Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant.

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.

Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.

The second messenger cyclic-di-adenosine monophosphate (c-di-AMP) plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c-di-AMP synthesizing diadenylate cyclase (CdaA); however, little is known about signals and effectors controlling CdaA activity and c-di-AMP levels. In this study, a genetic screen was employed to identify components which affect the c-di-AMP level in Lactococcus. We characterized suppressor mutations that restored osmoresistance to spontaneous c-di-AMP phosphodiesterase gdpP mutants, which contain high c-di-AMP levels. Loss-of-function and gain-of-function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c-di-AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM, which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c-di-AMP level and a reduction in the key peptidoglycan precursor UDP-N-acetylglucosamine in L. lactis. C-di-AMP synthesis by CdaA was shown to be inhibited by GlmM(I154F) more than GlmM and GlmM(I154F) was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c-di-AMP level modulating protein and provide a direct connection between c-di-AMP synthesis and peptidoglycan biosynthesis.

Isolation and Evaluation of Anti-Listeria Lactococcus lactis from Vegetal Sources.

This chapter describes methods used to isolate, identify, and partially characterize lactic acid bacteria (LAB) which exhibit inhibitory activity against Listeria monocytogenes from foods. Vegetal (plant based) sources are rich in naturally occurring LAB and therefore provide an easily accessible source of strains with potential antimicrobial activity for use in food-processing applications. From our previous work, the majority of LAB with inhibitory activity against L. monocytogenes were identified as generally recognized as safe (GRAS) Lactococcus lactis. Although these bacteria are most commonly known for their role in industrial dairy fermentations, they are believed to have originally derived from natural plant-based habitats. These isolates with anti-Listeria activity were all found to carry the genes involved in the production of nisin, which is an approved food-grade preservative (E234). These isolates may find various applications for in situ production of nisin allowing control of L. monocytogenes in various fermented and non-fermented foods and other environments.

Cystathionine gamma-lyase is a component of cystine-mediated oxidative defense in Lactobacillus reuteri BR11.

Lactobacillus reuteri BR11 possesses a novel mechanism of oxidative defense involving an abundant cystine ABC transporter encoded by the cyuABC gene cluster. Large amounts of thiols, including H(2)S, are secreted upon cystine uptake by the CyuC transporter. A cystathionine gamma-lyase (cgl) gene is cotranscribed with the cyu genes in several L. reuteri strains and was hypothesized to participate in cystine-mediated oxidative defense by producing reducing equivalents. This hypothesis was tested with L. reuteri BR11 by constructing a cgl mutant (PNG901) and comparing it to a similarly constructed cyuC mutant (PNG902). Although Cgl was required for H(2)S production from cystine, it was not crucial for oxidative defense in de Mann-Rogosa-Sharpe medium, in contrast to CyuC, whose inactivation resulted in lag-phase arrest in aerated cultures. The importance of Cgl in oxidative defense was seen only in the presence of hemin, which poses severe oxidative stress. The growth defects in aerated cultures of both mutants were alleviated by supplementation with cysteine (and cystine in the cgl mutant) but not methionine, with the cyuC mutant showing a much higher concentration requirement. We conclude that L. reuteri BR11 requires a high concentration of exogenous cysteine/cystine to grow optimally under aerobic conditions. This requirement is fulfilled by the abundant CyuC transporter, which has probably arisen due to the broad substrate specificity of Cgl, resulting in a futile pathway which degrades cystine taken up by the CyuC transporter to H(2)S. Cgl plays a secondary role in oxidative defense by its well-documented function of cysteine biosynthesis.

The genetic basis underlying variation in production of the flavour compound diacetyl by Lactobacillus rhamnosus strains in milk.

Diacetyl and the closely related compound acetoin impart desirable buttery flavour and odour to many foods including cheese and are generated through the metabolism of citrate by lactic acid bacteria (LAB). To increase the levels of these compounds, adjunct cultures capable of producing them can be added to cheese fermentations. In this study, we compared the diacetyl and acetoin producing abilities of 13 Lactobacillus rhamnosus strains from cheese sources. Diacetyl and acetoin production was found to be a common feature of Lb. rhamnosus grown in milk, with 12 strains producing these compounds. Whole genome sequencing of four strains revealed that genes encoding the citrate metabolising pathway present in other LAB are conserved in Lb. rhamnosus. One strain was, however, totally defective in diacetyl and acetoin production. This was likely due to an inability to produce the diacetyl/acetoin precursor compound acetolactate resulting from a frameshift mutation in the acetolactate synthase (als) gene. Complementation of this defective strain with a complete als gene from a diacetyl producing strain restored production of diacetyl and acetoin to levels equivalent to naturally high producing strains. Introduction of the same als-containing plasmid into the probiotic Lb. rhamnosus strain GG also increased diacetyl and acetoin levels. In model cheesemaking experiments, the als-complemented strain produced very high levels of diacetyl and acetoin over 35days of ripening. These findings identify the genetic basis for natural variation in production of a key cheese flavour compound in Lb. rhamnosus strains.

Culture-independent bacterial community profiling of carbon dioxide treated raw milk.

Due to technical simplicity and strong inhibition against the growth of psychrotrophic bacteria in milk, CO2 treatment has emerged as an attractive processing aid to increase the storage time of raw milk before downstream processing. However, it is yet to be adopted by the industry. In order to further explore the suitability of CO2 treatment for raw milk processing, the bacterial populations of carbonated raw milk collected locally from five different sources in Australia were analysed with next-generation sequencing. Growth inhibition by CO2 was confirmed, with spoilage delayed by at least 7days compared with non-carbonated controls. All non-carbonated controls were spoiled by Gammaproteobacteria, namely Pseudomonas fluorescens group bacteria, Serratia and Erwinia. Two out of the five carbonated samples shared the same spoilage bacteria as their corresponding controls. The rest of the three carbonated samples were spoiled by the lactic acid bacterium (LAB) Leuconostoc. This is consistent with higher tolerance of LAB towards CO2 and selection of LAB in meat products stored in CO2-enriched modified atmosphere packaging. No harmful bacteria were found to be selected by CO2. LAB are generally regarded as safe (GRAS), thus the selection for Leuconostoc by CO2 in some of the samples poses no safety concern. In addition, we have confirmed previous findings that 454 pyrosequencing and Illumina sequencing of 16S rRNA gene amplicons from the same sample yield highly similar results. This supports comparison of results obtained with the two different sequencing platforms, which may be necessary considering the imminent discontinuation of 454 pyrosequencing.

Inhibition of bacterial growth in sweet cheese whey by carbon dioxide as determined by culture-independent community profiling.

Whey is a valuable co-product from cheese making that serves as a raw material for a wide range of products. Its rich nutritional content lends itself to rapid spoilage, thus it typically needs to be pasteurised and refrigerated promptly. Despite the extensive literature on milk spoilage bacteria, little is known about the spoilage bacteria of whey. The utility of carbon dioxide (CO2) to extend the shelf-life of raw milk and cottage cheese has been well established, but its application in whey preservation has not yet been explored. This study aims to characterise the microbial populations of fresh and spoiled sweet whey by culture-independent community profiling using 454 pyrosequencing of 16S rRNA gene amplicons and to determine whether carbonation is effective in inhibiting bacterial growth in sweet whey. The microbiota of raw Cheddar and Mozzarella whey was dominated by cheese starter bacteria. After pasteurisation, two out of the three samples studied became dominated by diverse environmental bacteria from various phyla, with Proteobacteria being the most dominant. Diverse microbial profiles were maintained until spoilage occurred, when the entire population was dominated by just one or two genera. Whey spoilage bacteria were found to be similar to those of milk. Pasteurised Cheddar and Mozzarella whey was spoiled by Bacillus sp. or Pseudomonas sp., and raw Mozzarella whey was spoiled by Pseudomonas sp., Serratia sp., and other members of the Enterobacteriaceae family. CO2 was effective in inhibiting bacterial growth of pasteurised Cheddar and Mozzarella whey stored at 15°C and raw Mozzarella whey stored at 4°C. The spoilage bacteria of the carbonated samples were similar to those of the non-carbonated controls.

Draft Genome Sequence of Pseudomonas fluorescens SRM1, an Isolate from Spoiled Raw Milk.

Pseudomonas fluorescens is considered a major milk spoilage organism due to its psychrotrophic nature and ability to produce heat-stable proteases and lipases. Here, we report the draft genome and annotation of P. fluorescens SRM1 isolated from spoiled raw milk and the presence of an operon encoding spoilage enzymes.

Inhibition of Staphylococcus aureus growth on tellurite-containing media by Lactobacillus reuteri Is dependent on CyuC and thiol production.

Lactobacillus reuteri inhibits Staphylococcus aureus growth on Baird-Parker agar. This activity required the presence of tellurite and was not shared with other lactic acid bacteria or an L. reuteri mutant defective in cystine metabolism. Secreted products generated from L. reuteri cystine metabolism and thiols were shown to augment tellurite toxicity.