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24Resistance to gastrointestinal conditions is a requirement for bacteria to be 25 considered probiotics. In this work, we tested the resistance of six different Lactobacillus 26 strains and the effect of carbon source to four different gastrointestinal conditions: presence 27 of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides 28 synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides 29 synthesized from lactose (GOS-La) and lactulose were used as carbon sources and 30 compared with glucose. In general, all strains grew in all carbon sources, although after 24 31 h of fermentation the population of all Lactobacillus strains was higher for both types of 32 GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-33La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a 34 dependence on carbon source and Lactobacillus strain was observed for bile extract and 35 low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when 36 compared with other carbon sources. However, concentrations of lactic and acetic acid 37 were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that 38 the resistance to gastrointestinal conditions and hydrophobicity is directly related with the 39 carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and 40 lactulose could be an excellent alternative to monosaccharides to support growth of 41 probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract. 42 43

The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.

Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur-and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.