Novel Cluster AZ Arthrobacter phages Powerpuff, Lego, and YesChef exhibit close functional relationships with Microbacterium phages

Bacteriophages exhibit a vast spectrum of relatedness and there is increasing evidence of close genomic relationships independent of host genus. The variability in phage similarity at the nucleotide, amino acid, and gene content levels confounds attempts at quantifying phage relatedness, especially as more novel phages are isolated. This study describes three highly similar novel Arthrobacter globiformis phages–Powerpuff, Lego, and YesChef–which were assigned to Cluster AZ using a nucleotide-based clustering parameter. Phages in Cluster AZ, Microbacterium Cluster EH, and the former Microbacterium singleton Zeta1847 exhibited low nucleotide similarity. However, their gene content similarity was in excess of the recently adopted Microbacterium clustering parameter, which ultimately resulted in the reassignment of Zeta1847 to Cluster EH. This finding further highlights the importance of using multiple metrics to capture phage relatedness. Additionally, Clusters AZ and EH phages encode a shared integrase indicative of a lysogenic life cycle. In the first experimental verification of a Cluster AZ phage’s life cycle, we show that phage Powerpuff is a true temperate phage. It forms stable lysogens that exhibit immunity to superinfection by related phages, despite lacking identifiable repressors typically required for lysogenic maintenance and superinfection immunity. The ability of phage Powerpuff to undergo and maintain lysogeny suggests that other closely related phages may be temperate as well. Our findings provide additional evidence of significant shared phage genomic content spanning multiple actinobacterial host genera and demonstrate the continued need for verification and characterization of life cycles in newly isolated phages.

Closed Genome Sequence of Yavru, a Novel Arthrobacter globiformis Phage

We characterized the complete genome sequence of actinobacteriophage Yavru ( Siphoviridae ), a cluster FE bacteriophage infecting Arthrobacter globiformis NRRL B-2979; it was 89.5% identical to cluster FE phage Whytu, with a capsid width of 50 nm and a tail length of 90 nm. The genome was 15,193 bp in length, with 23 predicted protein-coding genes.

Genome Sequence of Bacteriophage Adumb2043, Isolated from Arthrobacter globiformis in Southern Colorado

We report the discovery and genome sequence of phage Adumb2043, a siphovirus infecting Arthrobacter globiformis , B2979-SEA. Adumb2043 was isolated from soil collected in Colorado Springs, Colorado. The genome has a length of 43,100 bp and contains 68 predicted protein-coding genes and no tRNA genes. Adumb2043 is related to actinobacteriophages Elezi and London.

Thermostable and Long-Circulating Albumin-Conjugated Arthrobacter globiformis Urate Oxidase

Urate oxidase derived from Aspergillus flavus has been investigated as a treatment for tumor lysis syndrome, hyperuricemia, and gout. However, its long-term use is limited owing to potential immunogenicity, low thermostability, and short circulation time in vivo. Recently, urate oxidase isolated from Arthrobacter globiformis (AgUox) has been reported to be thermostable and less immunogenic than the Aspergillus-derived urate oxidase. Conjugation of human serum albumin (HSA) to therapeutic proteins has become a promising strategy to prolong circulation time in vivo. To develop a thermostable and long-circulating urate oxidase, we investigated the site-specific conjugation of HSA to AgUox based on site-specific incorporation of a clickable non-natural amino acid (frTet) and an inverse electron demand Diels–Alder reaction. We selected 14 sites for frTet incorporation using the ROSETTA design, a computational stability prediction program, among which AgUox containing frTet at position 196 (Ag12) exhibited enzymatic activity and thermostability comparable to those of wild-type AgUox. Furthermore, Ag12 exhibited a high HSA conjugation yield without compromising the enzymatic activity, generating well-defined HSA-conjugated AgUox (Ag12-HSA). In mice, the serum half-life of Ag12-HSA was approximately 29 h, which was roughly 17-fold longer than that of wild-type AgUox. Altogether, this novel formulated AgUox may hold enhanced therapeutic efficacy for several diseases.

DEER and RIDME Measurements of the Nitroxide-Spin Labelled Copper-Bound Amine Oxidase Homodimer from Arthrobacter Globiformis

In the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin–spin distances at nanometre-scale by measuring dipole–dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO.

Novel Cluster AZ Arthrobacter phages Powerpuff, Lego, and YesChef exhibit close functional relationships with Microbacterium phages

Bacteriophages exhibit a vast spectrum of relatedness and there is increasing evidence of close genomic relationships independent of host genus. The variability in phage similarity at the nucleotide, amino acid, and gene content levels confounds attempts at quantifying phage relatedness, especially as more novel phages are isolated. This study describes three highly similar novel Arthrobacter globiformis phages–Powerpuff, Lego, and YesChef–which were assigned to Cluster AZ using a nucleotide-based clustering parameter. Phages in Cluster AZ and Microbacterium Cluster EH, as well as the former Microbacterium singleton Zeta1847, exhibited low nucleotide similarity but gene content similarity in excess of the recently adopted Microbacterium clustering parameter, which resulted in the reassignment of Zeta1847 to Cluster EH. Additionally, while Clusters AZ and EH phages lack identifiable repressors or partitioning systems typically required for lysogeny, they encode a shared integrase indicative of a lysogenic life cycle. In the first experimental verification of a Cluster AZ phage’s life cycle, we show that phage Powerpuff is a true temperate phage and forms stable lysogens. Moreover, we provide evidence that Clusters AZ and EH phages exhibit similar genome architectures in addition to their shared integrases, suggesting that these phages may all be temperate and undergo an unknown lysogeny mechanism. Our findings further highlight the importance of using multiple metrics to capture phage relatedness and provide additional evidence of significant shared phage genomic content spanning multiple actinobacterial host genera.

Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.

Characterization of novel Actinobacteriophage Giantsbane reveals potential genomic recombination hotspot and unexpected intra-cluster similarities

ABSTRACTBacteriophages that infect Arthrobacter, a genus of bacteria which play key ecological roles in soil, warrant further study. A novel Actinobacteriophage, Giantsbane, was isolated on Arthrobacter globiformis and purified. Particle stability was tested at various temperatures and salinity concentrations by observing titer differences; unpaired Student’s t-tests revealed these differences to be insignificant. Transmission electron microscopy and Illumina whole-genome sequencing revealed that Giantsbane’s morphology and genome length, respectively, were characteristic of Siphoviridae phages. 94 putative open reading frames were determined using Glimmer, GeneMark, and manual review; none were associated with lysogeny. Giantsbane was placed into phage cluster AU, and SplitsTree and batch ANI analyses revealed similarities with other AU phages. The annotated genome was further analyzed using Phamerator and MEME-Suite, which identified repeated motifs present in several other phages. These findings help further our understanding of the physiological and genomic aspects of phage biology.