The NtrYX Two-Component System Regulates the Bacterial Cell Envelope

Abstract

Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides, loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria. IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

A majority of Rhodobacter sphaeroides promoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation

“…Based on the purified CarD Rsp standards run in parallel ( Fig. 7 A ) and a volume of 0.87 μm 3 for aerobically grown log-phase cells ( 45 ), we estimate that the concentrations of CarD are ∼1.1 µM in log phase and <100 nM in stationary phase (calculation provided in SI Appendix , Expanded Materials and Methods ), in the range of the concentrations of CarD that affected transcription in vitro. Taken together, our data indicate that CarD concentrations change in vivo and thus have the potential to regulate transcription in cells.…”

Genome-Wide Identification of Transcription Start Sites in Two Alphaproteobacteria , Rhodobacter sphaeroides 2.4.1 and Novosphingobium aromaticivorans DSM 12444

“…Three replicates of R. sphaeroides 2.4.1 or N. aromaticivorans DSM 12444 ΔsacB cultures were grown, and RNA was isolated as previously described ( 8 , 18 , 19 ). TSS-seq libraries were produced using RppH, which converts the 5′ triphosphates on unprocessed mRNA species to monophosphates, making them a substrate for ligation of the Illumina adapters ( 20 ).…”

“…The FASTQ files were split using the index barcode sequences to separate the sequences for the samples treated with or without RppH (RppH + and RppH − , respectively) using fastx_barcode_splitter.pl version 0.0.13.2 ( http://hannonlab.cshl.edu/fastx_toolkit/ ). The sequences were trimmed to remove any remaining adapter-derived bases using Trimmomatic version 0.3 (HEADCROP, 6; MINLEN, 25) ( 19 ) and were aligned to the R. sphaeroides genome (assembly ASM1290v2, GenBank accession number GCF_000012905.2 ) or the N. aromaticivorans genome (assembly ASM1332v1, GenBank accession number GCF_000013325.1 ) using Bowtie 2 version 2.3.5.1 ( 21 ), allowing for one mismatch (38,571,087 total aligned reads for R. sphaeroides and 29,552,504 total aligned reads for N. aromaticivorans ) ( Table 1 ). The aligned Bowtie 2 file was further processed with Picard tools version 2.10.0 ( https://broadinstitute.github.io/picard/ ) and SAMtools ( 22 ).…”

The ChvG–ChvI and NtrY–NtrX two-component systems coordinately regulate growth ofCaulobacter crescentus

“…Like ChvGI, NtrYX (consisting of the NtrY HK and NtrX RR) is conserved in many aproteobacteria, including multiple pathogens and symbionts (35)(36)(37)(38)(39). Although early studies primarily showed that NtrYX regulates nitrogen metabolism, recent work suggests that, in certain ⍺-proteobacteria, it also affects exopolysaccharide biosynthesis, cell motility, and cell envelope composition (25,35,39,(40)(41)(42)(43)(44). These processes are also regulated by ChvGI, suggesting that ChvGI and NtrYX might act coordinately.…”