LuxS-independent formation of AI-2 from ribulose-5-phosphate

<p>Abstract</p> <p>Background</p> <p>In many bacteria, the signal molecule AI-2 is generated from its precursor <it>S</it>-ribosyl-L-homocysteine in a reaction catalysed by the enzyme LuxS. However, generation of AI-2-like activity has also been reported for organisms lacking the <it>luxS </it>gene and the existence of alternative pathways for AI-2 formation in <it>Escherichia coli </it>has recently been predicted by stochastic modelling. Here, we investigate the possibility that spontaneous conversion of ribulose-5-phosphate could be responsible for AI-2 generation in the absence of <it>luxS</it>.</p> <p>Results</p> <p>Buffered solutions of ribulose-5-phosphate, but not ribose-5-phosphate, were found to contain high levels of AI-2 activity following incubation at concentrations similar to those reported <it>in vivo</it>. To test whether this process contributes to AI-2 formation by bacterial cells <it>in vivo</it>, an improved <it>Vibrio harveyi </it>bioassay was used. In agreement with previous studies, culture supernatants of <it>E. coli </it>and <it>Staphylococcus aureus luxS </it>mutants were found not to contain detectable levels of AI-2 activity. However, low activities were detected in an <it>E. coli pgi-eda-edd-luxS </it>mutant, a strain which degrades glucose entirely via the oxidative pentose phosphate pathway, with ribulose-5-phosphate as an obligatory intermediate.</p> <p>Conclusion</p> <p>Our results suggest that LuxS-independent formation of AI-2, via spontaneous conversion of ribulose-5-phosphate, may indeed occur <it>in vivo</it>. It does not contribute to AI-2 formation in wildtype <it>E. coli </it>and <it>S. aureus </it>under the conditions tested, but may be responsible for the AI-2-like activities reported for other organisms lacking the <it>luxS </it>gene.</p

AI-2 does not function as a quorum sensing molecule in Campylobacter jejuni during exponential growth in vitro

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni </it>contains a homologue of the <it>luxS </it>gene shown to be responsible for the production of the signalling molecule autoinducer-2 (AI-2) in <it>Vibrio harveyi </it>and <it>Vibrio cholerae</it>. The aim of this study was to determine whether AI-2 acted as a diffusible quorum sensing signal controlling <it>C. jejuni </it>gene expression when it is produced at high levels during mid exponential growth phase.</p> <p>Results</p> <p>AI-2 activity was produced by the parental strain NCTC 11168 when grown in rich Mueller-Hinton broth (MHB) as expected, but interestingly was not present in defined Modified Eagles Medium (MEM-α). Consistent with previous studies, the <it>luxS </it>mutant showed comparable growth rates to the parental strain and exhibited decreased motility halos in both MEM-α and MHB. Microarray analysis of genes differentially expressed in wild type and <it>luxS </it>mutant strains showed that many effects on mRNA transcript abundance were dependent on the growth medium and linked to metabolic functions including methionine metabolism. Addition of exogenously produced AI-2 to the wild type and the <it>luxS </it>mutant, growing exponentially in either MHB or MEM-α did not induce any transcriptional changes as analysed by microarray.</p> <p>Conclusion</p> <p>Taken together these results led us to conclude that there is no evidence for the role of AI-2 in cell-to-cell communication in <it>C. jejuni </it>strain NCTC 11168 under the growth conditions used, and that the effects of the <it>luxS </it>mutation on the transcriptome are related to the consequential loss of function in the activated methyl cycle.</p

Improving carbon monoxide tolerance of Cupriavidus necator H16 through adaptive laboratory evolution

Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution.Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions.Conclusion:C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes

The Lanthipeptide Synthetase-like Protein CA_C0082 Is an Effector of Agr Quorum Sensing in Clostridium acetobutylicum

Lanthipeptide synthetases are present in all domains of life. They catalyze a crucial step during lanthipeptide biosynthesis by introducing thioether linkages during posttranslational peptide modification. Lanthipeptides have a wide range of functions, including antimicrobial and morphogenetic activities. Intriguingly, several Clostridium species contain lanthipeptide synthetase-like genes of the class II (lanM) family but lack other components of the lanthipeptide biosynthetic machinery. In all instances, these genes are located immediately downstream of putative agr quorum sensing operons. The physiological role and mode of action of the encoded LanM-like proteins remain uncertain as they lack conserved catalytic residues. Here we show for the industrial organism Clostridium acetobutylicum that the LanM-like protein CA_C0082 is not required for the production of active AgrD-derived signaling peptide but nevertheless acts as an effector of Agr quorum sensing. Expression of CA_C0082 was shown to be controlled by the Agr system and is a prerequisite for granulose (storage polymer) formation. The accumulation of granulose, in turn, was shown to be required for maximal spore formation but also to reduce early solvent formation. CA_C0082 and its putative homologs appear to be closely associated with Agr systems predicted to employ signaling peptides with six-membered ring structures and may represent a new subfamily of LanM-like proteins. This is the first time their contribution to bacterial Agr signaling has been described

Cationic polymer mediated bacterial clustering: cell-adhesive properties of homo- and copolymers

New anti-infective materials are needed urgently as alternatives to conventional biocides. It has recently been established that polymer materials designed to bind to the surface of bacteria can induce the formation of cell clusters which enhance the expression of quorum sensing controlled phenotypes. These materials are relevant for anti-infective strategies as they have the potential to inhibit adhesion while at the same time modulating Quorum Sensing (QS) controlled virulence. Here we carefully evaluate the role that charge and catechol moieties in these polymers play on the binding. We investigate the ability of the cationic polymers poly(N-[3-(dimethylamino)propyl] methacrylamide) (pDMAPMAm, P1), poly(N-dopamine methacrylamide-co-N-[3-(dimethylamino)propyl] methacrylamide) (pDMAm-co-pDMAPMAm, P2) and p(3,4-dihydroxy-l-phenylalanine methacrylamide), p(l-DMAm, P3) to cluster a range of bacteria, such as Staphylococcus aureus (Gram-positive), Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa (Gram-negative) under conditions of varying pH (6, 7 and 8) and polymer concentration (0.1 and 0.5 mg/mL). We identify that clustering ability is strongly dependent on the balance between charge and hydrophobicity. Moreover, our results suggest that catechol moieties have a positive effect on adhesive properties, but only in the presence of cationic residues such as for P2. Overall, our results highlight the subtle interplay between dynamic natural surfaces and synthetic materials, as well as the need to consider synergistic structure–property relationship when designing antimicrobial polymers

The carbonic anhydrase of Clostridium autoethanogenum represents a new subclass of β-carbonic anhydrases

Carbonic anhydrase catalyses the interconversion of carbon dioxide and water to bicarbonate and protons. It was unknown if the industrial relevant acetogen Clostridium autoethanogenum possesses these enzymes. We identified two putative carbonic anhydrase genes in its genome, one of the β class and one of the γ class. Carbonic anhydrase activity was found for the purified β class enzyme, but not the γ class candidate. Functional complementation of an Escherichia coli carbonic anhydrase knock-out mutant showed that the β class carbonic anhydrase could complement this activity, but not the γ class candidate gene. Phylogenetic analysis showed that the β class carbonic anhydrase of Clostridium autoethanogenum represents a novel sub-class of β class carbonic anhydrases that form the F-clade. The members of this clade have the shortest primary structure of any known carbonic anhydrase

Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum

Acetogenic bacteria can play a major role in achieving Net Zero through their ability to convert CO2 into industrially relevant chemicals and fuels. Full exploitation of this potential will be reliant on effective metabolic engineering tools, such as those based on the Streptococcus pyogenes CRISPR/Cas9 system. However, attempts to introduce cas9-containing vectors into Acetobacterium woodii were unsuccessful, most likely as a consequence of Cas9 nuclease toxicity and the presence of a recognition site for an endogenous A. woodii restriction–modification (R-M) system in the cas9 gene. As an alternative, this study aims to facilitate the exploitation of CRISPR/Cas endogenous systems as genome engineering tools. Accordingly, a Python script was developed to automate the prediction of protospacer adjacent motif (PAM) sequences and used to identify PAM candidates of the A. woodii Type I-B CRISPR/Cas system. The identified PAMs and the native leader sequence were characterized in vivo by interference assay and RT-qPCR, respectively. Expression of synthetic CRISPR arrays, consisting of the native leader sequence, direct repeats, and adequate spacer, along with an editing template for homologous recombination, successfully led to the creation of 300 bp and 354 bp in-frame deletions of pyrE and pheA, respectively. To further validate the method, a 3.2 kb deletion of hsdR1 was also generated, as well as the knock-in of the fluorescence-activating and absorption-shifting tag (FAST) reporter gene at the pheA locus. Homology arm length, cell density, and the amount of DNA used for transformation were found to significantly impact editing efficiencies. The devised workflow was subsequently applied to the Type I-B CRISPR/Cas system of Clostridium autoethanogenum, enabling the generation of a 561 bp in-frame deletion of pyrE with 100% editing efficiency. This is the first report of genome engineering of both A. woodii and C. autoethanogenum using their endogenous CRISPR/Cas systems

Functional genetic elements for controlling gene expression in Cupriavidus necator H16

A robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements such as constitutive and inducible promoters, as well as ribosome binding sites (RBSs), are required. In this study, we design, build and test promoters and RBSs for controlling gene expression in the model lithoautotroph Cupriavidus necator H16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within more than 700-fold dynamic range is compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-L-arabinose and RhaRS/PrhaBAD-L-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems are evaluated. By supplying different concentrations of inducers, over a 1000-fold range of gene expression levels is achieved. Application of inducible systems for controlling expression of isoprene synthase gene ispS leads to isoprene yields that exhibit a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements such as mRNA stem-loop structure and A/U- rich sequence on the gene expression is also evaluated. A second-order polynomial relationship is observed between the RBS activities and isoprene yields. This study presents quantitative data on regulatory genetic elements and expands the genetic toolbox of C. necator

Metabolic modeling-based drug repurposing in Glioblastoma

The manifestation of intra- and inter-tumor heterogeneity hinders the development of ubiquitous cancer treatments, thus requiring a tailored therapy for each cancer type. Specifically, the reprogramming of cellular metabolism has been identified as a source of potential drug targets. Drug discovery is a long and resource-demanding process aiming at identifying and testing compounds early in the drug development pipeline. While drug repurposing efforts (i.e., inspecting readily available approved drugs) can be supported by a mechanistic rationale, strategies to further reduce and prioritize the list of potential candidates are still needed to facilitate feasible studies. Although a variety of ‘omics’ data are widely gathered, a standard integration method with modeling approaches is lacking. For instance, flux balance analysis is a metabolic modeling technique that mainly relies on the stoichiometry of the metabolic network. However, exploring the network’s topology typically neglects biologically relevant information. Here we introduce Transcriptomics-Informed Stoichiometric Modelling And Network analysis (TISMAN) in a recombinant innovation manner, allowing identification and validation of genes as targets for drug repurposing using glioblastoma as an exemplar