Complete Genome Sequence of a Halophilic Bacterium, Halomonas sp. Strain NyZ770, from Mariana Trench Sediment

Halomonas sp. strain NyZ770 is a bacterium that was isolated from Mariana Trench sediment. Here, the complete genome sequence of this strain is reported. The genome was sequenced with the Illumina NovaSeq and Pacific Biosciences Sequel sequencing platforms and consists of a single chromosome of 4,024,853 bp, with a G+C content of 60.21%.

The Second Chromosome Promotes the Adaptation of the Genus Flammeovirga to Complex Environments

For decades, the typical bacterial genome has been thought to contain a single chromosome and a few small plasmids carrying nonessential genes. However, an increasing number of secondary chromosomes have been identified in various bacteria (e.g., plant symbiotic bacteria and human pathogens).

Co-occurrence of blaNDM–1 and mcr-9 in a Conjugative IncHI2/HI2A Plasmid From a Bloodstream Infection-Causing Carbapenem-Resistant Klebsiella pneumoniae

Spread of the carbapenemase-encoding and mobilized colistin resistance (mcr) genes among Enterobacteriales poses a great threat to global public health, especially when the both genes are transferred by a single plasmid. Here, we identified a blaNDM–1- and mcr-9-co-encoding plasmid harbored by a clinical isolate of Klebsiella pneumoniae (KPN710429). KPN710429 was recovered from a blood sample from an inpatient in a tertiary hospital in China, and antimicrobial susceptibility testing showed that it was multidrug-resistant and only susceptible to aztreonam, colistin, and tigecycline. KPN710429 belongs to sequence type (ST) 1308 and capsular serotype KL144. The string test of KPN710429 was negative, and this strain didn’t exhibit a hypervirulent phenotype according to serum-killing and Galleria mellonella lethality assessments. Whole-genome sequencing revealed the KPN710429 genome comprises a single chromosome and three plasmids. All virulence associated genes were harbored by chromosome. Most of its antimicrobial resistance genes, including blaNDM–1 and mcr-9 were carried by plasmid pK701429_2, belonging to the incompatibility (Inc) HI2/HI2A group and ST1. Comparative genomics assays indicates that pK710429_2 could be a hybrid plasmid, formed by a Tn6696-like blaNDM–1 region inserting into a mcr-9-positive-IncHI2/HI2A plasmid. pK710429_2 contained the conjugative transfer gene regions, Tra1 and Tra2, with some structural variations, and conjugation assays revealed that pK710429_2 was transferable. Although pK710429_2 lacked the qseB-qseC regulatory genes, mcr-9 expression was upregulated after pretreatment with colistin for 6 h, leading to colistin resistance in KPN710429. To our knowledge, this is the first report of a blaNDM–1- and mcr-9-co-encoding transferable plasmid harbored by a bloodstream-infection-causing K. pneumoniae strain in China. Effective surveillance should be implemented to assess the prevalence of the plasmid co-harboring carbapenemase-encoding gene and mcr-9.

SMCKAT, a Sequential Multi-Dimensional CNV Kernel-Based Association Test

Copy number variants (CNVs) are the most common form of structural genetic variation, reflecting the gain or loss of DNA segments compared with a reference genome. Studies have identified CNV association with different diseases. However, the association between the sequential order of CNVs and disease-related traits has not been studied, to our knowledge, and it is still unclear that CNVs function individually or whether they work in coordination with other CNVs to manifest a disease or trait. Consequently, we propose the first such method to test the association between the sequential order of CNVs and diseases. Our sequential multi-dimensional CNV kernel-based association test (SMCKAT) consists of three parts: (1) a single CNV group kernel measuring the similarity between two groups of CNVs; (2) a whole genome group kernel that aggregates several single group kernels to summarize the similarity between CNV groups in a single chromosome or the whole genome; and (3) an association test between the CNV sequential order and disease-related traits using a random effect model. We evaluate SMCKAT on CNV data sets exhibiting rare or common CNVs, demonstrating that it can detect specific biologically relevant chromosomal regions supported by the biomedical literature. We compare the performance of SMCKAT with MCKAT, a multi-dimensional kernel association test. Based on the results, SMCKAT can detect more specific chromosomal regions compared with MCKAT that not only have CNV characteristics, but the CNV order on them are significantly associated with the disease-related trait.

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

Background Topologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs has been shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing lead to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains, which are highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains. Results Here we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components and transcription mostly do not affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains. Conclusions In sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

1221. Genomic Factors Affecting the Efficacy of Antimicrobial Therapy in Daptomycin-, Linezolid-, Vancomycin-Resistant Enterococcus faecium (DLVRE)

Background Nosocomial acquisition of vancomycin-resistant Enterococcus (VRE) is one of the most challenging problems in healthcare. As Enterococcus isolates are increasingly resistant to vancomycin, clinicians now rely on alternative antimicrobial therapies including linezolid and daptomycin (DAP) to treat infections. For multidrug-resistant (MDR) VRE, combination therapy with beta-lactams and daptomycin has been shown to be effective. Methods Following initiation of empiric DAP and ceftaroline (CPT) for an MDR E. faecium bloodstream infection (VRE_001), we aimed to determine if there existed in vitro synergy between both agents that supported their clinical use. Combination synergy testing was performed using E-test strips and minimal inhibitory concentrations (MICs) were read at 24 hours. For whole genome sequence-based analysis (WGS), genomic DNA from VRE_001 was used for both short read (Illumina MiSeq) and long-read sequencing (MinION, Nanopore). The complete genome was assembled and the NCBI AMRFinderPlus program used to identify known resistance mechanisms. Results Original MICs of VRE_001 from the clinical microbiology laboratory at Northwestern Memorial revealed an MDR E. faecium (Table 1). Combination synergy testing in the experimental laboratory revealed only modest amounts of synergy between CPT and DAP (Table 2). Following WGS, VRE_001 was identified as an ST-584 E. faecium with a 3.2 Mbp genome, including a single chromosome and five plasmids. WGS analysis revealed several mechanisms of antimicrobial resistance (Table 3) genetically supporting the observed MDR-DLVRE phenotype. Conclusion Our investigational antimicrobial testing allowed for real-time in vitro analysis of synergistic MICs in a case of DLVRE bacteremia. Despite the fact that in vitro testing of CPT and DAP did not support the clinical usage of combination antimicrobial therapy, the patient cleared their blood cultures. WGS of VRE_001 revealed a plethora of antimicrobial resistance mechanisms including three mutations that explain high levels of DAP resistance. Synergy testing is not routinely available in most clinical laboratories, but rapid implementation of investigational MIC testing paired with genomic analysis may one day successfully support real-time clinical decision making. Disclosures All Authors: No reported disclosures

Behavioural and molecular characterisation of the Dlg2 haploinsufficiency rat model of genetic risk for psychiatric disorder

Genetic studies implicate disruption to the DLG2 gene in copy number variants as increasing risk for schizophrenia, autism spectrum disorders and intellectual disability. To investigate psychiatric endophenotypes associated with DLG2 haploinsufficiency (and concomitant PSD-93 protein reduction) a novel clinically relevant Dlg2+/- rat was assessed for abnormalities in anxiety, sensorimotor gating, hedonic reactions, social behaviour, and locomotor response to the N-Methyl-D-aspartic acid receptor antagonist phencyclidine. Dlg gene and protein expression were also investigated to assess model validity. Reductions in PSD-93 messenger RNA and protein were observed in the absence of compensation by other related genes or proteins. Behaviourally Dlg2+/- rats show potentiated locomotor response to phencyclidine, as is typical of psychotic disorder models, in the absence of deficits in the other behavioural phenotypes assessed here. This shows that the behavioural effects of Dlg2 haploinsufficiency may specifically relate to psychosis vulnerability but are subtle, providing a contrast to the gross deficits in Dlg2 homozygous models (Winkler, et al., 2018; Yoo et al., 2020a) which do not so specifically model the single chromosome DLG2 deletion in carriers of risk-associated copy number variants.

A single chromosome strain of S. cerevisiae exhibits diminished ethanol metabolism and tolerance

Background Eukaryotic organisms, like the model yeast S. cerevisiae, have linear chromosomes that facilitate organization and protection of nuclear DNA. A recent work described a stepwise break/repair method that enabled fusion of the 16 chromosomes of S. cerevisiae into a single large chromosome. Construction of this strain resulted in the removal of 30 of 32 telomeres, over 300 kb of subtelomeric DNA, and 107 subtelomeric ORFs. Despite these changes, characterization of the single chromosome strain uncovered modest phenotypes compared to a reference strain. Results This study further characterized the single chromosome strain and found that it exhibited a longer lag phase, increased doubling time, and lower final biomass concentration compared with a reference strain when grown on YPD. These phenotypes were amplified when ethanol was added to the medium or used as the sole carbon source. RNAseq analysis showed poor induction of genes involved in diauxic shift, ethanol metabolism, and fatty-acid ß-oxidation during growth on ethanol compared to the reference strain. Enzyme-constrained metabolic modeling identified decreased flux through the enzymes that are encoded by these poorly induced genes as a likely cause of diminished biomass accumulation. The diminished growth on ethanol for the single chromosome strain was rescued by nicotinamide, an inhibitor of sirtuin family deacetylases, which have been shown to silence gene expression in heterochromatic regions. Conclusions Our results indicate that sirtuin-mediated silencing in the single chromosome strain interferes with growth on non-fermentable carbon sources. We propose that the removal of subtelomeric DNA that would otherwise be bound by sirtuins leads to silencing at other loci in the single chromosome strain. Further, we hypothesize that the poorly induced genes in the single chromosome strain during ethanol growth could be silenced by sirtuins in wildtype S. cerevisiae during growth on glucose.

Whole genome sequencing of Enterobacter mori, an emerging pathogen of kiwifruit and the potential genetic adaptation to pathogenic lifestyle

Members of the Enterobacter genus are gram-negative bacteria, which are used as plant growth-promoting bacteria, and increasingly recovered from economic plants as emerging pathogens. A new Enterobacter mori strain, designated CX01, was isolated as an emerging bacterial pathogen of a recent outbreak of kiwifruit canker-like disease in China. The main symptoms associated with this syndrome are bleeding cankers on the trunk and branch, and brown leaf spots. The genome sequence of E. mori CX01 was determined as a single chromosome of 4,966,908 bp with 4640 predicted open reading frames (ORFs). To better understand the features of the genus and its potential pathogenic mechanisms, five available Enterobacter genomes were compared and a pan-genome of 4870 COGs with 3158 core COGs were revealed. An important feature of the E. mori CX01 genome is that it lacks a type III secretion system often found in pathogenic bacteria, instead it is equipped with type I, II, and VI secretory systems. Besides, the genes encoding putative virulence effectors, two-component systems, nutrient acquisition systems, proteins involved in phytohormone synthesis, which may contribute to the virulence and adaption to the host plant niches are included. The genome sequence of E. mori CX01 has high similarity with that of E. mori LMG 25,706, though the rearrangements occur throughout two genomes. Further pathogenicity assay showed that both strains can either invade kiwifruit or mulberry, indicating they may have similar host range. Comparison with a closely related isolate enabled us to understand its pathogenesis and ecology.