Xer recombination for the automatic deletion of selectable marker genes from plasmids in enteric bacteria.

Antibiotic resistance genes are widely used to select bacteria transformed with plasmids and to prevent plasmid loss from cultures, yet antibiotics represent contaminants in the biopharmaceutical manufacturing process, and retaining antibiotic resistance genes in vaccines and biological therapies is discouraged by regulatory agencies. To overcome these limitations, we have developed X-mark™, a novel technology that leverages Xer recombination to generate selectable marker gene-free plasmids for downstream therapeutic applications. Using this technique, X-mark plasmids with antibiotic resistance genes flanked by XerC/D target sites are generated in Escherichia coli cytosol aminopeptidase (E. coli pepA) mutants, which are deficient in Xer recombination on plasmids, and subsequently transformed into enteric bacteria with a functional Xer system. This results in rapid deletion of the resistance gene at high resolution (100%) and stable replication of resolved plasmids for more than 40 generations in the absence of antibiotic selective pressure. This technology is effective in both Escherichia coli and Salmonella enterica bacteria due to the high degree of homology between accessory sequences, including strains that have been developed as oral vaccines for clinical use. X-mark effectively eliminates any regulatory and safety concerns around antibiotic resistance carryover in biopharmaceutical products, such as vaccines and therapeutic proteins. Graphical Abstract.

Dental Emergencies and Coronavirus Disease-2019: Scoping Review of the Literature and Single Centre Experience.

Understanding the impact of the COVID-19 pandemic on dental emergencies. A systematic review of the literature (PubMed/Scopus) searching for articles on COVID-19 and dental abscess and a retrospective cohort study with quantitative/qualitative data analysis of our hospital E.R. patients admitted for cervico-facial abscess of dental origin were performed. Thirteen studies could be included in the review, concerning characteristics/management of patients with dental emergencies in hospitals/private practices, generally with poor evidence. For the retrospective analysis, 232 consecutive patients were included (100 study vs. 132 control). The prevalence of dental emergencies (abscess) and relative complications (mediastinitis, exitus) increased. Dental care availability was limited, with strong heterogeneity amongst regions/nations. At-risk (aerosol-generating) procedures were generally avoided, and hospitalization length reduced. Comorbidity patients and males seem less likely to restore regular dentist attendance during the post-lockdown pandemic. Despite the poor scientific evidence, COVID-19 seems to have impacted dental emergencies through limited routine dental care availability and influence on physicians' and patients' behaviour.

Dental Trauma in Children with Autistic Disorder: A Retrospective Study.

BACKGROUND: The oral health care of autistic children is elaborated; they often fail to define dental problems, and a family-centered approach can be useful to improve and intercept these disorders. AIM: To assess the oral status of autistic children, comparing it with no autistic patients. MATERIALS AND METHODS: A retrospective study analyzed the oral health status of 70 children, 35 with autism and 35 without the disorder. Conditions assessed were dental trauma type, periodontal tissue injuries, soft tissue lip injuries, different treatments carried out, associated soft tissue findings and disorders, and the long-term management. All patients (≤15 years of age) were chosen consecutively. RESULTS: Females (57%) suffered more traumatic injuries than males (43%) in the autistic group, whereas males affected by dental trauma (54%) are predominant in the control group. The enamel fracture was the main finding among the dental trauma types in both groups followed by enamel/dentin/pulp fracture (31%), root fracture (11%), and avulsions (3%) in the autistic group and by avulsions (20%), root fracture (11%), and enamel/dentin/pulp fracture (6%) in the control group. The comparison of all variables of the two groups showed a statistically significant difference (P < 0.012). The lower lip was statistically more injured than the upper lip (P < 0.005). CONCLUSIONS: The composite restorative technique was the most common approach carried out; the long-term evaluation, when possible, was predominantly managed through root canal therapy in the control group (81%), and root canal therapy (50%) and tooth extraction (50%) in the sample group.

Identification of new developmentally regulated genes involved in Streptomyces coelicolor sporulation.

BACKGROUND: The sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation. RESULTS: We report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity. CONCLUSION: Aerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.

One of the two genes encoding nucleoid-associated HU proteins in Streptomyces coelicolor is developmentally regulated and specifically involved in spore maturation.

Streptomyces genomes encode two homologs of the nucleoid-associated HU proteins. One of them, here designated HupA, is of a conventional type similar to E. coli HUalpha and HUbeta, while the other, HupS, is a two-domain protein. In addition to the N-terminal part that is similar to that of HU proteins, it has a C-terminal domain that is similar to the alanine- and lysine-rich C termini of eukaryotic linker histones. Such two-domain HU proteins are found only among Actinobacteria. In this phylum some organisms have only a single HU protein of the type with a C-terminal histone H1-like domain (e.g., Hlp in Mycobacterium smegmatis), while others have only a single conventional HU. Yet others, including the streptomycetes, produce both types of HU proteins. We show here that the two HU genes in Streptomyces coelicolor are differentially regulated and that hupS is specifically expressed during sporulation, while hupA is expressed in vegetative hyphae. The developmental upregulation of hupS occurred in sporogenic aerial hyphal compartments and was dependent on the developmental regulators whiA, whiG, and whiI. HupS was found to be nucleoid associated in spores, and a hupS deletion mutant had an average nucleoid size in spores larger than that in the parent strain. The mutant spores were also defective in heat resistance and spore pigmentation, although they possessed apparently normal spore walls and displayed no increased sensitivity to detergents. Overall, the results show that HupS is specifically involved in sporulation and may affect nucleoid architecture and protection in spores of S. coelicolor.

Osmoregulation in Streptomyces coelicolor: modulation of SigB activity by OsaC.

As free-living non-motile saprophytes, Streptomyces need to adapt to a wide range of environmental conditions and this is reflected by an enormous diversity of regulatory proteins encoded by, for example, the genome of the model streptomycete Streptomyces coelicolor. In this organism, we have identified a new osmoregulation gene, osaC, encoding a member of a novel family of regulatory proteins. Members of the family have a predicted domain composition consisting of an N-terminal kinase domain related to anti-sigma factors, sensory Pas and Gaf domains, and a C-terminal phosphatase domain. osaC is linked to the response regulator gene osaB; expression analysis of the latter revealed that it is induced after osmotic stress in a sigma(B)-dependent manner. OsaC is required to return osaB and sigB expression back to constitutive levels after osmotic stress. From analysis of the activities of OsaC(DeltaPho), lacking the C-terminal phosphatase domain, and OsaC(N92A), with a substitution of a critical asparagine residue in the kinase domain, we infer that this N-terminal domain functions as a sigma(B) anti-sigma factor. Indeed, co-purification experiments indicate association of OsaC and sigma(B). These results support a model for post-osmotic stress modulation of sigma(B) activity by OsaC.

The Streptomyces coelicolor dnaK operon contains a second promoter driving the expression of the negative regulator hspR at physiological temperature.

HspR (heat shock protein regulator) acts as a negative regulator of different genes in many bacteria. In Streptomyces coelicolor hspR gene is part and the transcriptional repressor of the dnaK operon which encodes the DnaK, GrpE, DnaJ chaperone machines and HspR itself. Our experiments led us to the discovery of a second promoter, internal to dnaK operon, located upstream hspR gene. Transcription from this promoter was detected at 30 degrees C indicating that hspR could play a key physiological role.

Biological cost of hypermutation in Pseudomonas aeruginosa strains from patients with cystic fibrosis.

The high prevalence of hypermutable (mismatch repair-deficient) Pseudomonas aeruginosa strains in patients with cystic fibrosis (CF) is thought to be driven by their co-selection with adaptive mutations required for long-term persistence. Whether the increased mutation rate of naturally hypermutable strains is associated with a biological benefit or cost for the colonization of secondary environments is not known. Thirty-nine P. aeruginosa strains were collected from ten patients with CF during their course of chronic lung infections and screened for hypermutability. Seven hypermutable P. aeruginosa strains (18 %) isolated from six patients with CF (60 %) were identified and assigned to five different genotypes. Complementation and sequence analysis in the mutS, mutL and uvrD genes of these hypermutable P. aeruginosa strains revealed novel mutations. To understand the consequences of hypermutation for the fitness of the organisms, five pairs of clinical wild-type/hypermutable, clonally related P. aeruginosa strains and the laboratory strains PAO1/PAO1DeltamutS were subjected to competition in vitro and in the agar-beads mouse model of chronic airway infection. When tested in competition assay in vitro, the wild-type outcompeted four clinical hypermutable strains and the PAO1DeltamutS strain. In vivo, all of the hypermutable strains were less efficient at establishing lung infection than their wild-type clones. These results suggest that P. aeruginosa hypermutation is associated with a biological cost, reducing the potential for colonization of new environments and therefore strain transmissibility.

Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea.

Using an Escherichia coli-Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be used to facilitate the genomic analysis of this actinomycete.