Phenotypic Heterogeneity, a Phenomenon That May Explain Why Quorum Sensing Does Not Always Result in Truly Homogenous Cell Behavior.

Phenotypic heterogeneity describes the occurrence of "nonconformist" cells within an isogenic population. The nonconformists show an expression profile partially different from that of the remainder of the population. Phenotypic heterogeneity affects many aspects of the different bacterial lifestyles, and it is assumed that it increases bacterial fitness and the chances for survival of the whole population or smaller subpopulations in unfavorable environments. Well-known examples for phenotypic heterogeneity have been associated with antibiotic resistance and frequently occurring persister cells. Other examples include heterogeneous behavior within biofilms, DNA uptake and bacterial competence, motility (i.e., the synthesis of additional flagella), onset of spore formation, lysis of phages within a small subpopulation, and others. Interestingly, phenotypic heterogeneity was recently also observed with respect to quorum-sensing (QS)-dependent processes, and the expression of autoinducer (AI) synthase genes and other QS-dependent genes was found to be highly heterogeneous at a single-cell level. This phenomenon was observed in several Gram-negative bacteria affiliated with the genera Vibrio, Dinoroseobacter, Pseudomonas, Sinorhizobium, and Mesorhizobium. A similar observation was made for the Gram-positive bacterium Listeria monocytogenes. Since AI molecules have historically been thought to be the keys to homogeneous behavior within isogenic populations, the observation of heterogeneous expression is quite intriguing and adds a new level of complexity to the QS-dependent regulatory networks. All together, the many examples of phenotypic heterogeneity imply that we may have to partially revise the concept of homogeneous and coordinated gene expression in isogenic bacterial populations.

RNA sequencing analysis of the broad-host-range strain Sinorhizobium fredii NGR234 identifies a large set of genes linked to quorum sensing-dependent regulation in the background of a traI and ngrI deletion mutant.

The alphaproteobacterium Sinorhizobium fredii NGR234 has an exceptionally wide host range, as it forms nitrogen-fixing nodules with more legumes than any other known microsymbiont. Within its 6.9-Mbp genome, it encodes two N-acyl-homoserine-lactone synthase genes (i.e., traI and ngrI) involved in the biosynthesis of two distinct autoinducer I-type molecules. Here, we report on the construction of an NGR234-ΔtraI and an NGR234-ΔngrI mutant and their genome-wide transcriptome analysis. A high-resolution RNA sequencing (RNA-seq) analysis of early-stationary-phase cultures in the NGR234-ΔtraI background suggested that up to 316 genes were differentially expressed in the NGR234-ΔtraI mutant versus the parent strain. Similarly, in the background of NGR234-ΔngrI 466 differentially regulated genes were identified. Accordingly, a common set of 186 genes was regulated by the TraI/R and NgrI/R regulon. Coregulated genes included 42 flagellar biosynthesis genes and 22 genes linked to exopolysaccharide (EPS) biosynthesis. Among the genes and open reading frames (ORFs) that were differentially regulated in NGR234-ΔtraI were those linked to replication of the pNGR234a symbiotic plasmid and cytochrome c oxidases. Biotin and pyrroloquinoline quinone biosynthesis genes were differentially expressed in the NGR234-ΔngrI mutant as well as the entire cluster of 21 genes linked to assembly of the NGR234 type III secretion system (T3SS-II). Further, we also discovered that genes responsible for rhizopine catabolism in NGR234 were strongly repressed in the presence of high levels of N-acyl-homoserine-lactones. Together with nodulation assays, the RNA-seq-based findings suggested that quorum sensing (QS)-dependent gene regulation appears to be of higher relevance during nonsymbiotic growth rather than for life within root nodules.

Evidence of autoinducer-dependent and -independent heterogeneous gene expression in Sinorhizobium fredii NGR234.

Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single-cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-l-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-ΔtraI or the NGR234-ΔngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum-quenching genes dlhR and qsdR1 and the type IV pilus gene cluster located on pNGR234b suggested that factors other than AI molecules affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide the first evidence that plant root exudates have strong effects on the heterogeneity of AI synthase and quorum-quenching genes in NGR234. Therefore, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression.

Novel reaction of succinyl coenzyme A (Succinyl-CoA) synthetase: activation of 3-sulfinopropionate to 3-sulfinopropionyl-CoA in Advenella mimigardefordensis strain DPN7T during degradation of 3,3'-dithiodipropionic acid.

The sucCD gene of Advenella mimigardefordensis strain DPN7(T) encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3'-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7(T) disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7(T) activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCD(Am)) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCD(Am) is Mg²âº or Mn²âº dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (V(max) = 9.85 ± 0.14 µmol min⁻¹ mg⁻¹, K(m) = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (V(max) = 0.12 ± 0.01 µmol min⁻¹ mg⁻¹) and the affinity was 6-fold lower (K(m) = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCD(Am) is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP.

The Absence of the N-acyl-homoserine-lactone Autoinducer Synthase Genes traI and ngrI Increases the Copy Number of the Symbiotic Plasmid in Sinorhizobium fredii NGR234.

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades.

Polythioester synthesis in Ralstonia eutropha H16: novel insights into 3,3'-thiodipropionic acid and 3,3'-dithiodipropionic acid catabolism.

Ralstonia eutropha H16 is capable of utilizing 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) as precursor substrates for biosynthesis of a polythioester (PTE) heteropolymer consisting of 3-hydroxybutyric acid (3HB) and 3-mercaptopropionic acid (3MP). To elucidate the hitherto unknown catabolic pathways of TDP and DTDP in R. eutropha H16, 19 defined deletion mutants were generated based on extensive functional genome analyses. Deletions of two ABC-type transporter clusters (H16_A0357-0359, H16_A3658-3660) resulted in an alteration of poly(3HB-co-3MP) composition with TDP as precursor to only 10.2±1.9mol% 3MP in comparison to 15.1±5.5mol% in the wild type. A mutant strain of H16 lacking Bordetella uptake gene-like substrate binding proteins (H16_A2779, H16_A0337) incorporated only 7.4±3.8mol% 3MP into PTE heteropolymers with DTDP as precursor in comparison to 24.5±14.5mol% in the wild type. Therefore, both gene products are probably involved in transport processes of this compound into the cells. However, the most significant reduction in 3MP contents of the heteropolymers with DTDP as precursor occurred upon the deletion of a gene encoding the putative thiol-disulfide interchange protein DsbD (H16_A3455, 3.9±2.6mol% 3MP). DsbD is proposed to be involved in the reduction of DTDP into two molecules of 3MP, the common cleavage product of TDP and DTDP.