Search

Abstract 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.

Micrococcus luteus (NCTC2665, 'Fleming strain') has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to [beta]-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome. doi: 10.1128/JB.01254-09

There is some debate concerning the degree of availability of organic vs. inorganic forms of trace minerals. The answer depends on the specific mineral, the dietary conditions and the physiologic state of the animal. It is clear that for certain minerals (e.g., selenium, chromium or iron), organic forms are better utilized than inorganic forms (Hallberg and Rossander-Hulthen 1993, Mertz and Roginski 1971). For other minerals (e.g., zinc or copper), it is perhaps less clear. For example, there are as many studies that have failed to show increased bioavailability with organic zinc forms as there are studies demonstrating improved bioavailability. It has been demonstrated in livestock and fish (Paripatananont 1994, Wedekind et al. 1992 and 1994) that growth rate, calcium and phytate levels are factors that significantly affect zinc utilization and thus determine whether the use of organic zinc sources is beneficial. Viewing these data as a whole, results suggest that there is little or no benefit to be gained in using organic zinc in diets low in phytate and calcium (low calcium defined as calcium levels approximating NRC recommendations for respective species). However, as phytate and/or calcium increase, or demand for zinc increases (e.g., fast growth rate), zinc utilization is greater with organic zinc sources (parameters used to assess zinc bioavailability included one or more of the following: bone. zinc, immune response and growth rate). Furthermore, the faster growing the animal, the greater the benefit demonstrated for organic zinc [e.g., fish > chicks (broiler breed > leghorn-type) > pigs]. The efficacy decreased as the animal's age increased, suggesting that there may be less benefit in using organic zinc sources in foods for adult animals. The purpose of this study was to compare the bioavailability of an organic zinc source (zinc propionate) with that of an inorganic zinc source (ZnO) in puppies. These two zinc sources were compared under three dietary conditions as follows: 1) at 10 g Ca/kg diet [the Association of American Feed Control Officials (1997) recommendation for Ca during growth]; 2) 15 g Ca/kg diet: and 3) 15 g Ca/kg diet + 50 g/kg beet pulp. The two Zn sources were compared under varying levels of Ca and fiber to determine whether fiber or excessive Ca had differing effects on the bioavailability of Zn from organic or inorganic Zn. Materials and methods. Forty-two puppies (9 wk mean age at study start) were fed a Zn-deficient egg white/cornstarch/sucrose diet (Table 1) for 2 wk. At the end of this pretest period, puppies were allotted to one of seven dietary treatments. There were three replicates per treatment with two puppies per pen. The puppies were blocked by litter, sex and weight. Within each block, each treatment had a similar mean initial weight and weight distribution. Puppies were housed in stainless steel pens and were allowed free access to food and water. Puppies were fed the experimental diets for 3 wk. However, puppies receiving the control diet were removed from the test if signs of Zn deficiency were severe, and only one of the six puppies receiving that treatment (treatment 1) could continue through d 35 of the study. The experimental protocol was reviewed and approved by the institutional Animal Care and Use Committee. Feed consumption was measured daily and body weights measured weekly. The basal diet (Table 1) was formulated to be adequate in all nutrients except Zn (NRC 1985). Dietary additions of Zn, Ca and beet pulp were made at the expense of cornstarch with Zn added as Zn propionate (ZnP; KemZIN; provided by Kemin Industries, Des Moines, IA) or ZnO and Ca provided as CaC[O.sub.3]. Cellulose was present n the diet to improve stool quality, but has no antagonistic effects on Zn bioavailability and also contributes no phytate to the diet (Wedekind et al. 1995). Treatment 1 was the control with 10 g Ca/kg diet and no added Zn (5.4 mg Zn/kg diet, analyzed); treatments 2 (ZnP) and 3 (ZnO) contained 10 g Ca/kg diet; treatments 4 (ZnP) and 5 (ZnO) contained 15 g Ca/kg diet; and treatments 6 (ZnP) and 7 (ZnO) contained 15 g Ca/kg diet plus 50 g/kg beet pulp. The Zn-supplemented treatments were intended to be equal in zinc concentration (40 mg/kg added Zn); however, actual analyzed Zn concentrations (mg Zn/kg diet) differed between the ZnO and ZnP treatments as shown in Table 2. The basal diet was analyzed for moisture, protein, fat, fiber, ash, Ca, P and Zn; other treatments were analyzed for moisture, Ca and Zn. Blood samples were taken weekly during the pretest period and at the end of the experimental period (i.e., wk 0, 1, 2 and 5). Blood was collected using special-purpose (low Zn) trace element evacuated tubes. At the end of the experiment, dewclaws, one canine deciduous tooth and testes were collected for Zn analysis. The dewclaws were autoclaved to facilitate removal of skin and connective tissue. The bones, teeth and testes were dried at 105 [degrees] C overnight. All samples were dry-ashed overnight in a muffle furnace (650 [degrees] C for bone and teeth samples; 450 [degrees] C for testes) before preparation for Zn analysis. Zn was determined in all tissues by inductively coupled plasma-emission spectrophotometry. Bioavailability of Zn was determined for the Zn propionate and ZnO treatments via multiple regression slope ratio analysis. Plasma Zn was regressed on supplemental Zn intake by comparing the change in plasma Zn between d 35 and 14 (end of Zn-deficient pretest period) for treatments 2-7. The model included block and the amount (milligrams) of Zn fed from each of the Zn sources such that six regression lines with a common intercept were determined. There were six observations per treatments 2-7; treatment one was not used), yielding six data points for the construction of a line of best fit. The slopes were compared with the standard [arbitrarily set as treatment 2, (ZnP at 10 g Ca/kg diet)]. Analyzed Zn values from treatments 2-7 were used in the regression analysis; thus discrepancies in dietary Zn levels between treatments were corrected for. Figure 1 shows a graphical representation of the data. The intercept was calculated by assuming that block effects summed to zero. The slopes of treatments 2-7 were statistically compared using a heterogeneity of slopes procedure to determine if significant differences in slopes existed. Differences between individual slopes were then tested using t tests among all pairs only if F-tests for differences among slopes were significant (P < 0.05). All ANOVA and regression analyses were performed using the General Linear Modes procedure of SAS (SAS Institute, Cary, NC). Probability values of P < 0.05 between treatments were considered significant. Results. Plasma Zn decreased over time during the Zn-deficient pretest period from an average of 11.32 [+ or -] 0.40 (SEM) to 4.44 [+ or -] 0.18 [micro]mol/L. Signs of Zn deficiency occurred as early as 5 d into the pretest period. The symptoms were suggestive of parakeratosis (e.g., crusted plaques with erosions on face and feet). Other symptoms included lethargy and anorexia. No differences in weight gain were seen between the Zn-supplemented treatments, but puppies fed treatment 1 (no added Zn) lost weight over the 3-wk experimental period. Because of small sample size (i.e., < 0.1 g average), the analytical variation was high for Zn concentrations in dewclaws, teeth and testes (Table 2). Other studies have shown bone to be a sensitive measure of Zn status (Wedekind et al. 1992), but sample size in those studies exceeded 1 g. Despite the higher Zn intakes for puppies fed the ZnO treatments, the plasma Zn concentrations tended to be higher for puppies fed the Zn propionate treatment under each of the three dietary regimens (10 g/kg Ca, 15 g/kg Ca and 15 g/kg Ca + 50 g/kg beet pulp). With increasing dietary Ca, Zn concentrations in plasma, teeth and testes tended to decline. This tendency was observed for bot h Zn sources. When plasma Zn was regressed on supplemental Zn intake, thus correcting for differences in dietary Zn intake, significant differences (P < 0.05) in Zn bioavailability were observed between Zn propionate and ZnO (Table 3). When dietary Ca was at 10 g/kg, the bioavailability of Zn in Zn propionate was 80% greater (i.e., 0.01041/0.00575 = 1.8) than the Zn in ZnO. The bioavailability of both Zn sources decreased when dietary Ca was increased to 15 g/kg, but an advantage was seen for Zn propionate vs. ZnO regardless of dietary Ca level and/or presence of beet pulp. Zn utilization was not significantly affected by beet pulp addition. Discussion. The results of our study showed that the bioavailability of Zn in Zn propionate is 60-80% greater than that of the Zn in ZnO. However, it is clear from other studies (Wedekind et al. 1992 and 1994) that the Zn in ZnS[O.sub.4] is approximately twofold more available than that in ZnO. Thus, it would appear from this study that the efficacy of Zn propionate, an organic Zn source, is no greater than that of certain other inorganic Zn sources. Other studies have been conducted in dogs comparing Zn bioavailability between organic and inorganic Zn sources. Lowe et al. (1994) found that organic Zn sources resulted in greater Zn retention, higher concentrations of Zn in hair and greater hair growth compared with ZnO. It should be noted that the diet in that study did contain phytate (soybean meal was in the diet at 16%) and Ca levels were high (3.2%). Slope-ratio analysis was not used in that study; however, if one compares the hair Zn concentrations between the Zn sources as a ratio (i.e., 21.09/10.83), then a 95% advantage in Zn bioavailability is yielded for the zinc amino chelate vs. ZnO at 1.2% dietary Ca. This is similar to the 60-80% advantage we saw in this study between organic and inorganic Zn sources. When the zinc amino acid chelate is compared with ZnO at 3.2% dietary Ca, that advantage increased to 290% (21.15/7.28). The difference in Ca levels and phytate content between that study and this one may explain the greater bioavailability estimate obtained (at 3.2% dietary Ca) from the earlier study. Thus, in conclusion, organic Zn sources are efficacious only under certain dietary conditions and may not yield significant improvements in pet food diets that are low in Ca and/or phytate. KEY WORDS: * zinc * bioavailability * puppies * calcium * organic zinc

Kidney stones are complex, biological matrices often composed of mixtures of minerals and organic matter. In this study, we use FT-IR spectroscopy, an advanced ATR correction and a novel Multicomponent [...]