CbrA is an atypical sensor kinase found in Pseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of hut genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the hut operon. Genes encoding transporters were not identified, but mutations were repeatedly found in cbrA . This, coupled with the findings of [ 3 H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction. IMPORTANCE Nutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator in Pseudomonas represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction.

Role of the transporter-like sensor kinase CbrA in histidine uptake and signal transduction

CbrAB is a two-component system, unique to bacteria of the family Pseudomonaceae, capable of integrating signals and involved in a multitude of physiological processes that allow bacterial adaptation to a wide variety of varying environmental conditions. This regulatory system provides a great metabolic versatility that results in excellent adaptability and metabolic optimization. The two-component system (TCS) CbrA–CbrB is on top of a hierarchical regulatory cascade and interacts with other regulatory systems at different levels, resulting in a robust output. Among the regulatory systems found at the same or lower levels of CbrAB are the NtrBC nitrogen availability adaptation system, the Crc/Hfq carbon catabolite repression cascade in Pseudomonas, or interactions with the GacSA TCS or alternative sigma ECF factor, such as SigX. The interplay between regulatory mechanisms controls a number of physiological processes that intervene in important aspects of bacterial adaptation and survival. These include the hierarchy in the use of carbon sources, virulence or resistance to antibiotics, stress response or definition of the bacterial lifestyle. The multiple actions of the CbrAB TCS result in an important competitive advantage.

/pdf/the-regulatory-hierarchy-following-signal-integration-by-the-274flc5w.pdf

The Regulatory Hierarchy Following Signal Integration by the CbrAB Two-Component System: Diversity of Responses and Functions

Fluorescent pseudomonads represent one of the largest groups of bacteria inhabiting the surfaces of plants, but their genetic composition in planta is poorly understood. Here, we examined the population structure and diversity of fluorescent pseudomonads isolated from sugar beet grown at two geographic locations (Oxford, United Kingdom and Auckland, New Zealand). To seek evidence for niche adaptation, bacteria were sampled from three types of leaves (immature, mature, and senescent) and then characterized using a combination of genotypic and phenotypic analysis. We first performed multilocus sequence analysis (MLSA) of three housekeeping genes (gapA, gltA, and acnB) in a total of 152 isolates (96 from Oxford, 56 from Auckland). The concatenated sequences were grouped into 81 sequence types and 22 distinct operational taxonomic units (OTUs). Significant levels of recombination were detected, particularly for the Oxford isolates (rate of recombination to mutation (r/m) = 5.23 for the whole population). Subsequent ancestral analysis performed in STRUCTURE found evidence of six ancestral populations, and their distributions significantly differed between Oxford and Auckland. Next, their ability to grow on 95 carbon sources was assessed using the Biolog™ GN2 microtiter plates. A distance matrix was generated from the raw growth data (A 660) and subjected to multidimensional scaling (MDS) analysis. There was a significant correlation between substrate utilization profiles and MLSA genotypes. Both phenotypic and genotypic analyses indicated presence of a geographic structure for strains from Oxford and Auckland. Significant differences were also detected for MLSA genotypes between strains isolated from immature versus mature/senescent leaves. The fluorescent pseudomonads thus showed an ecotypic population structure, suggestive of adaptation to both geographic conditions and local plant niches.

Genotypic and phenotypic analyses reveal distinct population structures and ecotypes for sugar beet‐associated Pseudomonas in Oxford and Auckland

The emergence of next-generation sequencing (NGS) technologies has made it possible to not only sequence entire genomes, but also identify metabolic engineering targets across the pangenome of a microbial population. This study leverages NGS data as well as existing molecular biology and bioinformatics tools to identify and validate genomic signatures for improving phenazine biosynthesis in Pseudomonas chlororaphis. We sequenced a diverse collection of 34 Pseudomonas isolates using short- and long-read sequencing techniques and assembled whole genomes using the NGS reads. In addition, we assayed three industrially relevant phenotypes (phenazine production, biofilm formation, and growth temperature) for these isolates in two different media conditions. We then provided the whole genomes and phenazine production data to a unitig-based microbial genome-wide association study (mGWAS) tool to identify novel genomic signatures responsible for phenazine production in P. chlororaphis. Post-processing of the mGWAS analysis results yielded 330 significant hits influencing the biosynthesis of one or more phenazine compounds. Based on a quantitative metric (called the phenotype score), we elucidated the most influential hits for phenazine production and experimentally validated them in vivo in the most optimal phenazine producing strain. Two genes significantly increased phenazine-1-carboxamide (PCN) production: a histidine transporter (ProY_1), and a putative carboxypeptidase (PS__04251). A putative MarR-family transcriptional regulator decreased PCN titer when overexpressed in a high PCN producing isolate. Overall, this work seeks to demonstrate the utility of a population genomics approach as an effective strategy in enabling identification of targets for metabolic engineering of bioproduction hosts.

/pdf/population-genomics-guided-engineering-of-phenazine-2efg1u94.pdf

Population genomics-guided engineering of phenazine biosynthesis in Pseudomonas chlororaphis

The cultivation of the cellulolytic bacterium, Clostridium thermocellum, can have cost-effective cellulosic biomass utilizations, such as consolidated bioprocessing, simultaneous biological enzyme production and saccharification. However, these processes require a longer cultivation term of approximately 1 week. We demonstrate that constituents of the C. thermocellum membrane vesicle fraction significantly promoted the growth rate of C. thermocellum. Similarly, cell-free Bacillus subtilis broth was able to increase C. thermocellum growth rate, while several B. subtilis single-gene deletion mutants, e.g., yxeJ, yxeH, ahpC, yxdK, iolF, decreased the growth stimulation ability. Metabolome analysis revealed signal compounds for cell-cell communication in the C. thermocellum membrane vesicle fraction (ethyl 2-decenoate, ethyl 4-decenoate, and 2-dodecenoic acid) and B. subtilis broth (nicotinamide, indole-3-carboxaldehyde, urocanic acid, nopaline, and 6-paradol). These findings suggest that the constituents in membrane vesicles from C. thermocellum and B. subtilis could promote C. thermocellum growth, leading to improved efficiency of cellulosic biomass utilization.

/pdf/metabolome-analysis-of-constituents-in-membrane-vesicles-for-fu5aak851m.pdf

Metabolome Analysis of Constituents in Membrane Vesicles for Clostridium thermocellum Growth Stimulation.

Phenotypic variation is a fundamental requirement for evolution by natural selection. While evidence of phenotypic variation in natural populations abounds, its genetic basis is rarely understood. Here we report variation in the ability of plant-colonizing Pseudomonas to utilize histidine, and its derivative, urocanate, as sole sources of carbon and nitrogen. From a population of 164 phyllosphere-colonizing Pseudomonas strains, 77% were able to utilize both histidine and urocanate (His(+) , Uro(+) ) as growth substrates, whereas the remainder could utilize histidine, but not urocanate (His(+) , Uro(-) ), or vice versa (His(-) , Uro(+) ). An in silico analysis of the hut locus, which determines capacity to utilize both histidine and urocanate, from genome-sequenced Pseudomonas strains, showed significant variation in the number of putative transporters. To identify transporter genes specific for histidine and urocanate, we focused on a single genotype of Pseudomonas fluorescens, strain SBW25, which is capable of utilizing both substrates. Site-directed mutagenesis, combined with [(3) H]histidine transport assays, shows that hutT(u) encodes a urocanate-specific transporter; hutT(h) encodes the major high-affinity histidine transporter; and hutXWV encodes an ABC-type transporter that plays a minor role in histidine uptake. Introduction of cloned copies of hutT(h) and hutT(u) from SBW25 into strains incapable of utilizing either histidine, or urocanate, complemented the defect, demonstrating a lack of functional transporters in these strains. Taken together our data show that variation in transport systems, and not in metabolic genes, explains a naturally occurring phenotypic polymorphism.

Variation in transport explains polymorphism of histidine and urocanate utilization in a natural Pseudomonas population.

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Variation in transport explains polymorphism of histidine and urocanate utilization in a natural Pseudomonas population.

Genotypic and phenotypic analyses reveal distinct population structures and ecotypes for sugar beet‐associated Pseudomonas in Oxford and Auckland