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. 2021 May 27:12:656589.
doi: 10.3389/fmicb.2021.656589. eCollection 2021.

Transcriptome Analysis Reveals Cr(VI) Adaptation Mechanisms in Klebsiella sp. Strain AqSCr

Paloma Lara  1 , Leticia Vega-Alvarado  2 , Diana X Sahonero-Canavesi  3 , Michel Koenen  3 , Laura Villanueva  3   4 , Fernando Riveros-Mckay  1 , Enrique Morett  1 , Katy Juárez  1
Affiliations

Transcriptome Analysis Reveals Cr(VI) Adaptation Mechanisms in Klebsiella sp. Strain AqSCr

Paloma Lara et al. Front Microbiol. .

Abstract

Klebsiella sp. strain AqSCr, isolated from Cr(VI)-polluted groundwater, reduces Cr(VI) both aerobically and anaerobically and resists up 34 mM Cr(VI); this resistance is independent of the ChrA efflux transporter. In this study, we report the whole genome sequence and the transcriptional profile by RNA-Seq of strain AqSCr under Cr(VI)-adapted conditions and found 255 upregulated and 240 downregulated genes compared to controls without Cr(VI) supplementation. Genes differentially transcribed were mostly associated with oxidative stress response, DNA repair and replication, sulfur starvation response, envelope-osmotic stress response, fatty acid (FA) metabolism, ribosomal subunits, and energy metabolism. Among them, genes not previously associated with chromium resistance, for example, cybB, encoding a putative superoxide oxidase (SOO), gltA2, encoding an alternative citrate synthase, and des, encoding a FA desaturase, were upregulated. The sodA gene encoding a manganese superoxide dismutase was upregulated in the presence of Cr(VI), whereas sodB encoding an iron superoxide dismutase was downregulated. Cr(VI) resistance mechanisms in strain AqSCr seem to be orchestrated by the alternative sigma factors fecl, rpoE, and rpoS (all of them upregulated). Membrane lipid analysis of the Cr(IV)-adapted strain showed a lower proportion of unsaturated lipids with respect to the control, which we hypothesized could result from unsaturated lipid peroxidation followed by degradation, together with de novo synthesis mediated by the upregulated FA desaturase-encoding gene, des. This report helps to elucidate both Cr(VI) toxicity targets and global bacterial response to Cr(VI).

Keywords: Klebsiella sp. AqSCr; chromate reduction; chromate resistance; fatty acid desaturase; transcriptome.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HR-R declared a shared affiliation, with no collaboration, with one of the authors, LV-A, to the handling editor at the time of the review.

Figures

FIGURE 1
FIGURE 1
Cr(VI) resistance as indicated by growth of Klebsiella sp. strain AqSCr in LB medium at different pH. (A) pH 7, (B) pH 8, and (C) pH 9.
FIGURE 2
FIGURE 2
Cr(VI) reduction by Klebsiella sp. strain AqSCr. (A) Anaerobic chromate reduction in sodium citrate medium. (B) Aerobic chromate reduction in unbuffered LB medium, phosphate-buffered LB medium, and HEPES-buffered LB medium. (C) In vitro chromate reductase activity in soluble and membrane fractions of Klebsiella sp. strain AqSCr with and without NADH.
FIGURE 3
FIGURE 3
Growth of Klebsiella sp. strain AqSCr in LB medium (pH 8) with 11 mM Cr(VI), starting with cells with pre-exposure (blue triangles) and without pre-exposure (green circles).
FIGURE 4
FIGURE 4
TEM Micrographs of Klebsiella sp. strain AqSCr cells grown in (A) LB (pH 8) without Cr(VI) and (B) LB (pH 8) with 11 mM Cr(VI).
FIGURE 5
FIGURE 5
Functional assignment of differentially transcribed genes of strain AqSCr grown in the presence of 11 mM Cr(VI) in LB medium (pH 8) with respect to controls.
FIGURE 6
FIGURE 6
Volcano plot displaying differentially transcribed genes between Cr(VI)-adapted cells and controls. Each dot represents an ORF. ORFs with p-adj ≤ 0.01 and |Log2FC| > 1.5 were considered as differentially transcribed. Genes with putative functions previously associated with Cr(VI)-response in other bacteria are highlighted.
FIGURE 7
FIGURE 7
Schematic model of Klebsiella sp. strain AqSCr metabolic adaptation to 11 mM Cr(VI). Stress pathways elicited by Cr(VI) (yellow squares) and metabolic responses (written in red bold inside red circles) associated to the upregulated (green) and downregulated genes (blue) in the presence of Cr(VI) are highlighted. Gene abbreviations correspond to those in Supplementary Tables 5, 6. Genes underlines are those that were found to be differentially transcribed but outside of the cutoff parameters.
FIGURE 8
FIGURE 8
Relative distribution of fatty acid methyl esters (FAMEs) of Klebsiella sp. strain AqSCr cells adapted to 11 mM Cr(VI) or grown in LB (pH 8) (controls). Each column represents the mean ± SEM of three independent replicates. Significant difference for specific FAMEs compounds of Cr(VI)-treated cells vs. control (LB), using a two-way ANOVA followed by a Sidak’s multiple comparison test. ****p < 0.0001, **p < 0.0016, n = 3. Bars represent mean ± SEM.
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