Abolishment of morphology‑based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021−March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratifed by vote in 2022. Signifcant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved

Isolation and characterization of lytic proteus virus 309

Proteus mirabilis is frequently associated with complicated urinary tract infections (UTIs) and is the main cause of catheter-associated urinary tract infections (CAUTIs). Treatment of such infections is complicated and challenging due to the biofilm forming abilities of P. mirabilis. If neglected or mistreated, infections may lead to life-threating conditions such as cystitis, pyelonephritis, kidney failure, and bacteremia that may progress to urosepsis. Treatment with antibiotics, especially in cases of recurring and persistent infections, leads to the development of resistant strains. Recent insights into phage therapy and using phages to coat catheters have been evaluated with many studies showing promising results

Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

: There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood.: Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identifed 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-Larabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications

Metaviromics of Namib desert salt pans : a novel lineage of haloarchaeal salterproviruses and a rich source of ssDNA viruses

Viral communities of two different salt pans located in the Namib Desert, Hosabes and Eisfeld, were investigated using a combination of multiple displacement amplification of metaviromic DNA and deep sequencing, and provided comprehensive sequence data on both ssDNA and dsDNA viral community structures. Read and contig annotations through online pipelines showed that the salt pans harbored largely unknown viral communities. Through network analysis, we were able to assign a large portion of the unknown reads to a diverse group of ssDNA viruses. Contigs belonging to the subfamily Gokushovirinae were common in both environmental datasets. Analysis of haloarchaeal virus contigs revealed the presence of three contigs distantly related with His1, indicating a possible new lineage of salterproviruses in the Hosabes playa. Based on viral richness and read mapping analyses, the salt pan metaviromes were novel and most closely related to each other while showing a low degree of overlap with other environmental viromes.National Research Foundation (NRF) of South Africa, Claude Leon Foundation and the Vice Chancellor’s Postdoctoral Fellowship program of the University of Pretoria.http://www.mdpi.com/journal/viruseshb201

Identification and characterization of a novel Geobacillus thermoglucosidasius bacteriophage, GVE3

The study of extremophilicphages may reveal new phage families as well as different mechanisms of infection, propagation and lysis to those found in phages from temperate environments. We describe a novel siphovirus, GVE3, that infects the thermophileGeobacillusthermoglucosidasius. The genome size is 141298 bp(G+C 29.6%) making it the largest Geobacillusspp infecting phage known.GVE3 appears to be most closely related to the recently described Bacillus anthracis phage vB_BanS_Tsamsa, rather thanGeobacillus infecting phages described thus far.Tetranucleotide usage deviation analysis supports this relationship, showing that the GVE3 genome sequence correlates best with B. anthracis and Bacillus cereus genome sequences, rather than Geobacillusspp genome sequences.National Research Foundation (NRF) of South Africahttp://link.springer.com/journal/7052016-09-30hb201

Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

Background: The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. Results Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0–6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s− 1 and 219 s− 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside. Conclusion All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

This study reports the expression, purification and kinetic characterization of a PDC from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120μM at pH 5), high catalytic efficiency (4.75 x 105 M-1s-1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55°C (the highest yet reported for a bacterial PDC). Due to good in vitro thermostablity (approximately 40% enzyme activity retained after 30 minutes at 65°C) this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius. Initial studies using a variety of methods failed to detect activity at any growth temperature. However, the application of codon harmonization (i.e., mimicry of the heterogeneous host’s transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45°C (as determined by enzyme activity, Western blot, mRNA detection and ethanol productivity). Here we describe the successful expression of PDC in a true thermophile. Yields as high as 0.35 g/g ±0.04 ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights that the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription / translation coupling.National Research Foundation South Africahttp://link.springer.com/journal/253hb201

Metagenomic analysis of the viral community in Namib desert hypoliths

Hypolithic microbial communities are specialized desert communities inhabiting the underside of translucent rocks where they are sheltered from harsh environmental conditions. Here, we present the first study of the viral fraction of these communities isolated from the hyperarid Namib Desert (coastal South Western Africa). Using next-generation sequencing of the isolated viral fraction, the diversity and taxonomic composition of hypolith communities was mapped and a functional assessment of the sequences determined. Phylotypic analysis showed that bacteriophages belonging to the order Caudovirales with the family Siphoviridae were most prevalent. A major fraction of phage types was linked by database homologies to Bacillus or Geobacillus sp. as a host. Phylogenetic analyses of terL and phoH marker genes indicated that many of the sequences were novel and distinct from known isolates and environments, an observation supported by the class distribution of identified ribonucleotide reductases. The composition of the viral hypolith fraction was not completely consistent with Namib hypolith phylotypic surveys, in which the cyanobacterial genus Chroococcidiopsis was found to be dominant. This could be attributed to lacking sequence information about hypolith viruses/bacteria in public databases or the hypothesis that hypolithic communities actively recruit viruses from the surrounding open soil in which Bacillaceae-infecting phages are more commonly found.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1462-2920hb201

Taxonomy of prokaryotic viruses : 2017 update from the ICTV Bacterial and Archaeal Viruses Subcommittee

Peer reviewe

Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus

BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high catalytic efficiencies, pHopt of 5.5 and Topt at 45 degrees C. The enzyme is not thermostable (T of 18 minutes at 60 degrees C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 A for C? when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.IS