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. 2017 Nov 6;7:14587. doi: 10.1038/s41598-017-15035-y

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Validation of penicillin-induced alterations to glutamine metabolism gene expression in S. pneumoniae by qRT-PCR. Genes involved in glutamine metabolism (glnR, glnA, glnP and glnQ) found to be down-regulated after exposure to PEN by RNA-seq were validated by qRT-PCR. RNA levels were normalized based on the amplification signals of 16S ribosomal RNA. Graphs show the log₂ fold change of expression at time T3 (corresponding to 40 min for R6, 28 min for CCRI-21487 and 85 min for CCRI-8970) over T0 in untreated (white bars) and PEN-treated (black bars) S. pneumoniae isolates. Results are displayed as mean ± SD of three biological replicates and significant differences are identified as determined by split plot design and Fisher’s F-test (***p ≤ 0.001).