High-resolution CTCF footprinting reveals impact of chromatin state on cohesin extrusion dynamics.

DNA looping is vital for establishing many enhancer-promoter interactions. While CTCF is known to anchor many cohesin-mediated loops, the looped chromatin fiber appears to predominantly exist in a poorly characterized actively extruding state. To better characterize extruding chromatin loop structures, we used CTCF MNase HiChIP data to determine both CTCF binding at high resolution and 3D contact information. Here we present FactorFinder, a tool that identifies CTCF binding sites at near base-pair resolution. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF) state is rare genome-wide with locus-specific variation from ~1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics, and find that active enhancers and RNA Pol II impede cohesin extrusion, facilitating an enrichment of enhancer-promoter contacts in the partially extruded loop state. We propose a model of topological regulation whereby the transient, partially extruded states play active roles in transcription.

Long-term passive wastewater surveillance of SARS-CoV-2 for seven university dormitories in comparison to municipal surveillance.

Wastewater-based surveillance (WBS) has been an effective tool for monitoring and understanding potential SARS-CoV-2 transmission across small and large-scale communities. In this study at the University of Saskatchewan, the assessment of SARS-CoV-2 was done over eight months during the 2021-2022 academic year. Wastewater samples were collected using passive samplers that were deployed in domestic sewer lines near adjacent campus residences and extracted for viral RNA, followed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The results showed similar trends for SARS-CoV-2 detection frequencies and viral loads across university residences, the whole campus, and from related WBS at Saskatoon Wastewater Treatment Plant. The maximum daily detection frequency for seven dormitories considered was about 75 %, while maximum daily case numbers for the residences and campus-wide were about 11 and 75 people, respectively. In addition, self-reported rates of infection on campus peaked during similar time frames as increases in viral load were detected at the Saskatoon wastewater treatment plant. These similarities indicate the usefulness and cost-effectiveness of monitoring the spread of COVID-19 in small-scale communities using WBS.

Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer.

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

Clinical genetic testing in pediatric cardiomyopathy: Is bigger better?

BACKGROUND: For clinical genetic testing of cardiomyopathy (CMP), current guidelines do not address which gene panels to use: targeted panels specific to a CMP phenotype or expanded (panCMP) panels that include genes associated with multiple phenotypic subtypes. AIM: Our objective was to assess the clinical utility of targeted versus panCMP panel testing in pediatric CMPs. METHODS: 151 pediatric patients with primary hypertrophic (n = 66), dilated (n = 64), restrictive (n = 8), or left-ventricular non-compaction (n = 13) CMP who underwent clinical genetic panel testing at a single centre were included. PanCMP (n = 47) and targeted panel testing (n = 104) were compared for yield of pathogenic variants and variants of unknown significance (VUS). RESULTS: Pathogenic variants were identified in 26% of patients, 42% had indeterminate results (only VUS detected), and 32% had negative results. Yield was lower (15%) in panCMP vs. targeted panel testing (32%) (P = .03) in all CMP subtypes. VUS detection was higher with panCMP (87%) than targeted panel testing (30%) (P <.0001). PanCMP panel testing only identified pathogenic variants in genes that overlapped targeted panels. CONCLUSION: PanCMP testing did not increase diagnostic yield compared to targeted panel testing. Until accuracy of variant interpretation with panCMP panels improves, targeted panels may be suitable for clinical testing in pediatric CMP.

Utility of genetics for risk stratification in pediatric hypertrophic cardiomyopathy.

Children with hypertrophic cardiomyopathy (HCM) experience sudden cardiac death (SCD) and other life-threatening events. We assessed if affected gene and variant burden predict outcomes. Patients <18 years old with primary HCM with a pathogenic variant or variant of uncertain significance in cardiomyopathy genes were included. Association of gene and variant number and type with freedom from major adverse cardiac events (MACE), that is, ICD insertion, myectomy, aborted SCD, transplantation or death, was assessed by Cox regression. A total of 98 of 155 gene-tested patients carried a non-benign variant. The primary affected gene was MYH7 in 35% (MYH7+) and MYBPC3 in 49% (MYBPC3+). MYH7+ patients had earlier disease onset and higher risk of MACE (hazard ratio 2.7, 95% CI 1.3-5.7). Risk of MACE was also higher in patients with multiple variants (n = 16) (HR 2.5, CI: 1.1-5.9) compared to a propensity score-matched single variant subset, after adjustment for primary gene, and in patients with de novo (n = 18) vs inherited variants (HR 5.7, CI: 2.6-12.7). Affected gene (eg, MYH7), higher variant burden and de novo variant status are independently associated with earlier onset and higher frequency of adverse outcomes in pediatric HCM, highlighting the importance of genetic risk stratification in HCM.

Use of the DICE (Dual Integrase Cassette Exchange) System.

When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone. When a single recombinase is used for RMCE, recombination between undesired pairings of the sites can lead to a significant number of unwanted cell lines. To reduce the frequency with which these side products occur, several variants of RMCE that increase desired outcomes have been developed. Nevertheless, an important feature lacking from these improved RMCE methods is that none have fully utilized the recombinases that have been demonstrated to be the most robust and stringent at performing genomic integration in plants and animals, i.e., the phiC31 and Bxb1 phage integrases. To address this need, we have developed an RMCE protocol using these two serine integrases that we call dual integrase cassette exchange (DICE). Our DICE system provides a means to achieve high-precision DNA integration at a desired location and is especially well suited for repeated recombination into the same locus. In this chapter, we provide our most current protocols for using DICE in feeder-free human-induced pluripotent stem cells .

Genomic integration of the full-length dystrophin coding sequence in Duchenne muscular dystrophy induced pluripotent stem cells.

The plasmid vectors that express the full-length human dystrophin coding sequence in human cells was developed. Dystrophin, the protein mutated in Duchenne muscular dystrophy, is extraordinarily large, providing challenges for cloning and plasmid production in Escherichia coli. The authors expressed dystrophin from the strong, widely expressed CAG promoter, along with co-transcribed luciferase and mCherry marker genes useful for tracking plasmid expression. Introns were added at the 3' and 5' ends of the dystrophin sequence to prevent translation in E. coli, resulting in improved plasmid yield. Stability and yield were further improved by employing a lower-copy number plasmid origin of replication. The dystrophin plasmids also carried an attB site recognized by phage phiC31 integrase, enabling the plasmids to be integrated into the human genome at preferred locations by phiC31 integrase. The authors demonstrated single-copy integration of plasmid DNA into the genome and production of human dystrophin in the human 293 cell line, as well as in induced pluripotent stem cells derived from a patient with Duchenne muscular dystrophy. Plasmid-mediated dystrophin expression was also demonstrated in mouse muscle. The dystrophin expression plasmids described here will be useful in cell and gene therapy studies aimed at ameliorating Duchenne muscular dystrophy.

Effects on the transcriptome upon deletion of a distal element cannot be predicted by the size of the H3K27Ac peak in human cells.

Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for colorectal cancer (CRC). A molecular understanding of the functional consequences of this genetic variation is complicated because most GWAS SNPs are located in non-coding regions. We used epigenomic information to identify H3K27Ac peaks in HCT116 colon cancer cells that harbor SNPs associated with an increased risk for CRC. Employing CRISPR/Cas9 nucleases, we deleted a CRC risk-associated H3K27Ac peak from HCT116 cells and observed large-scale changes in gene expression, resulting in decreased expression of many nearby genes. As a comparison, we showed that deletion of a robust H3K27Ac peak not associated with CRC had minimal effects on the transcriptome. Interestingly, although there is no H3K27Ac peak in HEK293 cells in the E7 region, deletion of this region in HEK293 cells decreased expression of several of the same genes that were downregulated in HCT116 cells, including the MYC oncogene. Accordingly, deletion of E7 causes changes in cell culture assays in HCT116 and HEK293 cells. In summary, we show that effects on the transcriptome upon deletion of a distal regulatory element cannot be predicted by the size or presence of an H3K27Ac peak.

A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

Quantitative analysis of TALE-DNA interactions suggests polarity effects.

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN >> NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

Highly active zinc-finger nucleases by extended modular assembly.

Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three- to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.

The generation of zinc finger proteins by modular assembly.

The modular assembly (MA) method of generating engineered zinc finger proteins (ZFPs) was the first practical method for creating custom DNA-binding proteins. As such, MA has enabled a vast exploration of sequence-specific methods and reagents, ushering in the modern era of zinc finger-based applications that are described in this volume. The first zinc finger nuclease to cleave an endogenous site was created using MA, as was the first artificial transcription factor to enter phase II clinical trials. In recent years, other excellent methods have been developed that improved the affinity and specificity of the engineered ZFPs. However, MA is still used widely for many applications. This chapter will describe methods and give guidance for the creation of ZFPs using MA. Such ZFPs might be useful as starting materials to perform other methods described in this volume. Here, we also describe a single-strand annealing recombination assay for the initial testing of zinc finger nucleases.

Restricted spacer tolerance of a zinc finger nuclease with a six amino acid linker.

Zinc finger nucleases can be engineered to create highly efficient and precise changes to the genetic information within living cells. We report the investigation of an important parameter that defines the type of target site the nuclease can cleave. The active nuclease is a dimer, requiring that the DNA target site contain two zinc finger binding sites separated by a short spacer. Using a plasmid-based recombination assay in HEK 293T cells, we show that a 6 amino acid linker between the zinc finger DNA-binding domain and the FokI cleavage domain restricts nuclease activity to sites containing a 6 bp spacer. These observations concur with other recent studies, suggesting this information will be useful in the design of new potent and accurate zinc finger nucleases.

Twenty-year experience with heart transplantation for infants and children with restrictive cardiomyopathy: 1986-2006.

Idiopathic restrictive cardiomyopathy (RCM) is a rare cardiomyopathy in children notable for severe diastolic dysfunction and progressive elevation of pulmonary vascular resistance (PVR). Traditionally, those with pulmonary vascular resistance indices (PVRI) >6 W.U. x m(2) have been precluded from heart transplantation (HTX). The clinical course of all patients transplanted for RCM between 1986 and 2006 were reviewed. Preoperative, intraoperative and postoperative variables were evaluated. A total of 23 patients underwent HTX for RCM, with a mean age of 8.8 +/- 5.6 years and a mean time from listing to HTX of 43 +/- 60 days. Preoperative and postoperative (114 +/- 40 days) PVRI were 5.9 +/- 4.4 and 2.9 +/- 1.5 W.U. x m(2), respectively. At time of most recent follow-up (mean = 5.7 +/- 4.6 years), the mean PVRI was 2.0 +/- 1.0 W.U. x m(2). Increasing preoperative mean pulmonary artery pressure (PA) pressure (p = 0.04) and PVRI > 6 W.U. x m(2) (chi(2)= 7.4, p < 0.01) were associated with the requirement of ECMO postoperatively. Neither PVRI nor mean PA pressure was associated with posttransplant mortality; 30-day and 1-year actuarial survivals were 96% and 86%, respectively. Five of the seven patients with preoperative PVRI > 6 W.U. x m(2) survived the first postoperative year. We report excellent survival for patients undergoing HTX for RCM despite the high proportion of high-risk patients.

Hypertrophy signaling during peripartum cardiac remodeling.

UNLABELLED: Molecular signaling pathways that regulate peripartum cardiac remodeling are not well understood. Our objectives were to study the role of mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) in mediating pregnancy and postpartum (PP) cardiac remodeling. METHODS: Adult female Sprague-Dawley rats were divided into nonpregnant (n = 5), 18 days pregnant (n = 5), 0 days PP (n = 7), and 14 days PP (n = 8). Rats underwent echocardiography under sedation to measure left ventricle (LV) size and function, and Western blots were performed to measure myocardial protein expression of MAPKs (p38, JNK, ERK), Akt, and eNOS. RESULTS: 1) During pregnancy, there was an increase in LV mass (0.62 +/- 0.03 to 1.1 +/- 0.04 g, P < 0.001), mass/volume ratio (0.7 +/- 0.02 to 1.28 +/- 0.02 g/ml, P < 0.0001), and ejection fraction (EF) (64 +/- 3 to 74 +/- 2%). Whereas LV mass and mass/volume ratio returned to prepregnancy values in the PP period, EF remained below normal range (53 +/- 3%, P < 0.05). 2) The expression of anti-hypertrophic factors (p38, JNK, Akt) decreased during pregnancy and normalized PP, except JNK, which increased to higher than normal levels. eNOS also increased to higher than baseline levels PP. 3) Activation of p38 and JNK was directly correlated with lower LV mass/volume ratio (r = -0.81 and -0.71, respectively; P < 0.05). CONCLUSION: Pregnancy is associated with physiological cardiac hypertrophy. There is rapid reversal of hypertrophy in the PP period while recovery of cardiac function is delayed, possibly related to PP upregulation of JNK. A dysregulation of MAPK signaling may be an important determinant of PP cardiac dysfunction.

Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.

Cys2-His2 zinc fingers are one of the most common types of DNA-binding domains. Modifications to zinc-finger binding specificity have recently enabled custom DNA-binding proteins to be designed to a wide array of target sequences. We present here a 1.96 A structure of Aart, a designed six-zinc finger protein, bound to a consensus DNA target site. This is the first structure of a designed protein with six fingers, and was intended to provide insights into the unusual affinity and specificity characteristics of this protein. Most protein-DNA contacts were found to be consistent with expectations, while others were unanticipated or insufficient to explain specificity. Several were unexpectedly mediated by glycerol, water molecules or amino acid-base stacking interactions. These results challenge some conventional concepts of recognition, particularly the finding that triplets containing 5'A, C, or T are typically not specified by direct interaction with the amino acid in position 6 of the recognition helix.

Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India

The present study deals with the isolation and characterization of the moderately halophilic-alkaliphilic bacteria from a saline habitat in western India. Eight different bacterial strains were isolated using enrichment techniques at 20 percent (w/v) NaCl and pH 10. The isolates exhibited diversity towards gram's reaction, colony and cell morphology. They were able to grow and produce alkaline protease over a broad range of NaCl, 5-20 percent (w/v) and pH, 8-10. None of the isolates could grow at pH 7, and one could not grow even at pH 8. Crude and partially purified proteases from strain S5 were subjected to characterization with reference to pH, salt stability and protein folding. Optimum protease activity and stability was recorded at 10 percent salt and pH 9-9.5. Denaturation kinetics of S5 alkaline protease along with a reference protease was studied at 8M urea followed by renaturation. The S5 alkaline protease could be partially renatured up to 32 percent of the original activity. Despite of the fact that all the 8 isolates were from the same site, they displayed significant diversity with respect to their salt requirement for growth and enzyme secretion. While the effect of pH was less demarcated on growth, the protease production was significantly affected. Isolate S5 produced substantial amount of halotolerant and alkaline protease. The activity and stability of the alkaline protease in a broader range of pH and salt would definitely make this enzyme an important candidate for various industrial applications.

O presente estudo relata o isolamento e caracterização de bactérias moderadamente halofilicas e alcalífilicas de um habitat salino no oeste da índia. Oito cepas diferentes de bactérias foram isoladas empregando técnicas de enriquecimento em NaCl a 20 por cento (p/v) e pH 10. As cepas apresentaram diversidade em relação à coloração de Gram e à morfologia das colônias e células. As cepas foram capazes de multiplicar e produzir protease alcalina em uma ampla faixa de concentração de NaCl (5 a 20 por cento) e pH (8 a 10). Nenhuma das cepas foi capaz de se multiplicar em pH 7, e uma não se multiplicou nem em pH 8.0. Proteases naturais e parcialmente purificadas da cepa S5 foram submetidas à caracterização com relação ao pH, estabilidade salina, e estrutura protéica. Atividade e estabilidade ótimas da protease foram obtidas com 10 por cento de sal e pH 9-9,5. A cinética de denaturação da protease de S5, juntamente com uma protease de referencia, foi avaliada com uréia 8M seguida de renaturação. A protease alcalina de S5 foi renaturada a 32 por cento da atividade original. Apesar de provenientes do mesmo local, as oito cepas mostraram grande diversidade em relação à exigência de sal para multiplicação e secreção enzimática. Enquanto o efeito do pH na multiplicação foi menos marcante, o efeito na produção de protease foi significativamente afetada. A cepa S5 produziu uma quantidade substancial de protease alcalina e halotolerante. A atividade e estabilidade da protease alcalina em uma faixa mais ampla de pH e sal tornam essa enzima uma importante candidata para diversas aplicações industriais.

Bacillus okhensis sp. nov., a halotolerant and alkalitolerant bacterium from an Indian saltpan.

A strictly aerobic, rod-shaped bacterium (0.6-0.8 x2-3 microm), designated strain Kh10-101T, was isolated from a saltpan (22 degrees 15' N, 69 degrees 1' E) in the vicinity of Port Okha, India. The creamish pigmented colonies of strain Kh10-101T were round, flat and translucent with irregular margins and a smooth surface. The strain possessed up to three subpolar flagella, and was motile by a corkscrew motion. The strain grew optimally at 37 degrees C (temperature growth range 25-40 degrees C) in a complex glucose-containing medium with 5 % NaCl (NaCl growth range 0-10 %) at pH 9 (pH growth range pH 7-10), indicating that it was a mesophilic halotolerant alkaliphile. The strain was sensitive to lincomycin, meticillin, cefuroxime and cephalexin, but resistant to gentamicin, tetracycline and cotrimazine. Spores were not detected and cells were heat sensitive. The isolate metabolized a range of carbohydrates and hydrolysed casein, gelatin and starch. Growth was not observed on aromatic compounds, Tween 40 or Tween 80. Nitrate was not reduced and catalase was produced. Electron microscopic examination of thin sections revealed a single thick Gram-positive cell wall. The DNA G+C content was 41+/-1 mol%. Phylogenetic analyses of the 16S rRNA gene sequence revealed that strain Kh10-101T was a member of the sixth rRNA group of the genus Bacillus, which includes alkalitolerant, alkaliphilic and halotolerant species. The halotolerant obligate alkaliphile Bacillus krulwichiae is the closest relative of strain Kh10-101T (96 % similarity) but a number of phenotypic differences suggest that strain Kh10-101T (=JCM 13040T=ATCC BAA-1137T) should be designated the type strain of a new species, for which the name Bacillus okhensis sp. nov. is proposed.