F. B. Rudolph

Intracellular Butyryl Phosphate and Acetyl Phosphate Concentrations in Clostridium acetobutylicum and Their Implications for Solvent Formation

ABSTRACT It has been suggested (L. H. Harris, R. P. Desai, N. E. Welker, and E. T. Papoutsakis, Biotechnol. Bioeng. 67:1-11, 2000) that butyryl phosphate (BuP) is a regulator of solventogenesis in Clostridium acetobutylicum. Here, we determined BuP and acetyl phosphate (AcP) levels in fermentations of C. acetobutylicum wild type (WT), degenerate strain M5, a butyrate kinase (buk) mutant, and a phosphotransacetylase (pta) mutant. A sensitive method was developed to measure BuP and AcP in the same sample. Compared to the WT, the buk mutant had higher levels of BuP and AcP; the BuP levels were high during the early exponential phase, and there was a peak corresponding to solvent production. Consistent with this, solvent formation was initiated significantly earlier and was much stronger in the buk mutant than in all other strains. For all strains, initiation of butanol formation corresponded to a BuP peak concentration that was more than 60 to 70 pmol/g (dry weight), and higher and sustained levels corresponded to higher butanol formation fluxes. The BuP levels never exceeded 40 to 50 pmol/g (dry weight) in strain M5, which produces no solvents. The BuP profiles were bimodal, and there was a second peak midway through solventogenesis that corresponded to carboxylic acid reutilization. AcP showed a delayed single peak during late solventogenesis corresponding to acetate reutilization. As expected, in the pta mutant the AcP levels were very low, yet this strain exhibited strong butanol production. These data suggest that BuP is a regulatory molecule that may act as a phosphodonor of transcriptional factors. DNA array-based transcriptional analysis of the buk and M5 mutants demonstrated that high BuP levels corresponded to downregulation of flagellar genes and upregulation of solvent formation and stress genes.

Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824

Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added. Journal of Industrial Microbiology & Biotechnology (2001) 27, 220–227.

Immunohemopoietic effects of dietary nucleotide restriction in mice

The influence of dietary sources of nucleotides on host in vivo and in vitro immuno-hematologic responses in BALB/c (NCI) mice was studied. Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in popliteal lymph nodes undergoing proliferative response to syngeneic and allogeneic in vivo stimulation. Supplementation of a nucleotide-free (NF) diet with yeast RNA (NFR) or uracil (NFU) significantly enhanced the host PLN immune response as compared with NF and NF supplemented with adenine (NFA) diets. Levels of ADA and PNP enzymes in the PLNs increased with the alloimmune PLN response of host, and immunosuppression was associated with decreased ADA and PNP activities in lymphocytes following antigenic stimulation. The induction of these enzymes during immune response appears to require dietary sources of certain nucleotides. When bone marrow cells from control chow fed animals were cultured with supernatants (sups) from mitogen activated splenocytes of animals on each dietary group, NF sups significantly decreased (P less than 0.05) the BM proliferative response compared with the response observed with NFR sups, and similar to NFA or NFU sups. When stimulated with purified IL-3, NFR BM cells had higher levels of Thy1.2 or Lyt 1 surface markers as compared with other test groups. In the in vivo splenic colony formation-CFUs assay, spleens from NFR- and NFU-fed animals had a significantly higher number of colonies than spleens from NF- or NFA-fed mice. Thus, NF diet decreases both in vivo lymphoproliferation response to alloantigen and hemopoietic growth factor production, rendering the host splenic environment deficient for stem cell growth. These adverse effects are reversed by RNA supplementation of NF diet. These nutritional studies demonstrate a critical and regulatory role for dietary nucleotides in immunohemopoiesis.

Suppression of opiate withdrawal by cyclosporin A and dietary modification

It has been demonstrated in a murine model that a defined diet (Purina Basal Diet 5755) has immunosuppressive effects similar to cyclosporin A (CsA). It was also shown that CsA treatment in opiate dependent rats can attenuate the severity of opiate withdrawal. In this study, an opiate dependence model was established in Balb/c mice to assess the effects of the 5755 diet and CsA on morphine withdrawal - a CNS mediated phenomenon. Three groups of mice were used; a chow-fed control group (Purina 5008), a chow fed CsA treated group, and a group maintained on the 5755 diet. Morphine dependence was established by subcutaneous implantation of a 100 mg morphine base pellet under ether anesthesia. Seventy-two hours after pellet implantation, withdrawal was precipitated by a single injection of the opiate antagonist naloxone (2 mg/kg ip). Two indicators of withdrawal were assessed; jumping and diarrhea. The data demonstrated that both CsA and the 5755 diet resulted in significant attenuation of withdrawal symptoms with the 5755 diet being the most effective of the two. These findings suggest that immune modulation elicited by the 5755 diet and CsA treatment has a direct impact on the CNS opioid function.

Nutritional supplementation of nucleotides restores opioid CNS-mediated phenomena in mice

Previous experiments have demonstrated that suppression of immune function by either cyclosporin A or by a nucleotide free (NF) diet results in attenuation of morphine withdrawal symptoms in mice suggesting that immune status impacts CNS opioid-related phenomena. The present study elaborates on these initial findings by examining the effects of repletion of the NF diet with nucleotides or their precursors on opiate withdrawal. Female Balb/c mice were divided into six groups: a control group (C) given a standard lab chow diet and five experimental groups each given one of the following diets: a nucleotide free diet (NF); the NF supplemented with 0.25% RNA (NFR 0.25); the NF supplemented with 2.5% RNA (NFR 2.5) the NF supplemented with 0.06% uracil (NFU 0.06); the NF supplemented with 0.6% uracil (NFU 0.6). The mice were made morphine dependent by subcutaneous implantation of morphine pellets. Seventy-two hours after morphine pellet implantation, withdrawal was precipitated with naloxone (2 mg/kg). The mice were then observed and two indicators of withdrawal scored: jumping and diarrhea. The NF, NFR 0.25, NFR 2.5 and NFU 0.06 groups demonstrated significantly attenuation of the withdrawal signs relative to control animals. The NFU 0.6 group, however, had withdrawal scores restored to near control levels for both jumping and diarrhea. This suggests that nucleotides, particularly uracil, may play an important role in the immune-to-brain signaling pathway.