Development of Microbiome Biobanks - Challenges and Opportunities.

The microbiome research field is rapidly evolving, but the required biobanking infrastructure is currently fragmented and not prepared for the biobanking of microbiomes. The rapid advancement of technologies requires an urgent assessment of how biobanks can underpin research by preserving microbiome samples and their functional potential.

The critical role of urease in yogurt fermentation with various combinations of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

The protocooperation between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus relies on metabolite exchanges that accelerate acidification during yogurt fermentation. Conflicting results have been obtained in terms of the effect of the Strep. thermophilus urease and the NH3 and CO2 that it generates on the rate of acidification in yogurt fermentation. It is difficult to perform a systematic study of the effects of urease on protocooperation because it is necessary to distinguish among the direct, indirect, and strain-specific effects resulting from the combination of the strains of both species. To evaluate the direct effects of urease on protocooperation, we generated 3 urease-deficient mutants (ΔureC) of fast- and slow-acidifying Strep. thermophilus strains and observed the effects of NH3 or CO2 supplementation on acidification by the ΔureC strains. Further, we examined 5 combinations of 3 urease-deficient ΔureC strains with 2 CO2-responsive or CO2-unresponsive strains of L. bulgaricus. Urease deficiency induced a shortage of ammonia nitrogen and CO2 for the fast- and slow-acidifying Strep. thermophilus and for the CO2-responsive L. bulgaricus, respectively. Notably, the shortage of ammonia nitrogen had more severe effects than that of CO2 on yogurt fermentation, even if coculture with L. bulgaricus masked the effect of urease deficiency. Our work established (1) that urease deficiency inhibits the fermentative acceleration of protocooperation regardless of the Strep. thermophilus and L. bulgaricus strain combinations, and (2) that urease is an essential factor for effective yogurt acidification.

Induction of heavy-metal-transporting CPX-type ATPases during acid adaptation in Lactobacillus bulgaricus.

Lactobacillus bulgaricus is a lactic acid bacteria (LAB) that, through the production of lactic acid, gradually acidifies its environment during growth. In the course of this process, L. bulgaricus acquires an improved tolerance to acidity. A survey of the recently established genome sequence shows that this bacterium possesses few of the pH control functions that have been described in other LAB and raises the question of what other mechanisms could be involved in its adaptation to the decreasing environmental pH. In some bacteria other than LAB, ion transport systems have been implicated in acid adaptation. We therefore studied the expression of this type of transport system during acid adaptation in L. bulgaricus by reverse transcription and real-time quantitative PCR and mapped transcription start sites. Intriguingly, the most significantly induced were three ATPases carrying the CPX signature of heavy-metal transporters. Protein homology and the presence of a conserved sequence motif in the promoter regions of the genes encoding these proteins strongly suggest that they are involved in copper homeostasis. Induction of this system is thought to assist in avoiding indirect damage that could result from medium acidification.

AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system.

We have implemented a genome annotation system for prokaryotes called AGMIAL. Our approach embodies a number of key principles. First, expert manual annotators are seen as a critical component of the overall system; user interfaces were cyclically refined to satisfy their needs. Second, the overall process should be orchestrated in terms of a global annotation strategy; this facilitates coordination between a team of annotators and automatic data analysis. Third, the annotation strategy should allow progressive and incremental annotation from a time when only a few draft contigs are available, to when a final finished assembly is produced. The overall architecture employed is modular and extensible, being based on the W3 standard Web services framework. Specialized modules interact with two independent core modules that are used to annotate, respectively, genomic and protein sequences. AGMIAL is currently being used by several INRA laboratories to analyze genomes of bacteria relevant to the food-processing industry, and is distributed under an open source license.

The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

Characterization of urease genes cluster of Streptococcus thermophilus.

AIMS: The milk acidification rate of Streptococcus thermophilus strains can be affected by several factors, one of which is the hydrolysis of urea by the urease complex. To evaluate the technological suitability of S. thermophilus strains deprived of urease activity in milk fermentation, the genetic cluster related to urease enzymatic activity has been characterized in the type strain DSM 20167T. METHODS AND RESULTS: Amplification of the urease genes of S. thermophilus DSM 20167T was developed on the basis of the urease gene cluster of the phylogenetically related S. salivarius. Nucleotide sequencing revealed the presence of eight open reading frames, which were most homologous to ureABC (structural genes) and ureI, ureEFGD (accessory genes) of S. salivarius and other ureolytic bacteria. Reverse transcriptase PCR experiments were in agreement with an operon organization for the eight genes (ureIABCEFGD). A food grade mutant A16 (DeltaureC3) with a 693 bp in-frame deletion in ureC gene exhibited a urease negative (Ure-) phenotype. Unlike the wild-type strain, the acidification rate of the mutant in reconstituted skimmed milk was not affected by the presence of urea or nickel ions. A small-scale yoghurt fermentation trials were carried out using the wild-type or the Ure- mutant A16 (DeltaureC3) in co-culture with Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 in presence of urea. The result obtained underlines that when the Ure- mutant was used as a co-starter the acidification rate was higher than that obtained using the wild-type strain. CONCLUSIONS: The study provides the first genetic characterization and the technological implication of S. thermophilus DSM 20617T urease activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The detrimental effect of ureolytic activity on the rate of milk acidification was evaluated and superseded using a food-grade Ure- recombinant strain. Small-scale yoghurt production trials highlighted the positive role of a Ure-S. thermophilus mutant as a co-starter in milk fermentations. Moreover, the vector pMI108 developed for the construction of the Ure- strain, should be considered as a potential tool for the generation of Ure- dairy S. thermophilus strains selected for other relevant technological properties but characterized by the undesirable ureolytic phenotype.

Production of growth-inhibiting factors by Lactobacillus delbrueckii.

AIMS: The detection of growth-inhibiting factors produced by Lactobacillus delbrueckii. METHODS AND RESULTS: A bioscreen assay was developed to study the effect of Lact. delbrueckii culture supernatant fluids on the growth of phylogenically or functionally related bacteria in broth cultures. Several growth-inhibiting factors could be distinguished based on differential effects on different test strains, separation by ultrafiltration and sensitivity to heat, proteinase treatment or catalase addition. CONCLUSION: Lactobacillus delbrueckii strain VI1007 was found to produce at least three growth-inhibiting factors, other than lactic acid, when grown under microaerobic conditions in MRS broth. These included H2O2 and a bacteriocin-like, heat- and proteinase-sensitive bactericidal molecule or complex with a molecular weight greater than 50 kDa. A third factor inhibited the growth of Streptococcus thermophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay system used allows the detection of subtle interactions between strains, that are likely to be of ecological importance in mixed cultures but would go unnoticed in classical agar diffusion tests.

Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynthesis genes hasA, hasB, and hasC in Streptococcus uberis.

The hyaluronic acid capsule of Streptococcus uberis has been implicated in conferring resistance to phagocytosis by bovine neutrophils. Construction of a bank of random insertion mutants of S. uberis (strain 0140J) was achieved using the pGh9::ISS1 mutagenesis system (22). Phenotypic screening of approximately 5,000 clones enabled the isolation of 11 acapsular mutants. Southern hybridization indicated that two mutants carried a lesion within a group of genes similar to those involved in the assembly of the hyaluronic acid capsule found in the group A Streptococcus (GAS) has operon. The DNA sequence flanking the points of insertion confirmed the presence of homologues of GAS hasA and hasB in S. uberis. The DNA sequence flanking the ISS1 insertion in another mutant identified a homologue of hasC in S. uberis. The GAS hasABC operon structure was not conserved in S. uberis, and two discrete loci comprising homologues of either hasAB or hasC were identified. Disruption of S. uberis hasA or hasC resulted in the complete cessation of hyaluronic acid capsule production. Correspondingly, these mutants were found to have lost their resistance to phagocytosis by bovine neutrophils. The bactericidal action of bovine neutrophils on S. uberis 0140J was shown unequivocally to depend upon the capsule status of the bacterium.

Physiological study of Lactobacillus delbrueckii subsp. bulgaricus strains in a novel chemically defined medium.

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h(-1). MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.

Identification of stress-inducible proteins in Lactobacillus delbrueckii subsp. bulgaricus.

Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a homofermentative bacterium that produces lactic acid during growth. We adapted the two-dimensional electrophoresis (2-DE) technique to study the response of this bacterium to acidity. De novo protein synthesis was monitored by [35S]methionine labeling of exponentially growing cultures under standard (pH 6) and acidic (pH 4.75) conditions. After 2-DE separation, the protein patterns were compared. The protein spots showing increased radioactivity levels under acid conditions were considered acid-induced. We determined the N-terminal amino acid sequence of three highly induced proteins; comparing these proteins to databases we identified them to be the well-known heat shock proteins GroES, GroEL, and DnaK. Their induction levels were measured and compared. This is the first study by 2-DE of stress response in L. bulgaricus. We established the method and present a protein map which will be useful for future studies.

Developmental risk factors for youth violence.

PURPOSE: To replicate earlier research findings on risk factors for youth violence and to explore the effects on violent behavior of constructs shown to increase risk for other problem behaviors, within a developmental frame. METHODS: Data were from the Seattle Social Development Project (SSDP), a prospective study involving a panel of youths followed since 1985. Potential risk factors for violence at age 18 years were measured at ages 10, 14, and 16 years. Bivariate relationships involving risk factor constructs in the individual, family, school, peer and community domains and violence were examined at each age to assess changes in their strength of prediction over time. Attention was also given to the additive strength of increasing numbers of risk factors in the prediction of violence at age 18 years. A final set of analyses explored the extent to which youths were correctly classified as having committed a violent act (or not) at age 18 years on the basis of their overall level of risk at ages 10, 14, and 16 years. RESULTS: At each age, risk factors strongly related to later violence were distributed among the five domains. Ten of 15 risk factors constructs measured at age 10 years were significantly predictive of violence at age 18 years. Twenty of 25 constructs measured at age 14 years and 19 of 21 constructs measured at age 16 years were significantly predictive of later violence. Many constructs predicted violence from more than one developmental point. Hyperactivity (parent rating), low academic performance, peer delinquency, and availability of drugs in the neighborhood predicted violence from ages 10, 14, and 16 years. Analyses of the additive effects of risk factors revealed that youths exposed to multiple risks were notably more likely than others to engage in later violence. The odds for violence of youths exposed to more than five risk factors compared to the odds for violence of youths exposed to fewer than two risk factors at each age were seven times greater at age 10 years, 10 times greater at age 14 years, and nearly 11 times greater at age 16 years. However, despite information gained from all significant risk factors, the overall accuracy in predicting youths who would go on to commit violent acts was limited. CONCLUSIONS: Findings from the study have important implications for preventive intervention programs. Prevention efforts must be comprehensive and developmentally sensitive, responding to large groups or populations exposed to multiple risks.

Acid- and multistress-resistant mutants of Lactococcus lactis : identification of intracellular stress signals.

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L. lactis strain MG1363. Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.

Exploring the effects of age of alcohol use initiation and psychosocial risk factors on subsequent alcohol misuse.

OBJECTIVE: This study examines whether the age of initiation of alcohol use mediates the effects of other variables that predict alcohol misuse among adolescents and also whether the age of initiation of alcohol use accounts for known gender differences in the severity of alcohol misuse. METHOD: Data were taken from an ethnically diverse sample of 808 (412 male) students who were recruited in grade 5 at age 10-11 and followed prospectively on an annual basis for the next 7 years to age 17-18. State-of-the-art missing data methodology was used to address nonresponse due to noninitiation of alcohol use. Structural equation modeling was used to examine hypotheses for the prediction of alcohol misuse. RESULTS: A younger age of alcohol initiation was strongly related to a higher level of alcohol misuse at age 17-18 and fully mediated the effects of parent drinking, proactive parenting, school bonding, peer alcohol initiation and ethnicity, all measured at age 10-11, and perceived harmfulness of alcohol use, measured at age 10-11 and age 11-12. However, age of alcohol initiation did not fully account for gender differences in the level of alcohol misuse at age 17-18. To further examine the role of gender, interactions between gender and school bonding, and gender and friend's alcohol initiation, were evaluated. However, neither of the interaction terms had direct effects on either age of initiation or level of alcohol-related problems. CONCLUSIONS: Most measured risk factors for alcohol misuse were mediated through age of alcohol initiation. Only gender differences in alcohol misuse at age 17-18 were not mediated by age of alcohol initiation. Variables associated with these differences require further study. The results of this study indicate the importance of prevention strategies to delay the age of initiation of alcohol use.

Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance.

Lactobacillus fermentum is a lactic acid bacterial species commonly found in the digestive tracts of pigs and rodents and also present in man. We characterized a 5.7-kb plasmid, pLEM3, conferring erythromycin resistance, which was isolated from a porcine strain of L. fermentum. Plasmid pLEM3 established efficiently in L. fermentum, conferred high-level erythromycin resistance (MIC > 1 mg/ml), and was segregationally stable. A deletion derivative of pLEM3, called pLEM5, was constructed and found to be as genetically stable as the parent. A multiple cloning site was inserted into pLEM5, generating plasmid pLEM7. Nucleotide sequence determination of pLEM5 revealed similarities with known genes. The replicon itself is a member of the pC194 family of rolling circle plasmids. The region responsible for erythromycin resistance was 98.2% identical to the erm gene of conjugative transposon Tn1545.

Alcohol, drugs, and violence in children's lives.

This chapter reviews the current state of knowledge concerning the interrelationship between the cycle of alcohol and other drugs (AOD) use and the cycle of violence. This issue is framed in terms of two questions. The first is the extent to which AOD use by the perpetrator is related to the perpetration of violence toward children, defined here as including both physical and sexual abuse. The second question is whether the experience of abuse during childhood is related to the subsequent development of the abuse of alcohol and other drugs. The review indicates that parental AOD abuse is related to physical and sexual abuse. However, because most perpetrators are not parents, the relationship is not yet clear. The data do support the link between experiencing childhood violence and the development of later AOD abuse. Theoretical explanations for each link are reviewed and mediating variables are identified. The review concludes with a presentation of methodological issues and the directions for future research.

Lactococcus lactis and stress.

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis. Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase. To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37 degrees C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.

Efficient insertional mutagenesis in lactococci and other gram-positive bacteria.

In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.

Entry of Listeria monocytogenes into hepatocytes requires expression of inIB, a surface protein of the internalin multigene family.

The intracellular bacterium Listeria monocytogenes can invade several types of normally non-phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800-amino-acid protein, and inIB, which encodes a 630-amino-acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB in invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.