Impact of lactose starvation on the physiology of Lactobacillus casei GCRL163 in the presence or absence of tween 80.

The global proteomic response of the nonstarter lactic acid bacteria Lactobacillus casei strain GCRL163 under carbohydrate depletion was investigated to understand aspects of its survival following cessation of fermentation. The proteome of L. casei GCRL163 was analyzed quantitatively after growth in modified MRS (with and without Tween 80) with different levels of lactose (0% lactose, starvation; 0.2% lactose, growth limiting; 1% lactose, non-growth-limited control) using gel-free proteomics. Results revealed that carbohydrate starvation lead to suppression of lactose and galactose catabolic pathways as well as pathways for nucleotide and protein synthesis. Enzymes of the glycolysis/gluconeogenesis pathway, amino acid synthesis, and pyruvate and citrate metabolism become more abundant as well as other carbohydrate catabolic pathways, suggesting increased optimization of intermediary metabolism and scavenging. Tween 80 did not affect growth yield; however, proteins related to fatty acid biosynthesis were repressed in the presence of Tween 80. The data suggest that L. casei adeptly switches to a scavenging mode, using both citrate and Tween 80, and efficiently adjusts energetic requirements when carbohydrate starved and thus can sustain survival for weeks to months. Explaining the adaptation of L. casei during lactose starvation will assist efforts to maintain viability of L. casei and extend its utility as a beneficial dietary adjunct and fermentation processing aid.

MudPIT profiling reveals a link between anaerobic metabolism and the alkaline adaptive response of Listeria monocytogenes EGD-e.

Listeria monocytogenes is a foodborne human pathogen capable of causing life-threatening disease in susceptible populations. Previous proteomic analysis we performed demonstrated that different strains of L. monocytogenes initiate a stringent response when subjected to alkaline growth conditions. Here, using multidimensional protein identification technology (MudPIT), we show that in L. monocytogenes EGD-e this response involves an energy shift to anaerobic pathways in response to the extracellular pH environment. Importantly we show that this supports a reduction in relative lag time following an abrupt transition to low oxygen tension culture conditions. This has important implications for the packaging of fresh and ready-to-eat foods under reduced oxygen conditions in environments where potential exists for alkaline adaptation.

Investigation of the Listeria monocytogenes Scott A acid tolerance response and associated physiological and phenotypic features via whole proteome analysis.

The global proteomic responses of the foodborne pathogen Listeria monocytogenes strain Scott A, during active growth and transition to the stationary growth phase under progressively more acidic conditions, created by addition of lactic acid and HCl, were investigated using label-free liquid chromatography/tandem mass spectrometry. Approximately 56% of the Scott A proteome was quantitatively assessable, and the data provides insight into its acquired acid tolerance response (ATR) as well as the relation of the ATR to the growth phase transition. Alterations in protein abundance due to acid stress were focused in proteins belonging to the L. monocytogenes common genome, with few strain-dependent proteins involved. However, one of the two complete prophage genomes appeared to enter lysogeny. During progressive acidification, the growth rate and yield were reduced 55% and 98%, respectively, in comparison to nonacidified control cultures. The maintenance of the growth rate was determined to be connected to activation of cytoplasmic pH homeostatic mechanisms while cellular reproductive-related and cell component turnover proteins were markedly more abundant in acid stressed cultures. Cell biomass accumulation was impeded predominantly due to repression of phosphodonor-linked enzymes involved with sugar phosphotransfer, glycolysis, and cell wall polymer biosynthesis. Acidification caused a shift from heterofermentation to an oxidatively stressed state in which ATP appears to be generated mainly through the pyruvate dehydrogenase/pyruvate oxidase/phosphotransacetylase/acetate kinase and branched chain acid dehydrogenase pathways. Analysis of regulons indicated energy conservation occurs due to repression by the GTP/isoleucine sensor CodY and also the RelA mediated stringent response. Whole proteome analysis proved to be an effective way to highlight proteins involved with the acquisition of the ATR.

MudPIT analysis of alkaline tolerance by Listeria monocytogenes strains recovered as persistent food factory contaminants.

Alkaline solutions are used to clean food production environments but the role of alkaline resistance in persistent food factory contamination by Listeria monocytogenes is unknown. We used shotgun proteomics to characterise alkaline adapted L. monocytogenes recovered as persistent and transient food factory contaminants. Three unrelated strains were studied including two persistent and a transient food factory contaminant determined using multilocus sequence typing (MLST). The strains were adapted to growth at pH 8.5 and harvested in exponential phase. Protein extracts were analysed using multidimensional protein identification technology (MudPIT) and protein abundance compared by spectra counting. The strains elicited core responses to alkaline growth including modulation of intracellular pH, stabilisation of cellular processes and reduced cell-division, independent to lineage, MLST or whether the strains were transient or persistent contaminants. Alkaline adaptation by all strains corresponded to that expected in stringent-response induced cells, with protein expression supporting metabolic shifts concordant with elevated alarmone production and indicating that the alkaline-stringent response results from energy rather than nutrient limitation. We believe this is the first report describing induction of a stringent response in different L. monocytogenes strains by alkaline pH under non-limiting growth conditions. The work emphasises the need for early intervention to avoid persistent food factory contamination by L. monocytogenes.

Variability in biofilm production by Listeria monocytogenes correlated to strain origin and growth conditions.

This study aimed to identify factors that influence the development of biofilm by Listeria monocytogenes strains and to determine the extent to which biofilm production protects against quaternary ammonium compound (QAC) disinfectant challenge. A total of 95 L. monocytogenes strains were studied and biofilm production was assessed as a function of incubation temperature, media pH, strain origin, serotype, and environmental persistence status. Attachment and biofilm development (inferred by the level of attached biomass) were measured in vitro using a colourimetric 96-well microtitre plate method in nutritive media (Brain-Heart Infusion). Increased biofilm production correlated with increasing temperature and the most acidic, or most alkaline, growth conditions tested. Clinical and environmental (food factory) strains were observed to increase biofilm production at higher and lower incubation temperatures respectively, independent of their rate of planktonic growth. Serotype 1/2a strains produced significantly more biofilm. Biofilm maturity, rather than strain, was correlated with resistance to QAC. Carbohydrate containing exopolymeric material could not be detected in the biofilm of representative strains, and no correlation between strains recovered as persistent food factory contaminants and biofilm production was identified. Although limited to in vitro inference based on the assay system used, our results suggest that environmental conditions determine the level of biofilm production by L. monocytogenes strains, independent of the rate of planktonic growth, and that this may manifest from selection pressures to which a given strain grows optimally.

Utility of gel-free, label-free shotgun proteomics approaches to investigate microorganisms.

This review will examine the current situation with label-free, quantitative, shotgun-oriented proteomics technology and discuss the advantages and limitations associated with its capability in capturing and quantifying large portions of proteomes of microorganisms. Such an approach allows (1) comparisons between physiological or genetic states of organisms at the protein level, (2) 'painting' of proteomic data onto genome data-based metabolic maps, (3) enhancement of the utility of genomic data and finally (4) surveying of non-genome sequenced microorganisms by taking advantage of available inferred protein data in order to gain new insights into strain-dependent metabolic or physiological capacities. The technology essentially is a powerful addition to systems biology with a capacity to be used to ask hypothesis-driven 'top-down' questions or for more empirical 'bottom-up' exploration.