A feed-forward relay integrates the regulatory activities of Bicoid and Orthodenticle via sequential binding to suboptimal sites.

The K50 (lysine at amino acid position 50) homeodomain (HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In Drosophila melanogaster, another K50HD protein, Bicoid (Bcd), has evolved to replace Otd's ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes, including otd Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. Here we define three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd and the presence or absence of sites for cofactors that interact with these proteins. Our results define specific patterning roles for Bcd and Otd and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.

Impacts of the ubiquitous factor Zelda on Bicoid-dependent DNA binding and transcription in Drosophila.

In vivo cross-linking studies suggest that the Drosophila transcription factor Bicoid (Bcd) binds to several thousand sites during early embryogenesis, but it is not clear how many of these binding events are functionally important. In contrast, reporter gene studies have identified >60 Bcd-dependent enhancers, all of which contain clusters of the consensus binding sequence TAATCC. These studies also identified clusters of TAATCC motifs (inactive fragments) that failed to drive Bcd-dependent activation. In general, active fragments showed higher levels of Bcd binding in vivo and were enriched in predicted binding sites for the ubiquitous maternal protein Zelda (Zld). Here we tested the role of Zld in Bcd-mediated binding and transcription. Removal of Zld function and mutations in Zld sites caused significant reductions in Bcd binding to known enhancers and variable effects on the activation and spatial positioning of Bcd-dependent expression patterns. Also, insertion of Zld sites converted one of six inactive fragments into a Bcd-responsive enhancer. Genome-wide binding experiments in zld mutants showed variable effects on Bcd-binding peaks, ranging from strong reductions to significantly enhanced levels of binding. Increases in Bcd binding caused the precocious Bcd-dependent activation of genes that are normally not expressed in early embryos, suggesting that Zld controls the genome-wide binding profile of Bcd at the qualitative level and is critical for selecting target genes for activation in the early embryo. These results underscore the importance of combinatorial binding in enhancer function and provide data that will help predict regulatory activities based on DNA sequence.

Air and waterborne microbiome of a pharmaceutical plant provide insights on spatiotemporal variations and community resilience after disturbance.

BACKGROUND: The presence of microrganisms in pharmaceutical production plant environments is typically monitored by cultural methods, however these cannot detect the unculturable fraction of the microbial community. To get more accurate information on the composition of these indoor microbial communities, both water and air microbiome from a pharmaceutical production plant were profiled by 16S amplicon sequencing. RESULTS: In the water system, we found taxa which typically characterize surface freshwater, groundwater and oligotrophic environments. The airborne microbiome resulted dominated by taxa usually found in outdoor air in combination with human-associated taxa. The alpha- and beta- diversity values showed that the heat-based sanitization process of the water plant affects the composition of the water microbiome by transiently increasing both diversity and evenness. Taxonomic compositional shifts were also detected in response to sanitization, consisting in an increase of Firmicutes and α-Proteobacteria. On the other hand, seasonality seems to be the main driver of bacterial community composition in air of this work environment. CONCLUSIONS: This approach resulted useful to describe the taxonomy of these indoor microbiomes and could be further applied to other built environments, in which the knowledge of the microbiome composition is of relevance. In addition, this study could assist in the design of new guidelines to improve microbiological quality control in indoor work environments.

Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1) is one of the largest genomic islands of this important opportunistic human pathogen. Previous studies have shown that PAPI-1 encodes several putative virulence factors, including a major regulator of biofilm formation and antibiotic-resistance traits. PAPI-1 is horizontally transferable into recipient strains lacking this island via conjugation mediated by the specialized type IV pilus. The PAPI-1 encodes a cluster of ten genes associated with the synthesis and assembly of the type IV pilus. The PAPI-1 acquisition mechanism is currently not well understood. RESULTS: In this study, we performed a series of conjugation experiments and identified determinants of PAPI-1 acquisition by analyzing transfer efficiency between the donor and a series of mutant recipient strains. Our data show that common polysaccharide antigen (CPA) lipopolysaccharide (LPS), a homopolymer of D-rhamnose, is required for initiating PAPI-1 transfer, suggesting that this structure acts as a receptor for conjugative type IV pilus in recipient strains. These results were substantiated by experimental evidence from PAPI-1 transfer assay experiments, in which outer membrane or LPS preparations from well-defined LPS mutants were added to the transfer mix to assess the role of P. aeruginosa LPS in PAPI-1 transfer and in vitro binding experiments between pilin fusion protein GST-pilV2' and immobilized LPS molecules were performed. Our data also showed that P. aeruginosa strains that had already acquired a copy of PAPI-1 were unable to import additional copies of the island, and that such strains produced proportionally lower amounts of CPA LPS compared to the strains lacking PAPI-1. CONCLUSIONS: These results suggest that a PAPI-1 exclusion mechanism exists in P. aeruginosa that might serve to regulate the avoidance of uncontrolled expansions of the bacterial genome.

RiboAbacus: a model trained on polyribosome images predicts ribosome density and translational efficiency from mammalian transcriptomes.

Fluctuations in mRNA levels only partially contribute to determine variations in mRNA availability for translation, producing the well-known poor correlation between transcriptome and proteome data. Recent advances in microscopy now enable researchers to obtain high resolution images of ribosomes on transcripts, providing precious snapshots of translation in vivo. Here we propose RiboAbacus, a mathematical model that for the first time incorporates imaging data in a predictive model of transcript-specific ribosome densities and translational efficiencies. RiboAbacus uses a mechanistic model of ribosome dynamics, enabling the quantification of the relative importance of different features (such as codon usage and the 5' ramp effect) in determining the accuracy of predictions. The model has been optimized in the human Hek-293 cell line to fit thousands of images of human polysomes obtained by atomic force microscopy, from which we could get a reference distribution of the number of ribosomes per mRNA with unmatched resolution. After validation, we applied RiboAbacus to three case studies of known transcriptome-proteome datasets for estimating the translational efficiencies, resulting in an increased correlation with corresponding proteomes. RiboAbacus is an intuitive tool that allows an immediate estimation of crucial translation properties for entire transcriptomes, based on easily obtainable transcript expression levels.

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer.

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo.

Anterior-posterior positional information in the absence of a strong Bicoid gradient.

The Bicoid (Bcd) transcription factor is distributed as a long-range concentration gradient along the anterior posterior (AP) axis of the Drosophila embryo. Bcd is required for the activation of a series of target genes, which are expressed at specific positions within the gradient. Here we directly tested whether different concentration thresholds within the Bcd gradient establish the relative positions of its target genes by flattening the gradient and systematically varying expression levels. Genome-wide expression profiles were used to estimate the total number of Bcd target genes, and a general correlation was found between the Bcd concentration required for activation and the positions where target genes are expressed in wild-type embryos. However, concentrations required for target gene activation in embryos with flattened Bcd were consistently lower than those present at each target gene's position in the wild-type gradient, suggesting that Bcd is in excess at every position along the AP axis. Also, several Bcd target genes were positioned in correctly ordered stripes in embryos with flattened Bcd, and we suggest that these stripes are normally regulated by interactions between Bcd and the terminal patterning system. Our findings argue strongly against the strict interpretation of the Bcd morphogen hypothesis, and support the idea that target gene positioning involves combinatorial interactions that are mediated by the binding site architecture of each gene's cis-regulatory elements.

Functional interaction between the Drosophila knirps short range transcriptional repressor and RPD3 histone deacetylase.

Knirps and other short range transcriptional repressors play critical roles in patterning the early Drosophila embryo. These repressors are known to bind the C-terminal binding protein corepressor, but their mechanism of action is poorly understood. We purified functional recombinant Knirps protein from transgenic embryos to identify possible cofactors that contribute to the activity of this protein. The protein migrates in a complex of approximately 450 kDa and was found to copurify with the Rpd3 histone deacetylase protein during a double affinity purification procedure. Association of Rpd3 with Knirps was dependent on the presence of the C-terminal binding protein-dependent repression domain of Knirps. Previous studies of an rpd3 mutant had not shown defects in the pattern of expression of even-skipped, a target of the Knirps repressor. However, in embryos doubly heterozygous for knirps and rpd3, a marked increase in the frequency of defects in the Knirps-regulated posterior domain of even-skipped expression was found, indicating that Rpd3 contributes to Knirps repression activity in vivo. This finding implicates deacetylation in the mechanism of short range repression in Drosophila.

Quantitative contributions of CtBP-dependent and -independent repression activities of Knirps.

The Drosophila Knirps protein is a short-range transcriptional repressor that locally inhibits activators by recruiting the CtBP co-repressor. Knirps also possesses CtBP-independent repression activity. The functional importance of multiple repression activities is not well understood, but the finding that Knirps does not repress some cis-regulatory elements in the absence of CtBP suggested that the co-factor may supply a unique function essential to repress certain types of activators. We assayed CtBP-dependent and -independent repression domains of Knirps in Drosophila embryos, and found that the CtBP-independent activity, when provided at higher than normal levels, can repress an eve regulatory element that normally requires CtBP. Dose response analysis revealed that the activity of Knirps containing both CtBP-dependent and -independent repression activities is higher than that of the CtBP-independent domain alone. The requirement for CtBP at certain enhancers appears to reflect the need for overall higher levels of repression, rather than a requirement for an activity unique to CtBP. Thus, CtBP contributes quantitatively, rather than qualitatively, to overall repression function. The finding that both repression activities are simultaneously deployed suggests that the multiple repression activities do not function as cryptic 'backup' systems, but that each contributes quantitatively to total repressor output.

Rhizobium sullae sp. nov. (formerly 'Rhizobium hedysari'), the root-nodule microsymbiont of Hedysarum coronarium L.

This work is the completion of a series of reports describing the nitrogen-fixing bacterial symbionts of sulla (Hedysarum coronarium L., Leguminosae) and providing the grounds for their proposal as a new taxon. The introduction summarizes a large amount of previous evidence gathered on the physiology, genetics and ecology of such organisms, which have in the past been referred to provisionally as 'Rhizobium hedysari'. Upon adding 16S RNA sequencing, amplified rDNA restriction analysis of the rrn operon, DNA-DNA hybridization homology and analysis of low-molecular-mass RNA species, it is concluded that the group of strains that specifically nodulate sulla consists of a coherent set of isolates that differ from previously described rhizobia to an extent that warrants the constitution of the species boundary. The name Rhizobium sullae sp. nov. is proposed, with isolate 1S123T (=USDA 4950T = DSM 14623T) as the type strain.