTING: Nationwide study in Denmark, 1995. SUBJECTS: Random sample from the civil registration system. A total of 852 men and 870 women aged 18-80 y participated, a response rate of 58%. INTERVENTIONS: A 7 day estimated dietary record was used to obtain information about the diet. Information about social background was gained through face-to-face interview. RESULTS: The intake of fruit and vegetables and the percentage energy (E%) from fat in the diet were significantly associated with the educational level of both men and women. For men with only basic schooling, the mean intake of vegetables and fat was 84 g/10 MJ and 41 E% respectively. Men with long higher education had a mean intake of 119 g/10 MJ of vegetables and 37 E% of fat. For women, the corresponding figures for the intake of vegetables and fat were 131 g/10 MJ and 38 E% and 175 g/10 MJ and 37 E%, respectively. For women, age, income and household composition were also significantly associated with the intake of fruit and vegetables. CONCLUSIONS: Education seems to be the most important social variable to explain social differences in dietary habits. Additional variables are needed to explain dietary habits of women. Differences are seen for both foods and nutrients. SPONSORSHIP: The data analysis was financially supported by the Health Insurance Fund.

Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: evolution, structure, and genome organization of lactococcal bacteriophages.

A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution. Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool.

Identification of a replication protein and repeats essential for DNA replication of the temperate lactococcal bacteriophage TP901-1.

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by orf13 and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within orf13 and was shown to confer a TP901-1 resistance phenotype when present in trans. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by orf13 and the repeats within orf13 were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that orf13 encodes a replication protein and that the repeats within the gene are the origin of replication.

Hand eczema in twins: a questionnaire investigation.

Hand eczema in a population-based twin cohort of 6666 persons aged 20-44 years was investigated by means of a questionnaire regarding skin symptoms on the hands and self- or physician-diagnosed hand eczema. Genetic influence was observed by significant differences between identical and fraternal twins regarding casewise concordance rate and correlations in liability under the threshold model. The casewise concordance rates were almost twice as high in identical compared with fraternal twins in both sexes. By extension of the threshold model a joint analysis could be performed, resulting in a heritability estimate of 0.65. Tendencies towards stronger genetic influence for men and for younger individuals were present, but neither was statistically significant. No particular symptom pattern expressed especially strong or weak genetic influence. Hereditary factors for hand eczema were observed despite a presumably substantial individual-specific environmental variation. The hypothesis that hereditary risk factors may play a significant part in the development of hand eczema in the general population, when no extreme environmental exposure exists, seems justifiable. The relevance of known individual risk factors such as atopic dermatitis or contact allergy in relation to heredity remains to be analysed. The possible importance of age and temporal change also needs further consideration.

The genetic switch regulating activity of early promoters of the temperate lactococcal bacteriophage TP901-1.

A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters p(R) and p(L). ORF4 (180 amino acids) was identified as a phage repressor necessary for repression of both promoters. Furthermore, the presence of ORF4 confers immunity of the host strain to TP901-1. ORF5 (72 amino acids) was found to be able to inhibit repression of the lytic promoter p(L) by ORF4. Upon transformation with a plasmid containing both ORF4 and ORF5 and their cognate promoters, clonal variation is observed: in each transformant, either p(L) is open and p(R) is closed or vice versa. The repression is still dependent on ORF4, and the presence of ORF5 is needed for the clonal variation. Induction of a repressed p(L) fusion containing orf4 and orf5 was obtained by addition of mitomycin C, and the induction was also shown to be dependent on the presence of the RecA protein, even though ORF4 does not contain a recognizable autocleavage site. Our results suggest that the relative amounts of the two proteins ORF4 and ORF5 determine the decision between lytic or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages.

Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1.

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated into attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.

Design of limiting dilution analysis experiments for helper T lymphocyte precursor frequency determination in the context of allogeneic bone marrow transplantation.

Helper, interleukin 2 (IL-2) producing, T lymphocyte precursor (HTLp) frequency determination by limiting dilution analysis (LDA) is of value for quantifying alloreactivity in allogeneic bone marrow transplantation (BMT). LDA assays are labour-intensive and time-consuming to perform and the numbers of donor and recipient cells available are limited. It is therefore important that the design of the experiment yields reliable frequencies with a minimum of effort and a realistic cell requirement. We have critically evaluated the methods proposed for LDA design by Strijbosch et al. [Strijbosch, L.W., Buurman, W.A., Does, R.J., Zinken, P.H., Groenewegen, G., 1987. Limiting dilution assays. Experimental design and statistical analysis. J. Immunol. Methods 97, 133] and by Blackett and Gordon [Blackett, N.M., Gordon, M.Y., 1996. Optimizing limiting dilution assays: frequency and 'ability' measurements of haemopoietic progenitor cells. Br. J. Haematol. 92, 507 (see comments)] and found them inadequate for this application. The estimation of the HTLp frequency is traditionally based on the single-hit Poisson model and the adequacy of this model was compared with that of a double-hit model. The results were in favour of the single-hit model. Ten different LDA experimental designs were explored by Monte Carlo simulations. The optimal design exploits the maximal numbers of cells that can be obtained for analysis to estimate HTLp frequencies in the range 1:1,000,000-1:20,000 with a coefficient of variation of 10-20% and with a minimum of manual labour.

Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis.

Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp. cremoris (B. Christiansen, L. Brondsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164-5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site on the L. lactis subsp. cremoris chromosome. Furthermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L. lactis subsp. cremoris when the orf1 gene is donated in trans. The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages. Based on the deletion analysis, several repeats located within the attP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA or lacLM, encoding beta-glucuronidase or beta-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of L. lactis subsp. cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site attB used by bacteriophage TP901-1.

Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation.

The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation. The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5. Both operons are targets for the transcriptional activator AppY. In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS. Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY. The appY gene also showed sigmaS-dependent induction by carbon starvation. The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift. Like the cbd operon, the hya operon was highly induced by phosphate starvation. For both operons the induction was strongly dependent on AppY. The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons.

A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1.

The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp. cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M. G. Johnsen, E. Stenby, F. K. Vogensen, and K. Hammer, J. Bacteriol. 176:1069-1076, 1994). The phage-encoded elements necessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames, Orf1, Orf2, and Orf3 By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process. Furthermore, it was found that Orf1, attP, and a 425-bp region upstream of the orf1 gene are sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 44% similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages. The constructed integration vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20% of the transformation efficiency.

The histone-like protein H-NS acts as a transcriptional repressor for expression of the anaerobic and growth phase activator AppY of Escherichia coli.

The transcriptional activator AppY is required for anaerobic and stationary-phase induction of the cyx-appA and hya operons of Escherichia coli, and expression of the appY gene itself is induced by these environmental conditions. The sequence of the appY gene and its promoter region is unusually AT rich. The nucleoid-associated protein H-NS has a DNA-binding specificity for intrinsically curved AT-rich DNA. Using a single-copy transcriptional appY-lacZ fusion, we have shown that appY gene expression is derepressed in hns mutants during aerobic exponential growth. In the hns mutant, growth phase and growth rate regulation under aerobic conditions was maintained, while ArcA-dependent anaerobic induction was greatly diminished. Judged by two-dimensional gel electrophoresis, the appY promoter fragment exhibits the features characteristic of curved DNA. Gel retardation assays showed that purified H-NS protein bound with high affinity to two different segments of the appY promoter region. The role of H-NS in the AppY regulatory cascade is discussed and compared with its function in the regulatory cascades of the AppY homologs CfaD and VirF.

Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli.

The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene have been investigated under different environmental conditions with single-copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase. ArcA and AppY activated transcription of the cyx-appA operon during entry into stationary phase and under anaerobic growth conditions. The expression of the cyx-appA operon was affected by the anaerobic energy metabolism. The presence of the electron acceptors nitrate and fumarate repressed the expression of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high level of expression of the operon was obtained in glucose medium supplemented with formate, in which E. coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented in this paper indicate a clear difference in the regulation of the cyx-appA operon and that of the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function under even more-oxygen-limiting conditions than cytochrome d oxidase. The expression of the appY gene is induced immediately by anaerobiosis, and this anaerobic induction is independent of Fnr, and AppY, but dependent on ArcA. The expression of the appY gene is not affected significantly by the anaerobic energy metabolism, i.e., fermentation versus anaerobic respiration. A model incorporating the anaerobic regulation of the appY gene and the two operons which are controlled by AppY, the hydrogenase 1 (hya) operon and the acid phosphatase (cyx-appA) operon, is presented. The expression of the appY gene is inversely correlated with the growth rate and is induced by phosphate starvation as well as during entry into stationary phase. During oxygen-limiting conditions the stationary-phase induction is partially dependent on ArcA. The alternative sigma factor sigma S has limited influence on the transcription of the appY gene during entry into stationary phase and no effect on the induction by phosphate starvation.

Role of the transcriptional activator AppY in regulation of the cyx appA operon of Escherichia coli by anaerobiosis, phosphate starvation, and growth phase.

Transcriptional lacZ fusions have been used to analyze the regulation of the appA operon of Escherichia coli. The appA operon contains the genes cyxA and cyxB, coding for the putative third cytochrome oxidase, and appA, encoding acid phosphatase. The analysis showed that the cyxAB and the appA genes are cotranscribed from a potentially strong promoter, Pcyx, located immediately upstream of cyxA and that the operon in addition contains an internal promoter, PappA, contributing significantly to the transcription of the appA gene. The two promoters were both induced by starvation for Pi and by entry into stationary phase. The cyx promoter was in addition found to be activated by anaerobic growth conditions. The product of the previously identified appY gene, which when present on a high-copy-number plasmid stimulates synthesis of acid phosphatase, was shown to activate the cyx promoter. An insertion mutation in the appY gene was constructed in vitro and recombined into the chromosome. The appY mutation eliminated induction of the cyx promoter by anaerobiosis and severely reduced induction of this promoter by phosphate starvation and upon entry into stationary phase but had no effect on induction of the appA promoter. The appY mutation had no effect on survival in stationary phase, nor did it have any effect on growth rate or yield under aerobic or anaerobic conditions. The possibility that AppY is a third global regulator of energy metabolism genes is discussed.

Anaerobic regulation of the hydrogenase 1 (hya) operon of Escherichia coli.

Using a transcriptional fusion to the lacZ gene, we have analyzed the anaerobic regulation of the hydrogenase 1 (hya) operon in response to different anaerobic growth conditions and to mutations in regulatory genes. We found that the transcription of the hya operon was induced when the growth condition was changed from aerobic to anaerobic and that this induction was independent of Fnr but dependent on regulators AppY and ArcA. Furthermore, we found that the transcription of the hya operon was not regulated by the cyclic AMP-cyclic AMP receptor protein complex. Investigation of the effects of different anaerobic growth conditions on the expression of the hya operon showed that expression was induced by formate and repressed by nitrate. Formate induction was not mediated by the fhlA gene product, and nitrate repression was not mediated by the narL gene product. We found a high level of anaerobic expression of the hya operon in glucose medium supplemented with formate and in glycerol medium supplemented with fumarate, suggesting that hydrogenase isoenzyme 1 has a function during both fermentative growth and anaerobic respiration.