Anne Elizabeth Hill

Identification and characterization of a novel cytochrome c(3) from Shewanella frigidimarina that is involved in Fe(III) respiration.

Shewanella frigidimarina NCIMB400 is a non-fermenting, facultative anaerobe from the gamma group of proteobacteria. When grown anaerobically this organism produces a wide variety of periplasmic c-type cytochromes, mostly of unknown function. We have purified a small, acidic, low-potential tetrahaem cytochrome with similarities to the cytochromes c(3) from sulphate-reducing bacteria. The N-terminal sequence was used to design PCR primers and the cctA gene encoding cytochrome c(3) was isolated and sequenced. The EPR spectrum of purified cytochrome c(3) indicates that all four haem irons are ligated by two histidine residues, a conclusion supported by the presence of eight histidine residues in the polypeptide sequence, each of which is conserved in a related cytochrome c(3) and in the cytochrome domains of flavocytochromes c(3). All four haems exhibit low midpoint redox potentials that range from -207 to -58 mV at pH 7; these values are not significantly influenced by pH changes. Shewanella cytochrome c(3) consists of a mere 86 amino acid residues with a predicted molecular mass of 11780 Da, including the four attached haem groups. This corresponds closely to the value of 11778 Da estimated by electrospray MS. To examine the function of this novel cytochrome c(3) we constructed a null mutant by gene disruption. S. frigidimarina lacking cytochrome c(3) grows well aerobically and its growth rate under anaerobiosis with a variety of electron acceptors is indistinguishable from that of the wild-type parent strain, except that respiration with Fe(III) as sole acceptor is severely, although not completely, impaired.

Wort FAN – its characteristics and importance during fermentation

Wort FAN (free amino nitrogen) is the sum of the individual amino acids and small peptides (mainly di-, tripeptides). A further source of assimilable nitrogen is ammonium ions. This study has substantiated previous reports that both lager and ale yeast strains can simultaneously use wort amino acids and small peptides followed by ammonium ions. It is confirmed that amino acids are taken up in a distinct order. However, it is proposed that methionine, isoleucine and leucine be reclassified from group B to group A. Further studies are necessary to confirm that wort with enhanced L-methionine levels reduced fermentation rate and extent and lower beer VDK levels. Also the reason why fermenting wort containing supplemented lysine levels produced elevated VDK concentrations is unclear, but it is probably due to the enhanced biomass formation compared to the unsupplimented wort. Proteolytic enzymes secreted/excreted by viable yeast cells during fermentation are responsible for degrading larger wort peptides into smaller molecules and provides the yeast with an additional source of assimilable nitrogen and this hydrolysis has a negative effect on beer foam stability.

Microbiology and Microbiological Control in the Brewery

CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608 Wild Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608 Molds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611 Gram-Positive Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612 Lactobacillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612 Pediococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613 Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613 Acetic Acid Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613 Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614 Zymomonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616 Anaerobic Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 616 Microbiological Quality Assurance and Quality Control . . . . . . . . . . . . 618 Setting Microbiological Standards and Sampling . . . . . . . . . . . . . . . . 619 Traditional Microbiological Procedures . . . . . . . . . . . . . . . . . . . . . . . . . 621 Rapid Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622 ATP Bioluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622 Direct Epifluorescence Filter Technique . . . . . . . . . . . . . . . . . . . . . . . 623 Antibody DEFT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623 Fluorescence In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . 624 Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624 The Microbiological Laboratory within the Brewery . . . . . . . . . . . . . . . . 625 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

Survey of restriction-modification systems and transformation in Mannheimia haemolytica and Pasteurella trehalosi

A significant obstacle to molecular studies of Mannheimia (Pasteurella) haemolytica, has been its resistance to genetic transformation. The lack of competence of many M. haemolytica strains has been attributed to the presence of restriction modification systems. In this study, representative strains of 12 M. haemolytica serotypes and four Pasteurella trehalosi serotypes were successfully transformed by electroporation using a recombinant vector derived from the native M. haemolytica A1 serotype plasmid pNSF2176. Transformation was achieved despite PCR-based evidence for the presence of genes encoding a type I restriction enzyme, phaI, and a type II restriction enzyme hsdM, in each of the M. haemolytica strains.