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Staphylococcus aureus is one of the principal causative agents of bacteremia which can progress to sepsis. Rapid diagnostic tests for identification and antibiotic resistance profiling of S. aureus would improve patient outcomes and antibiotic stewardship, but existing methods require a lengthy culture step to obtain enough material for testing. Complexity of the host matrix, where pathogenic microbes are often present, also interferes with many diagnostic methods. Here, we describe a straightforward and rapid method for enriching viable S. aureus using bio-orthogonal, or "click," chemistry methods. Bacteria labeled in this manner can potentially be cultured, interrogated using molecular methods for pathogen identification, or used to test antibiotic susceptibility.

Aberrant de novo methylation of DNA is considered an important mediator of tumorigenesis. To investigate the role of de novo DNA methyltransferase 3a (Dnmt3a) in intestinal tumor development, we analyzed the expression of Dnmt3a in murine colon crypts, murine colon adenomas and human colorectal cancer using RNA fluorescence in situ hybridization (FISH), quantitative PCR and immunostaining. Following conditional deletion of Dnmt3a in the colon of APC((Min/+)) mice, we analyzed tumor numbers, genotype of macroadenomas and laser dissected microadenomas, global and regional DNA methylation and gene expression. Our results showed increased Dnmt3a expression in colon adenomas of APC((Min/+)) mice and human colorectal cancer samples when compared with control tissue. Interestingly, in tumor tissue, RNA FISH analysis showed highest Dnmt3a expression in Lgr5-positive stem/progenitor cells. Deletion of Dnmt3a in APC((Min/+)) mice reduced colon tumor numbers by ~40%. Remaining adenomas and microadenomas almost exclusively contained the non-recombined Dnmt3a allele; no tumors composed of the inactivated Dnmt3a allele were detected. DNA methylation was reduced at the Oct4, Nanog, Tff2 and Cdkn1c promoters and expression of the tumor-suppressor genes Tff2 and Cdkn1c was increased. In conclusion, our results show that Dnmt3a is predominantly expressed in the stem/progenitor cell compartment of tumors and that deletion of Dnmt3a inhibits the earliest stages of intestinal tumor development.

Recently, researchers have uncovered the presence of many long noncoding RNAs (lncRNAs) in embryonic stem cells and believe they are important regulators of the differentiation process. However, there are only a few examples explicitly linking lncRNA activity to transcriptional regulation. Here, we used transcript counting and spatial localization to characterize a lncRNA (dubbed linc-HOXA1) located ∼50 kb from the Hoxa gene cluster in mouse embryonic stem cells. Single-cell transcript counting revealed that linc-HOXA1 and Hoxa1 RNA are highly variable at the single-cell level and that whenever linc-HOXA1 RNA abundance was high, Hoxa1 mRNA abundance was low and vice versa. Knockdown analysis revealed that depletion of linc-HOXA1 RNA at its site of transcription increased transcription of the Hoxa1 gene cis to the chromosome and that exposure of cells to retinoic acid can disrupt this interaction. We further showed that linc-HOXA1 RNA represses Hoxa1 by recruiting the protein PURB as a transcriptional cofactor. Our results highlight the power of transcript visualization to characterize lncRNA function and also suggest that PURB can facilitate lncRNA-mediated transcriptional regulation.

Twelve genes encoding key components of Clostridium cellulolyticum cellulosomes are clustered. Among them, the first, second, and fifth genes encode the assembly factor CipC and the two major cellulases Cel48F and Cel9E, respectively. Cellulolytic clones were selected from the noncellulolytic cipC insertional mutant trans -complemented with a cipC expression vector, in which one homologous recombination event between the 3′ end of the chromosomal cipC gene and the plasmidic cipC gene has restored the cluster continuity. The absence of the enzymes encoded by the cluster in the cipC mutant was thus only due to a strong polar effect, indicating that all genes were transcriptionally linked. Two large transcripts were detected in cellulose-grown cells by Northern hybridization: a 14-kb messenger which carries the cipC-cel48F-cel8C-cel9G-cel9E coding sequences and, in a smaller amount, a 12-kb messenger which carries the genes located in the 3′ part of the cluster. Four smaller transcripts were found in large amounts: a cipC-cel48F bicistronic one and three monocistronic ones, cipC , cel48F , and cel9E . The cipC-cel48F and cel48F messengers were shown to be stable. Analysis by reverse transcription-PCR suggested transcriptional linkage of all of the open reading frames. The production of a primary very large transcript covering the entire cluster was hypothesized. Primer extension analysis has identified two putative transcriptional start sites located 638/637 and 194 nucleotides upstream of the cipC translational start. The processing of the primary transcript would lead to the production of several secondary messengers displaying different stabilities, contributing to fine tuning of expression of individual genes of the operon.

High expression of the transcriptional activator ComK occurs in 10-20% of the cells in stationary phase cultures of Bacillus subtilis strain 168. ComK drives the expression of more than 100 genes constituting the semidormant K-state, distinct from sporulation and vegetative growth. Among the genes so activated are those that permit competence for genetic transformation. We have addressed the origin of bistability in expression of ComK. We show that bistability requires positive autoregulation at the promoter of comK, but not a potential toggle switch, in which ComK represses the promoter of rok and Rok represses the promoter of comK. We further address the source of the noise that results in the stochastic selection of cells that will express comK. A revised model for the regulation of comK expression is proposed that partially explains bistability.

Summary The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.

Summary Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large ‘cel’ gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component. Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose. It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the ‘cel’ cluster. The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125. Their N-termini did not match any of the known cellulase sequences from C. cellulolyticum. A large amount of entire CipC produced in the cipCMut1 strain by trans-complementation with plasmid pSOScipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes. The complexes produced in the mutant and complemented strains contained at least 12 different dockerin-containing proteins encoded by genes located outside of the ‘cel’ cluster. The disturbances observed in the mutant and trans-complemented strains were the result of a strong polar effect resulting from the cipC gene disruption. In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the ‘cel’ cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.

© Published by The Company of Biologists Ltd. All rights reserved., Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state., This work was supported by Fundação para a Ciência e Tecnologia, Portugal [SFRH/ BPD/78313/2011 to E.A., SFRH/BD/80191/2011 to A.M.V.G. and PTDC/SAUOBD/100664/2008]. M.M. and P.S. were supported by The Academy of Sciences of the Czech Republic [project M200521202]. P.S. is a member of the Centre for RNA Biology funded by the Czech Science Foundation [GACR P305/12/G034].

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV cm−1 field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV cm−1 pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 105 and 107 transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively. Journal of Industrial Microbiology & Biotechnology (2001) 27, 271–274.

International audience; Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 59-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression.

High expression of the transcriptional activator ComK occurs in 10–20% of the cells in stationary phase cultures of Bacillus subtilis strain 168. ComK drives the expression of more than 100 genes constituting the semidormant K-state, distinct from sporulation and vegetative growth. Among the genes so activated are those that permit competence for genetic transformation. We have addressed the origin of bistability in expression of ComK. We show that bistability requires positive autoregulation at the promoter of comK, but not a potential toggle switch, in which ComK represses the promoter of rok and Rok represses the promoter of comK. We further address the source of the noise that results in the stochastic selection of cells that will express comK. A revised model for the regulation of comK expression is proposed that partially explains bistability.

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.

Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large 'cel' gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component. Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose. It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the 'cel' cluster. The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125. Their N-termini did not match any of the known cellulase sequences from C. cellulolyticum. A large amount of entire CipC produced in the cipCMut1 strain by trans-complementation with plasmid pSOScipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes. The complexes produced in the mutant and complemented strains contained at least 12 different dockerin-containing proteins encoded by genes located outside of the 'cel' cluster. The disturbances observed in the mutant and trans-complemented strains were the result of a strong polar effect resulting from the cipC gene disruption. In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the 'cel' cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.

Two new insertion sequences, ISCce 1 and ISCce 2 , were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum . In these insertional mutants, the cipC gene was disrupted either by ISCce 1 alone or by both ISCce 1 and ISCce 2 . ISCce 1 is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS 481 and IS 3 families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce 1 . ISCce 2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS 256 family. Upon transposition, both ISCce 1 and ISCce 2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce 1 is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce 2 was found to be mostly inserted into ISCce 1 . In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C. cellulolyticum strain used.