Quorum-Sensing Systems in the Plant Growth-Promoting Bacterium Paraburkholderia phytofirmans PsJN Exhibit Cross-Regulation and Are Involved in Biofilm Formation.

Quorum-sensing systems play important roles in host colonization and host establishment of Burkholderiales species. Beneficial Paraburkholderia species share a conserved quorum-sensing (QS) system, designated BraI/R, that controls different phenotypes. In this context, the plant growth-promoting bacterium Paraburkholderia phytofirmans PsJN possesses two different homoserine lactone QS systems BpI.1/R.1 and BpI.2/R.2 (BraI/R-like QS system). The BpI.1/R.1 QS system was previously reported to be important to colonize and produce beneficial effects in Arabidopsis thaliana plants. Here, we analyzed the temporal variations of the QS gene transcript levels in the wild-type strain colonizing plant roots. The gene expression patterns showed relevant differences in both QS systems compared with the wild-type strain in the unplanted control treatment. The gene expression data were used to reconstruct a regulatory network model of QS systems in P. phytofirmans PsJN, using a Boolean network model. Also, we examined the phenotypic traits and transcript levels of genes involved in QS systems, using P. phytofirmans mutants in homoserine lactone synthases genes. We observed that the BpI.1/R.1 QS system regulates biofilm formation production in strain PsJN and this phenotype was associated with the lower expression of a specific extracytoplasmic function sigma factor ecf26.1 gene (implicated in biofilm formation) in the bpI.1 mutant strain.

Hierarchy of Carbon Source Utilization in Soil Bacteria: Hegemonic Preference for Benzoate in Complex Aromatic Compound Mixtures Degraded by Cupriavidus pinatubonensis Strain JMP134.

Cupriavidus pinatubonensis JMP134, like many other environmental bacteria, uses a range of aromatic compounds as carbon sources. Previous reports have shown a preference for benzoate when this bacterium grows on binary mixtures composed of this aromatic compound and 4-hydroxybenzoate or phenol. However, this observation has not been extended to other aromatic mixtures resembling a more archetypal context. We carried out a systematic study on the substrate preference of C. pinatubonensis JMP134 growing on representative aromatic compounds channeled through different catabolic pathways described in aerobic bacteria. Growth tests of nearly the entire set of binary combinations and in mixtures composed of 5 or 6 aromatic components showed that benzoate and phenol were always the preferred and deferred growth substrates, respectively. This pattern was supported by kinetic analyses that showed shorter times to initiate consumption of benzoate in aromatic compound mixtures. Gene expression analysis by real-time reverse transcription-PCR (RT-PCR) showed that, in all mixtures, the repression by benzoate over other catabolic pathways was exerted mainly at the transcriptional level. Additionally, inhibition of benzoate catabolism suggests that its multiple repressive actions are not mediated by a sole mechanism, as suggested by dissimilar requirements of benzoate degradation for effective repression in different aromatic compound mixtures. The hegemonic preference for benzoate over multiple aromatic carbon sources is not explained on the basis of growth rate and/or biomass yield on each single substrate or by obvious chemical or metabolic properties of these aromatic compounds.

Comparative genomics of Stutzerimonas balearica (Pseudomonas balearica): diversity, habitats, and biodegradation of aromatic compounds.

Stutzerimonas balearica (Pseudomonas balearica) has been found principally in oil-polluted environments. The capability of S. balearica to thrive from the degradation of pollutant compounds makes it a species of interest for potential bioremediation applications. However, little has been reported about the diversity of S. balearica. In this study, genome sequences of S. balearica strains from different origins were analyzed, revealing that it is a diverse species with an open pan-genome that will continue revealing new genes and functionalities as the genomes of more strains are sequenced. The nucleotide signatures and intra- and inter-species variation of the 16S rRNA genes of S. balearica were reevaluated. A strategy of screening 16S rRNA gene sequences in public databases enabled the detection of 158 additional strains, of which only 23% were described as S. balearica. The species was detected from a wide range of environments, although mostly from aquatic and polluted environments, predominantly related to petroleum oil. Genomic and phenotypic analyses confirmed that S. balearica possesses varied inherent capabilities for aromatic compounds degradation. This study increases the knowledge of the biology and diversity of S. balearica and will serve as a basis for future work with the species.

Identification of a Phylogenetically Divergent Vanillate O-Demethylase from Rhodococcus ruber R1 Supporting Growth on Meta-Methoxylated Aromatic Acids.

Rieske-type two-component vanillate O-demethylases (VanODs) catalyze conversion of the lignin-derived monomer vanillate into protocatechuate in several bacterial species. Currently, VanODs have received attention because of the demand of effective lignin valorization technologies, since these enzymes own the potential to catalyze methoxy group demethylation of distinct lignin monomers. In this work, we identified a phylogenetically divergent VanOD from Rhodococcus ruber R1, only distantly related to previously described homologues and whose presence, along with a 3-hydroxybenzoate/gentisate pathway, correlated with the ability to grow on other meta-methoxylated aromatics, such as 3-methoxybenzoate and 5-methoxysalicylate. The complementation of catabolic abilities by heterologous expression in a host strain unable to grow on vanillate, and subsequent resting cell assays, suggest that the vanAB genes of R1 strain encode a proficient VanOD acting on different vanillate-like substrates; and also revealed that a methoxy group in the meta position and a carboxylic acid moiety in the aromatic ring are key for substrate recognition. Phylogenetic analysis of the oxygenase subunit of bacterial VanODs revealed three divergent groups constituted by homologues found in Proteobacteria (Type I), Actinobacteria (Type II), or Proteobacteria/Actinobacteria (Type III) in which the R1 VanOD is placed. These results suggest that VanOD from R1 strain, and its type III homologues, expand the range of methoxylated aromatics used as substrates by bacteria.

Strict and direct transcriptional repression of the pobA gene by benzoate avoids 4-hydroxybenzoate degradation in the pollutant degrader bacterium Cupriavidus necator JMP134.

As other environmental bacteria, Cupriavidus necator JMP134 uses benzoate as preferred substrate in mixtures with 4-hydroxybenzoate, strongly inhibiting its degradation. The mechanism underlying this hierarchical use was studied. A C. necator benA mutant, defective in the first step of benzoate degradation, is unable to metabolize 4-hydroxybenzoate when benzoate is also included in the medium, indicating that this substrate and not one of its catabolic intermediates is directly triggering repression. Reverse transcription polymerase chain reaction analysis revealed that 4-hydroxybenzoate 3-hydroxylase-encoding pobA transcripts are nearly absent in presence of benzoate and a fusion of pobA promoter to lacZ reporter confirmed that benzoate drastically decreases the transcription of this gene. Expression of pobA driven by a heterologous promoter in C. necator benA mutant, allows growth on 4-hydroxybenzoate in presence of benzoate, overcoming its repressive effect. In contrast with other bacteria, regulators of benzoate catabolism do not participate in repression of 4-hydroxybenzoate degradation. Moreover, the effect of benzoate on pobA promoter can be observed in heterologous strains with the sole presence of PobR, the transcriptional activator of pobA gene, indicating that PobR is enough to fully reproduce the phenomenon. This novel mechanism for benzoate repression is probably mediated by direct action of benzoate over PobR.

Widespread distribution of hmf genes in Proteobacteria reveals key enzymes for 5-hydroxymethylfurfural conversion.

Furans represent a class of promising chemicals, since they constitute valuable intermediates in conversion of biomass into sustainable products intended to replace petroleum-derivatives. Conversely, generation of furfural and 5-hydroxymethylfurfural (HMF) as by-products in lignocellulosic hydrolysates is undesirable due its inhibitory effect over fermentative microorganisms. Therefore, the search for furans-metabolizing bacteria has gained increasing attention since they are valuable tools to solve these challenging issues. A few bacterial species have been described at genetic level, leading to a proposed HMF pathway encoded by a set of genes termed hmf/psf, although some enzymatic functions are still elusive. In this work we performed a genomic analysis of major subunits of furoyl-CoA dehydrogenase orthologues, revealing that the furoic acid catabolic route, key intermediate in HMF biodegradation, is widespread in proteobacterial species. Additionally, presence/absence profiles of hmf/psf genes in selected proteobacterial strains suggest parallel and/or complementary roles of enzymes with previously unclear function that could be key in HMF conversion. The furans utilization pattern of selected strains harboring different hmf/psf gene sets provided additional support for bioinformatic predictions of the relevance of some enzymes. On the other hand, at least three different types of transporter systems are clustered with hmf/psf genes, whose presence is mutually exclusive, suggesting a core and parallel role in furans transport in Proteobacteria. This study expands the number of bacteria that could be recruited in biotechnological processes for furans biodetoxification and predicts a core set of genes required to establish a functional HMF pathway in heterologous hosts for metabolic engineering endeavors.

Identification of a self-sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2-hydroxyphenylacetic acid catabolism, via homogentisate pathway.

The self-sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self-sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a ß-proteobacterium model for biodegradative pathways, catalyses the conversion of 2-hydroxyphenylacetic acid (2-HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2-HPA is degraded through the well-known homogentisate pathway requiring a 2-HPA 5-hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate-binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2-HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and α-, ß- and γ-proteobacteria lineages indicating a widespread distribution of 2-HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B self-sufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450-based biocatalytic processes.

Complete Multipartite Genome Sequence of the Cupriavidus basilensis Type Strain, a 2,6-Dichlorophenol-Degrading Bacterium.

We report the complete 8.94-Mb genome sequence of the type strain of Cupriavidus basilensis (DSM 11853T = CCUG 49340T = RK1T), formed by two chromosomes and six putative plasmids, which offers insights into its chloroaromatic-biodegrading capabilities.

Complete Genome Sequence of Rhodococcus ruber R1, a Novel Strain Showing a Broad Catabolic Potential toward Lignin-Derived Aromatics.

Rhodococcus ruber R1 was isolated from a pulp mill wastewater treatment plant because of its ability to use methoxylated aromatics as growth substrates. We report the 5.56-Mb genome sequence of strain R1, which can provide insights into the biodegradation of lignin-derived phenolic monomers and potentially support processes for lignocellulose conversion.

Draft Genome Sequences of Two Pseudomonas Strains That Are Able To Use Furan Derivatives as Their Sole Carbon Source.

Pseudomonas sp. strains ALS1279 and ALS1131 were isolated from wastewater treatment facilities on the basis of their ability to use furfural, a key lignocellulose-derived inhibitor, as their only carbon source. Here, we present the draft genome sequences of both strains, which can shed light on catabolic pathways for furan compounds in pseudomonads.

Genuine genetic redundancy in maleylacetate-reductase-encoding genes involved in degradation of haloaromatic compounds by Cupriavidus necator JMP134.

Maleylacetate reductases (MAR) are required for biodegradation of several substituted aromatic compounds. To date, the functionality of two MAR-encoding genes (tfdF(I) and tfdF(II)) has been reported in Cupriavidus necator JMP134(pJP4), a known degrader of aromatic compounds. These two genes are located in tfd gene clusters involved in the turnover of 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-CB). The C. necator JMP134 genome comprises at least three other genes that putatively encode MAR (tcpD, hqoD and hxqD), but confirmation of their functionality and their role in the catabolism of haloaromatic compounds has not been assessed. RT-PCR expression analyses of C. necator JMP134 cells exposed to 2,4-D, 3-CB, 2,4,6-trichlorophenol (2,4,6-TCP) or 4-fluorobenzoate (4-FB) showed that tfdF(I) and tfdF(II) are induced by haloaromatics channelled to halocatechols as intermediates. In contrast, 2,4,6-TCP only induces tcpD, and any haloaromatic compounds tested did not induce hxqD and hqoD. However, the tcpD, hxqD and hqoD gene products showed MAR activity in cell extracts and provided the MAR function for 2,4-D catabolism when heterologously expressed in MAR-lacking strains. Growth tests for mutants of the five MAR-encoding genes in strain JMP134 showed that none of these genes is essential for degradation of the tested compounds. However, the role of tfdF(I)/tfdF(II) and tcpD genes in the expression of MAR activity during catabolism of 2,4-D and 2,4,6-TCP, respectively, was confirmed by enzyme activity tests in mutants. These results reveal a striking example of genetic redundancy in the degradation of aromatic compounds.