Xanthomonas euvesicatoria pv. rosa strain Xer07 causes a leaf spot on a Rosa sp. and is closely related to X. euvesicatoria pv. euvesicatoria (Xee) and X. perforans (Xp), causal agents of bacterial spot of tomato. However, Xer07 is not pathogenic on tomato and elicits a hypersensitive reaction (HR). We compared the genomes of the three bacterial species to identify the factors that limit Xer07 on tomato. Comparison of pathogenicity associated factors including the type III secretion systems identified two genes, xopA and xer3856, in Xer07 that have lower sequence homology in tomato pathogens. xer3856 is a homolog of genes in X. citri (xac3856) and X. fuscans pv. aurantifolii, both of which have been reported to elicit HRs in tomato. When xer3856 was expressed in X. perforans and infiltrated in tomato leaflets, the transconjugant elicited an HR and significantly reduced bacterial populations compared to the wildtype X. perforans strain. When xer3856 was mutated in Xer07, the mutant strain still triggered an HR in tomato leaflets. The second gene identified codes for type III secreted effector XopA, which contains a harpin domain that is distinct from the xopA homologs in Xee and Xp. The Xer07-xopA, when expressed in X. perforans, did not elicit an HR in tomato leaflets, but significantly reduced bacterial populations. This indicates that xopA and xer3856 genes in combination with an additional factor(s) limit Xer07 in tomato.

Internal dynamics of proteins can play a critical role in the biological function of some proteins. Several well documented instances have been reported such as MBP, DHFR, hTS, DGCR8, and NSP1 of the SARS-CoV family of viruses. Despite the importance of internal dynamics of proteins, there currently are very few approaches that allow for meaningful separation of internal dynamics from structural aspects using experimental data. Here we present a computational approach named REDCRAFT that allows for concurrent characterization of protein structure and dynamics. Here, we have subjected DHFR (PDB-ID 1RX2), a 159-residue protein, to a fictitious, mixed mode model of internal dynamics. In this simulation, DHFR was segmented into 7 regions where 4 of the fragments were fixed with respect to each other, two regions underwent rigid-body dynamics, and one region experienced uncorrelated and melting event. The two dynamical and rigid-body segments experienced an average orientational modification of 7 degrees and 12 degrees respectively. Observable RDC data for backbone C '-N, N-H-N, and C '-H-N were generated from 102 uniformly sampled frames that described the molecular trajectory. The structure calculation of DHFR with REDCRAFT by using traditional Ramachandran restraint produced a structure with 29 angstrom of structural difference measured over the backbone atoms (bb-rmsd) over the entire length of the protein and an average bb-rmsd of more than 4.7 angstrom over each of the dynamical fragments. The same exercise repeated with context-specific dihedral restraints generated by PDBMine produced a structure with bb-rmsd of 21 angstrom over the entire length of the protein but with bb-rmsd of less than 3 angstrom over each of the fragments. Finally, utilization of the Dynamic Profile generated by REDCRAFT allowed for the identification of different dynamical regions of the protein and the recovery of individual fragments with bb-rmsd of less than 1 angstrom. Following the recovery of the fragments, our assembly procedure of domains (larger segments consisting of multiple fragments with a common dynamical profile) correctly assembled the four fragments that are rigid with respect to each other, categorized the two domains that underwent rigid-body dynamics, and identified one dynamical region for which no conserved structure could be defined. In conclusion, our approach was successful in identifying the dynamical domains, recovery of structure where it is meaningful, and relative assembly of the domains when possible.

Mycobacterium phages Mikro, Yorick, Virgeve, and MelsMeow were isolated from soil in Rock Hill, South Carolina. Mikro is a myovirus with a comparatively large genome of 157,166bp. The remainder are siphoviruses with genome lengths ranging from 59,227bp to 68,563bp. All phages were isolated on Mycobacterium smegmatis.

Extracellular Vesicles, also referred to as EVs, are spherical lipid membrane-bound vesicles produced by both Gram positive and Gram negative bacteria. These vesicles are secreted into the extracellular space and play important roles in cellular and host communication, elimination of competitors, virulence, detoxification of environmental stress, and nutrient sensing. They are often packed with proteins, enzymes, lipids, nucleic acids and other biomolecules. Streptococcus thermophilus is a lactic acid bacteria (LAB), inhabiting the human digestive tract, that has been shown to produce EVs. The bacterial florae are known to influence the host immune system, metabolism, and neurological processes, but little is known about the biochemical pathways involved. Since EVs are involved in host communication, they may play a key role in affecting the biochemical processes of the host. To study the content and potential effects of EVs, we have grown different LAB strains under anaerobic conditions, including S. thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus. Our goal is to isolate EVs from these LAB strains and identify small regulatory RNA (sRNA) molecules that mediate communication and biochemical effects on the host systems. As part of this study, we have begun to identify sRNA genes in LAB strains that may mediate communication with other bacteria or the host. In S. thermophilus, we have focused on the AsdS sRNA transcript, that is 152 base pairs in length, and is involved in quorum sensing. This gene is conserved among other streptococcal species and in the human pathogen, S. pyogenes, a homologous sRNA, known as MarS, is associated with the regulation of virulence factors. Since S. thermophilus is a non-pathogenic species, the AsdS gene cannot be involved in virulence, but based on functional bioinformatics predictions, it may be responsible for intraspecies communication, biofilm formation, and transport processes. Our current studies aim to identify additional sRNA transcripts that may be found in EVs and mediate effects on the host systems. © FASEB.

Streptococcus pyogenes is a human pathogen that is also known as group A Streptococcus (GAS), which is responsible for the development of sore throats and skin infections as well as the more severe condition, necrotizing fasciitis. The streptolysin associated gene A, sagA, codes for the protein streptolysin A (SagA), which is modified and exported as the cytotoxin streptolysin S (SLS). SLS is a virulence factor that inhibits the response of neutrophils during necrotizing fasciitis. The sagA gene is contained within the pleiotropic effect locus (pel). Previous studies have identified sagA as both the gene for the streptolysin S peptide, and the small regulatory RNA (sRNA), known as Pel, which influences expression of multiple virulence genes. The 5' UTR of sagA/Pel contains the regions necessary for both transcription initiation and ribosomal binding for translation. Focusing on the 5' UTR of sagA, we hypothesize that the RNA structure of this region affects the transcription of sagA and the initiation of translation. Our goal is to characterize the secondary and 3D structure of the sagA/Pel mRNA/sRNA, which will allow for a better understanding for the role it plays in streptococcal pathogenesis. In this study, RNA constructs containing predicted structured regions were produced by in vitro transcription. Secondary structures were predicted using the mfold and RNAfold webserver based programs. The predicted secondary structures were used to generate three-dimensional models with the program FARFAR through the ROSIE webserver. Differential scanning fluorimetry was also used to assess the structural stability of the RNA constructs and to screen a variety of conditions to be used in crystallization studies. These experiments provided evidence that the 5' UTR of sagA/Pel contains significant structured regions and optimal conditions were identified for future structural studies. Potential interactions between the sagA/Pel sRNA and regulated mRNA targets were predicted using the TargetRNA2 webserver. The potential sites for recognition were used to evaluate the functional significance of the structured regions within the 5' UTR of sagA/Pel. Identification of structural motifs necessary for regulating the expression of virulence genes will aid in the design of therapeutic strategies to inhibit the production of the virulence factors, such as SLS. In future studies, we plan to use structural techniques such as NMR spectroscopy and X-ray crystallography to help further understand the role that sagA/Pel plays in streptococcal diseases. © FASEB.