Isolation of a Dissimilatory Iodate-Reducing Aromatoleum sp. From a Freshwater Creek in the San Francisco Bay Area

Recent reports of dissimilatory iodate-reducing microorganisms (DIRM) have arisen from studies of bacteria in marine environments. These studies described the physiology and distribution of DIRM while also demonstrating their presence in iodine-rich marine environments. We posited that despite lower iodine concentrations, terrestrial and freshwater ecosystems should also harbor DIRM. We established numerous enrichments from coastal and freshwater environments that actively remove amended iodate. We describe the physiology and genome of a new DIRM isolate, Aromatoleum toluclasticum sp. TC-10, emerging from a freshwater creek microcosm. Like other DIRM, A. toluclasticum sp. TC-10 couples acetate oxidation to iodate reduction with a concomitant increase in the OD600. Our results indicate that A. toluclasticum sp. TC-10 performs dissimilatory iodate reduction (DIR) using the recently described iodate reductase (Idr). We provide further evidence of horizontal gene transfer of the idr genes by demonstrating the lack of Idr in the closely related (99.93% 16S rDNA sequence identity) A. toluclasticum sp. MF63 and describe the heterogeneity of the accessory proteins associated with the iodate reduction island (IRI). These observations provide additional evidence that DIR is a horizontally acquired metabolism with broad environmental distribution beyond exclusively marine environments.

Four Novel Mycoviruses from the Hypovirulent Botrytis cinerea SZ-2-3y Isolate from Paris polyphylla: Molecular Characterisation and Mitoviral Sequence Transboundary Entry into Plants

A hypovirulent SZ-2-3y strain isolated from diseased Paris polyphylla was identified as Botrytis cinerea. Interestingly, SZ-2-3y was coinfected with a mitovirus, two botouliviruses, and a 3074 nt fusarivirus, designated Botrytis cinerea fusarivirus 8 (BcFV8); it shares an 87.2% sequence identity with the previously identified Botrytis cinerea fusarivirus 6 (BcFV6). The full-length 2945 nt genome sequence of the mitovirus, termed Botrytis cinerea mitovirus 10 (BcMV10), shares a 54% sequence identity with Fusarium boothii mitovirus 1 (FbMV1), and clusters with fungus mitoviruses, plant mitoviruses and plant mitochondria; hence BcMV10 is a new Mitoviridae member. The full-length 2759 nt and 2812 nt genome sequences of the other two botouliviruses, named Botrytis cinerea botoulivirus 18 and 19 (BcBoV18 and 19), share a 40% amino acid sequence identity with RNA-dependent RNA polymerase protein (RdRp), and these are new members of the Botoulivirus genus of Botourmiaviridae. Horizontal transmission analysis showed that BcBoV18, BcBoV19 and BcFV8 are not related to hypovirulence, suggesting that BcMV10 may induce hypovirulence. Intriguingly, a partial BcMV10 sequence was detected in cucumber plants inoculated with SZ-2-3y mycelium or pXT1/BcMV10 agrobacterium. In conclusion, we identified a hypovirulent SZ-2-3y fungal strain from P. polyphylla, coinfected with four novel mycoviruses that could serve as potential biocontrol agents. Our findings provide evidence of cross-kingdom mycoviral sequence transmission.

New Insights into the Genome Organization of Yeast Double-Stranded RNA LBC Viruses

The yeasts Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) may show a killer phenotype that is encoded in dsRNA M viruses (V-M), which require the helper activity of another dsRNA virus (V-LA or V-LBC) for replication. Recently, two TdV-LBCbarr genomes, which share sequence identity with ScV-LBC counterparts, were characterized by high-throughput sequencing (HTS). They also share some similar characteristics with Sc-LA viruses. This may explain why TdV-LBCbarr has helper capability to maintain M viruses, whereas ScV-LBC does not. We here analyze two stretches with low sequence identity (LIS I and LIS II) that were found in TdV-LBCbarr Gag-Pol proteins when comparing with the homologous regions of ScV-LBC. These stretches may result from successive nucleotide insertions or deletions (indels) that allow compensatory frameshift events required to maintain specific functions of the RNA-polymerase, while modifying other functions such as the ability to bind V-M (+)RNA for packaging. The presence of an additional frameshifting site in LIS I may ensure the synthesis of a certain amount of RNA-polymerase until the new compensatory indel appears. Additional 5′- and 3′-extra sequences were found beyond V-LBC canonical genomes. Most extra sequences showed high identity to some stretches of the canonical genomes and can form stem-loop structures. Further, the 3′-extra sequence of two ScV-LBC genomes contains rRNA stretches. The origin and possible functions of these extra sequences are here discussed.

Using syncmers improves long-read mapping

As sequencing datasets keep growing larger, time and memory efficiency of read mapping are becoming more critical. Many clever algorithms and data structures were used to develop mapping tools for next generation sequencing, and in the last few years also for third generation long reads. A key idea in mapping algorithms is to sketch sequences with their minimizers. Recently, syncmers were introduced as an alternative sketching method that is more robust to mutations and sequencing errors. Here we introduce parameterized syncmer schemes, and provide a theoretical analysis for multi-parameter schemes. By combining these schemes with downsampling or minimizers we can achieve any desired compression and window guarantee. We introduced syncmer schemes into the popular minimap2 and Winnowmap2 mappers. In tests on simulated and real long read data from a variety of genomes, the syncmer-based algorithms reduced unmapped reads by 20-60% at high compression while using less memory. The advantage of syncmer-based mapping was even more pronounced at lower sequence identity. At sequence identity of 65-75% and medium compression, syncmer mappers had 50-60% fewer unmapped reads, and ∼ 10% fewer of the reads that did map were incorrectly mapped. We conclude that syncmer schemes improve mapping under higher error and mutation rates. This situation happens, for example, when the high error rate of long reads is compounded by a high mutation rate in a cancer tumor, or due to differences between strains of viruses or bacteria.

RAA enzyme is a new family of class A extended-spectrum β-lactamase from the Riemerella anatipestifer RCAD0122 strain

Whole genome sequencing of Riemerella anatipestifer isolate RCAD0122 revealed a chromosomally-located β-lactamases gene, bla RAA-1 , which encoded a novel class A extended-spectrum β-lactamases (ESBL), RAA-1. The RAA-1 shared ≤ 65% amino acid sequence identity with other characterized β-lactamases. The kinetic assay of native purified RAA-1 revealed ESBL-like hydrolysis activity. Furthermore, bla RAA-1 could be transferred to a homologous strain by natural transformation. However, the epidemiological study showed that the bla RAA-1 gene is not prevalent currently.

Crystal structure of a putative short-chain dehydrogenase/reductase from Paraburkholderia xenovorans

Paraburkholderia xenovorans degrades organic wastes, including polychlorinated biphenyls. The atomic structure of a putative dehydrogenase/reductase (SDR) from P. xenovorans (PxSDR) was determined in space group P21 at a resolution of 1.45 Å. PxSDR shares less than 37% sequence identity with any known structure and assembles as a prototypical SDR tetramer. As expected, there is some conformational flexibility and difference in the substrate-binding cavity, which explains the substrate specificity. Uniquely, the cofactor-binding cavity of PxSDR is not well conserved and differs from those of other SDRs. PxSDR has an additional seven amino acids that form an additional unique loop within the cofactor-binding cavity. Further studies are required to determine how these differences affect the enzymatic functions of the SDR.

Crystal structures of FolM alternative dihydrofolate reductase 1 from Brucella suis and Brucella canis

Members of the bacterial genus Brucella cause brucellosis, a zoonotic disease that affects both livestock and wildlife. Brucella are category B infectious agents that can be aerosolized for biological warfare. As part of the structural genomics studies at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), FolM alternative dihydrofolate reductases 1 from Brucella suis and Brucella canis were produced and their structures are reported. The enzymes share ∼95% sequence identity but have less than 33% sequence identity to other homologues with known structure. The structures are prototypical NADPH-dependent short-chain reductases that share their highest tertiary-structural similarity with protozoan pteridine reductases, which are being investigated for rational therapeutic development.

Cross-reactive and mono-reactive SARS-CoV-2 CD4+ T cells in prepandemic and COVID-19 convalescent individuals

Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct ex vivo staining of PBMC with tetramer reagents was used to define immunodominant and subdominant T cell epitopes and estimate the frequencies of these T cells in SARS-CoV-2 exposed and naïve individuals. Majority of SARS-CoV-2 epitopes identified have <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies.

First Report of citrus virus A in Texas associated with oak leaf patterns in Citrus sinensis

In 2018, Navarro et al. (2018a, b) identified two new negative sense coguviruses in citrus, citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA). Since then, the members of the genus Coguvirus have been also detected in other plant species (Xin et al. 2017; Wright et al. 2018; Svanella-Duma et al. 2019). In 2016, leaf flecking with oak leaf patterns were observed in five plants among embryo-rescued navel orange (NO) (Citrus sinensis (L.) Osbeck) trees grafted on C22 (C. sunki x Poncirus trifoliata) rootstock, maintained in a shade house. Madam Vinous (MV) sweet orange trees graft-inoculated with blind buds from the symptomatic NO plants developed the same symptoms in the new growth. These symptoms were similar to those on the citrus concave gum (CG) source tree of the California isolate CG301, one of the standard citrus disease isolates used as a positive control for biological indexing (Roistacher et al. 2000). None of the trees with oak leaf symptoms tested positive with reverse transcription (RT)-PCR for a panel of viruses and viroids commonly infecting citrus (Table S1) . In this study, CG301 leaf RNA-Seq data was used as a platform to identify any viral agent(s) associated with the oak leaf symptoms observed in the symptomatic NO trees (Fig. S1). Of ~162.8 million paired-end CG301 RNA-Seq reads (150 bp), de novo assembly of ~9.6 million reads, not mapped to C. sinensis genome (v.1.1), yielded 5,375 contigs. BLASTn using NCBI virus database (txid 10239) identified two contigs, #49 (6,715 nt) and #20 (2,764 nt), which exhibited ~96% sequence identity, respectively, to RNA1 and 2 of CiVA isolate W4 (MG764565; MG764566) and 71-73% identity to that of CCGaV isolate CGW2 (KX960112; KX960111). 5'-Nuclease assay developed based on contig #20 detected coguviral sequences in the five symptomatic NO and graft-inoculated MV trees as well as in CG301 but not in 44 asymptomatic field trees located near the shade house where the symptomatic NO trees had been kept. A full genomic sequence of the coguviruses present in CG301 and a symptomatic NO tree was reconstructed by RT-PCR. Both CG301 and NO isolate have a 6689 nt long negative sense RNA1 (MT922052; MK689372) encoding RNA-dependent RNA polymerase (RdRp) and a 2739 nt long ambisense RNA2 (MT922053; MK689373) encoding movement protein (MP) and nucleocapsid protein (NP). The isolate CG301 and NO share ~96% nucleotide sequence identity. The genome of both CG301 and NO isolate share 95.4-97.8 % sequence identity to that of CiVA isolate W4 and 70-72.9 % sequence identity to CCGaV isolate CGW2. BLASTp showed that RdRp of CG301 and NO isolate have 96.3-97.7 % sequence identity to CiVA W4 RdRp and ~77 % to CCGaV CGW2 RdRp. These data indicated the presence of CiVA in the symptomatic NO trees and in the concave gum source tree CG301. Recent reports of CiVA in South Africa and Greece indicated a potential wider distribution of CiVA in various citrus growing regions that may be associated with two graft-transmissible citrus diseases, citrus concave gum and impietratura disease (Roistacher et al. 2000; Velázquez et al. 2019; Beris et al. 2021; Bester et al. 2021). Although the source of CiVA in the symptomatic NO trees and the degree of CiVA prevalence in Texas had not been determined yet, a possible involvement of vector(s) or other means of spread (e.g. seed transmission) cannot be ruled out (Timmer et al. 2017). The current study demonstrated the need for further studies to determine the level of threat of coguviruses for citrus production in Texas.

Clustering of Aromatic Amino Acid Residues around Methionine in Proteins

Short-range, non-covalent interactions between amino acid residues determine protein structures and contribute to protein functions in diverse ways. The interactions of the thioether of methionine with the aromatic rings of tyrosine, tryptophan, and/or phenylalanine has long been discussed and such interactions are favorable on the order of 1–3 kcal mol−1. Here, we carry out a new bioinformatics survey of known protein structures where we assay the propensity of three aromatic residues to localize around the [-CH2-S-CH3] of methionine. We term these groups “3-bridge clusters”. A dataset consisting of 33,819 proteins with less than 90% sequence identity was analyzed and such clusters were found in 4093 structures (or 12% of the non-redundant dataset). All sub-classes of enzymes were represented. A 3D coordinate analysis shows that most aromatic groups localize near the CH2 and CH3 of methionine. Quantum chemical calculations support that the 3-bridge clusters involve a network of interactions that involve the Met-S, Met-CH2, Met-CH3, and the π systems of nearby aromatic amino acid residues. Selected examples of proposed functions of 3-bridge clusters are discussed.