Protective Cultures in Food Products: From Science to Market.

An ultimate goal in food production is to guarantee food safety and security. Fermented food products benefit from the intrinsic capabilities of the applied starter cultures as they produce organic acids and bactericidal compounds such as hydrogen peroxide that hamper most food pathogens. In addition, highly potent small peptides, bacteriocins, are being expelled to exert antibiotic effects. Based on ongoing scientific efforts, there is a growing market of food products to which protective cultures are added exclusively for food safety and for prolonged shelf life. In this regard, most genera from the order Lactobacillales play a prominent role. Here, we give an overview on protective cultures in food products. We summarize the mode of actions of antibacterial mechanisms. We display the strategies for the isolation and characterization of protective cultures in order to have them market-ready. A survey of the growing market reveals promising perspectives. Finally, a comprehensive chapter discusses the current legislation issues concerning protective cultures, leading to the conclusion that the application of protective cultures is superior to the usage of defined bacteriocins regarding simplicity, economic costs, and thus usage in less-developed countries. We believe that further discovery of bacteria to be implemented in food preservation will significantly contribute to customer's food safety and food security, badly needed to feed world's growing population but also for food waste reduction in order to save substantial amounts of greenhouse gas emissions.

Multiple allosteric effectors control the affinity of DasR for its target sites.

The global transcriptional regulator DasR connects N-acetylglucosamine (GlcNAc) utilization to the onset of morphological and chemical differentiation in the model actinomycete Streptomyces coelicolor. Previous work revealed that glucosamine-6-phosphate (GlcN-6P) acts as an allosteric effector which disables binding by DasR to its operator sites (called dre, for DasR responsive element) and allows derepression of DasR-controlled/GlcNAc-dependent genes. To unveil the mechanism by which DasR controls S. coelicolor development, we performed a series of electromobility shift assays with histidine-tagged DasR protein, which suggested that N-acetylglucosamine-6-phosphate (GlcNAc-6P) could also inhibit the formation of DasR-dre complexes and perhaps even more efficiently than GlcN-6P. The possibility that GlcNAc-6P is indeed an efficient allosteric effector of DasR was further confirmed by the high and constitutive activity of the DasR-repressed nagKA promoter in the nagA mutant, which lacks GlcNAc-6P deaminase activity and therefore accumulates GlcNAc-6P. In addition, we also observed that high concentrations of organic or inorganic phosphate enhanced binding of DasR to its recognition site, suggesting that the metabolic status of the cell could determine the selectivity of DasR in vivo, and hence its effect on the expression of its regulon.

Genome-wide analysis of in vivo binding of the master regulator DasR in Streptomyces coelicolor identifies novel non-canonical targets.

Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon of DasR extends well beyond what was previously predicted and includes a large number of genes with functions far removed from N-acetylglucosamine metabolism, such as genes for small RNAs and DNA transposases. Conversely, the DasR regulon during vegetative growth largely correlates to the presence of canonical DasR-responsive elements. The changes in DasR binding in vivo following N-acetylglucosamine induction were studied in detail and a possible molecular mechanism by which the influence of DasR is extended is discussed. Discussion of DasR binding was further informed by a parallel transcriptome analysis of the respective cultures. Evidence is provided that DasR binds directly to the promoters of all genes encoding pathway-specific regulators of antibiotic production in S. coelicolor, thereby providing an exquisitely simple link between nutritional control and secondary metabolism.

Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis.

BACKGROUND: As other bacteria, Mycobacterium smegmatis needs adaption mechanisms to cope with changing nitrogen sources and to survive situations of nitrogen starvation. In the study presented here, transcriptome analyses were used to characterize the response of the bacterium to nitrogen starvation and to elucidate the role of specific transcriptional regulators. RESULTS: In response to nitrogen deprivation, a general starvation response is induced in M. smegmatis. This includes changes in the transcription of several hundred genes encoding e.g. transport proteins, proteins involved in nitrogen metabolism and regulation, energy generation and protein turnover. The specific nitrogen-related changes at the transcriptional level depend mainly on the presence of GlnR, while the AmtR protein controls only a small number of genes. CONCLUSIONS: M. smegmatis is able to metabolize a number of different nitrogen sources and nitrogen control in M. smegmatis is similar to control mechanisms characterized in streptomycetes, while the master regulator of nitrogen control in corynebacteria, AmtR, is plays a minor role in this regulatory network.

Regulon of the N-acetylglucosamine utilization regulator NagR in Bacillus subtilis.

N-Acetylglucosamine (GlcNAc) is the most abundant carbon-nitrogen biocompound on earth and has been shown to be an important source of nutrients for both catabolic and anabolic purposes in Bacillus species. In this work we show that the GntR family regulator YvoA of Bacillus subtilis serves as a negative transcriptional regulator of GlcNAc catabolism gene expression. YvoA represses transcription by binding a 16-bp sequence upstream of nagP encoding the GlcNAc-specific EIIBC component of the sugar phosphotransferase system involved in GlcNAc transport and phosphorylation, as well as another very similar 16-bp sequence upstream of the nagAB-yvoA locus, wherein nagA codes for N-acetylglucosamine-6-phosphate deacetylase and nagB codes for the glucosamine-6-phosphate (GlcN-6-P) deaminase. In vitro experiments demonstrated that GlcN-6-P acts as an inhibitor of YvoA DNA-binding activity, as occurs for its Streptomyces ortholog, DasR. Interestingly, we observed that the expression of nag genes was still activated upon addition of GlcNAc in a ΔyvoA mutant background, suggesting the existence of an auxiliary transcriptional control instance. Initial computational prediction of the YvoA regulon showed a distribution of YvoA binding sites limited to nag genes and therefore suggests renaming YvoA to NagR, for N-acetylglucosamine utilization regulator. Whole-transcriptome studies showed significant repercussions of nagR deletion for several major B. subtilis regulators, probably indirectly due to an excess of the crucial molecules acetate, ammonia, and fructose-6-phosphate, resulting from complete hydrolysis of GlcNAc. We discuss a model deduced from NagR-mediated gene expression, which highlights clear connections with pathways for GlcNAc-containing polymer biosynthesis and adaptation to growth under oxygen limitation.

The permease gene nagE2 is the key to N-acetylglucosamine sensing and utilization in Streptomyces coelicolor and is subject to multi-level control.

The availability of nutrients is a major determinant for the timing of morphogenesis and antibiotic production in the soil-dwelling bacterium Streptomyces coelicolor. Here we show that N-acetylglucosamine transport, the first step of an important nutrient signalling cascade, is mediated by the NagE2 permease of the phosphotransferase system, and that the activity of this permease is linked to nutritional control of development and antibiotic production. The permease serves as a high-affinity transporter for N-acetylglucosamine (K(m) of 2.6 microM). The permease complex was reconstituted with individually purified components. This showed that uptake of N-acetylglucosamine requires a phosphoryl group transfer from phosphoenolpyruvate via the phosphotransferases EI, HPr and IIA(Crr) to NagF, which in turn phosphorylates N-acetylglucosamine during transport. Transcription of the nagF and nagE2 genes is induced by N-acetylglucosamine. Nutrient signalling by N-acetylglucosamine that triggers the onset of development was abolished in the nagE2 and nagF mutants. nagE2 is subject to multi-level control by the global transcription factor DasR and the activator AtrA that also stimulates genes for antibiotic actinorhodin biosynthesis. Hence, it is apparent that streptomycetes tightly control the nutritional state in a complex manner to ensure the correct timing for the developmental programme.

Common patterns - unique features: nitrogen metabolism and regulation in Gram-positive bacteria.

Gram-positive bacteria have developed elaborate mechanisms to control ammonium assimilation, at the levels of both transcription and enzyme activity. In this review, the common and specific mechanisms of nitrogen assimilation and regulation in Gram-positive bacteria are summarized and compared for the genera Bacillus, Clostridium, Streptomyces, Mycobacterium and Corynebacterium, with emphasis on the high G+C genera. Furthermore, the importance of nitrogen metabolism and control for the pathogenic lifestyle and virulence is discussed. In summary, the regulation of nitrogen metabolism in prokaryotes shows an impressive diversity. Virtually every phylum of bacteria evolved its own strategy to react to the changing conditions of nitrogen supply. Not only do the transcription factors differ between the phyla and sometimes even between families, but the genetic targets of a given regulon can also differ between closely related species.

Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA.

YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-A crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix-turn-helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122 degrees rotation of the DNA-binding domains that is best described as a jumping-jack-like motion.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of YvoA from Bacillus subtilis.

The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50 A resolution. The crystals belonged to the monoclinic space group C2 and preliminary analysis of the diffraction data indicated the presence of approximately 12 molecules per asymmetric unit.

Identification of a glucose permease from Mycobacterium smegmatis mc2 155.

We report here the molecular identification of a glucose permease from Mycobacterium smegmatis,a model organism for our understanding of the life patterns of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae. A computer-based search of the available genome of M. smegmatis mc(2) 155 with the sequences of well-characterized glucose transporters revealed the gene msmeg4187 as a possible candidate. The deduced protein belongs to the major facilitator superfamily of proton symporters and facilitators and exhibits up to 53% of amino acid identity to other members of this family. Heterologous expression of msmeg4187 in an Escherichia coli glucose-negative mutant led to the restoration of growth on glucose. The determination of the biochemical features characterize MSMEG4187 (GlcP) as a high affinity (K(m) of 19 microM), glucose-specific permease. The results represent the first molecular characterization of a sugar permease in mycobacteria, and thus supply fundamental data for further in-depth analysis on the nutritional lifestyle of these bacteria.

A genomic view on nitrogen metabolism and nitrogen control in mycobacteria.

Knowledge about nitrogen metabolism and control in the genus Mycobacterium is sparse, especially compared to the state of knowledge in related actinomycetes like Streptomyces coelicolor or the close relative Corynebacterium glutamicum. Therefore, we screened the published genome sequences of Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium ssp. paratuberculosis and Mycobacterium leprae for genes encoding proteins for uptake of nitrogen sources, nitrogen assimilation and nitrogen control systems, resulting in a detailed comparative genomic analysis of nitrogen metabolism-related genes for all completely sequenced members of the genus. Transporters for ammonium, nitrate, and urea could be identified, as well as enzymes crucial for assimilation of these nitrogen sources, i.e. glutamine synthetase, glutamate dehydrogenase, glutamate synthase, nitrate reductase, nitrite reductase, and urease proteins. A reduction of genes encoding proteins for nitrogen transport and metabolism was observed for the pathogenic mycobacteria, especially for M. leprae. Signal transduction components identified for the different species include adenylyl- and uridylyltransferase and a P(II)-type signal transduction protein. Exclusively for M. smegmatis, two homologs of putative nitrogen regulatory proteins were found, namely GlnR and AmtR, while in other mycobacteria, AmtR was absent and GlnR seems to be the nitrogen transcription regulator protein.

Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors.

The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures.

Nitrogen control in Mycobacterium smegmatis: nitrogen-dependent expression of ammonium transport and assimilation proteins depends on the OmpR-type regulator GlnR.

The effect of nitrogen regulation on the level of transcriptional control has been investigated in a variety of bacteria, such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, and Streptomyces coelicolor; however, until now there have been no data for mycobacteria. In this study, we found that the OmpR-type regulator protein GlnR controls nitrogen-dependent transcription regulation in Mycobacterium smegmatis. Based on RNA hybridization experiments with a wild-type strain and a corresponding mutant strain, real-time reverse transcription-PCR analyses, and DNA binding studies using cell extract and purified protein, the glnA (msmeg_4290) gene, which codes for glutamine synthetase, and the amtB (msmeg_2425) and amt1 (msmeg_6259) genes, which encode ammonium permeases, are controlled by GlnR. Furthermore, since glnK (msmeg_2426), encoding a PII-type signal transduction protein, and glnD (msmeg_2427), coding for a putative uridylyltransferase, are in an operon together with amtB, these genes are part of the GlnR regulon as well. The GlnR protein binds specifically to the corresponding promoter sequences and functions as an activator of transcription when cells are subjected to nitrogen starvation.

Ins and outs of glucose transport systems in eubacteria.

Glucose is the classical carbon source that is used to investigate the transport, metabolism, and regulation of nutrients in bacteria. Many physiological phenomena like nutrient limitation, stress responses, production of antibiotics, and differentiation are inextricably linked to nutrition. Over the years glucose transport systems have been characterized at the molecular level in more than 20 bacterial species. This review aims to provide an overview of glucose uptake systems found in the eubacterial kingdom. In addition, it will highlight the diverse and sophisticated regulatory features of glucose transport systems.

Feast or famine: the global regulator DasR links nutrient stress to antibiotic production by Streptomyces.

Members of the soil-dwelling prokaryotic genus Streptomyces produce many secondary metabolites, including antibiotics and anti-tumour agents. Their formation is coupled with the onset of development, which is triggered by the nutrient status of the habitat. We propose the first complete signalling cascade from nutrient sensing to development and antibiotic biosynthesis. We show that a high concentration of N-acetylglucosamine-perhaps mimicking the accumulation of N-acetylglucosamine after autolytic degradation of the vegetative mycelium-is a major checkpoint for the onset of secondary metabolism. The response is transmitted to antibiotic pathway-specific activators through the pleiotropic transcriptional repressor DasR, the regulon of which also includes all N-acetylglucosamine-related catabolic genes. The results allowed us to devise a new strategy for activating pathways for secondary metabolite biosynthesis. Such 'cryptic' pathways are abundant in actinomycete genomes, thereby offering new prospects in the fight against multiple drug-resistant pathogens and cancers.

The chitobiose-binding protein, DasA, acts as a link between chitin utilization and morphogenesis in Streptomyces coelicolor.

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such as cellulose, xylan and chitin for the colonization of their environmental niche and therefore these polysaccharides may play a critical role in monitoring the global nutritional status of the environment. In this work we analysed the role of DasA, the sugar-binding component of the chitobiose ATP-binding cassette transport system, in informing the cell of environmental conditions, and its role in the onset of development and in ensuring correct sporulation. The chromosomal interruption of dasA resulted in a carbon-source-dependent vegetative arrest phenotype, and we identified a second DasR-dependent sugar transporter, in addition to the N-acetylglucosamine phosphotransferase system (PTS(GlcNAc)), that relates primary metabolism to development. Under conditions that allowed sporulation, highly aberrant spores with many prematurely produced germ tubes were observed. While GlcNAc locks streptomycetes in the vegetative state, a high extracellular concentration of the GlcNAc polymer chitin has no effect on development. The striking distinction is due to a difference in the transporters responsible for the import of GlcNAc, which enters via the PTS, and of chitin, which enters as the hydrolytic product chitobiose (GlcNAc(2)) through the DasABC transporter. A model explaining the role of these two essentially different transport systems in the control of development is provided.

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis.

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.

PREDetector: a new tool to identify regulatory elements in bacterial genomes.

In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory networks in bacteria. The main difficulty once a regulon prediction is available is to estimate its reliability prior to start expensive experimental validations and therefore trying to find a way how to identify true positive hits from an endless list of potential target genes of a regulatory protein. Here we introduce PREDetector (Prokaryotic Regulatory Elements Detector), a tool developed for predicting regulons of DNA-binding proteins in bacterial genomes that, beside the automatic prediction, scoring and positioning of potential binding sites and their respective target genes in annotated bacterial genomes, it also provides an easy way to estimate the thresholds where to find reliable possible new target genes. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php.

Sugar transport systems of Bifidobacterium longum NCC2705.

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.