dDuring construction of several gene deletion mutants in Lactococcus lactis MG1363 which involved a high-temperature (37.5°C) incubation step, additional spontaneous mutations were observed which resulted in stable heat resistance and in some cases salthypersensitive phenotypes. Whole-genome sequencing of one strain which was both heat resistant and salt hypersensitive, followed by PCR and sequencing of four other mutants which shared these phenotypes, revealed independent mutations in llmg_1816 in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family, members of which exhibit cyclic dimeric AMP (c-di-AMP)-specific phosphodiesterase activity. Mutations were predicted to lead to single amino acid substitutions or protein truncations. An independent llmg_1816 mutant (⌬1816), created using a suicide vector, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid excision. L. lactis ⌬1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High-temperature incubation of a wild-type industrial L. lactis strain also resulted in spontaneous mutation of llmg_1816 and heat-resistant and salt-hypersensitive phenotypes, suggesting that this is not a strain-specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by the llmg_1816-altered strain was inhibited by lower salt concentrations than the parent strain. This study demonstrates that spontaneous mutations can occur during high-temperature growth of L. lactis and that inactivation of llmg_1816 leads to temperature resistance and salt hypersensitivity.

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In Pakistan, milk adulteration is a growing issue of significant importance. This problem persists at every level; from production point to consumption. Adulteration poses ill effects on the public health. This study was designed to screen the various adulterants in the market milk sold in the vicinity of Mirpurkhas district of Sindh, Pakistan during the year 2013. A total of 100 milk samples were collected. Twenty milk samples (n= 20) from each of milk producers (MP), milk collectors (MC), middlemen (MM), processors (P) and dairy shops (DS) were examined for various adulterants. Among these adulterants water (73%) was found in majority of milk samples, followed by detergent (32%), cane sugar (22%), caustic soda (20%), rice flour (17%), sodium chloride and skimmed milk powder (15%), hydrogen peroxide (13%), starch (12%), formalin (11%), urea and vegetable oil (10%), boric acid (8%), ammonium sulfate (6%), glucose (5%), sorbitol (4%) and arrowroot (1%), respectively. Freezing point of 65% milk samples of MP, 75% of MC, 75% of MM, 80% of P and 85%% of DS appeared towards 0 o C rather than that of control milk, and assumed to be adulterated with extraneous water. The averaged extent of water in milk sold by P remarkably higher than that of other milk marketing channels, but statistically non-significant (P>0.05) with all milk intermediaries; MP, MC, MM and DS. The risk of adulteration at DS was found to be significantly higher (P<0.05) than MC and MP, but not significant (P>0.05) with P and MM.