sRNA-mediated RNA processing regulates bacterial cell division.

Tight control of cell division is essential for survival of most organisms. For prokaryotes, the regulatory mechanisms involved in the control of cell division are mostly unknown. We show that the small non-coding sRNA StsR has an important role in controlling cell division and growth in the alpha-proteobacterium Rhodobacter sphaeroides. StsR is strongly induced by stress conditions and in stationary phase by the alternative sigma factors RpoHI/HII, thereby providing a regulatory link between cell division and environmental cues. Compared to the wild type, a mutant lacking StsR enters stationary phase later and more rapidly resumes growth after stationary phase. A target of StsR is UpsM, the most abundant sRNA in the exponential phase. It is derived from partial transcriptional termination within the 5' untranslated region of the mRNA of the division and cell wall (dcw) gene cluster. StsR binds to UpsM as well as to the 5' UTR of the dcw mRNA and the sRNA-sRNA and sRNA-mRNA interactions lead to a conformational change that triggers cleavage by the ribonuclease RNase E, affecting the level of dcw mRNAs and limiting growth. These findings provide interesting new insights into the role of sRNA-mediated regulation of cell division during the adaptation to environmental changes.

Incoherent dual regulation by a SAM-II riboswitch controlling translation at a distance.

In Sinorhizobium meliloti, the methionine biosynthesis genes metA and metZ are preceded by S-adenosyl-L-methionine (SAM) riboswitches of the SAM-II class. Upon SAM binding, structural changes in the metZ riboswitch were predicted to cause transcriptional termination, generating the sRNA RZ. By contrast, the metA riboswitch was predicted to regulate translation from an AUG1 codon. However, downstream of the metA riboswitch, we found a putative Rho-independent terminator and an in-frame AUG2 codon, which may contribute to metA regulation. We validated the terminator between AUG1 and AUG2, which generates the sRNA RA1 that is processed to RA2. Under high SAM conditions, the activities of the metA and metZ promoters and the steady-state levels of the read-through metA and metZ mRNAs were decreased, while the levels of the RZ and RA2 sRNAs were increased. Under these conditions, the sRNAs and the mRNAs were stabilized. Reporter fusion experiments revealed that the Shine-Dalgarno (SD) sequence in the metA riboswitch is required for translation, which, however, starts 74 nucleotides downstream at AUG2, suggesting a novel translation initiation mechanism. Further, the reporter fusion data supported the following model of RNA-based regulation: Upon SAM binding by the riboswitch, the SD sequence is sequestered to downregulate metA translation, while the mRNA is stabilized. Thus, the SAM-II riboswitches fulfil incoherent, dual regulation, which probably serves to ensure basal metA and metZ mRNA levels under high SAM conditions. This probably helps to adapt to changing conditions and maintain SAM homoeostasis.

A major checkpoint for protein expression in Rhodobacter sphaeroides during heat stress response occurs at the level of translation.

Temperature above the physiological optimum is a stress condition frequently faced by bacteria in their natural environments. Here, we were interested in the correlation between levels of RNA and protein under heat stress. Changes in RNA and protein levels were documented in cultures of Rhodobacter sphaeroides using RNA sequencing, quantitative mass spectrometry, western blot analysis, in vivo [35 S] methionine-labelling and plasmid-borne reporter fusions. Changes in the transcriptome were extensive. Strikingly, the proteome remained unchanged except for very few proteins. Examples include a heat shock protein, a DUF1127 protein of unknown function and sigma factor proteins from leaderless transcripts. Insight from this study indicates that R. sphaeroides responds to heat stress by producing a broad range of transcripts while simultaneously preventing translation from nearly all of them, and that this selective production of protein depends on the untranslated region of the transcript. We conclude that measurements of transcript abundance are insufficient to understand gene regulation. Rather, translation can be an important checkpoint for protein expression under certain environmental conditions. Furthermore, during heat shock, regulation at the level of transcription might represent preparation for survival in an unpredictable environment while regulation at translation ensures production of only a few proteins.

A novel LuxR-type solo of Sinorhizobium meliloti, NurR, is regulated by the chromosome replication coordinator, DnaA and activates quorum sensing.

The genome of Sinorhizobium meliloti, a model for studying plant-bacteria symbiosis, contains eight genes coding for LuxR-like proteins. Two of these, SinR and ExpR, are essential for quorum sensing (QS). Roles and regulation surrounding the others are mostly unknown. Here, we reveal the DNA recognition sequence and regulon of the LuxR-like protein SMc00877. Unlike ExpR, which uses the long-chain acyl homoserine lactones (AHLs) as inducers, SMc00877 functioned independently of AHLs and was even functional in Escherichia coli. A target of SMc00877 is SinR, the major regulator of AHL production in S. meliloti. Disruption of SMc00877 decreased AHL production. A weaker production of AHLs resulted in smaller microcolonies, starting from single cells, and delayed AHL-dependent regulation. SMc00877 was expressed only in growing cells in the presence of replete nutrients. Therefore, we renamed it NurR (nutrient sensitive LuxR-like regulator). We traced this nutrient-sensitive expression to transcription control by the DNA replication initiation factor, DnaA, which is essential for growth. These results indicate that NurR has a role in modulating the threshold of QS activation according to growth. We propose growth behavior as an additional prerequisite to population density for the activation of QS in S. meliloti.

Individual therapeutic DAS28-dcrit responses differentiate between effectiveness of rheumatoid arthritis therapies and reflect patient-reported outcomes: retrospective analysis of DAS28 responses in comparative tocilizumab studies.

Assessment of individual therapeutic responses provides valuable information concerning treatment benefits in individual patients. We evaluated individual therapeutic responses as determined by the Disease Activity Score-28 joints critical difference for improvement (DAS28-dcrit) in rheumatoid arthritis (RA) patients treated with intravenous tocilizumab or comparator anti-tumor necrosis factor (TNF) agents. The previously published DAS28-dcrit value [DAS28 decrease (improvement) ≥ 1.8] was retrospectively applied to data from two studies of tocilizumab in RA, the 52-week ACT-iON observational study and the 24-week ADACTA randomized study. Data were compared within (not between) studies. DAS28 was calculated with erythrocyte sedimentation rate as the inflammatory marker. Stability of DAS28-dcrit responses and European League Against Rheumatism (EULAR) good responses was determined by evaluating repeated responses at subsequent timepoints. A logistic regression model was used to calculate p values for differences in response rates between active agents. Patient-reported outcomes (PROs; pain, global health, function, and fatigue) in DAS28-dcrit responder versus non-responder groups were compared with an ANCOVA model. DAS28-dcrit individual response rates were 78.2% in tocilizumab-treated patients and 58.2% in anti-TNF-treated patients at week 52 in the ACT-ion study (p = 0.0001) and 90.1% versus 59.1% at week 24 in the ADACTA study (p < 0.0001). DAS28-dcrit responses showed greater stability over time (up to 52 weeks) than EULAR good responses. For both active treatments, DAS28-dcrit responses were associated with statistically significant improvements in mean PRO values compared with non-responders. The DAS28-dcrit response criterion provides robust assessments of individual responses to RA therapy and may be useful for discriminating between active agents in clinical studies and guiding treat-to-target decisions in daily practice.

Genome sequence of the endophytic strain Enterobacter sp. J49, a potential biofertilizer for peanut and maize.

Enterobacter sp. J49 is a plant growth promoting endophytic strain that promotes the growth of peanut and maize crops. This strain promotes plant growth by different mechanisms with the supply of soluble phosphorus being one of the most important. Enterobacter sp. J49 not only increases the phosphorus content in the plant but also in the soil favoring the nutrition of other plants usually used in rotation with these crops. The aims of this study were to analyze the genome sequence of Enterobacter sp. J49 in order to deepen our knowledge regarding its plant growth promoting traits and to establish its phylogenetic relationship with other species of Enterobacter genus. Genome sequence of Enterobacter sp. J49 is a valuable source of information to continuing the research of its potential industrial production as a biofertilizer of peanut, maize and other economically important crops.

Comparative analyses of the variation of the transcriptome and proteome of Rhodobacter sphaeroides throughout growth.

BACKGROUND: In natural environments, bacteria must frequently cope with extremely scarce nutrients. Most studies focus on bacterial growth in nutrient replete conditions, while less is known about the stationary phase. Here, we are interested in global gene expression throughout all growth phases, including the adjustment to deep stationary phase. RESULTS: We monitored both the transcriptome and the proteome in cultures of the alphaproteobacterium Rhodobacter sphaeroides, beginning with the transition to stationary phase and at different points of the stationary phase and finally during exit from stationary phase (outgrowth) following dilution with fresh medium. Correlation between the transcriptomic and proteomic changes was very low throughout the growth phases. Surprisingly, even in deep stationary phase, the abundance of many proteins continued to adjust, while the transcriptome analysis revealed fewer adjustments. This pattern was reversed during the first 90 min of outgrowth, although this depended upon the duration of the stationary phase. We provide a detailed analysis of proteomic changes based on the clustering of orthologous groups (COGs), and compare these with the transcriptome. CONCLUSIONS: The low correlation between transcriptome and proteome supports the view that post-transcriptional processes play a major role in the adaptation to growth conditions. Our data revealed that many proteins with functions in transcription, energy production and conversion and the metabolism and transport of amino acids, carbohydrates, lipids, and secondary metabolites continually increased in deep stationary phase. Based on these findings, we conclude that the bacterium responds to sudden changes in environmental conditions by a radical and rapid reprogramming of the transcriptome in the first 90 min, while the proteome changes were modest. In response to gradually deteriorating conditions, however, the transcriptome remains mostly at a steady state while the bacterium continues to adjust its proteome. Even long after the population has entered stationary phase, cells are still actively adjusting their proteomes.

Adaptation of the Alphaproteobacterium Rhodobacter sphaeroides to stationary phase.

Exhaustion of nutritional resources stimulates bacterial populations to adapt their growth behaviour. General mechanisms are known to facilitate this adaptation by sensing the environmental change and coordinating gene expression. However, the existence of such mechanisms among the Alphaproteobacteria remains unclear. This study focusses on global changes in transcript levels during growth under carbon-limiting conditions in a model Alphaproteobacterium, Rhodobacter sphaeroides, a metabolically diverse organism capable of multiple modes of growth including aerobic and anaerobic respiration, anaerobic anoxygenic photosynthesis and fermentation. We identified genes that showed changed transcript levels independently of oxygen levels during the adaptation to stationary phase. We selected a subset of these genes and subjected them to mutational analysis, including genes predicted to be involved in manganese uptake, polyhydroxybutyrate production and quorum sensing and an alternative sigma factor. Although these genes have not been previously associated with the adaptation to stationary phase, we found that all were important to varying degrees. We conclude that while R. sphaeroides appears to lack a rpoS-like master regulator of stationary phase adaptation, this adaptation is nonetheless enabled through the impact of multiple genes, each responding to environmental conditions and contributing to the adaptation to stationary phase.

Front-propagation in bacterial inter-colony communication.

Many bacterial species exchange signaling molecules to coordinate population-wide responses. For this process, known as quorum sensing, the concentration of the respective molecules is crucial. Here, we consider the interaction between spatially distributed bacterial colonies so that the spreading of the signaling molecules in space becomes important. The exponential growth of the signal-producing populations and the corresponding increase in signaling molecule production result in an exponential concentration profile that spreads with uniform speed. The theoretical predictions are supported by experiments with different strains of the soil bacterium Sinorhizobium meliloti that display fluorescence when either producing or responding to the signaling molecules.

RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti.

The ribonucleases (RNases) E and J play major roles in E. coli and Bacillus subtilis, respectively, and co-exist in Sinorhizobium meliloti. We analysed S. meliloti 2011 mutants with mini-Tn5 insertions in the corresponding genes rne and rnj and found many overlapping effects. We observed similar changes in mRNA levels, including lower mRNA levels of the motility and chemotaxis related genes flaA, flgB and cheR and higher levels of ndvA (important for glucan export). The acyl-homoserine lactone (AHL) levels were also higher during exponential growth in both RNase mutants, despite no increase in the expression of the sinI AHL synthase gene. Furthermore, several RNAs from both mutants migrated aberrantly in denaturing gels at 300 V but not under stronger denaturing conditions at 1300 V. The similarities between the two mutants could be explained by increased levels of the key methyl donor S-adenosylmethionine (SAM), since this may result in faster AHL synthesis leading to higher AHL accumulation as well as in uncontrolled methylation of macromolecules including RNA, which may strengthen RNA secondary structures. Indeed, we found that in both mutants the N6-methyladenosine content was increased almost threefold and the SAM level was increased at least sevenfold. Complementation by induced ectopic expression of the respective RNase restored the AHL and SAM levels in each of the mutants. In summary, our data show that both RNase E and RNase J are needed for SAM homeostasis in S. meliloti.

The Sinorhizobium fredii HH103 Genome: A Comparative Analysis With S. fredii Strains Differing in Their Symbiotic Behavior With Soybean.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

The minus-end actin capping protein, UNC-94/tropomodulin, regulates development of the Caenorhabditis elegans intestine.

BACKGROUND: Tropomodulins are actin-capping proteins that regulate the stability of the slow-growing, minus-ends of actin filaments. The C. elegans tropomodulin homolog, UNC-94, has sequence and functional similarity to vertebrate tropomodulins. We investigated the role of UNC-94 in C. elegans intestinal morphogenesis. RESULTS: In the embryonic C. elegans intestine, UNC-94 localizes to the terminal web, an actin- and intermediate filament-rich structure that underlies the apical membrane. Loss of UNC-94 function results in areas of flattened intestinal lumen. In worms homozygous for the strong loss-of-function allele, unc-94(tm724), the terminal web is thinner and the amount of F-actin is reduced, pointing to a role for UNC-94 in regulating the structure of the terminal web. The non-muscle myosin, NMY-1, also localizes to the terminal web, and we present evidence that increasing actomyosin contractility by depleting the myosin phosphatase regulatory subunit, mel-11, can rescue the flattened lumen phenotype of unc-94 mutants. CONCLUSIONS: The data support a model in which minus-end actin capping by UNC-94 promotes proper F-actin structure and contraction in the terminal web, yielding proper shape of the intestinal lumen. This establishes a new role for a tropomodulin in regulating lumen shape during tubulogenesis.

RNase E affects the expression of the acyl-homoserine lactone synthase gene sinI in Sinorhizobium meliloti.

Quorum sensing of Sinorhizobium meliloti relies on N-acyl-homoserine lactones (AHLs) as autoinducers. AHL production increases at high population density, and this depends on the AHL synthase SinI and two transcriptional regulators, SinR and ExpR. Our study demonstrates that ectopic expression of the gene rne, coding for RNase E, an endoribonuclease that is probably essential for growth, prevents the accumulation of AHLs at detectable levels. The ectopic rne expression led to a higher level of rne mRNA and a lower level of sinI mRNA independently of the presence of ExpR, the AHL receptor, and AHLs. In line with this, IPTG (isopropyl-ß-D-thiogalactopyranoside)-induced overexpression of rne resulted in a shorter half-life of sinI mRNA and a strong reduction of AHL accumulation. Moreover, using translational sinI-egfp fusions, we found that sinI expression is specifically decreased upon induced overexpression of rne, independently of the presence of the global posttranscriptional regulator Hfq. The 28-nucleotide 5' untranslated region (UTR) of sinI mRNA was sufficient for this effect. Random amplification of 5' cDNA ends (5'-RACE) analyses revealed a potential RNase E cleavage site at position +24 between the Shine-Dalgarno site and the translation start site. We postulate therefore that RNase E-dependent degradation of sinI mRNA from the 5' end is one of the steps mediating a high turnover of sinI mRNA, which allows the Sin quorum-sensing system to respond rapidly to changes in transcriptional control of AHL production.

Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus.

Bacteria use CRISPR Cas systems to defend against invading foreign nucleic acids, e.g., phage genomes, plasmids or mobile genetic elements. Some CRISPR Cas systems were reported to have physiological importance under a variety of abiotic stress conditions. We used physiological tests under different stress conditions and RNA-seq analyses to address the possible function of the RNA-targeting class 2 type VI CRISPR Cas system of the facultative phototrophic α-proteobacterium Rhodobacter capsulatus. Expression of the system was low under exponential non-stress conditions and high during oxidative stress, membrane stress and in stationary phase. Induction of the CRISPR Cas system in presence of a target protospacer RNA resulted in a growth arrest of R. capsulatus. RNA-seq revealed a strong alteration of the R. capsulatus transcriptome when cas13a was induced in presence of a target protospacer. RNA 5' end mapping indicated that the CRISPR Cas-dependent transcriptome remodeling is accompanied by fragmentation of cellular RNAs, e.g., for mRNAs originating from a genomic locus which encodes multiple ribosomal proteins and the RNA polymerase subunits RpoA, RpoB and RpoC. The data suggest a function of this CRISPR Cas system in regulated growth arrest, which may prevent the spread of phages within the population.

Temporal expression program of quorum sensing-based transcription regulation in Sinorhizobium meliloti.

The Sin quorum sensing (QS) system of S. meliloti activates exopolysaccharide and represses flagellum production. The system consists of an N-acyl-homoserine lactone (AHL) synthase, SinI, and at least two LuxR-type regulators, SinR and ExpR. SinR appears to be independent of AHLs for its control of sinI expression, while ExpR is almost completely dependent upon AHLs. In this study, we confirmed 7 previously detected ExpR-DNA binding sites and used the consensus sequence to identify another 26 sites, some of which regulate genes previously not known to be members of the ExpR/AHL regulon. The activities of promoters dependent upon ExpR/AHL were titrated against AHL levels, with varied outcomes in AHL sensitivity. The data suggest a type of temporal expression program whereby the activity of each promoter is subject to a specific range of AHL concentrations. For example, genes responsible for exopolysaccharide production are activated at lower concentrations of AHLs than those required for the repression of genes controlling flagellum production. Several features of ExpR-regulated promoters appear to determine their response to AHLs. The location of the ExpR-binding site with respect to the relevant transcription start within each promoter region determines whether ExpR/AHL activates or represses promoter activity. Furthermore, the strength of the response is dependent upon the concentration of AHLs. We propose that this differential sensitivity to AHLs provides a bacterial colony with a transcription control program that is dynamic and precise.

Quorum sensing influences growth and photosynthetic membrane production in high-cell-density cultivations of Rhodospirillum rubrum.

BACKGROUND: The facultative anoxygenic photosynthetic bacterium Rhodospirillum rubrum exhibits versatile metabolic activity allowing the adaptation to rapidly changing growth conditions in its natural habitat, the microaerobic and anoxic zones of stagnant waters. The microaerobic growth mode is of special interest as it allows the high-level expression of photosynthetic membranes when grown on succinate and fructose in the dark, which could significantly simplify the industrial production of compounds associated with PM formation. However, recently we showed that PM synthesis is no longer inducible when R. rubrum cultures are grown to high cell densities under aerobic conditions. In addition a reduction of the growth rate and the continued accumulation of precursor molecules for bacteriochlorophyll synthesis were observed under high cell densities conditions. RESULTS: In the present work, we demonstrate that the cell density-dependent effects are reversible if the culture supernatant is replaced by fresh medium. We identified six N-acylhomoserine lactones and show that four of them are produced in varying amounts according to the growth phase and the applied growth conditions. Further, we demonstrate that N-acylhomoserine lactones and tetrapyrrole compounds released into the growth medium affect the growth rate and PM expression in high cell density cultures. CONCLUSIONS: In summary, we provide evidence that R. rubrum possesses a Lux-type quorum sensing system which influences the biosynthesis of PM and the growth rate and is thus likely to be involved in the phenotypes of high cell density cultures and the rapid adaptation to changing environmental conditions.

Genetic Engineering of Agrobacterium Increases Curdlan Production through Increased Expression of the crdASC Genes.

Curdlan is a water-insoluble polymer that has structure and gelling properties that are useful in a wide variety of applications such as in medicine, cosmetics, packaging and the food and building industries. The capacity to produce curdlan has been detected in certain soil-dwelling bacteria of various phyla, although the role of curdlan in their survival remains unclear. One of the major limitations of the extensive use of curdlan in industry is the high cost of production during fermentation, partly because production involves specific nutritional requirements such as nitrogen limitation. Engineering of the industrially relevant curdlan-producing strain Agrobacterium sp. ATTC31749 is a promising approach that could decrease the cost of production. Here, during investigations on curdlan production, it was found that curdlan was deposited as a capsule. Curiously, only a part of the bacterial population produced a curdlan capsule. This heterogeneous distribution appeared to be due to the activity of Pcrd, the native promoter responsible for the expression of the crdASC biosynthetic gene cluster. To improve curdlan production, Pcrd was replaced by a promoter (PphaP) from another Alphaproteobacterium, Rhodobacter sphaeroides. Compared to Pcrd, PphaP was stronger and only mildly affected by nitrogen levels. Consequently, PphaP dramatically boosted crdASC gene expression and curdlan production. Importantly, the genetic modification overrode the strict nitrogen depletion regulation that presents a hindrance for maximal curdlan production and from nitrogen rich, complex media, demonstrating excellent commercial potential for achieving high yields using cheap substrates under relaxed fermentation conditions.

Design-Build-Test of Synthetic Promoters for Inducible Gene Regulation in Alphaproteobacteria.

Inducible gene expression is useful for biotechnological applications and for studying gene regulation and function in bacteria. Many inducible systems that perform in model organisms such as the Gammaproteobacterium Escherichia coli do not perform well in other bacteria that are of biotechnological interest. Typical problems include weak or leaky expression. Here, we describe an invention named ACIT (Alphaproteobacteria chromosomally integrating transcription-control cassette) that is carried on a suicide plasmid to enable insertion into the chromosome of the host. ACIT consists of multiple DNA fragments specifically arranged in a cassette that allows tight transcription control over any gene or gene cluster of interest following homologous recombination. At the heart of the invention is the ability to modify or exchange parts, e.g., promoters, to suit particular bacteria and growth conditions, allowing for customized gene expression control. Furthermore, ACIT provides a basis for a design-build-test approach for controlling gene expression in less studied bacteria. We describe examples of its control over pigment and exopolysaccharide production, growth, cell form, and social behavior in various Alphaproteobacteria.

Real-Time Monitoring of Grazing Cattle Using LORA-WAN Sensors to Improve Precision in Detecting Animal Welfare Implications via Daily Distance Walked Metrics.

Animal welfare monitoring relies on sensor accuracy for detecting changes in animal well-being. We compared the distance calculations based on global positioning system (GPS) data alone or combined with motion data from triaxial accelerometers. The assessment involved static trackers placed outdoors or indoors vs. trackers mounted on cows grazing on pasture. Trackers communicated motion data at 1 min intervals and GPS positions at 15 min intervals for seven days. Daily distance walked was determined using the following: (1) raw GPS data (RawDist), (2) data with erroneous GPS locations removed (CorrectedDist), or (3) data with erroneous GPS locations removed, combined with the exclusion of GPS data associated with no motion reading (CorrectedDist_Act). Distances were analyzed via one-way ANOVA to compare the effects of tracker placement (Indoor, Outdoor, or Animal). No difference was detected between the tracker placement for RawDist. The computation of CorrectedDist differed between the tracker placements. However, due to the random error of GPS measurements, CorrectedDist for Indoor static trackers differed from zero. The walking distance calculated by CorrectedDist_Act differed between the tracker placements, with distances for static trackers not differing from zero. The fusion of GPS and accelerometer data better detected animal welfare implications related to immobility in grazing cattle.