First identification and characterization of porcine enterovirus G in the United States

Porcine enterovirus G (EV-G) is a member of the family Picornavirdae, genus Enterovirus. To date, eleven EV-G types (EV-G1 through EV-G11) have been identified in pigs from Asia and Europe however they have never been reported in North America. In this study, we isolated and characterized the complete genome of NP/2013/USA, an EV-G from a porcine diarrhea sample from the United States. The complete genome consists of 7,390 nucleotides excluding the 3′ poly(A) tail, and has an open reading frame that encodes a 2,169 amino acid polyprotein. NP/2013/USA was most similar at the nucleotide (84%) and amino acid (95%) level to the HM131607, an EV-G1 type isolated from China in 2012

Transcriptional regulation of a bacteriophage encoded extracellular DNase (Spd-3) by Rgg in Streptococcus pyogenes.

The Streptococcus pyogenes transcriptional regulator Rgg controls the expression of virulence-associated genes encoded both within the core genome and within horizontally transmissible DNA such as temperate bacteriophage. Previously, we showed that Rgg binds to the non-coding DNA upstream of the bacteriophage gene encoding an extracellular DNase Spd-3. In the current study, we further characterized Rgg-mediated regulation of spd-3 expression. Two spd-3 transcripts were identified by northern blotting. The 5' ends were 27 and 594 nucleotides upstream of the start codon as determined with primer extension analysis and 5' RACE (rapid amplification of c-DNA ends), respectively. Results obtained with gel shift assays showed that purified Rgg bound specifically to non-coding DNA containing the promoters of both transcripts. Transcriptional fusion analyses confirmed the presence of Rgg-repressible promoters within these DNA regions. In addition, repression was associated with direct DNA binding by Rgg as determined with chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (qPCR). The results show that the chromosomally encoded transcriptional regulator, Rgg, directly represses both bacteriophage promoters controlling the expression of Spd-3. The results provide new information regarding the regulation of prophage encoded virulence factors of S. pyogenes and highlight the complex evolutionary history of S. pyogenes and temperate bacteriophage

Rgg binds specifically to P2.

<p>Rgg binding to the non-coding DNA upstream of <i>spd-3</i> containing P2 was assessed by incubating with 0, 6.5, 26, 39 and 52 pmoles (Lanes 1–6) of purified Rgg with radiolabeled target DNA. Lane 7) Specific un-labeled competing DNA was added to the reaction. Lane 8) Labeled non-specific DNA (<i>groEL)</i> was added to the reaction.</p

Detection of <i>spd-3</i> transcripts.

<p>Northern blotting using an <i>spd-3</i> specific probe and RNA isolated from the wild type (wt) and the <i>rgg</i> mutant (<i>rgg</i>) strains showed two transcripts in the sample obtained from the mutant strain. The approximate sizes were determined by using an RNA ladder (not shown).</p

Rgg binds to episomal P2 containing DNA.

<p>The amount of the non-coding DNA upstream of <i>spd-3</i> cloned into a transcriptional reporter fusion plasmid was measured by quantitative PCR, as a negative control, <i>groEL</i> in ChIP samples obtained from the <i>rgg</i> mutant and strain SA6. Experiments were conducted at least three times, and the means and standard deviations are shown.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Primer extension analysis of <i>spd-3</i> transcripts.

<p>RNA isolated from the wt (lane 5) and <i>rgg</i> mutant strain (lane 6) was used to identify the 5′ end of the <i>spd-3</i> transcript. The DNA sequence (G A T C) generated from the non-coding DNA upstream of <i>spd-3</i> (lanes 1–4) is shown. The antisense strand DNA sequence proximal to the 5′ end is shown to the right and bold type indicates the start of the transcript.</p

Rgg binds <i>in vivo</i> to <i>spd-3</i> promoter regions P1 and P2.

<p>ChIP and qPCR was used to measure the amount of P1 (Panel A) and P2 (Panel B) containing DNA bound by Rgg. Experiments were conducted at least three times, and the means and standard deviations are shown.</p

Inactivation of the Rgg2 transcriptional regulator ablates the virulence of Streptococcus pyogenes.

Streptococcus pyogenes adapts to different niches encountered in the human host via the activity of numerous regulatory proteins including the Rgg family of transcriptional regulators. The S. pyogenes chromosome encodes four Rgg paralogues designated Rgg1 (RopB), Rgg2 (MutR), Rgg3, and Rgg4 (ComR). In order to understand the role of the Rgg2 protein in the regulation of metabolic and virulence-associated properties of S. pyogenes, the rgg2 gene was inactivated in the M1 serotype strain SF370. Inactivation of rgg2 increased the growth yield of S. pyogenes in THY broth, increased biofilm formation, and increased production of SIC, which is an important virulence factor that inhibits complement mediated lysis. To identify Rgg2-regulated genes, the transcriptomes of SF370 and the rgg2 mutant strains were compared in the middle-exponential and post-exponential phases of growth. Rgg2 was found to control the expression of dozens of genes primarily in the exponential phase of growth, including genes associated with virulence (sse, scpA, slo, nga, mf-3), DNA transformation, and nucleotide metabolism. Inactivation of rgg2 decreased the ability of S. pyogenes to adhere to epithelial cells. In addition, the mutant strain was more sensitive to killing when incubated with human blood and avirulent in a murine bacteremia model. Finally, inoculation of mice with the avirulent rgg2 mutant of S. pyogenes SF370 conferred complete protection to mice subsequently challenged with the wild-type strain. Restoration of an intact rgg2 gene in mutant strain restored the wild-type phenotypes. Overall, the results demonstrate that Rgg2 is an important regulatory protein in S. pyogenes involved in controlling genes associated with both metabolism and virulence