Electron microscopic analysis of dairy microbes inactivated by ultrasound.

Ultrasonication is a non-thermal method of food preservation that has the advantage of inactivating microbes in food without causing the common side-effects associated with conventional heat treatments, such as nutrient and flavour loss. The aim of this study was to evaluate the use of ultrasound as an alternative to heat pasteurisation and to assess cell damage using transmission electron microscopy (TEM). Three spoilage microbes, previously isolated from pasteurised milk, were used as "test" microbes. Saline solution (SSS) and UHT milk were used as suspension media and were inoculated with exponential growth phase "test" microbes at a microbial concentration of 1 x 10(4) cfu ml(-1). The samples were subjected to power ultrasound (20 kHz, 750 W), at 100%/124 microm wave amplitude for different time intervals. Both Escherichia coli and Saccharomyces cerevisiae were reduced by >99% (for both suspension media) after ultrasonication and Lactobacillus acidophilus was reduced by 72% and 84% in SSS and milk, respectively. Transmission electron microscope micrographs showed that ultrasonication inflicts extensive microbicidal/microbistatic external and internal damage on all three "test" microbes. In E. coli, sonication-induced emulsification caused the formation of unique minute lipopolysaccharide vesicles from the fragmenting cell envelope.

Co-regulation of the nitrogen-assimilatory gene cluster in Clostridium saccharobutylicum.

Nitrogen assimilation is important during solvent production by Clostridium saccharobutylicum NCP262, as acetone and butanol yields are significantly affected by the nitrogen source supplied. Growth of this bacterium was dependent on the concentration of organic nitrogen supplied and the expression of the assimilatory enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT), was shown to be induced in nitrogen-limiting conditions. The regions flanking the gene encoding GS, glnA, were isolated from C. saccharobutylicum genomic DNA, and DNA sequencing revealed that the structural genes encoding the GS (glnA) and GOGAT (gltA and gltB) enzymes were clustered together with the nitR gene in the order glnA-nitR-gltAB. RNA analysis showed that the glnA-nitR and the gltAB genes were co-transcribed on 2.3 and 6.2 kb RNA transcripts respectively, and that all four genes were induced under the same nitrogen-limiting conditions. Complementation of an Escherichia coli gltD mutant, lacking a GOGAT small subunit, was achieved only when both the C. saccharobutylicum gltA and gltB genes were expressed together under anaerobic conditions. This is believed to be the first functional analysis of a gene cluster encoding the key enzymes of nitrogen assimilation, GS and GOGAT. A similar gene arrangement is seen in Clostridium beijerinckii NCIMB 8052, and based on the common regulatory features of the promoter regions upstream of the glnA operons in both species, we suggest a model for their co-ordinated regulation by an antitermination mechanism as well as antisense RNA.