The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism and the symbiotic interaction with alfalfa

Abstract

Background: The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored.

Results: Two independent S. meliloti mutants, 2011-3.4 and 1021Deltahfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Deltahfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Deltahfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein.

Conclusions: Our results support that the S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.

Figure 1

Mutational analysis of the S. meliloti hfq gene. (a) Arrangement of the genomic hfq region, multiple amino acid sequence alignment of Hfq proteins encoded by enterobacterial and α-proteobacterial genomes and details of the hfq mutants. The genetic map is drawn to scale. Numbering denotes the gene coordinates in the S. meliloti genome database. In the 1021Δhfq mutant the full-length Hfq ORF was replaced by a HindIII site. The DNA fragment cloned on complementation plasmid pJBHfq is indicated. In the alignment, Hfq sequences are denoted by the species abbreviation as follows: Ecol, E. coli; Stiph, Salmonella tiphymurium; Bsu, Brucella suis; Bmel, B. melitensis; Acaul, Azorhizobium caulinodans; Atum, Agrobacterium tumefaciens; Mlot, Mesorhizobium loti; Rleg, Rhizobium leguminosarum; Smel, S. meliloti. Species belonging to the α-subdivision of the proteobacteria are indicated to the left. Shadowed are the amino acid residues conserved in at least 80% sequences and boxed are the conserved amino acids within the C-terminal extension of Hfq proteins encoded by enterobacteria. The two conserved Sm-like domains are indicated. Double arrowheads indicate the integration sites of pK18mobsacB in 2011-3.4 and 2011-1.2 derivatives. (b) Growth curves in TY broth of the S. meliloti wild-type strains 2011 (left panel) and 1021 (right panel) and their respective hfq mutant derivatives as determined by OD₆₀₀ readings of triplicate cultures in 2 h intervals. Graphs legends: 2011, wild-type strain; 1.2, 2011-1.2 control strain; 3.4, 2011-3.4 derivative; 3.4(pJBHfq), 2011-3.4 complemented with plasmid pJBHfq; 1021, reference wild-type strain; Δhfq, 1021 hfq deletion mutant; Δhfq(pJBHfq), Δhfq complemented with pJBHfq.

Figure 2

Hfq-dependent alteration of the S. meliloti transcriptome and proteome. Differentially expressed transcripts (upper graphs) and proteins (lower graphs) in the S. meliloti hfq knock-out mutants. Histograms show the number of differentially expressed genes and their distribution in the three S. meliloti replicons: chromosome (Chrom.), pSymA and pSymB. The distribution of annotated ORFs in the genome is indicated as reference. The adscription of these genes to functional categories according to the KEGG and S. meliloti databases is shown to the right in circle charts (see text for web pages of the referred databases). In brackets the number of genes belonging to each category.

Figure 3

Hfq influences central metabolic pathways in S. meliloti. Functional distribution of down- and up-regulated transcripts (upper graphs) and proteins (lower graphs) in the S. meliloti hfq mutants. In brackets is the number of genes in each category. Histograms detail the subdivision of transport and metabolic genes.

Figure 4

Symbiotic phenotype of the S. meliloti hfq knock-out mutants. (a) Nodule formation kinetics of the S. meliloti 1021 wild-type strain and its mutant derivative 1021Δhfq determined as the number of nodules per plant (left plot) and % nodulated plants (right plot). Each point represents the mean ± standard error of determinations in two independent sets of 24 plants grown hydroponically in test tubes. Dpi, days post inoculation. (b) Competition assays between the S. meliloti wild-type strain 2011 and its hfq insertion mutant derivative 2011-3.4. Nodule occupancy (expressed as % of invaded nodules by each strain) was determined in plants grown in either Leonard assemblies or agar plates and co-inoculated with both strains at 1:1 ratio. (c) Symbiotic efficiency of the 1021 and 1021Δhfq strains. Left histogram, % nitrogen fixing nodules induced by each strain in plants grown either in test tubes (two sets of 24 plants) or agar plates (5 plates of 10 plants) 30 dpi. Right panels: growth of 1021- and 1021Δhfq-inoculated plants 30 dpi in Leonard jars and dry-weigh of the same plants expressed as the mean ± standard error from measurements in 24 individual plants. Ni, not inoculated.

Figure 5

The 1021Δhfq mutant is impaired in the survival within the nodule cells. Representative enlarged images of nodules induced in alfalfa plants by the 1021 (a) and 1021Δhfq (e) strains. Bright-field microscopy of longitudinal sections of the same nodules (b and f); the zones characterizing the histology of nitrogen-fixing indeterminate nodules are indicated in (b). Merged images of the same nodule sections observed with green and blue filters (520 nm and 470 nm, respectively) (c and g). Magnification of the images of central nodule tissues (d and h); 1021Δhfq-induced nodules are scarcely invaded by bacteria and show signs of premature senescence: degradation of leghemoglobin (arrows) and cell debris (double arrowheads). Scale bars, 250 μm.

Figure 6

Hfq contributes to the regulation of nifA and fixK expression. RT-PCR analysis on RNA extracted from the wild-type strain 1021 (lanes 1 and 3) and the hfq mutant (lanes 2 and 4) before (lanes 1 and 2) and after (lanes 3 and 4) culture incubation for 4 h in microaerobiosis (2% O₂). 16S was amplified as constitutive control of expression. Mock-treated (no RT) RNA samples were also PCR amplified with the same primer combinations to check for absence of DNA contamination (not shown).

Figure 7

Binding of S. meliloti sRNAs to a FLAG-epitope tagged Hfq protein. Western-blot showing the specific recognition of the chromosomally encoded 3 × FLAG tagged Hfq protein by ANTI-FLAG M2^® monoclonal antibodies in total protein extracts of two independent 1021hfq^FLAG strains (i.e. two different clones arising from the second cross-over event) (left panel); and Northern analysis of CoIP RNA from the 1021hfq^FLAG and wild-type strains for the detection of the Smr sRNAs (right panel). Lane 1 shows the expression pattern of the corresponding sRNAs in the wild-type strain.

Figure 8

Summary of pathways and phenotypes linked to an hfq mutation in S. meliloti. Double arrowheads denote favoured pathways and blocked arrows unfavoured pathways in the absence of Hfq. +O₂, aerobic conditions; -O₂, microaerobic conditions.