The D-ring, Not the A-ring, Rotates in Synechococcus OS-B' Phytochrome

Phytochrome photoreceptors in plants and microorganisms switch photochromically between two states, controlling numerous important biological processes. Although this phototransformation is generally considered to involve rotation of ring D of the tetrapyrrole chromophore, Ulijasz et al. (Ulijasz, A. T., Cornilescu, G., Cornilescu, C. C., Zhang, J., Rivera, M., Markley, J. L., and Vierstra, R. D. (2010) Nature 463, 250–254) proposed that the A-ring rotates instead. Here, we apply magic angle spinning NMR to the two parent states following studies of the 23-kDa GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain fragment of phytochrome from Synechococcus OS-B′. Major changes occur at the A-ring covalent linkage to the protein as well as at the protein residue contact of ring D. Conserved contacts associated with the A-ring nitrogen rule out an A-ring photoflip, whereas loss of contact of the D-ring nitrogen to the protein implies movement of ring D. Although none of the methine bridges showed a chemical shift change comparable with those characteristic of the D-ring photoflip in canonical phytochromes, denaturation experiments showed conclusively that the same occurs in Synechococcus OS-B′ phytochrome upon photoconversion. The results are consistent with the D-ring being strongly tilted in both states and the C15=C16 double bond undergoing a Z/E isomerization upon light absorption. More subtle changes are associated with the A-ring linkage to the protein. Our findings thus disprove A-ring rotation and are discussed in relation to the position of the D-ring, photoisomerization, and photochromicity in the phytochrome family

Deletion of the Zinc Transporter Lipoprotein AdcAII Causes Hyperencapsulation of Streptococcus pneumoniae Associated with Distinct Alleles of the Type I Restriction-Modification System.

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated ΔadcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the ΔadcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated ΔadcAII strains. However, transformation of ΔadcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated ΔadcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of ΔadcAII Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the ΔadcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and ΔadcAII, further investigation of which could further characterize mechanisms of capsule regulation

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus

Structural basis for the photoconversion of a phytochrome to the activated far-red light-absorbing form

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red light-absorbing, ground state Pr and a far-red light-absorbing, photoactivated state Pfr1,2. While the structures of several phytochromes as Pr have been determined3-7, little is known about the structure of Pfr and how it initiates signaling. Here, we describe the three-dimensional solution structure of the bilin-binding domain as Pfr using the cyanobacterial phytochrome from Synechococcus OSB’. Contrary to predictions, light-induced rotation of the A but not the D pyrrole ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intra- and interdomain contact sites within the phytochrome dimer. From our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer

Transcriptional Regulation of the Capsular Polysaccharide Biosynthesis Locus of Streptococcus Pneumoniae: a Bioinformatic Analysis

The polysaccharide capsule of Streptococcus pneumoniae is the main virulence factor, which makes the bacterium resistant to phagocytosis. Expression of capsular polysaccharide must be adjusted at different stages of pneumococcal infection, thus, their transcriptional regulation appears to be crucial. To get insight into the existence of regulatory mechanisms common to most serotypes, a bioinformatic analysis of the DNA region located upstream of the capsular locus was performed. With the exception of serotype 37, the capsular locus is located between dexB and aliA on the pneumococcal chromosome. Up to 26 different sequence organizations were found among pneumococci synthesizing their capsule through a Wzy-polymerase-dependent mechanism, mostly varying according to the presence/absence of distinct insertion elements. As a consequence, only ∼250 bp (including a 107 bp RUP_A element) was conserved in 86 sequences, although only a short (ca. 87 bp) region located immediately upstream of cpsA was strictly conserved in all the sequences analyzed. An exhaustive search for possible operator sequences was done. Interestingly, although the promoter region of serotype 3 isolates completely differs from that of other serotypes, most of the proteins proposed to regulate transcription in serotype 3 pneumococci were also predicted to function as possible regulators in non-serotype 3 S. pneumoniae isolates

Structure-function analysis for the development of peptide inhibitors for a Gram-positive quorum sensing system

open access articleThe Streptococcus pneumoniae Rgg144/SHP144 regulator-peptide quorum sensing (QS) system is critical for nutrient utilization, oxidative stress response, and virulence. Here, we characterized this system by assessing the importance of each residue within the active short hydrophobic peptide (SHP) by alanine-scanning mutagenesis and testing the resulting peptides for receptor binding and activation of the receptor. Interestingly, several of the mutations had little effect on binding to Rgg144 but reduced transcriptional activation appreciably. In particular, a proline substitution (P21A) reduced transcriptional activation by 29-fold but bound with a 3-fold higher affinity than the wild-type SHP. Consistent with the function of Rgg144, the mutant peptide led to decreased utilization of mannose and increased susceptibility to superoxide generator paraquat. Pangenome comparison showed full conservation of P21 across SHP144 allelic variants. Crystallization of Rgg144 in the absence of peptide revealed a comparable structure to the DNA bound and free forms of its homologs suggesting similar mechanisms of activation. Together, these analyses identify key interactions in a critical pneumococcal QS system. Further manipulation of the SHP has the potential to facilitate the development of inhibitors that are functional across strains. The approach described here is likely to be effective across QS systems in multiple species

Reciprocal Regulation of Cephalosporin Resistance in Enterococcus faecalis

Antibiotic-resistant enterococci are major causes of hospital-acquired infections and therefore represent a serious public health problem. One well-known risk factor for the acquisition of hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporin antibiotics. Enterococci can proliferate in patients undergoing cephalosporin therapy due to intrinsic cephalosporin resistance, a characteristic of the genus Enterococcus. However, the molecular basis for cephalosporin resistance in E. faecalis has yet to be adequately elucidated. Previously we determined that a putative Ser/Thr kinase, IreK (formerly PrkC), is required for intrinsic cephalosporin resistance in E. faecalis. Here we show that kinase activity is required for cephalosporin resistance and, further, that resistance in E. faecalis is reciprocally regulated by IreK and IreP, a PP2C-type protein phosphatase encoded immediately upstream of IreK. Mutants of two divergent lineages of E. faecalis lacking IreP exhibit remarkable hyperresistance to cephalosporins but not to antibiotics targeting other cellular processes. Further genetic analyses indicate that hyperresistance of the IreP mutant is mediated by the IreK kinase. Additionally, competition experiments reveal that hyperresistant ΔireP mutants exhibit a substantial fitness defect in the absence of antibiotics, providing an evolutionary rationale for the use of a complex signaling system to control intrinsic cephalosporin resistance. These results support a model in which IreK and IreP act antagonistically via protein phosphorylation and dephosphorylation as part of a signal transduction circuit to regulate cellular adaptation to cephalosporin-induced stress

Tightening the knot in phytochrome by single molecule atomic force microscopy

A growing number of proteins have been shown to adopt knotted folds. Yet the biological roles and biophysical properties of these knots remain poorly understood. We have used protein engineering and atomic force microscopy to explore single-molecule mechanics of the figure-of-eight knot in the chromophore-binding domain of the red/far red photoreceptor, phytochrome. Under load, apo phytochrome unfolds at forces of ~47 pN, while phytochrome carrying its covalently bound tetrapyrrole chromophore unfolds at ~73 pN. These forces are among the lowest measured in mechanical protein unfolding, hence the presence of the knot does not automatically indicate a super-stable protein. Our experiments reveal a stable intermediate along the mechanical unfolding pathway, reflecting sequential unfolding of two distinct subdomains in phytochrome, potentially the GAF and PAS domains. For the first time, our experiments allow direct determination of knot size under load. In the unfolded chain, the tightened knot is reduced to 17 amino acids, resulting in apparent shortening of the polypeptide chain by 6.2 nm. Steered molecular dynamics simulations corroborate this number. Finally, we found that covalent phytochrome dimers created for these experiments retain characteristic photoreversibility, unexpectedly arguing against dramatic rearrangement of the native GAF dimer interface upon photoconversion.Comment: 12 pages plus five figures; has been submitted to Biophysical J. Replacement on 9/16 is ONLY to correct a typo in the meta data; the uploaded file is identical to first versio

Interplay between manganese and iron in pneumococcal pathogenesis: role of the orphan response regulator RitR

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that is carried asymptomatically in the nasopharynx by up to 70% of the human population. Translocation of the bacteria into internal sites can cause a range of diseases, such as pneumonia, otitis media, meningitis, and bacteremia. This transition from nasopharynx to growth at systemic sites means that the pneumococcus needs to adjust to a variety of environmental conditions, including transition metal ion availability. Although it is an important nutrient, iron potentiates oxidative stress, and it is established that in S. pneumoniae, expression of iron transport systems and proteins that protect against oxidative stress are regulated by an orphan response regulator, RitR. In this study, we investigated the effect of iron and manganese ion availability on the growth of a ritR mutant. Deletion of ritR led to impaired growth of bacteria in high-iron medium, but this phenotype could be suppressed with the addition of manganese. Measurement of metal ion accumulation indicated that manganese prevents iron accumulation. Furthermore, the addition of manganese also led to a reduction in the amount of hydrogen peroxide produced by bacterial cells. Studies of virulence in a murine model of infection indicated that RitR was not essential for pneumococcal survival and suggested that derepression of iron uptake systems may enhance the survival of pneumococci in some niches

Cyanobacteria in motion

This work was supported by a grant from the DFG (WI 2014/7-1) to A.W