Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage.

BACKGROUND: Whole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species. RESULTS: Here we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc2155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered. The genomic DNA from pure populations of the BGlluviae phage and the clear plaque mutant were sequenced. A single substitution, at amino acid 54 (I to T), in the immunity repressor protein resulted in a clear plaque phenotype. CONCLUSIONS: This substitution is predicted to be located at the subunit interaction interface of the repressor protein, and thus prevents the establishment of lysogeny.

Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes.

UNLABELLED: Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

The SKP1-Cul1-F-box and leucine-rich repeat protein 4 (SCF-FbxL4) ubiquitin ligase regulates lysine demethylase 4A (KDM4A)/Jumonji domain-containing 2A (JMJD2A) protein.

Chromatin-modifying enzymes play a fundamental role in regulating chromatin structure so that DNA replication is spatially and temporally coordinated. For example, the lysine demethylase 4A/Jumonji domain-containing 2A (KDM4A/JMJD2A) is tightly regulated during the cell cycle. Overexpression of JMJD2A leads to altered replication timing and faster S phase progression. In this study, we demonstrate that degradation of JMJD2A is regulated by the proteasome. JMJD2A turnover is coordinated through the SKP1-Cul1-F-box ubiquitin ligase complex that contains cullin 1 and the F-box and leucine-rich repeat protein 4 (FbxL4). This complex interacted with JMJD2A. Ubiquitin overexpression restored turnover and blocked the JMJD2A-dependent faster S phase progression in a cullin 1-dependent manner. Furthermore, increased ubiquitin levels decreased JMJD2A occupancy and BrdU incorporation at target sites. This study highlights a finely tuned mechanism for regulating histone demethylase levels and emphasizes the need to tightly regulate chromatin modifiers so that the cell cycle occurs properly.

Genomic diversity of bacteriophages infecting Microbacterium spp.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

At least four distinct circadian regulatory mechanisms are required for all phases of rhythms in mRNA amount.

Since the advent of techniques to investigate gene expression on a large scale, numerous circadian rhythms in mRNA amount have been reported. These rhythms generally differ in amplitude and phase. The authors investigated how far a parameter not regulated by the circadian clock can influence the phase of a rhythm in RNA amount arising from a circadian rhythm of transcription. Using a discrete-time approach, they modeled a sinusoidal rhythm in transcription with various constant exponential RNA decay rates. They found that the slower the RNA degradation, the later the phase of the RNA amount rhythm compared with the phase of the transcriptional rhythm. However, they also found that the phase of the RNA amount rhythm is limited to a timeframe spanning the first quarter of the period following the phase of the transcriptional rhythm. This finding is independent of the amplitude and vertical shift of the transcriptional rhythm or even of the way constant RNA degradation is modeled. The authors confirmed their results with a continuous-time model, which allowed them to derive a simple formula relating the phase of the RNA amount rhythm solely to the phase and period of its sinusoidal transcriptional rhythm and its constant RNA half-life. This simple formula even holds true for the best sinusoidal approximations of a nonsinusoidal rhythm of transcription and RNA amount. When expanding the model to include additional events with constant exponential kinetics, such as RNA processing, they found that each event expands the phase limit by another quarter of the period when it occurs in sequence but not when it occurs as a competing process. However, the limit expansion comes at the price of minuscule amplitudes. When using a discrete-time approach to model constant rates of transcription with a sinusoidal RNA half-life, the authors found that the phase of the RNA amount rhythm is unaffected by changes in the constant rate of transcription. In summary, their data show that at least 4 distinct circadian regulatory mechanisms are required to allow for all phases in rhythms of RNA amount, one for each quarter of the period.

Complete Genome Sequences of 44 Arthrobacter Phages.

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae, Myoviridae, and Podoviridae) are represented.

Comprehensive study of excess phosphate response reveals ethylene mediated signaling that negatively regulates plant growth and development.

Excess Phosphorus (P) in agriculture is causing serious environmental problems like eutrophication of lakes and rivers. Unlike the enormous information available for phosphate starvation response (P0), very few information is available for the effect of excess phosphate Pi on plants. Characterization of Excess Phosphate Response (EPiR) is essential for designing strategies to increase phosphate accumulation and tolerance. We show a significant modulation in the root developmental plasticity under the increasing supply of excess Pi. An excess supply of 20 mM Pi (P20) produces a shallow root system architecture (RSA), reduces primary root growth, root apical meristem size, and meristematic activity in Arabidopsis. The inhibition of primary root growth and development is indeterminate in nature and caused by the decrease in number of meristematic cortical cells due to EPiR. Significant changes occurred in metal nutrients level due to excess Pi supply. A comparative microarray investigation of the EPiR response reveals a modulation in ethylene biosynthesis and signaling, metal ions deficiency response, and root development related genes. We used ethylene-insensitive or sensitive mutants to provide more evidence for ethylene-mediated signaling. A new role of EPiR in regulating the developmental responses of plants mediated by ethylene has been demonstrated.

Genome Sequences of Subcluster K5 Mycobacteriophages AlleyCat, Edugator, and Guillsminger.

Bacteriophages AlleyCat, Edugator, and Guillsminger were isolated on Mycobacterium smegmatis mc2155 from enriched soil samples. All are members of mycobacteriophage subcluster K5, with genomes of 62,112 to 63,344 bp. Each genome contains 92 to 99 predicted protein-coding genes and one tRNA. Guillsminger is the first mycobacteriophage to carry an IS1380 family transposon.

Prophage-mediated defence against viral attack and viral counter-defence.

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defence promotes phage co-evolution.

Chlamydomonas reinhardtii strain CC-124 is highly sensitive to blue light in addition to green and red light in resetting its circadian clock, with the blue-light photoreceptor plant cryptochrome likely acting as negative modulator.

The unicellular green alga Chlamydomonas reinhardtii has long served as model organism for studies on the circadian clock. This clock is present in all eukaryotes and some prokaryotes allowing them to anticipate and take advantage of the daily oscillations in the environment. Although much is known about the circadian clock in C. reinhardtii, the photoreceptors mediating entrainment of the clock to the daily changes of light remain obscure. Based on its circadian rhythm of phototaxis as a reporter of the clock's phase, we show here that C. reinhardtii strain CC-124 is highly sensitive to blue light of 440 nm when resetting its circadian clock upon light pulses. Thus, CC-124 differs in this respect from what was previously reported for a cell wall-deficient strain. An action spectrum analysis revealed that CC-124 also responds with high sensitivity to green (540 nm), red (640-660 nm), and possibly UV-A (≤400 nm) light, and therefore shows similarities as well to what has been reported for the cell wall-deficient strain. We also investigated two RNA interference strains with reductions in the level of the blue light photoreceptor plant cryptochrome (CPH1). One of them, the strain with the greater reduction, surprisingly showed an increased sensitivity in clock resetting upon blue light pulses of 440 nm. This increase in sensitivity reverted to wild-type levels when the RNA interference strain reverted to wild-type protein levels. It suggests that plant cryptochrome in C. reinhardtii could function as negative rather than positive modulator of circadian clock resetting.

Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas.

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii.