Most Sinorhizobium meliloti Extracytoplasmic Function Sigma Factors Control Accessory Functions

Abstract

Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF σs) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like σs (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF σs. We discovered new targets for individual σs, confirmed others by overexpressing individual ECF σs, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF σ gene and its demonstrated or putative anti-σ gene and also a strain in which all 11 ECF σ and anti-σ genes were deleted. This all-ECF σ deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF σs were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF σ and anti-σ genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [σs]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.

Keywords: Rhizobium; Sinorhizobium; microarrays; sigma factors; symbiosis.

FIG 1

Genomic context of ECF σ genes in S. meliloti. σs are grouped according to the classification described in reference . Genomic context is anchored by blue ECF σs and red adjacent known and putative anti-σs. Abbreviated S. meliloti gene names indicate the replicon on which they are found: a, pSymA; b, pSymB; and c, chromosome. The likely rpoE5 anti-σ lies downstream and in the same orientation as its partner σ; the overlapping arrow in the opposite orientation indicates an ORF with striking similarity to that of rsiA1, the rpoE2 anti-σ (see Fig. S1 for more details). The anti-anti-σ (rsiB1) transcribed divergently from rsiA1 is shown in turquoise. RpoE9 likely encodes its own anti-σ domain in the C-terminal half of its ORF. The flags dispersed throughout indicate the location and orientation of promoter motifs discussed in the text. The remaining colors follow the Riley classification convention found on the INRA Sinorhizobium meliloti 1021 website (https://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi), with several exceptions for clarity. The purple ORFs flanking rpoE8 indicate small molecule metabolism, light gray indicates hypothetical partial homology, green indicates hypothetical global homology, white indicates unknown function, mauve indicates a not classified regulator, light blue indicates central intermediary metabolism, brown indicates macromolecule metabolism, and yellow indicates cell processes.

FIG 2

Network of ECF σs and their putative target genes. The network diagram, created with the R igraph package (90) and the Fruchterman-Reingold layout algorithm (91), is based on transcriptome data from Data Set S1. RpoE1 to RpoE10 (E1 to E10) and FecI are represented by green circles. Arrows of arbitrary length from each ECF σ point to putative target genes (blue circles) whose expression appears dependent on that particular σ. Light blue circles indicate target genes of only one σ, while dark blue circles indicate target genes of more than one σ. Since each ECF σ was overexpressed from an exogenous promoter, ECF σs are not included on the diagram as targets, even if demonstrated to autoregulate their own expression in other studies. Because the layout algorithm places features somewhat arbitrarily, some green circles such as those for RpoE5 and RpoE6 are partially obscured by blue circles representing their unique and shared target genes. The numbers of direct and indirect target genes for each σ as a result of this study are as follows: RpoE1, 3; RpoE2, 320; RpoE3, 4; RpoE5, 1; RpoE6, 40; RpoE7, 7; RpoE8, 0; RpoE9, 1; and RpoE10, 6.

FIG 3

ECF σ −35 and −10 consensus promoter motifs. Motifs were identified from sequences upstream of transcription start sites (TSSs) of ECF σ-dependent target genes as described in Materials and Methods (Table 1). Sequence logos for predicted promoters were generated with WebLogo (https://weblogo.berkeley.edu). Promoters of genes that showed cross-regulation by multiple ECF σs were included in the sequence logo for only the ECF σ that showed the highest increase in expression of that target gene. The height of each letter in the sequence logo is proportional to the frequency of that nucleotide at that position, while the height of the entire stack is proportional to the sequence conservation at that position. Thus, logos generated from two sequences (RpoE4, RpE7, and RpoE8) will have blank spaces where no conservation is observed and letters of full height at the other positions. Similarly, logos generated from one sequence (RpoE3 and RpoE9) will have letters of full height at all positions. ECF σs are listed by their ECF group numbers; the number of upstream sequences used to develop each motif and their spacer lengths are indicated in the charts next to each logo. Sequences corresponding to the cross-species consensus motifs previously identified within the −35 and −10 regions are underlined in boldface (31). 5′-RACE mapping and RNA-Seq (22) of SMb20593, downstream of rpoE8, failed to identify its TSS; thus, a near perfect match to the putative RpoE8 promoter motif identified upstream of rpoE8, which is similar to consensus motifs identified for ECF29 family σs (31), was used to define the consensus.

FIG 4

ECFσs are not required for symbiosis on M. sativa and M. truncatula. Nodulation assays were performed as described in Materials and Methods. The y axis indicates the average number of nodules per plant, 21 days after inoculation. The number of putative nitrogen-fixing nodules is indicated in red, and the number of small, white (nonfixing) nodules observed for each of the three bacterial strains is indicated in pale blue. Nodules formed by the nonfixing nifD mutant (CL309) were small and either white or very pale pink. The total number of nodules is shown above each column. The numbers of M. sativa plants assayed for each strain in this representative experiment are as follows: CL150, 20; RFF625c, 20; and CL309, 10. The numbers of M. truncatula plants assayed are as follows: CL150, 19; RFF625c, 20; and CL309, 19.

FIG 5

Comparison of the all-ECF σ deletion strain RFF625c to WT CL150 by Biolog Phenotype MicroArray and cell viability assays. Biolog kinetic plots, generated by Biolog OmniLog PM software, are shown in panels A to D for selected cultivation conditions. The conditions tested were growth in the presence of (A) 100 mM NaNO₃ (Biolog plate PM09, well H06), (B) domiphen bromide (plate PM15, well D06), (C) iodonitrotetrazolium violet (INT; plate PM19, well D05), and (D) d-melezitose as the sole carbon source (plate PM02, well C04). Lines of the same color represent two biological replicates for CL150 (green) and RFF625c (purple). (E to G) Relative cell viabilities, determined as described in Materials and Methods, in the presence of domiphen bromide (E), benzethonium chloride (F), and INT (G). Cell viability measurements were normalized to the untreated CL150 control. Results showing statistically significant differences between the two strains, using a heteroscedastic, two-tailed t test, are indicated by asterisks (*, P  < 0.05; **, P  < 0.01; ***, P  < 0.001). Error bars indicate standard deviation from four replicates.