Detection of IncP replicon-specific regions in DNA from Antarctic microbiota

Abstract Plasmids capable of horizontal transfer contribute to the adaptability of bacteria, as they may provide genes that enable their hosts to cope with different selective pressures. Only limited information is available on plasmids from Antarctic habitats, and up until now surveys have only used traditional methods of endogenous plasmid isolation. The method based on primer systems, designed on the basis of published sequences for plasmids from different incompatibility (Inc) groups, is appropriate to detect the replicon-specific regions of corresponding plasmids in cultured bacteria, or in total community DNA, which share sufficient DNA similarity with reference plasmids at the amplified regions. In this study, we applied broad-host-range plasmid-specific primers to DNA from microbial samples collected at six different locations in Northern Victoria Land (Antarctica). DNA preparations were used as targets for PCR (polymerase chain reaction) amplification with primers for the IncP (trfA2) and IncQ (oriV ) groups. PCR products were Southern blotted and hybridized with PCR-derived probes for trfA2 and oriV regions. This approach detected the occurrence of IncP-specific sequences in eight out of fifteen DNA samples, suggesting a gene-mobilizing capacity within the original habitats

Co-haplotyping symbiont and host to unravel invasion pathways of the exotic pest Halyomorpha halys in Italy

The brown marmorated stink bug Halyomorpha halys (Stål) is a globally invasive species that harbors a primary bacterial symbiont ‘Candidatus Pantoea carbekii’. In this work, P. carbekii was used here as another genetic marker to investigate the biodiversity and biogeographical patterns of this important pest, in native and newly invaded areas, especially in Italy. The correlation between the genetic structure of the symbiont and that of its host was studied through the analyses of one bacterial and one host marker, the putative pseudogene ΔybgF and the mitochondrial gene COI, respectively. As a result, five new P. carbekii haplotypes were identified, and an association pattern between host-symbiont haplotypes was observed. Host species showed higher haplotype diversity than symbiont, which can be expected in a long term host-symbiont association. Populations from the north-eastern Italy showed the highest values of genetic diversity for both markers, highlighting that this particular Italian area could be the result of multiple ongoing introductions. Moreover, some of the symbiont-host haplotypes observed were shared only by populations from north-eastern Italy and native areas, especially Japan, suggesting further introductions from this native country to Italy. Overall, our findings improve the understanding of the potential origin of multiple accidental introductions of H. halys in Italy

A connection between stress and development in the multicelular prokaryote Streptomyces coelicolor

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2–egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2–egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis

Sex difference and intra-operative tidal volume: Insights from the LAS VEGAS study

BACKGROUND: One key element of lung-protective ventilation is the use of a low tidal volume (VT). A sex difference in use of low tidal volume ventilation (LTVV) has been described in critically ill ICU patients.OBJECTIVES: The aim of this study was to determine whether a sex difference in use of LTVV also exists in operating room patients, and if present what factors drive this difference.DESIGN, PATIENTS AND SETTING: This is a posthoc analysis of LAS VEGAS, a 1-week worldwide observational study in adults requiring intra-operative ventilation during general anaesthesia for surgery in 146 hospitals in 29 countries.MAIN OUTCOME MEASURES: Women and men were compared with respect to use of LTVV, defined as VT of 8 ml kg-1 or less predicted bodyweight (PBW). A VT was deemed 'default' if the set VT was a round number. A mediation analysis assessed which factors may explain the sex difference in use of LTVV during intra-operative ventilation.RESULTS: This analysis includes 9864 patients, of whom 5425 (55%) were women. A default VT was often set, both in women and men; mode VT was 500 ml. Median [IQR] VT was higher in women than in men (8.6 [7.7 to 9.6] vs. 7.6 [6.8 to 8.4] ml kg-1 PBW, P < 0.001). Compared with men, women were twice as likely not to receive LTVV [68.8 vs. 36.0%; relative risk ratio 2.1 (95% CI 1.9 to 2.1), P < 0.001]. In the mediation analysis, patients' height and actual body weight (ABW) explained 81 and 18% of the sex difference in use of LTVV, respectively; it was not explained by the use of a default VT.CONCLUSION: In this worldwide cohort of patients receiving intra-operative ventilation during general anaesthesia for surgery, women received a higher VT than men during intra-operative ventilation. The risk for a female not to receive LTVV during surgery was double that of males. Height and ABW were the two mediators of the sex difference in use of LTVV.TRIAL REGISTRATION: The study was registered at Clinicaltrials.gov, NCT01601223

Dominant symbiotic bacteria associated with wild medfly populations reveal a bacteriocin-like killing phenotype: a ‘cold-case’ study

The gut of the agricultural pest Ceratitis capitata hosts a varied community of bacteria, mainly Enterobacteriaceae, that were implicated in several processes that increase the fitness of the insect. In this study, we investigated the antagonistic activity in vitro of Klebsiella oxytoca strains isolated in the 1990s from the alimentary tract of wild medflies collected from different varieties of fruit trees at diverse localities. Assays were carried out against reference strains (representative of Gram-negative and -positive bacterial species) of the American Type Culture Collection (ATCC). Eight Klebsiella, out of 11, expressed a killing activity against Escherichia coli ATCC 23739, and Enterobacter cloacae ATCC 13047; among the eight strains, at least one showed activity against Salmonella typhimurium ATCC 23853. Genomic DNA derived from all Klebsiella strains was then subjected to PCR amplification using specific primer pairs designed from each of the four bacteriocin (KlebB, C, D, CCL) sequences found so far in Klebsiella. KlebD primer pairs were the only to produce a single product for all strains expressing the killing phenotype in vitro. One of the amplicons was cloned and sequenced; the DNA sequence shows 93% identity with a plasmid-carried colicin-D gene of a strain of Klebsiella michiganensis, and 86% identity with the sequence encoding for the klebicin D activity protein in K. oxytoca. Our work provides the first evidence that dominant symbiotic bacteria associated with wild medfly populations express a killing phenotype that may mediate inter and intraspecies competition among bacterial populations in the insect gut in vivo

Ceratotoxins, antibacterial sex-specific peptides from the medfly Ceratitis capitata.

Ctxs are a family of α-helical, cationic, 3 kDa antibacterial peptides secreted by the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. These peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers chemically synthesized Ctxs formed voltage-dependent ion channels according to the barrel-stave model. A synthetic truncated CtxA molecule resulted to permeabilize the outer and inner membranes of Escherichia coli cells

Polystyrene shaping effect on the enriched bacterial community from the plastic‑eating Alphitobius diaperinus (Insecta: Coleoptera)

Plastic pollution has become a serious issue of global concern, and biodegradation of plastic wastes is representing one attractive environment-friendly alternative to traditional disposal paths. It is known that insects are involved in the plastic polymer degradation process, with reported evidence of tenebrionid beetle larvae capable to degrade polystyrene (PS), one of the most used plastics worldwide. Recently, a ribosomal RNA based survey on the insect gut microbiota of the lesser mealworm Alphitobius diaperinus has revealed diferentially abundant microbial taxa between PS-fed larvae and control group. Following these fndings, an enrichment bacterial culture was set up in liquid carbon-free basal medium with PS flm as sole carbon source using PS-fed larvae of A. diaperinus as inoculum. After two-months the culture was analysed both by molecular and culture-based methods. Isolated bacteria which had become prevalent under the selective enrichment conditions resulted ascribable to three taxonomic groups: Klebsiella, Pseudomonas, and Stenothrophomonas. The predominance of these groups in PS-fed larvae was confrmed by using bacterial 16S rRNA gene amplicon sequencing, and it was consistent with the results of previous reports. Isolated bacteria were able to attach to PS surfaces and SEM observations showed the presence of thin fbrillar structures connecting the bacterial cells to the abiotic surface

Alicyclobacillus pohliae sp. nov., a novel thermophilic endospore-forming bacterium isolated from geothermal soil of the north-west slope of Mount Melbourne (Antarctica)

Gram-positive, rod-shaped, endospore-forming, thermophilic bacteria were isolated from a geothermal soil collected on the north-west slope of Mount Melbourne in Antarctica. They grew aerobically at 42–60°C (optimum 55°C) and at pH 4.5–7.5 (optimum pH 5.5). Phylogenetic analysis of 16S rRNA gene sequences showed that these isolates were related most closely to the type strain of Alicyclobacillus pomorum (91% similarity). Growth occurred in the presence of ferrous iron at micromolar concentrations and acid was produced from various sugars. Iso-branched fatty acids C15 : 0 (45.56 %) and C17 : 0 (35.81 %) were the most abundant cellular fatty acids. The DNA G+C content was 55.1 mol%. On the basis of phenotypic and phylogenetic characteristics, it is concluded that these strains represent a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus pohliae sp. nov. is proposed. The type strain is MP4T (=CIP 109385T =NCIMB 14276T)