Characterization of the integrated filamentous phage Pf5 and its involvement in small-colony formation

Bacteriophages play an important role in bacterial virulence and phenotypic variation. It has been shown that filamentous bacteriophage Pf4 of Pseudomonas aeruginosa strain PAO1 mediates the formation of small-colony variants (SCVs) in biofilms. This morphology type is associated with parameters of poor lung function in cystic fibrosis patients, and SCVs are often more resistant to antibiotics than wild-type cells. P. aeruginosa strain PA14 also contains a Pf1-like filamentous prophage, which is designated Pf5, and is highly homologous to Pf4. Since P. aeruginosa PA14 produces SCVs very efficiently in biofilms grown in static cultures, the role of Pf5 in SCV formation under these conditions was investigated. The presence of the Pf5 replicative form in total DNA from SCVs and wild-type cells was detected, but it was not possible to detect the Pf5 major coat protein by immunoblot analysis in PA14 SCV cultures. This suggests that the Pf5 filamentous phage is not present at high densities in the PA14 SCVs. Consistent with these results, we were unable to detect coaB expression in SCV cultures and SCV colonies. The SCV variants formed under static conditions were not linked to Pf5 phage activity, since Pf5 insertion mutants with decreased or no production of the Pf5 RF produced SCVs as efficiently as the wild-type strain. Finally, analysis of 48 clinical P. aeruginosa isolates showed no association between the presence of Pf1-like filamentous phages and the ability to form SCVs under static conditions; this suggests that filamentous phages are generally not involved in the emergence of P. aeruginosa SCVs

Cooperative secretions facilitate host range expansion in bacteria

The majority of emergent human pathogens are zoonotic in origin, that is, they can transmit to humans from other animals. Understanding the factors underlying the evolution of pathogen host range is therefore of critical importance in protecting human health. There are two main evolutionary routes to generalism: organisms can tolerate multiple environments or they can modify their environments to forms to which they are adapted. Here we use a combination of theory and a phylogenetic comparative analysis of 191 pathogenic bacterial species to show that bacteria use cooperative secretions that modify their environment to extend their host range and infect multiple host species. Our results suggest that cooperative secretions are key determinants of host range in bacteria, and that monitoring for the acquisition of secreted proteins by horizontal gene transfer can help predict emerging zoonoses

CapsID: a web-based tool for developing parsimonious sets of CAPS molecular markers for genotyping

BACKGROUND: Genotyping may be carried out by a number of different methods including direct sequencing and polymorphism analysis. For a number of reasons, PCR-based polymorphism analysis may be desirable, owing to the fact that only small amounts of genetic material are required, and that the costs are low. One popular and cheap method for detecting polymorphisms is by using cleaved amplified polymorphic sequence, or CAPS, molecular markers. These are also known as PCR-RFLP markers. RESULTS: We have developed a program, called CapsID, that identifies snip-SNPs (single nucleotide polymorphisms that alter restriction endonuclease cut sites) within a set or sets of reference sequences, designs PCR primers around these, and then suggests the most parsimonious combination of markers for genotyping any individual who is not a member of the reference set. The output page includes biologist-friendly features, such as images of virtual gels to assist in genotyping efforts. CapsID is freely available at . CONCLUSION: CapsID is a tool that can rapidly provide minimal sets of CAPS markers for molecular identification purposes for any biologist working in genetics, community genetics, plant and animal breeding, forensics and other fields

Repeat bleaching of a central Pacific coral reef over the past six decades (1960–2016)

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Communications Biology 1 (2018): 177, doi:10.1038/s42003-018-0183-7.The oceans are warming and coral reefs are bleaching with increased frequency and severity, fueling concerns for their survival through this century. Yet in the central equatorial Pacific, some of the world’s most productive reefs regularly experience extreme heat associated with El Niño. Here we use skeletal signatures preserved in long-lived corals on Jarvis Island to evaluate the coral community response to multiple successive heatwaves since 1960. By tracking skeletal stress band formation through the 2015-16 El Nino, which killed 95% of Jarvis corals, we validate their utility as proxies of bleaching severity and show that 2015-16 was not the first catastrophic bleaching event on Jarvis. Since 1960, eight severe (>30% bleaching) and two moderate (<30% bleaching) events occurred, each coinciding with El Niño. While the frequency and severity of bleaching on Jarvis did not increase over this time period, 2015–16 was unprecedented in magnitude. The trajectory of recovery of this historically resilient ecosystem will provide critical insights into the potential for coral reef resilience in a warming world.Funding for this study was provided by National Science Foundation awards OCE 1537338, OCE 1605365, and OCE 1031971 to A.L.C., and the Robertson Foundation to A.L.C., National Science Foundation Graduate Research Fellowships to T.M.D. and A.E.A., and a National Defense Science and Engineering Graduate Fellowship to H.E.R

QTL dynamics for fruit firmness and softening around an ethylene-dependent polygalacturonase gene in apple (Malus×domestica Borkh.)

Apple fruit are well known for their storage life, although a wide range of flesh softening occurs among cultivars. Loss of firmness is genetically coordinated by the action of several cell wall enzymes, including polygalacturonase (PG) which depolymerizes cell wall pectin. By the analysis of ‘Fuji’ (Fj) and ‘Mondial Gala’ (MG), two apple cultivars characterized by a distinctive ripening behaviour, the involvement of Md-PG1 in the fruit softening process was confirmed to be ethylene dependent by its transcript being down-regulated by 1-methylcyclopropene treatment in MG and in the low ethylene-producing cultivar Fj. Comparing the PG sequence of MG and Fj, a single nucleotide polymorphism (SNP) was discovered. Segregation of the Md-PG1SNP marker within a full-sib population, obtained by crossing Fj and MG, positioned Md-PG1 in the linkage group 10 of MG, co-located with a quantitative trait locus (QTL) identified for fruit firmness in post-harvest ripening. Fruit firmness and softening analysed in different stages, from harvest to post-storage, determined a shift of the QTL from the top of this linkage group to the bottom, where Md-ACO1, a gene involved in ethylene biosynthesis in apple, is mapped. This PG–ethylene-related gene has beeen positioned in the apple genome on chromosome 10, which contains several QTLs controlling fruit firmness and softening, and the interplay among the allelotypes of the linked loci should be considered in the design of a marker-assisted selection breeding scheme for apple texture

A High Throughput Amenable Arabidopsis-P. aeruginosa System Reveals a Rewired Regulatory Module and the Utility to Identify Potent Anti-Infectives

We previously demonstrated that in a metasystem consisting of Arabidopsis seedlings growing in liquid medium (in 96 well plates) even microbes considered to be innocuous such as laboratory strains of E. coli and B. subtilis can cause potent damage to the host. We further posited that such environment-induced adaptations are brought about by ‘system status changes’ (rewiring of pre-existing cellular signaling networks and components) of the host and the microbe, and that prolongation of such a situation could lead to the emergence of pathogenic states in real-life. Here, using this infection model, we show that the master regulator GacA of the human opportunistic pathogen P. aeruginosa (strain PA14) is dispensable for pathogenesis, as evidenced by three independent read-outs. The gene expression profile of the host after infection with wild type PA14 or the gacA mutant are also identical. GacA normally acts upstream of the quorum sensing regulatory circuit (that includes the regulator LasR) that controls a subset of virulence factors. Double mutants in gacA and lasR behave similar to the lasR mutant, as seen by abrogation of a characteristic cell type specific host cell damage caused by PA14 or the gacA mutant. This indicates that a previously unrecognized regulatory mechanism is operative under these conditions upstream of LasR. In addition, the detrimental effect of PA14 on Arabidopsis seedlings is resistant to high concentrations of the aminoglycoside antibiotic gentamicin. These data suggest that the Arabidopsis seedling infection system could be used to identify anti-infectives with potentially novel modes of action

Selection for antimicrobial resistance is reduced when embedded in a natural microbial community

This is the final version. Available from Springer Nature via the DOI in this record.Antibiotic resistance has emerged as one of the most pressing, global threats to public health. In single-species experiments selection for antibiotic resistance occurs at very low antibiotic concentrations. However, it is unclear how far these findings can be extrapolated to natural environments, where species are embedded within complex communities. We competed isogenic strains of Escherichia coli, differing exclusively in a single chromosomal resistance determinant, in the presence and absence of a pig faecal microbial community across a gradient of antibiotic concentration for two relevant antibiotics: gentamicin and kanamycin. We show that the minimal selective concentration was increased by more than one order of magnitude for both antibiotics when embedded in the community. We identified two general mechanisms were responsible for the increase in minimal selective concentration: an increase in the cost of resistance and a protective effect of the community for the susceptible phenotype. These findings have implications for our understanding of the evolution and selection of antibiotic resistance, and can inform future risk assessment efforts on antibiotic concentrations.Medical Research Council (MRC)European Commissio

Biofilm Induced Tolerance towards Antimicrobial Peptides

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms

Species Difference of CD137 Ligand Signaling in Human and Murine Monocytes

BACKGROUND: Stimulation of CD137 ligand on human monocytes has been shown to induce DC differentiation, and these CD137L-DCs are more potent than classical DCs, in stimulating T cell responses in vitro. To allow an in vivo evaluation of the potency of CD137L-DCs in murine models we aimed at generating murine CD137L-DCs. METHODOLOGY/PRINCIPAL FINDINGS: When stimulated through CD137 ligand murine monocytes responded just as human monocytes with an increased adherence, morphological changes, proliferation and an increase in viable cell numbers. But CD137 ligand signaling did not induce expression of inflammatory cytokines and costimulatory molecules in murine monocytes and these cells had no T cell stimulatory activity. Murine monocytes did not differentiate to inflammatory DCs upon CD137 ligand signaling. Furthermore, while CD137 ligand signaling induces maturation of human immature classical DCs it failed to do so with murine immature classical DCs. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that both human and murine monocytes become activated by CD137 ligand signaling but only human and not murine monocytes differentiate to inflammatory DCs

Gene-Boosted Assembly of a Novel Bacterial Genome from Very Short Reads

Recent improvements in technology have made DNA sequencing dramatically faster and more efficient than ever before. The new technologies produce highly accurate sequences, but one drawback is that the most efficient technology produces the shortest read lengths. Short-read sequencing has been applied successfully to resequence the human genome and those of other species but not to whole-genome sequencing of novel organisms. Here we describe the sequencing and assembly of a novel clinical isolate of Pseudomonas aeruginosa, strain PAb1, using very short read technology. From 8,627,900 reads, each 33 nucleotides in length, we assembled the genome into one scaffold of 76 ordered contiguous sequences containing 6,290,005 nucleotides, including one contig spanning 512,638 nucleotides, plus an additional 436 unordered contigs containing 416,897 nucleotides. Our method includes a novel gene-boosting algorithm that uses amino acid sequences from predicted proteins to build a better assembly. This study demonstrates the feasibility of very short read sequencing for the sequencing of bacterial genomes, particularly those for which a related species has been sequenced previously, and expands the potential application of this new technology to most known prokaryotic species