Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster.

The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in Pseudomonas putida KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ70-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. P. putida K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS-GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and P. putida K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.

DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway.

The facultative anaerobe Thauera aromatica strain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. The dbdR gene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR for p-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (P orf20 ) was only active in the presence of 3,5-DHB, the other two (P dbhL and P orf18 ) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the P dbdR promoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica AR-1, allowing a fast and optimized response to the presence of the aromatic.IMPORTANCEThauera aromatica AR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

The Azoarcus anaerobius 1,3-Dihydroxybenzene (Resorcinol) Anaerobic Degradation Pathway Is Controlled by the Coordinated Activity of Two Enhancer-Binding Proteins.

The anaerobic resorcinol degradation pathway in Azoarcus anaerobius is unique in that it uses an oxidative rather than a reductive strategy to overcome the aromatic ring stability in degradation of this compound, in a process that is dependent on nitrate respiration. We show that the pathway is organized in five transcriptional units, three of which are inducible by the presence of the substrate. Three σ54-dependent promoters located upstream from the three operons coding for the main pathway enzymes were identified, which shared a similar structure with conserved upstream activating sequences (UASs) located at 103 to 111 bp from the transcription start site. Expression of the pathway is controlled by the bacterial enhancer-binding proteins (bEBPs) RedR1 and RedR2, two homologous regulators that, despite their high sequence identity (97%), have nonredundant functions: RedR2, the master regulator which also controls RedR1 expression, is itself able to promote transcription from two of the promoters, while RedR1 activity is strictly dependent on the presence of RedR2. The two regulators were shown to interact with each other, suggesting that the natural mode of activation is by forming heterodimers, which become active in the presence of the substrate after its metabolization to hydroxybenzoquinone through the pathway enzymes. The model structure of the N-terminal domain of the proteins is composed of tandem GAF and PAS motifs; the possible mechanisms controlling the activity of the regulators are discussed.IMPORTANCEAzoarcus anaerobius is a strict anaerobe that is able to use 1,3-dihydroxybenzene as the sole carbon source in a process that is dependent on nitrate respiration. We have shown that expression of the pathway is controlled by two regulators of almost identical sequences: the bEBPs RedR1 and RedR2, which share 97% identity. These regulators control three promoters with similar structure. Despite their sequence identity, the two bEBPs are not redundant and are both required for maximum pathway expression. In fact, the two proteins function as heterodimers and require activation by the pathway intermediate hydroxyhydroquinone. The structure of the domain sensing the activation signal resembles that of regulators that are known to interact with other proteins.

Subtle sequence differences between two interacting σ54 -dependent regulators lead to different activation mechanisms.

In the strictly anaerobic nitrate reducing bacterium Aromatoleum anaerobium, degradation of 1,3-dihydroxybenzene (1,3-DHB, resorcinol) is controlled by two bacterial enhancer-binding proteins (bEBPs), RedR1 and RedR2, which regulate the transcription of three σ54 -dependent promoters controlling expression of the pathway. RedR1 and RedR2 are identical over their length except for their N-terminal tail which differ in sequence and length (six and eight residues, respectively), a single change in their N-terminal domain (NTD), and nine non-identical residues in their C-terminal domain (CTD). Their NTD is composed of a GAF and a PAS domain connected by a linker helix. We show that each regulator is controlled by a different mechanism: whilst RedR1 responds to the classical NTD-mediated negative regulation that is released by the presence of its effector, RedR2 activity is constitutive and controlled through interaction with BtdS, an integral membrane subunit of hydroxyhydroquinone dehydrogenase carrying out the second step in 1,3-DHB degradation. BtdS sequesters the RedR2 regulator to the membrane through its NTD, where a four-Ile track in the PAS domain, interrupted by a Thr in RedR1, and the N-terminal tail are involved. The presence of 1,3-DHB, which is metabolized to hydroxybenzoquinone, releases RedR2 from the membrane. Most bEBPs assemble into homohexamers to activate transcription; we show that hetero-oligomer formation between RedR1 and RedR2 is favoured over homo-oligomers. However, either an NTD-truncated version of RedR1 or a full-length RedR2 are capable of promoter activation on their own, suggesting they should assemble into homohexamers in vivo. We show that promoter DNA behaves as an allosteric effector through binding the CTD to control ΔNTD-RedR1 multimerization and activity. Overall, the regulation of the 1,3-DHB anaerobic degradation pathway can be described as a novel mode of bEBP activation and assembly.

Occurrence and diversity of the oxidative hydroxyhydroquinone pathway for the anaerobic degradation of aromatic compounds in nitrate-reducing bacteria.

The nitrate-reducing betaproteobacteria Azoarcus anaerobius and Thauera aromatica AR-1 use an oxidative mechanism to anaerobically degrade resorcinol and 3,5-dihydroxybenzoate (3,5-DHB), respectively, rendering hydroxyhydroquinone as intermediate. The first pathway step is performed by a dimethylsulphoxide-reductase family hydroxylase. The gene cluster coding for the pathway is homologous in these strains. Only these two Rhodocyclales are known to follow this anaerobic pathway, and nothing is known about its distribution in prokaryotes. To determine the relevance and diversity of this strategy in nature, we enriched for bacteria able to oxidize resorcinol or 3,5-DHB under denitrifying conditions. Nitrate-reducing bacteria able to degrade these compounds were present in soil, aquifer and marine sediments. We were able to isolate a number of strains with this capacity from soil and aquifer samples. Amplicon libraries of rehL, the gene encoding the first step of this pathway, showed an overall low diversity, most sequences clustering with either pathway enzyme. Isolates belonging to the Beta- and Gammaproteobacteria able to grow on these substrates revealed rehL homologues only in strains belonging to Thauera and Azoarcus. Analysis of sequenced genomes in the databases detected the presence of highly similar clusters in two additional betaproteobacteria and in the gammaproteobacterium Sedimenticola selenatireducens, although anaerobic growth on a dihydroxyaromatic could only be confirmed in Thauera chlorobenzoica 3CB-1. The presence of mobile elements in the flanking sequences of some of the clusters suggested events of horizontal gene transfer, probably contributing to expand the pathway to a broader host range within the Proteobacteria.

Bacterial nanocellulose production from naphthalene.

Polycyclic aromatic compounds (PAHs) are toxic compounds that are released in the environment as a consequence of industrial activities. The restoration of PAH-polluted sites considers the use of bacteria capable of degrading aromatic compounds to carbon dioxide and water. Here we characterize a new Xanthobacteraceae strain, Starkeya sp. strain N1B, previously isolated during enrichment under microaerophilic conditions, which is capable of using naphthalene crystals as the sole carbon source. The strain produced a structured biofilm when grown on naphthalene crystals, which had the shape of a half-sphere organized over the crystal. Scanning electron microscopy (SEM) and GC-MS analysis indicated that the biofilm was essentially made of cellulose, composed of several micron-long nanofibrils of 60 nm diameter. A cellulosic biofilm was also formed when the cells grew with glucose as the carbon source. Fourier transformed infrared spectroscopy (FTIR) confirmed that the polymer was type I cellulose in both cases, although the crystallinity of the material greatly depended on the carbon source used for growth. Using genome mining and mutant analysis, we identified the genetic complements required for the transformation of naphthalene into cellulose, which seemed to have been successively acquired through horizontal gene transfer. The capacity to develop the biofilm around the crystal was found to be dispensable for growth when naphthalene was used as the carbon source, suggesting that the function of this structure is more intricate than initially thought. This is the first example of the use of toxic aromatic hydrocarbons as the carbon source for bacterial cellulose production. Application of this capacity would allow the remediation of a PAH into such a value-added polymer with multiple biotechnological usages.