Genomic Analyses of Clonal Isolates Provide Clues to the Evolution of Streptococcus pneumoniae

A commentary on Rapid pneumococcal evolution in response to clinical intervention

High-Level Macrolide Resistance Due to the Mega Element [mef(E)/mel] in Streptococcus pneumoniae

Transferable genetic elements conferring macrolide resistance in Streptococcus pneumoniae can encode the efflux pump and ribosomal protection protein, mef(E)/mel, in an operon of the macrolide efflux genetic assembly (Mega) element- or induce ribosomal methylation through a methyltransferase encoded by erm(B). During the past 30 years, strains that contain Mega or erm(B) or both elements on Tn2010 and other Tn916-like composite mobile genetic elements have emerged and expanded globally. In this study, we identify and define pneumococcal isolates with unusually high-level macrolide resistance (MICs > 16 μg/ml) due to the presence of the Mega element [mef(E)/mel] alone. High-level resistance due to mef(E)/mel was associated with at least two specific genomic insertions of the Mega element, designated Mega-2.IVa and Mega-2.IVc. Genome analyses revealed that these strains do not possess erm(B) or known ribosomal mutations. Deletion of mef(E)/mel in these isolates eliminated macrolide resistance. We also found that Mef(E) and Mel of Tn2010-containing pneumococci were functional but the high-level of macrolide resistance was due to Erm(B). Using in vitro competition experiments in the presence of macrolides, high-level macrolide-resistant S. pneumoniae conferred by either Mega-2.IVa or erm(B), had a growth fitness advantage over the lower-level, mef(E)/mel-mediated macrolide-resistant S. pneumoniae phenotypes. These data indicate the ability of S. pneumoniae to generate high-level macrolide resistance by macrolide efflux/ribosomal protection [Mef(E)/Mel] and that high-level resistance regardless of mechanism provides a fitness advantage in the presence of macrolides

\u3ci\u3ePseudomonas syringae\u3c/i\u3e Type III Chaperones ShcO1, ShcS1, and ShcS2 Facilitate Translocation of Their Cognate Effectors and Can Substitute for Each Other in the Secretion of HopO1-1

The Pseudomonas syringae type III secretion system (TTSS) translocates effector proteins into plant cells. Several P. syringae effectors require accessory proteins called type III chaperones (TTCs) to be secreted via the TTSS. We characterized the hopO1-1, hopS1, and hopS2 operons in P. syringae pv. tomato DC3000; these operons encode three homologous TTCs, ShcO1, ShcS1, and ShcS2. ShcO1, ShcS1, and ShcS2 facilitated the type III secretion and/or translocation of their cognate effectors HopO1-1, HopS1, and HopS2, respectively. ShcO1 and HopO1-1 interacted with each other in yeast two-hybrid and coimmunoprecipitation assays. Interestingly, ShcS1 and ShcS2 were capable of substituting for ShcO1 in facilitating HopO1-1 secretion and translocation and each TTC was able to bind the other’s cognate effectors in yeast two-hybrid assays. Moreover, ShcO1, ShcS1, and ShcS2 all bound to the middle-third region of HopO1-1. The HopS2 effector possessed atypical P. syringae TTSS N-terminal characteristics and was translocated in low amounts. A site-directed HopS2 mutation that introduced a common N-terminal characteristic from other P. syringae type III secreted substrates increased HopS2 translocation, supporting the idea that this characteristic functions as a secretion signal. Additionally, hopO1-2 and hopT1-2 were shown to encode effectors secreted via the DC3000 TTSS. Finally, a DC3000 hopO1-1 operon deletion mutant produced disease symptoms similar to those seen with wild-type DC3000 but was reduced in its ability to multiply in Arabidopsis thaliana. The existence of TTCs that can bind to dissimilar effectors and that can substitute for each other in effector secretion provides insights into the nature of how TTCs function

Loss of coral reef growth capacity to track future increases in sea level

Water-depths above coral reefs is predicted to increase due to global sea-level rise (SLR). As ecological degradation inhibits the vertical accretion of coral reefs, it is likely that coastal wave exposure will increase but there currently exists a lack of data in projections concerning local rates of reef growth and local SLR. In this study we have aggregated ecological data of more than 200 tropical western Atlantic and Indian Ocean reefs and calculated their vertical growth which we have then compared with recent and projected rates of SLR across different Representative Concentration Pathway (RCP) scenarios. While many reefs currently show vertical growth that would be sufficient to keep-up with recent historic SLR, future projections under scenario RCP4.5 reveal that without substantial ecological recovery many reefs will not have the capacity to track SLR. Under RCP8.5, we predict that mean water depth will increase by over half a metre by 2100 across the majority of reefs. We found that coral cover strongly predicted whether a reef could track SLR, but that the majority of reefs had coral cover significantly lower than that required to prevent reef submergence. To limit reef submergence, and thus the impacts of waves and storms on adjacent coasts, climate mitigation and local impacts that reduce coral cover (e.g., local pollution and physical damage through development land reclamation) will be necessary

\u3ci\u3ePseudomonas syringae\u3c/i\u3e Type III Chaperones ShcO1, ShcS1, and ShcS2 Facilitate Translocation of Their Cognate Effectors and Can Substitute for Each Other in the Secretion of HopO1-1

The Pseudomonas syringae type III secretion system (TTSS) translocates effector proteins into plant cells. Several P. syringae effectors require accessory proteins called type III chaperones (TTCs) to be secreted via the TTSS. We characterized the hopO1-1, hopS1, and hopS2 operons in P. syringae pv. tomato DC3000; these operons encode three homologous TTCs, ShcO1, ShcS1, and ShcS2. ShcO1, ShcS1, and ShcS2 facilitated the type III secretion and/or translocation of their cognate effectors HopO1-1, HopS1, and HopS2, respectively. ShcO1 and HopO1-1 interacted with each other in yeast two-hybrid and coimmunoprecipitation assays. Interestingly, ShcS1 and ShcS2 were capable of substituting for ShcO1 in facilitating HopO1-1 secretion and translocation and each TTC was able to bind the other’s cognate effectors in yeast two-hybrid assays. Moreover, ShcO1, ShcS1, and ShcS2 all bound to the middle-third region of HopO1-1. The HopS2 effector possessed atypical P. syringae TTSS N-terminal characteristics and was translocated in low amounts. A site-directed HopS2 mutation that introduced a common N-terminal characteristic from other P. syringae type III secreted substrates increased HopS2 translocation, supporting the idea that this characteristic functions as a secretion signal. Additionally, hopO1-2 and hopT1-2 were shown to encode effectors secreted via the DC3000 TTSS. Finally, a DC3000 hopO1-1 operon deletion mutant produced disease symptoms similar to those seen with wild-type DC3000 but was reduced in its ability to multiply in Arabidopsis thaliana. The existence of TTCs that can bind to dissimilar effectors and that can substitute for each other in effector secretion provides insights into the nature of how TTCs function

Induction of Efflux-Mediated Macrolide Resistance in Streptococcus pneumoniae ▿

The antimicrobial efflux system encoded by the operon mef(E)-mel on the mobile genetic element MEGA in Streptococcus pneumoniae and other Gram-positive bacteria is inducible by macrolide antibiotics and antimicrobial peptides. Induction may affect the clinical response to the use of macrolides. We developed mef(E) reporter constructs and a disk diffusion induction and resistance assay to determine the kinetics and basis of mef(E)-mel induction. Induction occurred rapidly, with a >15-fold increase in transcription within 1 h of exposure to subinhibitory concentrations of erythromycin. A spectrum of environmental conditions, including competence and nonmacrolide antibiotics with distinct cellular targets, did not induce mef(E). Using 16 different structurally defined macrolides, induction was correlated with the amino sugar attached to C-5 of the macrolide lactone ring, not with the size (e.g., 14-, 15- or 16-member) of the ring or with the presence of the neutral sugar cladinose at C-3. Macrolides with a monosaccharide attached to C-5, known to block exit of the nascent peptide from the ribosome after the incorporation of up to eight amino acids, induced mef(E) expression. Macrolides with a C-5 disaccharide, which extends the macrolide into the ribosomal exit tunnel, disrupting peptidyl transferase activity, did not induce it. The induction of mef(E) did not require macrolide efflux, but the affinity of macrolides for the ribosome determined the availability for efflux and pneumococcal susceptibility. The induction of mef(E)-mel expression by inducing macrolides appears to be based on specific interactions of the macrolide C-5 saccharide with the ribosome that alleviate transcriptional attenuation of mef(E)-mel

Survival of GacS/GacA Mutants of the Biological Control Bacterium Pseudomonas aureofaciens 30-84 in the Wheat Rhizosphere

GacS/GacA comprises a two-component regulatory system that controls the expression of secondary metabolites required for the control of plant diseases in many pseudomonads. High mutation frequencies of gacS and gacA have been observed in liquid culture. We examined whether gacS/gacA mutants could competitively displace the wild-type populations on roots and thus pose a threat to the efficacy of biological control. The survival of a gac mutant alone and in competition with the wild type on roots was examined in the biological control strain Pseudomonas aureofaciens 30-84. In this bacterium, GacS/GacA controls the expression of phenazine antibiotics that are inhibitory to plant pathogenic fungi and enhance the competitive survival of the bacterium. Wheat seedlings were inoculated with strain 30-84, and bacteria were recovered from roots after 21 days in sterile or nonsterile soil to check for the presence of gacS or gacA mutants. Although no mutants were detected in the inoculum, gacS/gacA mutants were recovered from 29 out of 31 roots and comprised up to 36% of the total bacterial populations. Southern hybridization analysis of the recovered gacA mutants did not indicate a conserved mutational mechanism. Replacement series analysis on roots utilizing strain 30-84 and a gacA mutant (30-84.gacA) or a gacS mutant (30-84.A2) demonstrated that although the mutant population partially displaced the wild type in sterile soil, it did not do so in natural soil. In fact, in natural soil final rhizosphere populations of wild-type strain 30-84 starting from mixtures were at least 1.5 times larger than would be predicted from their inoculation ratio and generally were greater than or equal to the population of wild type alone despite lower inoculation rates. These results indicate that although gacS/gacA mutants survive in natural rhizosphere populations, they do not displace wild-type populations. Better survival of wild-type populations in mixtures with mutants suggests that mutants arising de novo or introduced within the inoculum may be beneficial for the survival of wild-type populations in the rhizosphere

Composite mobile genetic elements disseminating macrolide resistance in Streptococcus pneumoniae

Macrolide resistance in Streptococcus pneumoniae emerged in the US and globally during the early 1990’s. The RNA methylase encoded by erm(B) and the macrolide efflux genes mef(E) and mel were identified as the resistance determining factors. These genes are disseminated in the pneumococcus on mobile, often chimeric elements consisting of multiple smaller elements. To better understand the variety of elements encoding macrolide resistance and how they have evolved in the pre- and post-conjugate vaccine eras, the genomes of 121 invasive and ten carriage isolates from Atlanta from 1994-2011 were analyzed for mobile elements involved in the dissemination of macrolide resistance. The isolates were selected to provide broad coverage of the genetic variability of antibiotic resistant pneumococci and included 100 invasive isolates resistant to macrolides. Tn916-like elements carrying mef(E) and mel on the Macrolide Genetic Assembly (Mega) and erm(B) on the erm(B) element and Tn917 were integrated into the pneumococcal chromosome backbone and into larger Tn5253-like composite elements. The results reported here include identification of novel insertion sites for Mega and characterization of the insertion sites of Tn916-like elements in the pneumococcal chromosome and in larger composite elements were characterized. The data indicate that integration of elements by conjugation was infrequent compared to recombination. Thus it appears that conjugative mobile elements allow the pneumococcus to acquire DNA from distantly related bacteria, but once integrated into a pneumococcal genome, transformation and recombination is the primary mechanism for transmission of novel DNA throughout the pneumococcal population