Inductors and regulatory properties of the genomic island‐associated fru₂ metabolic operon of Streptococcus agalactiae

Abstract

The fru metabolic operon of Streptococcus agalactiae encodes the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) enzyme II complex Fru (EIIB , EIIA , and EIIC ); Fru R, a transcriptional activator with PTS regulatory domains (PRDs); a d-allulose-6-phosphate 3-epimerase; a transaldolase; and a transketolase. We showed that the transcription of fru is induced during the stationary phase of growth in complex media and during incubation in human cerebrospinal or amniotic fluids. d-allose and d-ribose are environmental signals governing this induction. PTS is involved in the activation of the fru promoter, and the histidine-67 of EIIA and the cysteine-9 of EIIB are important for this function. The activation of fru is also controlled by Fru R. The histidine-243 in the PRD domain, the histidine-323 in the PRD domain, the cysteine-400 in the EIIB-like domain, and the histidine-549 in the EIIA-like domain are important for the function of Fru R. Fru R binds to a DNA region containing palindromic sequences upstream of the identified transcriptional start site. EIIB interacts physically with the C-terminal part of Fru R (expressing the EIIB-like and EIIA-like motifs) and with EIIA . We propose a model of regulation of fru depending on the presence of an activatory carbohydrate in the growth medium.

Functional Study of the Type II-A CRISPR-Cas System of Streptococcus agalactiae Hypervirulent Strains

“…Assays of b-galactosidase were then performed as previously described. 27,28 To study cas9 transcription, S. agalactiae strains were grown in triplicate to midlog and stationary phases, and total RNA was extracted with a phenol/TRIzol-based purification method as previously described. 29 Quantitative reverse transcription PCR (RT-qPCR) was performed using the LightCycler Ò 480 SYBR Green I mix, with the associated instrument and software (Roche).…”

“…30 Construction of the cas9 deletion mutant (BM110Dcas9) was performed as previously described with a thermosensitive shuttle vector pG+host1 (pG1 ts ). 28,31 The primers used to construct this mutant are described in Supplementary Table S1. To complement this mutant, the entire coding sequence of cas9 was PCR-amplified and inserted into pG1 ts .…”

The Role of Regulator Catabolite Control Protein A (CcpA) in Streptococcus agalactiae Physiology and Stress Response

“…b-galactosidase transcriptional fusion assays. The promoter regions of SAK_0348, SAK_0902, and SAK_1689 were cloned in a pTCV-lacZ (80) vector to construct transcriptional fusions between the E. coli lacZ reporter gene and upstream regions of the genes as described by Patron et al (81). Briefly, promoter regions were amplified with the primers listed in Table S2.…”

“…Supernatants were used for assays. The absorbance at 595 nm (A 595 ) and the absorbance after addition of o-nitrophenyl-b-D-galactopyranoside at 420 nm (A 420 ) were measured as described by Patron et al (81). Protein concentration (C mg/mL ) was deduced from A 595 .…”

Bacterial and Host Determinants of Group B Streptococcal Vaginal Colonization and Ascending Infection in Pregnancy

“…Adhesins were downregulated, whereas, capsule, hemolysin, and IL-8 proteinase were upregulated, suggesting that GBS modulates its virulence factors in response to amniotic fluid (Sitkiewicz et al, 2009). Expression of the fru2 metabolic operon which encodes factors involved in fructose metabolism (Patron et al, 2017) was induced, suggesting that this carbon source might be important for GBS survival in this niche. Although these studies highlight adaptations that GBS might undergo following microbial invasion of the amniotic cavity, the relevance of these findings in vivo are unclear.…”