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MCB 301 Lab Manual supplemental Spring 2008 1 List of Possible Bacteria You will each be given one organism to inoculate a set of biochemical tests for Experiment 11. Organisms that have had their genomes fully sequenced are listed below with their genome id number. In several cases, a genome sequence is not available for the bacteria you will grow and test. Alternative genomes are listed in parentheses. Prior to running the biochemical tests, you will explore the genome of a given organism an
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  MCB 301 Lab Manual supplementalSpring 2008 List of Possible Bacteria You will each be given one organism to inoculate a set of biochemical tests for Experiment 11. Organisms that have had their genomes fully sequenced are listedbelow with their genome id number. In several cases, a genome sequence is notavailable for the bacteria you will grow and test. Alternative genomes are listed inparentheses. Prior to running the biochemical tests, you will explore the genome of agiven organism and predict the result of the biochemical phenotype on the basis of genotype.Gram Positive Bacilli Bacillus megaterium ( B. clausii    66692.3   ) Bacillus subtilis  224308.1 Lactobacillus plantarum  220668.1 Gram Positive Cocci Enterococcus faecium ( E. faecalis 226185.1) Staphylococcus aureus (NP) str. NCTC8325, 93061.3 Staphylococcus epidermidis str. RP62A,176279.3  Gram Negative Bacilli Salmonella Arizonae321314.4(aka S.enterica subsp. enterica ser. Choleraesuis) Proteus vulgaris ( P. mirabilis 584.1   ) Serratia marcescens (NP)615.1 Citrobacter freundii  ( Salmonellatyphimurium 99287.1   ) Hafnia alvei  ( Yersinia pseudotuberculosis 273123.1) Enterobacter cloacae ( Shigella flexneri   2a   str. 301 198214.1) Morganella morganii  ( Salmonella entericasubsp. enterica serovar Typhi Ty2   209261.1) Experiment 10 a-c: Bioinformatic Predictions, Summary Predictions will be made from annotated genomes available at the National MicrobialPathogen Data Resource,www.nmpdr.org. Whole genome sequences are availablethrough a variety of databases, but the NMPDR has many useful tools that will make iteasier for you to predict the biochemical test results. Most importantly, NMPDRcurators have organized the genome annotations into biological subsystems.In the list above, genome id numbers link to the respective organism's SubsystemSummary. This summary is a list of genes that have been classified according tofunction. Functional roles that meke up a pathway or complex are grouped together intosubsystems. Related subsystems are grouped together under a common heading, for example, the first set of subsystems is listed under the heading, Amino Acids andDerivatives. Subheadings further classify related subsystems. Subsystem names arelinked to a page that describes the subsystem. Proteins listed in each subsystem arelinked to pages that display their genomic context.The presence or absence of individual genes or groups of genes that form a completemetabolic pathway will be investigated using the subsystems of NMPDR. Thesegenotypes will predict the biochemical phenotype that results from each test. If youhave enough information about the genotype to predict the outcome with precision,record your prediction using the outcome notations listed for each biochemical test. If you only have an idea of positive or negative, list a plus or minus.1  MCB 301 Lab Manual supplementalSpring 2008 Experiment 10a: Carbohydrate Metabolism 1.Sugar Fermentations - Glucose, Lactose, Raffinose, Sucrose Bacterial cells are able to generate energy from nutrients through respiration or throughfermentation. Respiration uses an external electron acceptor, like oxygen (aerobicrespiration) or some other exogenous source (anaerobic respiration) to generate highyields of ATP through complete oxidation of an organic compound. Fermentation, onthe other hand, only partially oxidizes the substrate and generates a relatively smallamount of ATP. The terminal electron acceptor is usually produced as an intermediatein the pathway and so is internal instead of external.Different bacteria can ferment a wide variety of sugars and other compounds. Thedetermination of a fermentation pattern for a series of different energy/carbon sources(usually sugars) by an unknown bacterial species is often a central part in theidentification process. For example, sugar fermentation patterns are used in theidentification of enteric bacteria. Acid products, which may be produced from the fermentation of a sugar, will cause anoticeable color change in the pH indicator included in the medium. Sugar fermentationdoes not produce alkaline products. However, non-fermentative hydrolysis of aminoacids in the peptone, present in most fermentation media, may give an alkaline reaction,which will also cause a color change in the pH indicator. Gas production, H2 inparticular, can be determined by placing a small, inverted Durham tube in the testmedium. If gas is produced, it is trapped in the Durham tube and can be seen as abubble. Possible ResultsA/G: Both acid and gas have been produced. The medium has changed color from bluish-green to yellow and a gas bubble has formed in the Durhamtube. Note: reference to gas is for the glucose test only. A:  Acid has been produced. The medium has turned yellow. (A): A little bit of acid has been produced. The medium has turned lime greenor yellowish in the bottom of the tube and greenish at the top. 0 +: No acid or gas has been produced and growth is noted [shake the tube tolook for turbidity (cloudiness) indicating bacterial growth]. The medium isgreen. 0 +/B: No acid or gas has been produced and growth is noted. The mediumhas turned blue due to an alkaline reaction caused by amino acidutilization as a carbon source. This change will occur if the amount of alkaline end products made from the utilization of amino acids in themedium exceeds the amount of acidic end products from the sugar.2  MCB 301 Lab Manual supplementalSpring 2008 2.Sugar Fermentation - Mannitol Mannitol, a sugar alcohol, is widespread in plants and algae. It is the reduced form of the monosaccharide mannose, an aldose (sugar aldehyde). Mannitol has 14 hydrogenscompared to 12 for mannose. Possible ResultsA: Acid has been produced. The medium has changed color from purple toyellow. (A):  A little bit of acid may have been produced. The medium has changed to agrayish purple color (in between yellow and purple). 0 +: No acid has been produced. The medium remains purple and growth isnoted [shake the tube to look for turbidity (cloudiness) indicating bacterialgrowth].Control:Streptococcus mutans210007.1 uses all four sugars as well as mannitol.The net reaction observed in the fermentation test is usually the difference between theproduction of acid from a sugar and the production of alkaline end products, such asammonia, from peptone. The test result therefore is dependent on several factors. (1)Does the microorganism produce acid? If so, how much and at what rate? (2) Will themedium support growth? How highly is it buffered and how much alkaline product willbe generated in it? (3) How sensitive is the indicator?We can explore the metabolic capacity of the organisms to predict the answer to (1)above for each of the five separate sugars to be tested. Open the link above for thepositive control, and also open the link on page 1 that corresponds to your assignedorganism (control-click on this document). Subsystems describing the sugar utilizationor fermentation will be listed under the major heading, Carbohydrates. Subheadingscould include Central carbohydrate metabolism, Di- and oligosaccharides, Fermentations or Monosaccharides. 3   HOHmannosemannitol  MCB 301 Lab Manual supplementalSpring 2008 Scroll through the subsystem summary for the positive control. Note which subsystemsyou think are involved in fermentation and utilization of these sugars. Now scrollthrough the subsystem summary for your assigned genome. Record your predictionson the answer spreadsheet (last page), and include evidence supporting your predictionin the column labeled Why? For example, evidence could be absence of subsystemx or presence of gene y. QUESTIONS ON FERMENTATIONS 1.By looking only at the Subsystem Summary, is it possible to predict thefull range of biochemical test results, or simply plus or minus?2. In the Subsystem Summary for the positive control genome, S. mutans , click onthe Mannitol Utilization heading (or click here) to open the subsystem. The pagepresents a spreadsheet with genomes as rows and functional roles as columns.Genes that perform the listed functions are populated in the cells of thespreadsheet. Numbers with the same background color are in close proximity inthe genome. The abbreviated column headers are decoded in pop-up boxes if youpoint to them. The abbreviations are also listed in the table of functional roles,which you must scroll down to find. Below the table of functional roles are thecurator's notes. These notes will explain the variant codes and are frequently veryinformative. Scroll back up to the subsystem spreadsheet. Notice that there are missing genes in one strain of Streptococcus pyogenes. Which functions aremissing? Do you predict that this strain will ferment mannitol? Explain.4
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