Analysis of Small Non-coding RNAs as Signaling Intermediates of Environmentally Integrated Responses to Abiotic Stress.

Research to date on abiotic stress responses in plants has been largely focused on the plant itself, but current knowledge indicates that microorganisms can interact with and help plants during periods of abiotic stress. In our research, we aim to investigate the interkingdom communication between the plant root and the rhizo-microbiota. Our investigation showed that miRNA plays a pivotal role in this interkingdom communication. Here, we describe a protocol for the analysis of miRNA secreted by the plant root, which includes all of the steps from the isolation of the miRNA to the bioinformatics analysis. Because of their short nucleotide length, Next Generation Sequencing (NGS) library preparation from miRNAs can be challenging due to the presence of dimer adapter contaminants. Therefore, we highlight some strategies we adopt to inhibit the generation of dimer adapters during library preparation. Current screens of miRNA targets mostly focus on the identification of targets present in the same organism expressing the miRNA. Our bioinformatics analysis challenges the barrier of evolutionary divergent organisms to identify candidate sequences of the microbiota targeted by the miRNA of plant roots. This protocol should be of interest to researchers investigating interkingdom RNA-based communication between plants and their associated microorganisms, particularly in the context of holobiont responses to abiotic stresses.

Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression.

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in "beneficial" gut commensals.

Molecular analysis of the replication functions of the bifidobacterial conjugative megaplasmid pMP7017.

pMP7017 is a conjugative megaplasmid isolated from the gut commensal Bifidobacterium breve JCM7017 and was shown to encode two putative replicases, designated here as RepA and RepB. In the current work, RepB was identified as the pMP7017 replicative initiator, as the repB gene, and its surrounding region was shown to be sufficient to allow autonomous replication in two bifidobacterial species, B. breve and Bifidobacterium longum subsp. longum. RepB was shown to bind to repeat sequence downstream of its coding sequence and this region was determined to be essential for efficient replication. Based on our results, we hypothesize that pMP7017 is an iteron-regulated plasmid (IRP) under strict auto-regulatory control. Recombinantly produced and purified RepB was determined to exist as a dimer in solution, differing from replicases of other IRPs, which exist as a mix of dimers and monomers. Furthermore, a stable low-copy Bifidobacterium-E. coli shuttle vector, pRD1.3, was created which can be employed for cloning and expression of large genes, as was demonstrated by the cloning and heterologous expression of the 5.1 kb apuB gene encoding the extracellular amylopullulanase from B. breve UCC2003 into B. longum subsp. longum NCIMB8809.

A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis.

In Lactococcus lactis, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.

Bifidobacterium breve UCC2003 Employs Multiple Transcriptional Regulators To Control Metabolism of Particular Human Milk Oligosaccharides.

Bifidobacterial carbohydrate metabolism has been studied in considerable detail for a variety of both plant- and human-derived glycans, particularly involving the bifidobacterial prototype strain Bifidobacterium breve UCC2003. We recently elucidated the metabolic pathways by which the human milk oligosaccharide (HMO) constituents lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and lacto-N-biose (LNB) are utilized by B. breve UCC2003. However, to date, no work has been carried out on the regulatory mechanisms that control the expression of the genetic loci involved in these HMO metabolic pathways. In this study, we describe the characterization of three transcriptional regulators and the corresponding operator and associated (inducible) promoter sequences, with the latter governing the transcription of the genetic elements involved in LN(n)T/LNB metabolism. The activity of these regulators is dependent on the release of specific monosaccharides, which are believed to act as allosteric effectors and which are derived from the corresponding HMOs targeted by the particular locus.IMPORTANCE Human milk oligosaccharides (HMOs) are a key factor in the development of the breastfed-infant microbiota. They function as prebiotics, selecting for a specific range of microbes, including a number of infant-associated species of bifidobacteria, which are thought to provide a range of health benefits to the infant host. While much research has been carried out on elucidating the mechanisms of HMO metabolism in infant-associated bifidobacteria, to date there is very little understanding of the transcriptional regulation of these pathways. This study reveals a multicomponent transcriptional regulation system that controls the recently identified pathways of HMO metabolism in the infant-associated Bifidobacterium breve prototype strain UCC2003. This not only provides insight into the regulatory mechanisms present in other infant-associated bifidobacteria but also provides an example of a network of sequential steps regulating microbial carbohydrate metabolism.

Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage.

RNA dependent DNA-polymerases, reverse transcriptases, are key enzymes for retroviruses and retroelements. Their fidelity, including indel generation, is significant for their use as reagents including for deep sequencing. Here, we report that certain RNA template structures and G-rich sequences, ahead of diverse reverse transcriptases can be strong stimulators for slippage at slippage-prone template motif sequence 3' of such 'slippage-stimulatory' structures. Where slippage is stimulated, the resulting products have one or more additional base(s) compared to the corresponding template motif. Such structures also inhibit slippage-mediated base omission which can be more frequent in the absence of a relevant stem-loop. Slippage directionality, base insertion and omission, is sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The retrotransposon-derived enzyme TGIRT exhibits more slippage in vitro than the retroviral enzymes tested including that from HIV. Structure-mediated slippage may be exhibited by other polymerases and enrich gene expression. A cassette from Drosophila retrotransposon Dme1_chrX_2630566, a candidate for utilizing slippage for its GagPol synthesis, exhibits strong slippage in vitro. Given the widespread occurrence and importance of retrotransposons, systematic studies to reveal the extent of their functional utilization of RT slippage are merited.

Specific reverse transcriptase slippage at the HIV ribosomal frameshift sequence: potential implications for modulation of GagPol synthesis.

Synthesis of HIV GagPol involves a proportion of ribosomes translating a U6A shift site at the distal end of the gag gene performing a programmed -1 ribosomal frameshift event to enter the overlapping pol gene. In vitro studies here show that at the same shift motif HIV reverse transcriptase generates -1 and +1 indels with their ratio being sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The GGG sequence 3' adjacent to the U6A shift/slippage site, which is important for ribosomal frameshifting, is shown here to limit reverse transcriptase base substitution and indel 'errors' in the run of A's in the product. The indels characterized here have either 1 more or less A, than the corresponding number of template U's. cDNA with 5 A's may yield novel Gag product(s), while cDNA with an extra base, 7 A's, may only be a minor contributor to GagPol polyprotein. Synthesis of a proportion of non-ribosomal frameshift derived GagPol would be relevant in efforts to identify therapeutically useful compounds that perturb the ratio of GagPol to Gag, and pertinent to the extent in which specific polymerase slippage is utilized in gene expression.

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators.

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

Carbon dynamics, development and stress responses in Arabidopsis: involvement of the APL4 subunit of ADP-glucose pyrophosphorylase (starch synthesis).

An Arabidopsis thaliana T-DNA insertional mutant was identified and characterized for enhanced tolerance to the singlet-oxygen-generating herbicide atrazine in comparison to wild-type. This enhanced atrazine tolerance mutant was shown to be affected in the promoter structure and in the regulation of expression of the APL4 isoform of ADP-glucose pyrophosphorylase, a key enzyme of the starch biosynthesis pathway, thus resulting in decrease of APL4 mRNA levels. The impact of this regulatory mutation was confirmed by the analysis of an independent T-DNA insertional mutant also affected in the promoter of the APL4 gene. The resulting tissue-specific modifications of carbon partitioning in plantlets and the effects on plantlet growth and stress tolerance point out to specific and non-redundant roles of APL4 in root carbon dynamics, shoot-root relationships and sink regulations of photosynthesis. Given the effects of exogenous sugar treatments and of endogenous sugar levels on atrazine tolerance in wild-type Arabidopsis plantlets, atrazine tolerance of this apl4 mutant is discussed in terms of perception of carbon status and of investment of sugar allocation in xenobiotic and oxidative stress responses.

Transcriptional slippage controls production of type III secretion apparatus components in Shigella flexneri.

During transcription, series of approximately 9 As or Ts can direct RNA polymerase to incorporate into the mRNA nucleotides not encoded by the DNA, changing the reading frame downstream from the slippage site. We detected series of 9 or 10 As in spa13, spa33 and mxiA encoding type III secretion apparatus components. Analysis of cDNAs indicated that transcriptional slippage occurs in spa13, mxiA and spa33. Changes in the reading frame were confirmed by using plasmids carrying slippage sites in the 5' part of lacZ. Slippage is required for production of Spa13 from two overlapping reading frames and should lead to production of truncated MxiA and Spa33 proteins. Complementation of spa13 and mxiA mutants with plasmids carrying altered sites indicated that slippage in spa13 is required for assembly of the secretion apparatus and that slippage sites in spa13 and mxiA have not been selected to encode Lys residues or to produce two proteins endowed with different activities. The presence of slippage sites decreases production of Spa13 by 70%, of MxiA and Spa33 by 15% and of Spa32 (encoded downstream from spa13) by 50%. These results suggest that transcriptional slippage controls protein production by reducing the proportion of mRNA translated into functional proteins.

The pleiotropic Arabidopsis frd mutation with altered coordination of chloroplast biogenesis, cell size and differentiation, organ size and number.

In higher plants, plastid development must be tightly coordinated with cell and organ development. In this paper, a novel T-DNA-mutagenized Arabidopsis line showing chlorotic leaves and minute stature was identified in a genetic screen for altered chloroplast development. The mutation corresponded to a single locus on chromosome IV and was associated with insertion of the T-DNA. This locus was named FARFADET and resulted in pleiotropic effects on chloroplast biogenesis, cell size and differentiation, organ size and number. Thus, in contrast with previously described chlorotic mutants, frd mutants were affected not only in chloroplast development and chlorophyll accumulation, but also in cell and organ development. Alteration of differentiation affected different cell types such as leaf epidermal cells, trichomes, mesophyll cells, and columella cells. A major effect on mesophyll cell differentiation was the lack of palisadic parenchyma and absence of grana stacks. Moreover, meristem size and lateral meristem initiation were affected. Genetic and molecular characterisation showed that the T-DNA insertion generated 41 bp deletion in a potential miRNA precursor. The predicted miRNA target genes were involved in plant development and stress. It is therefore hypothesized that the frd mutation had affected coordination of cell developmental span and the control of the division-differentiation balance.

Transcriptional slippage in mxiE controls transcription and translation of the downstream mxiD gene, which encodes a component of the Shigella flexneri type III secretion apparatus.

The Shigella flexneri transcription activator MxiE is produced by transcriptional slippage from two overlapping open reading frames. By using plasmids encoding a mxiD-lacZ translational fusion, we showed that transcriptional slippage in mxiE influences both transcription and translation of the downstream mxiD gene encoding an essential component of the type III secretion apparatus.

A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri.

Bacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of approximately 15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an anti-activator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.

Frameshifting by transcriptional slippage is involved in production of MxiE, the transcription activator regulated by the activity of the type III secretion apparatus in Shigella flexneri.

Bacteria of Shigella spp. are responsible for shigellosis in humans. They use a type III secretion (TTS) system encoded by a 200 kb virulence plasmid to enter epithelial cells and trigger apoptosis in macrophages. This TTS system comprises a secretion apparatus, translocators and effectors that transit through this apparatus, cytoplasmic chaperones and specific transcription regulators. The TTS apparatus assembled during growth of Shigella flexneri in broth is activated upon contact with epithelial cells. Transcription of approximately 15 genes encoding effectors, including IpaH proteins, is regulated by the TTS apparatus activity and controlled by MxiE, a transcription activator of the AraC family, and IpgC, the chaperone of the translocators IpaB and IpaC. We present evidence that MxiE is produced by a frameshift between a 59-codon open reading frame (ORF) (mxiEa) containing the translation start site and a 214-codon ORF (mxiEb) encoding the DNA binding domain of the protein. The mxiEa encoded N-terminal part of MxiE is required for MxiE function. Frameshifting efficiency was approximately 30% during growth in broth and was not modulated by the activity of secretion or the coactivator IpgC. Frameshifting involves slippage of RNA polymerase during transcription of mxiE, which results in the incorporation of one additional nucleotide in the mRNA and places mxiEa and mxiEb in the same reading frame. Frameshifting might represent an additional means of controlling gene expression under specific environmental conditions.