Robust Markers Reflecting Phylogeny and Taxonomy of Rhizobia

Genomic ANI (Average Nucleotide Identity) has been found to be able to replace DNA-DNA hybridization in prokaryote taxonomy. The ANI of each of the core genes that has a phylogeny congruent with the reference species tree of rhizobia was compared to the genomic ANI. This allowed us to identify three housekeeping genes ({"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA) whose ANI reflected the intraspecies and interspecies genomic ANI among rhizobial strains, revealing an ANI gap (≥2%) between the inter- and intra-species comparisons. The intraspecies (96%) and interspecies (94%) ANI boundaries calculated from three genes ({"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA) provided a criterion for bacterial species definition and confirmed 621/629 of known interspecies relationships within Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium. Some widely studied strains should be renamed. The {"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA ANI also correlates well with the genomic ANI of strains in Agrobacterium, Methylobacterium, Ralstonia, Rhodopseudomonas, Cupriavidus and Burkholderia, suggesting their wide applicability in other bacteria.     

Role of Sphingomonas sp. strain Fr1 PhyR-NepR-σEcfG cascade in general stress response and identification of a negative regulator of PhyR

The general stress response in Alphaproteobacteria was recently described to depend on the alternative sigma factor σ(EcfG), whose activity is regulated by its anti-sigma factor NepR. The response regulator PhyR, in turn, regulates NepR activity in a partner-switching mechanism according to which phosphorylation of PhyR triggers sequestration of NepR by the sigma factor-like effector domain of PhyR. Although genes encoding predicted histidine kinases can often be found associated with phyR, little is known about their role in modulation of PhyR phosphorylation status. We demonstrate here that the PhyR-NepR-σ(EcfG) cascade is important for multiple stress resistance and competitiveness in the phyllosphere in a naturally abundant plant epiphyte, Sphingomonas sp. strain Fr1, and provide evidence that the partner switching mechanism is conserved. We furthermore identify a gene, designated phyP, encoding a predicted histidine kinase at the phyR locus as essential. Genetic epistasis experiments suggest that PhyP acts upstream of PhyR, keeping PhyR in an unphosphorylated, inactive state in nonstress conditions, strictly depending on the predicted phosphorylatable site of PhyP, His-341. In vitro experiments show that Escherichia coli inner membrane fractions containing PhyP disrupt the PhyR-P/NepR complex. Together with the fact that PhyP lacks an obvious ATPase domain, these results are in agreement with PhyP functioning as a phosphatase of PhyR, rather than a kinase.     

DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides

Singlet oxygen (¹O₂) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to ¹O₂ formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under ¹O₂ stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards ¹O₂. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to ¹O₂ resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to ¹O₂ exposure in R. sphaeroides was extended.     

Replicon-Dependent Differentiation of Symbiosis-Related Genes in Sinorhizobium Strains Nodulating Glycine max

In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes ({"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of {"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.     

The Sinorhizobium meliloti Essential Porin RopA1 Is a Target for Numerous Bacteriophages

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti harbors a gene, {"type":"entrez-protein","attrs":{"text":"SMc02396","term_id":"1174172413","term_text":"SMC02396"}}SMc02396, which encodes a predicted outer membrane porin that is conserved in many symbiotic and pathogenic bacteria in the order Rhizobiales. Here, this gene (renamed ropA1) is shown to be required for infection by two commonly utilized transducing bacteriophages (ΦM12 and N3). Mapping of S. meliloti mutations conferring resistance to ΦM12, N3, or both phages simultaneously revealed diverse mutations mapping within the ropA1 open reading frame. Subsequent tests determined that RopA1, lipopolysaccharide, or both are required for infection by all of a larger collection of Sinorhizobium-specific phages. Failed attempts to disrupt or delete ropA1 suggest that this gene is essential for viability. Phylogenetic analysis reveals that ropA1 homologs in many Rhizobiales species are often found as two genetically linked copies and that the intraspecies duplicates are always more closely related to each other than to homologs in other species, suggesting multiple independent duplication events.     

Multiple σEcfG and NepR Proteins Are Involved in the General Stress Response in Methylobacterium extorquens

In Alphaproteobacteria, the general stress response (GSR) is controlled by a conserved partner switch composed of the sigma factor σ^EcfG, its anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. Many species possess paralogues of one or several components of the system, but their roles remain largely elusive. Among Alphaproteobacteria that have been genome-sequenced so far, the genus Methylobacterium possesses the largest number of σ^EcfG proteins. Here, we analyzed the six σ^EcfG paralogues of Methylobacterium extorquens AM1. We show that these sigma factors are not truly redundant, but instead exhibit major and minor contributions to stress resistance and GSR target gene expression. We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR. Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system. Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.     

A highly conserved protein of unknown function is required by Sinorhizobium meliloti for symbiosis and environmental stress protection

We report here the first characterization of the Sinorhizobium meliloti open reading frame SMc01113. The SMc01113 protein is a member of a highly conserved protein family, universal among bacteria. We demonstrate that the SMc01113 gene is absolutely required for S. meliloti symbiosis with alfalfa and also for the protection of the bacterium from a wide range of environmental stresses.     

LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.     

Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021

Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.     

A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

Fatty acid‐releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase

Phospholipids are well known for their membrane‐forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD‐deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin‐like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003‐deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth.     

PdhS,an Old-Pole-Localized Histidine Kinase,Recruits the Fumarase FumC in Brucella abortus

The bacterial pathogen Brucella abortus was recently demonstrated to recruit the essential cytoplasmic histidine kinase PdhS to its old pole. Here, we report identification of the fumarase FumC as a specific partner for the N-terminal “sensing” domain of PdhS, using an ORFeome-based yeast two-hybrid screen. We observed that FumC and PdhS colocalize at the old pole of B. abortus, while the other fumarase FumA is not polarly localized. FumC is not required for PdhS localization, and polar FumC localization is not FumA dependent. FumC homologs are not polarly localized in Sinorhizobium meliloti and Caulobacter crescentus, suggesting that polar recruitment of FumC by PdhS is evolutionarily recent.It was previously reported that morphological asymmetry, here defined by the production of two sibling cells different in length at each cell cycle, is a common feature shared by at least four members of the alphaproteobacteria with diversified lifestyles, namely, the free-living model bacterium Caulobacter crescentus, the plant pathogen Agrobacterium tumefaciens, the plant symbiont Sinorhizobium meliloti, and the facultative intracellular pathogen Brucella abortus (5). In B. abortus, an essential cytoplasmic histidine kinase named PdhS is anchored to the old pole of the bacterium (6). PdhS interacts with the response regulator DivK (6).PdhS is a rather atypical histidine kinase. It is cytoplasmic and contains an N-terminal “sensing” domain with no predicted function, except for a PAS domain that remains to be characterized. Nevertheless, the N-terminal part preceding the PAS domain of PdhS is sufficient for polar localization in B. abortus, as well as in the alphaproteobacteria S. meliloti and C. crescentus (6). This suggests that a molecular mechanism and/or polar structure required for PdhS targeting is conserved in these alphaproteobacteria.