Contribution of teg49 Small RNA in the 5′ Upstream Transcriptional Region of sarA to Virulence in Staphylococcus aureus

High-throughput RNA sequencing technology has found the 5\u27 untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA

Contribution of teg49 Small RNA in the 5′ Upstream Transcriptional Region of sarA to Virulence in Staphylococcus aureus

High-throughput RNA sequencing technology has found the 5\u27 untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA

Multiplex SSR-PCR Analysis of Genetic Diversity and Redundancy in the Philippine Rice (Oryza sativa L.) Germplasm Collection

Rice germplasm conservation is vital to ensuring the availability of a rich gene pool for future varietal improvement programs. However, with limited resources for gene banking, there is a need to identify and prioritize unique accessions in the PhilRice gene bank for maximum resource utilization. A robust and unequivocal way to identify duplicates is through multiplex SSR-PCR DNA fingerprinting. The present study established the optimal concentrations and reaction conditions for the successful amplification of PCR products using a multiplex panel composed of rice simple sequence repeat (SSR) markers, namely RM312, RM316, RM514 and RM171. The panel was then used to analyze the genetic diversity and identify duplicates among the 427 rice germplasm accessions with similar or identical variety names from the PhilRice genebank. A total of 15 alleles were detected at the 4 SSR loci. The polymorphism information content (PIC) values of the SSR markers were moderately high ranging from 0.459 to 0.643. A dendrogram was constructed using the Dice similarity coefficient and the UPGMA algorithm. The multiplex SSR-PCR panel produced unique profiles of 31 accessions that, being genetically distinct, should be maintained as part of the main collection of the genebank. There were 17 accessions identified as possible redundants having a bootstrap value greater than 95%. Additional SSR and morphological markers will be required to further strengthen the evidence for redundancy, and hence justify removal of unnecessary duplicates from the collection

Coordinated Regulation by AgrA, SarA, and SarR To Control agr Expression in Staphylococcus aureus

The agr locus of Staphylococcus aureus is composed of two divergent transcripts (RNAII and RNAIII) driven by the P2 and P3 promoters. The P2-P3 intergenic region comprises the SarA/SarR binding sites and the four AgrA boxes to which AgrA binds. We reported here the role of AgrA, SarA, and SarR on agr P2 and P3 transcription. Using real-time reverse transcription (RT)-PCR and promoter fusion studies with selected single, double, triple, and complemented mutants, we showed that AgrA is indispensable to agr P2 and P3 transcription, whereas SarA activates and SarR represses P2 transcription. In vitro runoff transcription assays revealed that AgrA alone promoted transcription from the agr P2 promoter, with SarA enhancing it and SarR inhibiting agr P2 transcription in the presence of AgrA or with SarA and AgrA. Electrophoretic mobility shift assay (EMSA) analysis disclosed that SarR binds more avidly to the agr promoter than SarA and displaces SarA from the agr promoter. Additionally, SarA and AgrA bend the agr P2 promoter, whereas SarR does not. Collectively, these data indicated that AgrA activates agr P2 and P3 promoters while SarA activates the P2 promoter, presumably via bending of promoter DNA to bring together AgrA dimers to facilitate engagement of RNA polymerase (RNAP) to initiate transcription

The Global Regulator Spx Functions in the Control of Organosulfur Metabolism in Bacillus subtilis

Spx is a global transcriptional regulator of the oxidative stress response in Bacillus subtilis. Its target is RNA polymerase, where it contacts the α subunit C-terminal domain. Recently, evidence was presented that Spx participates in sulfate-dependent control of organosulfur utilization operons, including the ytmI, yxeI, ssu, and yrrT operons. The yrrT operon includes the genes that function in cysteine synthesis from S-adenosylmethionine through intermediates S-adenosylhomocysteine, ribosylhomocysteine, homocysteine, and cystathionine. These operons are also negatively controlled by CymR, the repressor of cysteine biosynthesis operons. All of the operons are repressed in media containing cysteine or sulfate but are derepressed in medium containing the alternative sulfur source, methionine. Spx was found to negatively control the expression of these operons in sulfate medium, in part, by stimulating the expression of the cymR gene. In addition, microarray analysis, monitoring of yrrT-lacZ fusion expression, and in vitro transcription studies indicate that Spx directly activates yrrT operon expression during growth in medium containing methionine as sole sulfur source. These experiments have uncovered additional roles for Spx in the control of gene expression during unperturbed, steady-state growth

The Staphylococcus-Specific Gene rsr Represses agr and Virulence in Staphylococcus aureus▿

The expression of virulence factors in Staphylococcus aureus is tightly coordinated by a vast network of regulatory molecules. In this report, we characterize a genetic locus unique to staphylococci called rsr that has a role in repressing two key virulence regulators, sarR and agr. Using strain SH1000, we showed that the transcription of virulence effectors, such as hla, sspA, and spa, is altered in an rsr mutant in a way consistent with agr upregulation. Analysis of RNAIII expression of the agr locus in rsr and rsr-sarR mutants indicated that rsr likely contributes to agr expression independently of SarR. We also provide evidence using a murine model of S. aureus skin infection that the effects mediated by rsr reduce disease progression