Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.

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.

Genome-wide RNA sequencing analysis of quorum sensing-controlled regulons in the plant-associated Burkholderia glumae PG1 strain.

Burkholderia glumae PG1 is a soil-associated motile plant-pathogenic bacterium possessing a cell density-dependent regulation system called quorum sensing (QS). Its genome contains three genes, here designated bgaI1 to bgaI3, encoding distinct autoinducer-1 (AI-1) synthases, which are capable of synthesizing QS signaling molecules. Here, we report on the construction of B. glumae PG1 ΔbgaI1, ΔbgaI2, and ΔbgaI3 mutants, their phenotypic characterization, and genome-wide transcriptome analysis using RNA sequencing (RNA-seq) technology. Knockout of each of these bgaI genes resulted in strongly decreased motility, reduced extracellular lipase activity, a reduced ability to cause plant tissue maceration, and decreased pathogenicity. RNA-seq analysis of all three B. glumae PG1 AI-1 synthase mutants performed in the transition from exponential to stationary growth phase revealed differential expression of a significant number of predicted genes. In comparison with the levels of gene expression by wild-type strain B. glumae PG1, 481 genes were differentially expressed in the ΔbgaI1 mutant, 213 were differentially expressed in the ΔbgaI2 mutant, and 367 were differentially expressed in the ΔbgaI3 mutant. Interestingly, only a minor set of 78 genes was coregulated in all three mutants. The majority of the QS-regulated genes were linked to metabolic activities, and the most pronounced regulation was observed for genes involved in rhamnolipid and Flp pilus biosynthesis and the type VI secretion system and genes linked to a clustered regularly interspaced short palindromic repeat (CRISPR)-cas gene cluster.

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.

Rhizobium sp. strain NGR234 possesses a remarkable number of secretion systems.

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.

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.

Phenotypic Heterogeneity Affects Stenotrophomonas maltophilia K279a Colony Morphotypes and ß-Lactamase Expression.

Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas maltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the L1 and L2 ß-lactamases in response to ß-lactam treatment. Here we report that the patient isolate S. maltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bla L1 and bla L2 were transcriptionally the most strongly upregulated genes. Promoter fusions of bla L1 and bla L2 genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla L2 expressing cells as identified by RNA-seq analysis. Overexpression of comE in S. maltophilia K279a reduced the level of cells that were in a bla L2-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including bla L1, bla L2, and comE.