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Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N 4-acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N 4-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (k cat /K m up to 2.8 × 10 6 M −1 s −1). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N 4-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N 4-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases. Human activating signal cointegrator 1 (ASC-1), otherwise known as the thyroid hormone receptor interactor protein 4 (ASC-1/TRIP4), interacts with a wide range of unrelated transcription factors to facilitate nuclear receptors-mediated transcription. It also plays a pivotal role in the transactivation of serum response factor (SRF), activating protein 1 (AP-1), and nuclear factor κB (NF-κB) 1,2. In 2006 it was shown that the C-terminal domain of ASC-1 defines a large ASC-1 homology (ASCH) domain superfamily 2. To date, the ASCH-containing proteins have been reported for a wide range of organisms representing all three kingdoms of life, and have also been found in viruses 3. Although it has long been suggested that this domain of ~110 residues may be responsible for RNA binding during transcription coactivation, RNA processing, and regulation of translation, the vast majority of ASCH proteins are small (~140 residues) hypothetical proteins, which at present have no function assigned to them 2. One such protein is the product of the yqfB gene in Escherichia coli. Herein we demonstrate that the 103-amino acid YqfB is a unique monomeric amidohydrolase responsible for the catabolism of the modified nucleoside, N 4-acetylcytidine (ac4C), in E. coli. More than 160 of differently modified nucleotides play a crucial role in various biological processes 1,2,4,5. The biosynthetic pathways of many modified bases, nucleosides and nucleotides are well understood 5,6 , but the catabolism or salvage of those compounds are scarcely studied. Just like the ASCH proteins, the modified nucleoside ac4C is found in organisms within all three domains of life 4-9. It prevents misreading of AUA isoleucine codons during protein synthes...

A cryptic plasmid from Arthrobacter rhombi PRH1, designated as pPRH, was sequenced and characterized. It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55-61% and 60-69% homology, respectively, with the RepA and RepB proteins of reported rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed. The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase.

SummaryA high‐throughput method (≥ 106 of clones can be analysed on a single agar plate) for the selection of ester‐hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2′,3′,5′‐O‐tri‐acetyluridine and 2′,3′,5′‐O‐tri‐hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.

Achromobacter spp. are ubiquitous in nature and are increasingly being recognized as emerging nosocomial pathogens. Nevertheless, to date, only 30 complete genome sequences of Achromobacter phages are available in GenBank, and nearly all of those phages were isolated on Achromobacter xylosoxidans. Here, we report the isolation and characterization of bacteriophage vB_AchrS_AchV4. To the best of our knowledge, vB_AchrS_AchV4 is the first virus isolated from Achromobacter spanius. Both vB_AchrS_AchV4 and its host, Achromobacter spanius RL_4, were isolated in Lithuania. VB_AchrS_AchV4 is a siphovirus, since it has an isometric head (64 ± 3.2 nm in diameter) and a non-contractile flexible tail (232 ± 5.4). The genome of vB_AchrS_AchV4 is a linear dsDNA molecule of 59,489 bp with a G+C content of 62.8%. It contains no tRNA genes, yet it includes 82 protein-coding genes, of which 27 have no homologues in phages. Using bioinformatics approaches, 36 vB_AchrS_AchV4 genes were given a putative function. A further four were annotated based on the results of LC–MS/MS. Comparative analyses revealed that vB_AchrS_AchV4 is a singleton siphovirus with no close relatives among known tailed phages. In summary, this work not only describes a novel and unique phage, but also advances our knowledge of genetic diversity and evolution of Achromobacter bacteriophages.

It has been recently shown that bioluminescence imaging can be usefully applied to provide new insights into bacterial self-organization. In this work we employ bioluminescence imaging to record images of nutrient rich liquid cultures of the lux-gene reporter Escherichia coli in microtiter plate wells. The images show that patterns of inhomogenous bioluminescence form along the three-phase contact lines. The paper analyzes the dependencies of the average number of luminous aggregates (clouds) on various environmental factors. In particular, our results show that optimal (neutral) pH and high aeration rates determine the highest mean number of clouds, and that spatiotemporal patterns do not form in the pH buffered suspensions. In addition, a sigmoidal (switch-like) dependence of the number of aggregates on the rate of aeration was observed. The obtained bioluminescence imaging data was interpreted by employing the Keller-Segel-Fisher (KSF) model of chemotaxis and logistic growth, adapted to systems of metabolically flexible (two-state) bacteria. The modified KSF model successfully simulated the observed switch-like responses. The results of the microtiter plate tests and their simulations indicate that the segregation of bacteria with different activities proceeds in the three-phase contact line region.

In this study, the development of a rapid, high-throughput method for the selection of amide-hydrolysing enzymes from the metagenome is described. This method is based on uridine auxotrophic Escherichia coli strain DH10B ∆pyrFEC and the use of N4-benzoyl-2’-deoxycytidine as a sole source of uridine in the minimal microbial M9 medium. The approach described here permits the selection of unique biocatalysts, e.g., a novel amidohydrolase from the activating signal cointegrator homology (ASCH) family and a polyethylene terephthalate hydrolase (PETase)-related enzyme.