A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production.

BACKGROUND: Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process. RESULTS: Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae. CONCLUSIONS: Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.

High Intrapatient Tacrolimus Variability Is Associated With Worse Outcomes in Renal Transplantation Using a Low-Dose Tacrolimus Immunosuppressive Regime.

BACKGROUND: High intrapatient tacrolimus variability has been associated with worse clinical outcomes postrenal transplantation. Theoretically, tacrolimus levels consistently outside the target therapeutic window may result in allograft dysfunction as subtherapeutic tacrolimus levels predispose to episodes of acute rejection, whereas supratherapeutic levels may cause nephrotoxicity. METHODS: We investigated the effect of tacrolimus variability in a "Symphony" style low-dose tacrolimus based regime, by collecting data from 432 patients over a 4-year period.Three hundred seventy-six patients were included, with a mean follow-up of 1495 days. Tacrolimus variability 6 to 12 months after renal transplantation was calculated, and outcomes were compared in low (n = 186) and high variability (n = 190) groups. RESULTS: High variability patients were found to be at increased risk of rejection during the first posttransplant year (P = 0.0054) and to have reduced rejection-free survival (hazard ratio, 1.953; 95% confidence interval, 1.234-3.093; P = 0.0054). High variability patients had significantly worse (P < 0.0001) glomerular filtration rates at 1, 2, 3, and 4 years posttransplant. High variability patients were at increased risk of allograft loss (hazard ratio, 4.928; 95% confidence interval, 2.050-11.85; P = 0.0004). CONCLUSIONS: This suggests that highly variable tacrolimus levels predict worse outcomes postrenal transplantation, although the causal nature of this relationship remains unclear. High tacrolimus variability may identify a subset of patients who warrant increased surveillance and patient education regarding dietary and medication compliance.

Budding yeast Dbf4 sequences required for Cdc7 kinase activation and identification of a functional relationship between the Dbf4 and Rev1 BRCT domains.

Cdc7-Dbf4 is a two-subunit kinase required for initiating DNA replication. The Dbf4 regulatory subunit is required for Cdc7 kinase activity. Previous studies have shown that the C termini of Dbf4 orthologs encode a single (putative) C(2)H(2) zinc (Zn) finger, referred to as "motif C." By mutational analysis we show that the Zn finger is not required for the essential function of Dbf4. However, deletion and point mutants altering conserved Zn-finger residues exhibit a substantially slowed S-phase, DNA damage sensitivity, and a hypo-mutagenic phenotype following UV irradiation. Using two-hybrid and biochemical assays, we show that the Dbf4 Zn finger interacts with Cdc7 and stimulates its kinase activity. However, a separable Dbf4 region also mediates an interaction with Cdc7 such that only the loss of both Cdc7-interacting regions results in lethality. In contrast, an N-terminal BRCT-like domain is not required for induced mutagenesis nor does it interact with Cdc7. By making chimeric Dbf4 proteins that contain known BRCT domains in Saccharomyces cerevisiae, we show that the BRCT domain from Rev1, a translesion DNA polymerase, can uniquely substitute for the Dbf4 BRCT domain. Thus, we have mapped regions on budding yeast Dbf4 required for binding and activating Cdc7 kinase. Our data also suggest that the Dbf4 and Rev1 BRCT domains interact with a common protein or structure, although the precise function of both domains and their binding partners remains elusive.