The effects of supplementation of riboflavin along with Bacillus subtilis was investigated on growth performance and intestinal health of broilers under coccidial challenge. Treatments are 3 × 2 × 2 factorial arrangement, which includes 3 levels of riboflavin (0.75, 6.6 (recommended), and 20 ppm), with or without Bacillus subtilis, and with or without coccidial challenge. Coccidial challenge impaired the intestinal morphology and reduced body weight (BW), body weight gain (BWG), and feed intake and increased FCR between d 14-28. However, the cocci impact on birds was reduced along with age, as the effects on internal organs was reduced, which allowed birds to recover. However, coccidiosis reduced overall BW and BWG and increased abdominal fat pad weight and slight woody breast incidence. In conclusion, riboflavin at tested level was unable to enhance growth performance and intestinal health, but its inclusion altered birds response to coccidial challege other than feeding Bacillus subtlis alone.

Purified Bacillus subtilis DNA taken up by one-day-old barley embryos and permanently fixed within the barley cell is shown to have maintained its biological activity in B. subtilis transformation. Cesuim chloride density gradient centrifugation of DNA isolated from the roots of barley embryos after the uptake and incorporation of radioactive, bromouracil-labelled B. subtilis DNA indicates the presence of the bacterial DNA in several discreet bands of well-defined density, whose density is changed by sonication. These preliminary findings concerning the fate of bacterial DNA taken up by barley roots are discussed in terms of the recombination model of Ledoux and Huart and further experiments are suggested.

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Adaptation to any undesirable change in the environment helps to ensure the survival of many microorganisms. During nutrient starvation, bacteria undergo a stringent response characterized by the accumulation of the alarmone (p)ppGpp. This results in the repression of stable RNA species and a change in colony morphology. In Gram-negative bacteria such as Escherichia coli, RelA and SpoT synthesize and hydrolyze these nucleotides, respectively, under conditions of nutrient starvation. In Gram-positive bacteria, the bifunctional enzyme Rei is responsible for the accumulation of (p)ppGpp. These enzymes catalyze the transfer of a pyrophosphate moiety from ATP to the 3' end of either GTP or GDP. The overproduction of (p)ppGpp has many diverse consequences on bacterial physiology such as sporulation, virulence, long term persistence of pathogenic bacteria, cell morphology, antibiotic synthesis and fatty acid metabolism. In Bacillus subtilis a novel (p)ppGpp synthase, YwaC, is also involved in the accumulation of (p)ppGpp but does not associate with the ribosome. Transcriptional analysis of ywaC has implicated it with cell wall stress especially associated with lesions in the teichoic acid biosynthetic pathway. The work described here includes a steady state kinetic analysis of the reaction catalyzed by YwaC. Recombinant YwaC was over-expressed in E. coli and purified to homogeneity. Steady-state kinetic experiments were performed utilizing a high-performance liquid chromatography assay. This examination yielded Km values for GDP and GTP of 5 J.1M and 6 J.1M respectively, while the kcat was measured to be 0.13 min"1 and 0.11 min"1 respectively. As is common with other (p)ppGpp synthases, the low activity ofYwaC may be increased in the presence of the appropriate effector molecule. To explore the functional phenotype associated with ywaC a deletion strain was made by replacing the gene on the chromosome of B. subtilis with a spectinomycin resistance cassette. A variety of antibiotics were used to probe the ywaC deletion strain in an attempt to detect antibiotic sensitivity in comparison to wildtype cells. In addition, the morphology of the ywaC deletion strain was investigated using phase contrast confocal microscopy. Length and shape remained the same in a ywaC knockout. Growth profiles performed over a 24-hour period showed that the knockout strain grew similarly to wildtype B. subtilis. Thus, the phenotype analysis described herein failed to further elucidate the function of YwaC. Nevertheless, rigorous biochemical analysis described here have established the enzymatic role of (p)ppGpp synthesis for YwaC, but there remains much room for further investigation. / Thesis / Master of Science (MSc)

Necrotic enteritis (NE) is a disease that negatively affects broiler performance and has increasingly become a problem with the reduced use of antibiotics. There is increased interest to utilize alternative methods including coccidial vaccines and direct fed microbials (DFM) to reduce or prevent NE. Additionally, it has been demonstrated that reduced dietary Ca can reduce NE mortality. The objective of the first study was to examine the effects of Bacillus subtilis (DFM) in combination with attenuated (AV) and non-attenuated (NV) coccidial vaccines in broilers with NE. The objective of the second study was to determine the effects of DFM, antibiotic (BMD) or negative control (NC) with standard dietary Ca (0.9%) or below standard Ca (0.75%, 0.6%) in broilers with NE. To induce a natural NE occurrence, birds were vaccinated with the appropriate coccidial vaccine and then placed on reused litter from a previous flock exhibiting NE lesions and mortality. In the first trial DFM reduced BW and BW gain (P ≤ 0.05) during the starter phase (0 to 14 d). The addition of a DFM in the AV group resulted in decreased (P ≤ 0.05) BW gain and BW at 42 d. The DFM in the NV group resulted in improved (P ≤ 0.05) mortality corrected feed conversion ratio from 0 to 42 d. Overall, with a natural NE occurrence, the effect of DFM was variable depending on what type of coccidial preventative was utilized and was beneficial when used with NV. In the second trial BW and BW gain were increased in broilers fed 0.60% dietary Ca and DFM compared to 0.90% Ca and NC (P ≤ 0.05). Broilers fed 0.60% Ca had reduced NE mortality compared to broilers fed 0.90% Ca (P ≤ 0.05). Reducing dietary Ca to below industry standards was able to lessen the severity of NE though the addition of DFM was needed to improve BW. Overall, with a natural NE occurrence, the DFM was beneficial when used with NV and in diets with 0.6% dietary Ca. / Doctor of Philosophy / Necrotic enteritis (NE) is caused by the bacterium Clostridium perfringens and is a disease that reduces growth of broiler chicks and has increasingly become a problem in the chicken industry with the reduced use of antibiotics. Coccidiosis is a protozoal disease that causes intestinal damage and predisposes broilers to developing NE. Prevention of coccidiosis can be through medication or coccidial vaccines. Probiotics or direct fed microbials (DFM) have demonstrated the potential to reduce both coccidiosis and NE symptoms. Additionally, it has been shown that reducing the amount of calcium (Ca) in the diet can reduce chick death from NE. The first study examined the effects of a beneficial bacterium, Bacillus subtilis (DFM) in combination with two types of coccidial vaccines, attenuated (AV) and non-attenuated (NV). The second study investigated DFM with standard dietary Ca (0.9%) or below standard Ca levels (0.75%, 0.6%). To induce a "natural" NE disease occurrence, birds were vaccinated with the appropriate coccidial vaccine and then placed into pens. Pens contained reused litter from a previous flock of birds with NE (contained spores of Clostridium perfringens). In the first trial DFM reduced body weight of broilers during the first two weeks (P ≤ 0.05). The DFM decreased (P ≤ 0.05) BW gain and BW in the AV group but improved feed efficiency in the NV group (P ≤ 0.05). In the second trial BW and BW gain were increased in broilers fed DFM and lower Ca diets (0.6%) compared to those fed the standard (0.90%) Ca diets without DFM (P ≤ 0.05). Dead birds were reduced when broilers were fed lower Ca diets (0.6%) compared to broilers fed standard (0.90%) Ca diets (P ≤ 0.05). In conclusion, the DFM was beneficial when used with NV and in diets with lower Ca (0.6%) during a natural NE occurrence.

M.S.

Peptidoglycan not only protects bacterial cells against intracellular pressure but also provides the cells with a defined morphology. Penicillin-binding proteins (PBPs) catalyze the polymerization of the peptidoglycan in Bacillus subtilis. PBP2a and PBP2b are class B PBPs which have been known to have transpeptidase activities and they localize at different positions on the cell membrane. PBP2a spreads around the cylindrical wall as well as some at the septum, and PPB2b localizes exclusively to the septum and some at the cell poles. Both PBP2a and PBP2b are composed of four domains: S, N, P, and C domains from the N- to C- terminus. A FLAG epitope was tagged to the C-terminal ends of PBP2a and PBP2b. Cells with FLAG tagged PBP2a or PBP2b grow as well as wild type strain. Expression of PBP2a-FLAG and PBP2b-FLAG can be detected by western blotting using anti FLAG antibody. The expression of wild type PBP2a/PBP2b in these strains was tightly controlled by a xylose promoter. The FLAG fusion didn't influence the normal membrane localizations of PBP2a or PBP2b. PBP2a/2b mutant strains with the S and/or N domains switched between PBP2a and PBP2b were constructed. All these domain-switch proteins were tagged with a FLAG at the C-terminus. The expression of these recombinant proteins can be detected by western blotting. None of these domain-switch proteins was able to complement the wild type PBP2a and PBP2b and cells with only these recombinant proteins but no wild type proteins were non-viable. Cellular localization of these domain switch proteins were visualized using immunofluorescence microscopy. Proteins containing the PBP2a S domain had the same localization patterns as wild type PBP2a. Proteins that have the PBP2b S domain localized specifically at the septum and cell poles, which is similar to the wild type PBP2b. These results indicate that the S domain is the determinant to direct PBP2a and PBP2b to their cellular destinations. / Master of Science