CodY of Streptococcus pneumoniae: link between nutritional gene regulation and colonization

CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.     

CodY-Regulated Aminotransferases AraT and BcaT Play a Major Role in the Growth of Lactococcus lactis in Milk by Regulating the Intracellular Pool of Amino Acids

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.     

Genome-Scale Model of Streptococcus thermophilus LMG18311 for Metabolic Comparison of Lactic Acid Bacteria

In this report, we describe the amino acid metabolism and amino acid dependency of the dairy bacterium Streptococcus thermophilus LMG18311 and compare them with those of two other characterized lactic acid bacteria, Lactococcus lactis and Lactobacillus plantarum. Through the construction of a genome-scale metabolic model of S. thermophilus, the metabolic differences between the three bacteria were visualized by direct projection on a metabolic map. The comparative analysis revealed the minimal amino acid auxotrophy (only histidine and methionine or cysteine) of S. thermophilus LMG18311 and the broad variety of volatiles produced from amino acids compared to the other two bacteria. It also revealed the limited number of pyruvate branches, forcing this strain to use the homofermentative metabolism for growth optimization. In addition, some industrially relevant features could be identified in S. thermophilus, such as the unique pathway for acetaldehyde (yogurt flavor) production and the absence of a complete pentose phosphate pathway.Lactic acid bacteria (LAB) are of great importance in the food industry because of their lactic acid production and their characteristic impact (e.g., texture, flavor) on the final product (19). LAB, as fastidious organisms, require a complex medium (such as milk) and are dependent on their proteolytic system for their supply of essential amino acids (34). Amino acids are not only the building blocks for proteins and peptides, but they also serve as precursors for many other biomolecules (1). Amino acids are also important for the final flavor of a product. Most amino acids do not directly influence the product flavor, but they will contribute indirectly to it because they are precursors of aromatic compounds (36). The conversion of amino acids to flavor compounds is initiated mainly by amino acid transamination, which uses an α-keto acid as an amino group acceptor for the aminotransferases (27). The presence (or absence) of the α-keto acid either by endogenous production or by addition to the medium is an important factor in flavor formation (13). The α-keto acids are decarboxylated into aldehydes, which are the precursors of other flavor compounds such as alcohols, esters, and carboxylic acids (27). A large variation in flavor formation between strains and species is observed. Different studies have reported this biodiversity (25, 27, 32, 33); van Hylckama Vlieg et al. studied, for instance, the difference between dairy and nondairy lactococcal strains, since the latter group has some unique flavor-forming activities (33).Amino acid catabolism and anabolism are complex processes, and thus, metabolic models will be helpful for their understanding. Genome-scale metabolic models provide an overview of all metabolic conversions in an organism based on its genome sequence and make it possible to visualize different metabolic pathways, such as amino acid metabolism. These models can be used to understand the metabolism and can then be applied for a directed study of functionality. For Lactobacillus plantarum and Lactococcus lactis, such genome-scale models have already been developed (18, 29); the construction of such a model for Streptococcus thermophilus LMG18311 is described in this paper. The characterization of the genome sequence of this S. thermophilus strain has revealed the presence of a large number of incomplete or truncated genes. These so-called pseudogenes amount to 10% of the total genes, and most of them relate to carbohydrate metabolism, transport, and regulation (2, 11). S. thermophilus is an important starter for the dairy industry. It is used in combination with Lactobacillus delbrueckii subsp. bulgaricus for the production of yogurt. It is also used for the manufacture of cheeses in which high cooking temperatures are applied (11). The objective of this paper is to study the metabolism of S. thermophilus with the use of genome-scale models and experimental data in a comparative way. This comparison with other LAB may reveal important differences. This study showed the simple primary metabolism and the extensive amino acid metabolism of S. thermophilus.     

thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyA_St and thyA_Lb, were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyA_St or thyA_Lb as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA_Lb as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.     

A static model to analyze carbon and nitrogen partitioning in the mammary gland of lactating sows

Quantitative estimates of mammary nutrient inputs, outputs and metabolism in sows are scarce, despite being critical elements to identify parameters controlling milk synthesis central for the feeding of lactating sows. The objective of this study was to quantify the mammary gland input and output of nutrients as well as the intramammary partitioning of carbon and nitrogen with the purpose to identify mechanisms controlling mammary nutrient inputs, metabolism and milk production in lactating sows. A data set was assembled by integration of results from four studies. The data set included data on litter performance, mammary arterial-venous concentration differences (AV-difference) of energy metabolites and amino acids, and the contents of lactose, fat and amino acids in milk. Milk yield was estimated based on average litter size and litter gain, and mammary plasma flow (MPF) was estimated using the sum of phenylalanine and tyrosine as internal flow markers. The yield and composition of milk were used to estimate mammary nutrient output in milk, and MPF and AV-difference were used to estimate net mammary input of carbon and nitrogen and output of CO₂. Carbon and nitrogen used for the synthesis of lactose, fat and protein in milk and CO₂-yielding processes were represented in a static nutrient partitioning model. The origin of mammary CO₂ output was calculated using theoretical estimates of carbon released in processes supporting mammary synthesis of de novo fat, protein and lactose in milk, mammary tissue protein turnover and transport of glucose and amino acids. Results indicated that total input of carbon from glucose and lactate was partitioned into lactose (36%), fat (31%) and CO₂-yielding processes (34%). Theoretical CO₂ estimates indicated that de novo fat synthesis, milk protein synthesis and mammary tissue protein turnover were the main processes related to mammary CO₂ production. More than 90% of mammary gland amino acid input was used for milk protein. The quadratic relationship between AV-difference and mammary input of essential amino acids indicated that both changes in AV-difference and MPF contributed to the regulation of mammary input of essential amino acids. The impact of the arterial supply of amino acids on mammary input may be greater for the branched-chain amino acids, arginine and phenylalanine than for other essential amino acids. In conclusion, relationships between input and output parameters indicate that AV-difference and MPF regulate mammary nutrient input to match the supply and demand of nutrients for the mammary gland.     

The thioredoxin system in the dental caries pathogen Streptococcus mutans and the food-industry bacterium Streptococcus thermophilus

The Streptococcus genus includes the pathogenic species Streptococcus mutans, the main responsible of dental caries, and the safe microorganism Streptococcus thermophilus, used for the manufacture of dairy products. These facultative anaerobes control the levels of reactive oxygen species (ROS) and indeed, both S. mutans and S. thermophilus possess a cambialistic superoxide dismutase, the key enzyme for a preventive action against ROS. To evaluate the properties of a crucial mechanism for repairing ROS damages, the molecular and functional characterization of the thioredoxin system in these streptococci was investigated. The putative genes encoding its protein components in S. mutans and S. thermophilus were analysed and the corresponding recombinant proteins were purified. A single thioredoxin reductase was obtained from either S. mutans (SmTrxB) or S. thermophilus (StTrxB1), whereas two thioredoxins were prepared from either S. mutans (SmTrxA and SmTrxH1) or S. thermophilus (StTrxA1 and StTrxA2). Both SmTrxB and StTrxB1 reduced the synthetic substrate DTNB in the presence of NADPH, whereas only SmTrxA and StTrxA1 accelerated the insulin reduction in the presence of DTT. To reconstitute an in vitro streptococcal thioredoxin system, the combined activity of the thioredoxin components was tested through the insulin precipitation in the absence of DTT. The assay functions with a combination of SmTrxB or StTrxB1 with either SmTrxA or StTrxA1. These results suggest that the streptococcal members of the thioredoxin system display a direct functional interaction between them and that these protein components are interchangeable within the Streptococcus genus. In conclusion, our data prove the existence of a functioning thioredoxin system even in these microaerophiles.     

Co-Inactivation of GlnR and CodY Regulators Impacts Pneumococcal Cell Wall Physiology

CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY ^- ami ⁺ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation of these regulators renders pneumococci sensitive to iron and PG-targeting agents.     

The initiation of GTP hydrolysis by the G-domain of FeoB: insights from a transition-state complex structure

The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoB^St) in complex with the activating potassium ion and a transition-state analogue, GDP⋅AlF₄ ⁻, reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoB^St, and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoB^St. Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.