A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.

In trypanosomatids, gene expression is regulated mainly by post-transcriptional mechanisms, which affect mRNA processing, translation and degradation. Currently, our understanding of factors that regulate either mRNA stability or translation is rather limited. We know that often, the regulators are proteins that bind to the 3'-untranslated region; they presumably interact with ribonucleases and translation factors. However, very few such proteins have been characterized in any detail. Here we describe a genome-wide screen to find proteins implicated in post-transcriptional regulation in Trypanosoma brucei. We made a library of random genomic fragments in a plasmid that was designed for expression of proteins fused to an RNA-binding domain, the lambda-N peptide. This was transfected into cells expressing mRNAs encoding a positive or negative selectable marker, and bearing the "boxB" lambda-N recognition element in the 3'-untranslated region. The screen identified about 300 proteins that could be implicated in post-transcriptional mRNA regulation. These included known regulators, degradative enzymes and translation factors, many canonical RNA-binding proteins, and proteins that act via multi-protein complexes. However there were also nearly 150 potential regulators with no previously annotated function, or functions unrelated to mRNA metabolism. Almost 50 novel regulators were shown to bind RNA using a targeted proteome array. The screen also provided fine structure mapping of the hit candidates' functional domains. Our findings not only confirm the key role that RNA-binding proteins play in the regulation of gene expression in trypanosomatids, but also suggest new roles for previously uncharacterized proteins.

Two hits in one: whole genome sequencing unveils LIG4 syndrome and urofacial syndrome in a case report of a child with complex phenotype.

BACKGROUND: Ligase IV syndrome, a hereditary disease associated with compromised DNA damage response mechanisms, and Urofacial syndrome, caused by an impairment of neural cell signaling, are both rare genetic disorders, whose reports in literature are limited. We describe the first case combining both disorders in a specific phenotype. CASE PRESENTATION: We report a case of a 7-year old girl presenting with a complex phenotype characterized by multiple congenital abnormalities and dysmorphic features, microcephaly, short stature, combined immunodeficiency and severe vesicoureteral reflux. Whole Genome Sequencing was performed and a novel ligase IV homozygous missense c.T1312C/p.Y438H mutation was detected, and is believed to be responsible for most of the clinical features of the child, except vesicoureteral reflux which has not been previously described for ligase IV deficiency. However, we observed a second rare damaging (nonsense) homozygous mutation (c.C2125T/p.R709X) in the leucine-rich repeats and immunoglobulin-like domains 2 gene that encodes a protein implicated in neural cell signaling and oncogenesis. Interestingly, this mutation has recently been reported as pathogenic and causing urofacial syndrome, typically displaying vesicoureteral reflux. Thus, this second mutation completes the missing genetic explanation for this intriguing clinical puzzle. We verified that both mutations fit an autosomal recessive inheritance model due to extensive consanguinity. CONCLUSIONS: We successfully identified a novel ligase IV mutation, causing ligase IV syndrome, and an additional rare leucine-rich repeats and immunoglobulin-like domains 2 gene nonsense mutation, in the context of multiple autosomal recessive conditions due to extensive consanguinity. This work demonstrates the utility of Whole Genome Sequencing data in clinical diagnosis in such cases where the combination of multiple rare phenotypes results in very intricate clinical pictures. It also reports a novel causative mutation and a clinical phenotype, which will help in better defining the essential features of both ligase IV and leucine-rich repeats and immunoglobulin-like domains 2 deficiency syndromes.

Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks.

The trypanosome zinc finger protein ZC3H11 binds to AU-rich elements in mRNAs. It is essential for survival of the mammalian-infective bloodstream form, where it stabilizes several mRNAs including some encoding chaperones, and is also required for stabilization of chaperone mRNAs during the heat-shock response in the vector-infective procyclic form. When ZC3H11 was artificially 'tethered' to a reporter mRNA in bloodstream forms it increased reporter expression. We here show that ZC3H11 interacts with trypanosome MKT1 and PBP1, and that domains required for both interactions are necessary for function in the bloodstream-form tethering assay. PBP1 interacts with MKT1, LSM12 and poly(A) binding protein, and localizes to granules during parasite starvation. All of these proteins are essential for bloodstream-form trypanosome survival and increase gene expression in the tethering assay. MKT1 is cytosolic and polysome associated. Using a yeast two-hybrid screen and tandem affinity purification we found that trypanosome MKT1 interacts with multiple RNA-binding proteins and other potential RNA regulators, placing it at the centre of a post-transcriptional regulatory network. A consensus interaction sequence, H(E/D/N/Q)PY, was identified. Recruitment of MKT1-containing regulatory complexes to mRNAs via sequence-specific mRNA-binding proteins could thus control several different post-transcriptional regulons.

Transcriptome-wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co-transcriptional degradation in determining mRNA levels.

African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control. Transcription is constitutive and polycistronic; individual mRNAs are excised by trans splicing and polyadenylation. We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq. Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5'-3' exoribonuclease XRNA. Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation. Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern. Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3 kb show unexpectedly low abundances. Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction.

The roles of 3'-exoribonucleases and the exosome in trypanosome mRNA degradation.

The degradation of eukaryotic mRNAs can be initiated by deadenylation, decapping, or endonuclease cleavage. This is followed by 5'-3' degradation by homologs of Xrn1, and/or 3'-5' degradation by the exosome. We previously reported that, in African trypanosome Trypanosoma brucei, most mRNAs are deadenylated prior to degradation, and that depletion of the major 5'-3' exoribonuclease XRNA preferentially stabilizes unstable mRNAs. We now show that depletion of either CAF1 or CNOT10, two components of the principal deadenylation complex, strongly inhibits degradation of most mRNAs. RNAi targeting another deadenylase, PAN2, or RRP45, a core component of the exosome, preferentially stabilized mRNAs with intermediate half-lives. RRP45 depletion resulted in a 5' bias of mRNA sequences, suggesting action by a distributive 3'-5' exoribonuclease. Results suggested that the exosome is involved in the processing of trypanosome snoRNAs. There was no correlation between effects on half-lives and on mRNA abundance.

Post-transcriptional regulation of the trypanosome heat shock response by a zinc finger protein.

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3'-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures.

Depletion of the Trypanosome Pumilio domain protein PUF2 or of some other essential proteins causes transcriptome changes related to coding region length.

Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Depletion of PUF2 inhibited growth of bloodstream-form trypanosomes, causing selective loss of mRNAs with long open reading frames and increases in mRNAs with shorter open reading frames. Reexamination of published RNASeq data revealed the same trend in cells depleted of some other proteins. We speculate that these length effects could be caused by inhibition of the elongation phase of transcription or by an influence of translation status or polysomal conformation on mRNA decay.

Exploratory focused pharmacogenetic testing reveals novel markers associated with risperidone pharmacokinetics in Saudi children with autism.

Background: Autism spectrum disorders (ASDs) encompass a broad range of phenotypes characterized by diverse neurological alterations. Genomic studies have revealed considerable overlap between the molecular mechanisms implicated in the etiology of ASD and genes involved in the pharmacokinetic (PK) and pharmacodynamic (PD) pathways of antipsychotic drugs employed in ASD management. Given the conflicting data originating from candidate PK or PD gene association studies in diverse ethnogeographic ASD populations, dosage individualization based on "actionable" pharmacogenetic (PGx) markers has limited application in clinical practice. Additionally, off-label use of different antipsychotics is an ongoing practice, which is justified given the shortage of approved cures, despite the lack of satisfactory evidence for its safety according to precision medicine. This exploratory study aimed to identify PGx markers predictive of risperidone (RIS) exposure in autistic Saudi children. Methods: This prospective cohort study enrolled 89 Saudi children with ASD treated with RIS-based antipsychotic therapy. Plasma levels of RIS and 9-OH-RIS were measured using a liquid chromatography-tandem mass spectrometry system. To enable focused exploratory testing, genotyping was performed with the Axiom PharmacoFocus Array, which included a collection of probe sets targeting PK/PD genes. A total of 720 PGx markers were included in the association analysis. Results: A total of 27 PGx variants were found to have a prominent impact on various RIS PK parameters; most were not located within the genes involved in the classical RIS PK pathway. Specifically, 8 markers in 7 genes were identified as the PGx markers with the strongest impact on RIS levels (p < 0.01). Four PGx variants in 3 genes were strongly associated with 9-OH-RIS levels, while 5 markers in 5 different genes explained the interindividual variability in the total active moiety. Notably, 6 CYP2D6 variants exhibited strong linkage disequilibrium; however, they significantly influenced only the metabolic ratio and had no considerable effects on the individual estimates of RIS, 9-OH-RIS, or the total active moiety. After correction for multiple testing, rs78998153 in UGT2B17 (which is highly expressed in the brain) remained the most significant PGx marker positively adjusting the metabolic ratio. For the first time, certain human leukocyte antigen (HLA) markers were found to enhance various RIS exposure parameters, which reinforces the gut-brain axis theory of ASD etiology and its suggested inflammatory impacts on drug bioavailability through modulation of the brain, gastrointestinal tract and/or hepatic expression of metabolizing enzymes and transporters. Conclusion: Our hypothesis-generating approach identified a broad spectrum of PGx markers that interactively influence RIS exposure in ASD children, which indicated the need for further validation in population PK modeling studies to define polygenic scores for antipsychotic efficacy and safety, which could facilitate personalized therapeutic decision-making in this complex neurodevelopmental condition.

The role of the 5'-3' exoribonuclease XRNA in transcriptome-wide mRNA degradation.

The steady-state level of each mRNA in a cell is a balance between synthesis and degradation. Here, we use high-throughput RNA sequencing (RNASeq) to determine the relationship between mRNA degradation and mRNA abundance on a transcriptome-wide scale. The model organism used was the bloodstream form of Trypanosoma brucei, a protist that lacks regulation of RNA polymerase II initiation. The mRNA half-lives varied over two orders of magnitude, with a median half-life of 13 min for total (rRNA-depleted) mRNA. Data for poly(A)+ RNA yielded shorter half-lives than for total RNA, indicating that removal of the poly(A) tail was usually the first step in degradation. Depletion of the major 5'-3' exoribonuclease, XRNA, resulted in the stabilization of most mRNAs with half-lives under 30 min. Thus, on a transcriptome-wide scale, degradation of most mRNAs is initiated by deadenylation. Trypanosome mRNA levels are strongly influenced by gene copy number and mRNA half-life: Very abundant mRNAs that are required throughout the life-cycle may be encoded by multicopy genes and have intermediate-to-long half-lives; those encoding ribosomal proteins, with one to two gene copies, are exceptionally stable. Developmentally regulated transcripts with a lower abundance in the bloodstream forms than the procyclic forms had half-lives around the median, whereas those with a higher abundance in the bloodstream forms than the procyclic forms, such as those encoding glycolytic enzymes, had longer half-lives.

A family harboring an MLKL loss of function variant implicates impaired necroptosis in diabetes.

Maturity-onset diabetes of the young, MODY, is an autosomal dominant disease with incomplete penetrance. In a family with multiple generations of diabetes and several early onset diabetic siblings, we found the previously reported P33T PDX1 damaging mutation. Interestingly, this substitution was also present in a healthy sibling. In contrast, a second very rare heterozygous damaging mutation in the necroptosis terminal effector, MLKL, was found exclusively in the diabetic family members. Aberrant cell death by necroptosis is a cause of inflammatory diseases and has been widely implicated in human pathologies, but has not yet been attributed functions in diabetes. Here, we report that the MLKL substitution observed in diabetic patients, G316D, results in diminished phosphorylation by its upstream activator, the RIPK3 kinase, and no capacity to reconstitute necroptosis in two distinct MLKL-/- human cell lines. This MLKL mutation may act as a modifier to the P33T PDX1 mutation, and points to a potential role of impairment of necroptosis in diabetes. Our findings highlight the importance of family studies in unraveling MODY's incomplete penetrance, and provide further support for the involvement of dysregulated necroptosis in human disease.

Circadian succession of molecular processes in living tissues.

BACKGROUND: Oscillations of different origin, period and amplitude play an important role in the regulation of cellular processes. Most widely studied is the circadian or approximately daily variation in gene expression activity. Timing of gene expression is controlled by internal molecular clock keeping steady periodic expression. In this study, we shift attention towards a broad range of periodically expressed genes involved in multiple cellular functions which may or may not be under direct control of the intrinsic circadian clock. Are all molecular functions represented in expressed genes at all times? Alternatively, are different molecular functions performed at different times? Is there a pattern of succession for molecular processes and functions throughout their daily activity period? RESULTS: To answer these questions, we re-analyzed a number of mouse circadian gene expression data available from public sources. These data represent the normal function of metabolically active peripheral tissues (white adipose tissue, brown adipose tissue, liver). We applied novel methods for the estimation of confidence in phase assignment to identify groups of synchronous genes peaking at the same time regardless of the amplitude or the absolute intensity of expression. Each synchronous group has been annotated to identify Gene Ontology (GO) terms and molecular pathways. Our analysis identified molecular functions specific to a particular time of the day in different tissues. CONCLUSION: Improved methodology for datamining allowed for the discovery of functions and biological pathways in groups of genes with synchronized peak expression time. In particular, such functions as oxidative phase of energy metabolism, DNA repair, mRNA processing, lipid biosynthesis and others are separated in time. This timewise compartmentalization is important for understanding the cellular circuitry and can be used to optimize the time of intervention with drug or genome medication.

C2H2-Type Zinc Finger Proteins: Evolutionarily Old and New Partners of the Nuclear Hormone Receptors.

Nuclear hormone receptors (NRs) are evolutionarily conserved ligand-dependent transcription factors. They are essential for human life, mediating the actions of lipophilic molecules, such as steroid hormones and metabolites of fatty acid, cholesterol, and external toxic compounds. The C2H2-type zinc finger proteins (ZNFs) form the largest family of the transcription factors in humans and are characterized by multiple, tandemly arranged zinc fingers. Many of the C2H2-type ZNFs are conserved throughout evolution, suggesting their involvement in preserved biological activities, such as general transcriptional regulation and development/differentiation of organs/tissues observed in the early embryonic phase. However, some C2H2-type ZNFs, such as those with the Krüppel-associated box (KRAB) domain, appeared relatively late in evolution and have significantly increased family members in mammals including humans, possibly modulating their complicated transcriptional network and/or supporting the morphological development/functions specific to them. Such evolutional characteristics of the C2H2-type ZNFs indicate that these molecules influence the NR functions conserved through evolution, whereas some also adjust them to meet with specific needs of higher organisms. We review the interaction between NRs and C2H2-type ZNFs by focusing on some of the latter molecules.

Genome-wide Regulatory Roles of the C2H2-type Zinc Finger Protein ZNF764 on the Glucocorticoid Receptor.

The C2H2-type zinc finger protein ZNF764 acts as an enhancer for several steroid hormone receptors, and haploinsufficiency of this gene may be responsible for tissue resistance to multiple steroid hormones including glucocorticoids observed in a patient with 16p11.2 microdeletion. We examined genome-wide regulatory actions of ZNF764 on the glucocorticoid receptor (GR) in HeLa cells as a model system. ZNF764- and GR-binding sites demonstrated similar distribution in various genomic features. They positioned predominantly around 50-500 kbs from the transcription start sites of their nearby genes, and were closely localized with each other, overlapping in ~37% of them. ZNF764 demonstrated differential on/off effects on GR-binding and subsequent mRNA expression: some genes were highly dependent on the presence/absence of ZNF764, but others were not. Pathway analysis revealed that these 3 gene groups were involved in distinct cellular activities. ZNF764 physically interacted with GR at ligand-binding domain through its KRAB domain, and both its physical interaction to GR and zinc finger domain appear to be required for ZNF764 to regulate GR transcriptional activity. Thus, ZNF764 is a cofactor directing GR transcriptional activity toward specific biologic pathways by changing GR binding and transcriptional activity on the glucocorticoid-responsive genes.

More robust detection of motifs in coexpressed genes by using phylogenetic information.

BACKGROUND: Several motif detection algorithms have been developed to discover overrepresented motifs in sets of coexpressed genes. However, in a noisy gene list, the number of genes containing the motif versus the number lacking the motif might not be sufficiently high to allow detection by classical motif detection tools. To still recover motifs which are not significantly enriched but still present, we developed a procedure in which we use phylogenetic footprinting to first delineate all potential motifs in each gene. Then we mutually compare all detected motifs and identify the ones that are shared by at least a few genes in the data set as potential candidates. RESULTS: We applied our methodology to a compiled test data set containing known regulatory motifs and to two biological data sets derived from genome wide expression studies. By executing four consecutive steps of 1) identifying conserved regions in orthologous intergenic regions, 2) aligning these conserved regions, 3) clustering the conserved regions containing similar regulatory regions followed by extraction of the regulatory motifs and 4) screening the input intergenic sequences with detected regulatory motif models, our methodology proves to be a powerful tool for detecting regulatory motifs when a low signal to noise ratio is present in the input data set. Comparing our results with two other motif detection algorithms points out the robustness of our algorithm. CONCLUSION: We developed an approach that can reliably identify multiple regulatory motifs lacking a high degree of overrepresentation in a set of coexpressed genes (motifs belonging to sparsely connected hubs in the regulatory network) by exploiting the advantages of using both coexpression and phylogenetic information.

Confidence in Phase Definition for Periodicity in Genes Expression Time Series.

Circadian oscillation in baseline gene expression plays an important role in the regulation of multiple cellular processes. Most of the knowledge of circadian gene expression is based on studies measuring gene expression over time. Our ability to dissect molecular events in time is determined by the sampling frequency of such experiments. However, the real peaks of gene activity can be at any time on or between the time points at which samples are collected. Thus, some genes with a peak activity near the observation point have their phase of oscillation detected with better precision then those which peak between observation time points. Separating genes for which we can confidently identify peak activity from ambiguous genes can improve the analysis of time series gene expression. In this study we propose a new statistical method to quantify the phase confidence of circadian genes. The numerical performance of the proposed method has been tested using three real gene expression data sets.

Methods to determine the transcriptomes of trypanosomes in mixtures with mammalian cells: the effects of parasite purification and selective cDNA amplification.

Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq). We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.

The silicon trypanosome: a test case of iterative model extension in systems biology.

The African trypanosome, Trypanosoma brucei, is a unicellular parasite causing African Trypanosomiasis (sleeping sickness in humans and nagana in animals). Due to some of its unique properties, it has emerged as a popular model organism in systems biology. A predictive quantitative model of glycolysis in the bloodstream form of the parasite has been constructed and updated several times. The Silicon Trypanosome is a project that brings together modellers and experimentalists to improve and extend this core model with new pathways and additional levels of regulation. These new extensions and analyses use computational methods that explicitly take different levels of uncertainty into account. During this project, numerous tools and techniques have been developed for this purpose, which can now be used for a wide range of different studies in systems biology.

Depletion of trypanosome CTR9 leads to gene expression defects.

The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1.

Inferring the transcriptional network of Bacillus subtilis.

The adaptation of bacteria to the vigorous environmental changes they undergo is crucial to their survival. They achieve this adaptation partly via intricate regulation of the transcription of their genes. In this study, we infer the transcriptional network of the Gram-positive model organism, Bacillus subtilis. We use a data integration workflow, exploiting both motif and expression data, towards the generation of condition-dependent transcriptional modules. In building the motif data, we rely on both known and predicted information. Known motifs were derived from DBTBS, while predicted motifs were generated by a de novo motif detection method that utilizes comparative genomics. The expression data consists of a compendium of microarrays across different platforms. Our results indicate that a considerable part of the B. subtilis network is yet undiscovered; we could predict 417 new regulatory interactions for known regulators and 453 interactions for yet uncharacterized regulators. The regulators in our network showed a preference for regulating modules in certain environmental conditions. Also, substantial condition-dependent intra-operonic regulation seems to take place. Global regulators seem to require functional flexibility to attain their roles by acting as both activators and repressors.

Functional analysis of luxS in the probiotic strain Lactobacillus rhamnosus GG reveals a central metabolic role important for growth and biofilm formation.

Quorum sensing is involved in the regulation of multicellular behavior through communication via small molecules. Given the high number and diversity of the gastrointestinal microbiota, it is postulated that members of this community communicate to coordinate a variety of adaptive processes. AI-2 is suggested to be a universal bacterial signaling molecule synthesized by the LuxS enzyme, which forms an integral part of the activated methyl cycle. We have previously reported that the well-documented probiotic strain Lactobacillus rhamnosus GG, a human isolate, produces AI-2-like molecules. In this study, we identified the luxS homologue of L. rhamnosus GG. luxS seems to be located in an operon with a yxjH gene encoding a putative cobalamin-independent methionine synthase. In silico analysis revealed a methionine-specific T box in the leader sequence of the putative yxjH-luxS operon. However, transcriptional analysis showed that luxS is expressed mainly as a monocistronic transcript. Construction of a luxS knockout mutant confirmed that the luxS gene is responsible for AI-2 production in L. rhamnosus GG. However, this mutation also resulted in pleiotropic effects on the growth of this fastidious strain. Cysteine, pantothenate, folic acid, and biotin could partially complement growth, suggesting a central metabolic role for luxS in L. rhamnosus GG. Interestingly, the luxS mutant also showed a defect in monospecies biofilm formation. Experiments with chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione, coculture with the wild type, and nutritional complementation suggested that the main cause of this defect has a metabolic nature. Moreover, our data indicate that suppressor mutations are likely to occur in luxS mutants of L. rhamnosus GG. Therefore, results of luxS-related studies should be carefully interpreted.