E. coli chromosomal-driven expression of NADK2 from A. thaliana: A preferable alternative to plasmid-driven expression for challenging proteins.

The expression and purification of large recombinant proteins or protein complexes is problematic for some biotechnology laboratories. Indeed, it is often difficult to obtain enough active proteins to perform biological characterization or reach commercialization, when large proteins or protein complexes are expressed in E. coli via the popular T7-based plasmid-driven expression system. There is also an industrial demand to decrease our dependence on plasmid-driven expression, because of its drawbacks, such as: i) the common use of antibiotics to maintain the plasmid, ii) the issue of plasmid copy number, and iii) the risk of overloading the expression system. Despite all these issues, alternative solutions, such as gene integration in the bacterial chromosome, are rarely employed and their advantages are still a matter of debate. Plant plastidial NAD kinases (NADK; ATP:NAD 2'-phosphotransferase, EC 2.7.1.23) are a classic example of proteins with high molecular weight, that are difficult to express and purify with traditional T7-based technology. We therefore compared plasmid-driven and chromosomal-driven expression of the Arabidopsis thaliana NADK2 protein, using a proprietary counter-selection tool, COLIBELT®, that allows scar-free and marker-free chromosomal modifications. Here we show that chromosomal-driven expression allowed recovery of more active NADK2 protein than classic T7 expression systems, as well as better production, thus confirming that expression from one single chromosomal copy is preferable to plasmid-driven expression and might be appealing for both basic and applied research.

New euchromatic variant dup(11)(p15.3p15.1) transmitted through two generations defined by low coverage whole genome sequencing.

We report on a 14-year old boy, his father, and his paternal uncle, all three carriers of a duplication of chromosomal region 11p15.3-p15.1. The aberration was transmitted by the grandmother, who is carrier of a balanced insertion 46,XX,ins(14;11)(q32.1;p15.3p15.1). In order to determine the precise molecular basis of this structural variant, we performed low-coverage whole genome sequencing on the boy's father. This approach allowed precise determination of the genomic breakpoints and revealed a duplication of 6.9 Mb, centromeric to the Beckwith-Wiedemann/Silver-Russell syndrome critical region in 11p15.5, that inserted in inverse orientation into 14q32.12 (according to HGVS nomenclature: NC_000014.8:g.92871000_92871001ins[NC_000011.9:g.12250642_19165928inv;T]). To our knowledge, this is the first report of a duplication of 11p15.3-p15.1 involving more than 40 genes and transmitted through two generations without apparent clinical effects.

Occurrence and persistence of multidrug-resistant Enterobacterales isolated from urban, industrial and surface water in Monastir, Tunisia.

The One Health approach of antimicrobial resistance highlighted the role of the aquatic environment as a reservoir and dissemination source of resistance genes and resistant bacteria, especially due to anthropogenic activities. Resistance to extended-spectrum cephalosporins (ESC) conferred by extended-spectrum beta-lactamases (ESBLs) in E. coli has been proposed as the major marker of the AMR burden in cross-sectoral approaches. In this study, we investigated wastewater, surface water and seawater that are subjected to official water quality monitoring in Monastir, Tunisia. While all but one sample were declared compliant according to the official tests, ESC-resistant bacteria were detected in 31 (19.1 %) samples. Thirty-nine isolates, coming from urban, industrial and surface water in Monastir, were collected and characterized using antibiograms and whole-genome sequencing. These isolates were identified as 27 Escherichia coli (69.3 %) belonging to 13 STs, 10 Klebsiella pneumoniae (25.6 %) belonging to six STs, and two Citrobacter freundii (5.1 %). We observed the persistence and dissemination of clones over time and in different sampling sites, and no typically human-associated pathogens could be identified apart from one ST131. All isolates presented a blaCTX-M gene - blaCTX-M-15 (n = 22) and blaCTX-M-55 (n = 8) being the most frequent variants - which were identified on plasmids (n = 20) or on the chromosome (n = 19). In conclusion, we observed ESC resistance in rather ubiquitous bacteria that are capable of surviving in the water environment. This suggests that including the total coliform count and the ESBL count as determined by bacterial growth on selective plates in the official monitoring would greatly improve water quality control in Tunisia.

Rare 15q21.1q22.31 Duplication Due to a Familial Chromosomal Insertion and Diagnostic Investigation in a Carrier of Balanced Chromosomal Rearrangement and Intellectual Disability.

Insertions are rare balanced chromosomal rearrangements with an increased risk of imbalances for the offspring. Moreover, balanced rearrangements in individuals with abnormal phenotypes may be associated to the phenotype by different mechanisms. This study describes a three-generation family with a rare chromosomal insertion. G-banded karyotype, chromosomal microarray analysis (CMA), whole-exome sequencing (WES), and low-pass whole-genome sequencing (WGS) were performed. Six individuals had the balanced insertion [ins(9;15)(q33;q21.1q22.31)] and three individuals had the derivative chromosome 9 [der(9)ins(9;15)(q33;q21.1q22.31)]. The three subjects with unbalanced rearrangement showed similar clinical features, including intellectual disability, short stature, and facial dysmorphisms. CMA of these individuals revealed a duplication of 19.3 Mb at 15q21.1q22.31. A subject with balanced rearrangement presented with microcephaly, severe intellectual disability, absent speech, motor stereotypy, and ataxia. CMA of this patient did not reveal pathogenic copy number variations and low-pass WGS showed a disruption of the RABGAP1 gene at the 9q33 breakpoint. This gene has been recently associated with a recessive disorder, which is not compatible with the mode of inheritance in this patient. WES revealed an 88 bp deletion in the MECP2 gene, consistent with Rett syndrome. This study describes the clinical features associated with the rare 15q21.1-q22.31 duplication and reinforces that searching for other genetic causes is warranted for individuals with inherited balanced chromosomal rearrangements and abnormal phenotypes.

A study of innate immune kinetics reveals a role for a chloride transporter in a virulent Francisella tularensis type B strain.

Tularemia is a zoonotic disease of global proportions. Francisella tularensis subspecies tularensis (type A) and holarctica (type B) cause disease in healthy humans, with type A infections resulting in higher mortality. Repeated passage of a type B strain in the mid-20th century generated the Live Vaccine Strain (LVS). LVS remains unlicensed, does not protect against high inhalational doses of type A, and its exact mechanisms of attenuation are poorly understood. Recent data suggest that live attenuated vaccines derived from type B may cross-protect against type A. However, there is a dearth of knowledge regarding virulent type B pathogenesis and its capacity to stimulate the host's innate immune response. We therefore sought to increase our understanding of virulent type B in vitro characteristics using strain OR96-0246 as a model. Adding to our knowledge of innate immune kinetics in macrophages following infection with virulent type B, we observed robust replication of strain OR96-0246 in murine and human macrophages, reduced expression of pro-inflammatory cytokine genes from "wild type" type B-infected macrophages compared to LVS, and delayed macrophage cell death suggesting that virulent type B may suppress macrophage activation. One disruption in LVS is in the gene encoding the chloride transporter ClcA. We investigated the role of ClcA in macrophage infection and observed a replication delay in a clcA mutant. Here, we propose its role in acid tolerance. A greater understanding of LVS attenuation may reveal new mechanisms of pathogenesis and inform strategies toward the development of an improved vaccine against tularemia.

Novel chromosomal insertions of ISEcp1-blaCTX-M-15 and diverse antimicrobial resistance genes in Zambian clinical isolates of Enterobacter cloacae and Escherichia coli.

BACKGROUND: The epidemiology of extended-spectrum ß-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types. blaCTX-M genes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given that blaCTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integrated blaCTX-M and the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements. METHODS: A total of 46 clinical isolates of cefotaxime-resistant Enterobacteriaceae (1 Enterobacter cloacae, 9 Klebsiella pneumoniae, and 36 Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes, blaCTX-M genes were categorized as either chromosomal or plasmid-borne. RESULTS: WGS-based genotyping identified 58 AMR genes, including four blaCTX-M alleles (i.e., blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination of blaCTX-M genes. Out of 45 blaCTX-M gene-carrying strains, 7 harbored the gene in their chromosome. In one E. cloacae and three E. coli strains, chromosomal blaCTX-M-15 was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1 at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles. CONCLUSION: Our study revealed the co-occurrence of ISEcp1-blaCTX-M-15 and multiple AMR genes on chromosomal insertions in E. cloacae and E. coli, suggesting that ISEcp1 may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among these Enterobacteriaceae species.

A novel insertion ins(18;5)(q21.1;q31.2q35.1) in acute myeloid leukemia associated with microdeletions at 5q31.2, 5q35.1q35.2 and 18q12.3q21.1 detected by oligobased array comparative genomic hybridization.

BACKGROUND: Nonrandom clonal chromosomal aberrations can be detected in approximately 55% of adult patients with acute myeloid leukemia (AML). Recurrent cytogenetic abnormalities play an important role in diagnosis, classification and prognosis of AML. However, several chromosomal abnormalities have not been completely determined or characterized, primarily because of their low incidence and limited amount of data. RESULTS: We characterized an AML patient with a novel apparently balanced insertion ins(18;5)(q21;q31.2q35.1) that was cryptic by G-banding. The rearrangement was further examined by molecular cytogenetic methods and oligobased high-resolution array CGH (oaCGH) analysis. We show that an approximately 31.8 Mb large segment from chromosome 5 bands q31.2 to q35.1 has been inserted, by a direct mechanism, into chromosome 18 between bands q12.3 and q21.1. The insertion was unbalanced with concurrent submicroscopic deletions at 5q31.2 (approximately 0.37 Mb in size), 5q35.1q35.2 (approximately 1.98 Mb in size), and 18q12.3q21.1 (approximately 2.07 Mb in size). The microdeletions affect genes on 5q and 18q that have been associated with hematological malignancy and other cancers. A novel juxtaposition of the genes NPM1 and HAUS1 at 5q35.1 and 18q21.1, respectively, was detected by FISH analysis. Searching the literature and the Mitelman database revealed no previously reported ins(18;5) cases. Interestingly, however, two AML patients with translocation t(5;18)(q35;q21) encompassing the 5q35 and 18q21 breakpoint regions as detected in our present ins(18;5) patient have been reported. CONCLUSIONS: It is well-known that cytogenetic abnormalities on the long arm of chromosome 5 affect hematopoiesis. However, the precise mechanism of their involvement in myeloid transformation is elusive. Our present data shed new light onto the frequent abnormalities on 5q as well as to the less frequent abnormalities observed on 18q in myeloid malignancies. In addition, we show that oaCGH analysis is a useful adjunct to revealing submicroscopic aberrations in regions of clinical importance. Reporting rare and nonrandom chromosomal abnormalities contribute to the identification of the whole spectrum of cytogenetic abnormalities in AML and their prognostic significance.

An interstitial, apparently-balanced chromosomal insertion in the etiology of Langer-Giedion syndrome in an Asian family.

Langer-Giedion syndrome (LGS; MIM 150230), also called trichorhinophalangeal syndrome type II (TRPS2), is a contiguous gene syndrome caused by a one-copy deletion in the chromosome 8q23-q24 region, spanning the genes TRPS1 and EXT1. We identified an LGS family with two affected and two unaffected siblings from unaffected parents. To investigate the etiology of recurrence of LGS in this family, array CGH was performed on all family members. We identified a 7.29 Mb interstitial deletion at chromosome region 8q23-q24 in the two affected siblings, but no such deletion in the unaffected family members. However, the mother and one of the two unaffected siblings carried a 1.29 Mb deletion at chromosome region 8q24.1, sharing the distal breakpoint with the larger deleted segment found in the affected siblings. Another unaffected sibling had a 6.0 Mb duplication, sharing the proximal breakpoint of the deletion in the affected siblings. Karyotypic and FISH analyses in the unaffected mother revealed an insertional translocation of 8q23-q24 genomic material into chromosome 13: 46,XX,ins(13;8)(q33;q23q24). This insertional translocation in the mother results in the recurrence of LGS in this family, highlighting the importance of submicroscopic rearrangements in the genetic counseling for LGS.