Unraveling and resolving inefficient glucolipid biosurfactants production through quantitative multiomics analyses of Starmerella bombicola strains.

Glucolipids (GLs) are glycolipid biosurfactants with promising properties. These GLs are composed of glucose attached to a hydroxy fatty acid through a ω and/or ω-1 glycosidic linkage. Up until today these interesting molecules could only be produced using an engineered Starmerella bombicola strain (∆ugtB1::URA3 G9) producing GLs instead of sophorolipids, albeit with a very low average productivity (0.01 g·L-1 ·h-1 ). In this study, we investigated the reason(s) for this via reverse-transcription quantitative polymerase chain reaction and Liquid chromatography-multireaction monitoring-mass spectrometry. We found that all glycolipid biosynthetic genes and enzymes were downregulated in the ∆ugtB1 G9 strain in comparison to the wild type. The underlying reason for this downregulation was further investigated by performing quantitative metabolome comparison of the ∆ugtB1 G9 strain with the wild type and two other engineered strains also tinkered in their glycolipid biosynthetic gene cluster. This analysis revealed a clear distortion of the entire metabolism of the ∆ugtB1 G9 strain compared to all the other strains. Because the parental strain of the former was a spontaneous ∆ura3 mutant potentially containing other "hidden" mutations, a new GL production strain was generated based on a rationally engineered ∆ura3 mutant (PT36). Indeed, a 50-fold GL productivity increase (0.51 g·L-1 ·h-1 ) was obtained with the new ∆ugtB1::URA3 PT36 strain compared with the G9-based strain (0.01 g·L-1 ·h-1 ) in a 10 L bioreactor experiment, yielding 118 g/L GLs instead of 8.39 g/L. Purification was investigated and basic properties of the purified GLs were determined. This study forms the base for further development and optimization of S. bombicola as a production platform strain for (new) biochemicals.

Small RNA NcS27 co-regulates utilization of carbon sources in Burkholderia cenocepacia J2315.

Small non-coding sRNAs have versatile roles in regulating bacterial metabolism. Four short homologous Burkholderia cenocepacia sRNAs strongly expressed under conditions of growth arrest were recently identified. Here we report the detailed investigation of one of these, NcS27. sRNA NcS27 contains a short putative target recognition sequence, which is conserved throughout the order Burkholderiales. This sequence is the reverse complement of the Shine-Dalgarno sequence of a large number of genes involved in transport and metabolism of amino acids and carbohydrates. Overexpression of NcS27 sRNA had a distinct impact on growth, attenuating growth on a variety of substrates such as phenylalanine, tyrosine, glycerol and galactose, while having no effect on growth on other substrates. Transcriptomics and proteomics of NcS27 overexpression and silencing mutants revealed numerous predicted targets changing expression, notably of genes involved in degradation of aromatic amino acids phenylalanine and tyrosine, and in transport of carbohydrates. The conserved target recognition sequence was essential for growth phenotypes and gene expression changes. Cumulatively, our data point to a role of NcS27 in regulating the shutdown of metabolism upon nutrient deprivation in B. cenocepacia. We propose Burkholderiadouble-hairpin sRNA regulator bdhR1 as designation for ncS27.

Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region.

AdeR-AdeS is a two-component regulatory system, which controls expression of the adeABC efflux pump involved in Acinetobacter baumannii multidrug resistance. AdeR is a response regulator consisting of an N-terminal receiver domain and a C-terminal DNA-binding-domain. AdeR binds to a direct-repeat DNA in the intercistronic region between adeR and adeABC. We demonstrate a markedly high affinity binding between unphosphorylated AdeR and DNA with a dissociation constant of 20 nM. In addition, we provide a 2.75 Å crystal structure of AdeR DNA-binding-domain complexed with the intercistronic DNA. This structure shows that the α3 and ß hairpin formed by ß5-ß6 interacts with the major and minor groove of the DNA, which in turn leads to the introduction of a bend. The AdeR receiver domain structure revealed a dimerization motif mediated by a gearwheel-like structure involving the D108F109-R122 motif through cation π stack interaction. The structure of AdeR receiver domain bound with magnesium indicated a conserved Glu19Asp20-Asp63 magnesium-binding motif, and revealed that the potential phosphorylation site Asp63OD1 forms a hydrogen bond with Lys112. We thus dissected the mechanism of how AdeR recognizes the intercistronic DNA, which leads to a diverse mode of response regulation. Unlocking the AdeRS mechanism provides ways to circumvent A. baumannii antibiotic resistance.

A Method for Comprehensive Proteomic Analysis of Human Faecal Samples to Investigate Gut Dysbiosis in Patients with Cystic Fibrosis.

BACKGROUND: This chapter reports the evaluation of two shotgun metaproteomic workflows. The methods were developed to investigate gut dysbiosis via analysis of the faecal microbiota from patients with cystic fibrosis (CF). We aimed to set up an unbiased and effective method to extract the entire proteome, i.e. to extract sufficient bacterial proteins from the faecal samples in combination with a maximum of host proteins giving information on the disease state. METHODS: Two protocols were compared; the first method involves an enrichment of the bacterial proteins while the second method is a more direct method to generate a whole faecal proteome extract. The different extracts were analysed using denaturing polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry aiming a maximal coverage of the bacterial protein content in faecal samples. RESULTS AND CONCLUSIONS: In all extracts, microbial proteins are detected, and in addition, nonbacterial proteins are detected in all samples providing information about the host status. Our study demonstrates the huge influence of the used protein extraction method on the obtained result and shows the need for a standardised and appropriate sample preparation for metaproteomic analysis. To address questions on the health status of the patients, a whole protein extract is preferred over a method to enrich the bacterial fraction. In addition, the method of the whole protein fraction is faster, which gives the possibility to analyse more biological replicates.

Proteomic Response to Thaxtomin Phytotoxin Elicitor Cellobiose and to Deletion of Cellulose Utilization Regulator CebR in Streptomyces scabies.

Streptomyces scabies is responsible for common scab disease on root and tuber vegetables. Production of its main phytotoxin thaxtomin A is triggered upon transport of cellulose byproducts cellotriose and cellobiose, which disable the repression of the thaxtomin biosynthesis activator gene txtR by the cellulose utilization regulator CebR. To assess the intracellular response under conditions where S. scabies develops a virulent behavior, we performed a comparative proteomic analysis of wild-type S. scabies 87-22 and its cebR null mutant (hyper-virulent phenotype) grown in the absence or presence of cellobiose. Our study revealed significant changes in abundance of proteins belonging to metabolic pathways known or predicted to be involved in pathogenicity of S. scabies. Among these, we identified proteins of the cello-oligosaccharide-mediated induction of thaxtomin production, the starch utilization system required for utilization of the carbohydrate stored in S. scabies's hosts, and siderophore synthesis utilization systems, which are key features of pathogens to acquire iron once they colonized the host. Thus, proteomic analysis supported by targeted mass spectrometry-based metabolite quantitative analysis revealed the central role of CebR as a regulator of virulence of S. scabies.

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity.

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50 kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be -170 mV, +133 mV and +210 mV, respectively, at pH 7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH 7.0 and millimolar range KM values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (KM = 0.6 ±â€¯0.2 and 1.8 ±â€¯0.5 mM H2O2, respectively), with similar turnover numbers (kcat = 19 ±â€¯2 and 13 ±â€¯2 s-1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV) = O species was observed upon incubation of fully oxidized YhjA with H2O2. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.

Targeting Protumoral Tumor-Associated Macrophages with Nanobody-Functionalized Nanogels through Strain Promoted Azide Alkyne Cycloaddition Ligation.

Tumor-associated macrophages (TAMs) with high expression levels of the Macrophage Mannose Receptor (MMR, CD206) exhibit a strong angiogenic and immune suppressive activity. Thus, they are a highly attractive target in cancer immunotherapy, with the aim to modulate their protumoral behavior. Here, we introduce polymer nanogels as potential drug nanocarriers which were site-specifically decorated with a Nanobody (Nb) specific for the MMR. Using azide-functionalized RAFT chain transfer agents, they provide access to amphiphilic reactive ester block copolymers that self-assemble into micelles and are afterwards core-cross-linked toward fully hydrophilic nanogels with terminal azide groups on their surface. MMR-targeting Nb can site-selectively be functionalized with one single cyclooctyne moiety by maleimide-cysteine chemistry under mildly reducing conditions which enables successful chemoorthogonal conjugation to the nanogels. The resulting Nb-functionalized nanogels were highly efficient in targeting MMR-expressing cells and TAMs both in vitro and in vivo. We believe that these findings pave the road for targeted eradication or modulation of pro-tumoral MMRhigh TAMs.

Elevated faecal ovotransferrin concentrations are indicative for intestinal barrier failure in broiler chickens.

Intestinal health is critically important for the welfare and performance of poultry. Enteric diseases that cause gut barrier failure result in high economic losses. Up till now there is no reliable faecal marker to measure gut barrier failure under field conditions. Therefore, the aim of the present study was to identify a faecal protein marker for diminished intestinal barrier function due to enteric diseases in broilers. To assess this, experimental necrotic enteritis and coccidiosis in broilers were used as models for gut barrier failure. Ovotransferrin was identified as a marker for gut barrier failure using a proteomics approach on samples from chickens with necrotic enteritis. These results were confirmed via ELISA on samples derived from both necrotic enteritis and coccidiosis trials, where faecal ovotransferrin levels were significantly correlated with the severity of gut barrier failure caused by either coccidiosis or necrotic enteritis. This indicates that faecal ovotransferrin quantification may represent a valuable tool to measure gut barrier failure caused by enteric pathogens.

The solution structure of the soluble form of the lipid-modified azurin from Neisseria gonorrhoeae, the electron donor of cytochrome c peroxidase.

Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277±5 mV, at pH7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the ß-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment.

Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress.

Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.

Unipept web services for metaproteomics analysis.

UNLABELLED: Unipept is an open source web application that is designed for metaproteomics analysis with a focus on interactive datavisualization. It is underpinned by a fast index built from UniProtKB and the NCBI taxonomy that enables quick retrieval of all UniProt entries in which a given tryptic peptide occurs. Unipept version 2.4 introduced web services that provide programmatic access to the metaproteomics analysis features. This enables integration of Unipept functionality in custom applications and data processing pipelines. AVAILABILITY AND IMPLEMENTATION: The web services are freely available at http://api.unipept.ugent.be and are open sourced under the MIT license. CONTACT: Unipept@ugent.be SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Understanding the response of Desulfovibrio desulfuricans ATCC 27774 to the electron acceptors nitrate and sulfate - biosynthetic costs modulate substrate selection.

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6h time point. Nitrite levels measured along the growth curve revealed a peak at 3h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates.

The unique peptidome: Taxon-specific tryptic peptides as biomarkers for targeted metaproteomics.

The Unique Peptide Finder (http://unipept.ugent.be/peptidefinder) is an interactive web application to quickly hunt for tryptic peptides that are unique to a particular species, genus, or any other taxon. Biodiversity within the target taxon is represented by a set of proteomes selected from a monthly updated list of complete and nonredundant UniProt proteomes, supplemented with proprietary proteomes loaded into persistent local browser storage. The software computes and visualizes pan and core peptidomes as unions and intersections of tryptic peptides occurring in the selected proteomes. In addition, it also computes and displays unique peptidomes as the set of all tryptic peptides that occur in all selected proteomes but not in any UniProt record not assigned to the target taxon. As a result, the unique peptides can serve as robust biomarkers for the target taxon, for example, in targeted metaproteomics studies. Computations are extremely fast since they are underpinned by the Unipept database, the lowest common ancestor algorithm implemented in Unipept and modern web technologies that facilitate in-browser data storage and parallel processing.

Intra- and Interspecies Effects of Outer Membrane Vesicles from Stenotrophomonas maltophilia on ß-Lactam Resistance.

The treatment ofStenotrophomonas maltophiliainfection with ß-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded ß-lactamases. Here, we show that these ß-lactamase-packed OMVs are capable of establishing extracellular ß-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating speciesPseudomonas aeruginosaandBurkholderia cenocepacia.

Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola.

Although sophorolipids (SLs) produced by S. bombicola are a real showcase for the industrialization of microbial biosurfactants, some important drawbacks are associated with this efficient biological process, e.g., the simultaneous production of acidic and lactonic SLs. Depending on the application, there is a requirement for the naturally produced mixture to be manipulated to give defined ratios of the components. Recently, the enzyme responsible for the lactonization of SLs was discovered. The discovery of the gene encoding this lactone esterase (sble) enabled the development of promising S. bombicola strains producing either solely lactonic (using a sble overexpression strain described in this paper: oe sble) or solely acidic SLs (using a sble deletion strain, which was recently described, but not characterized yet: Δsble). The new S. bombicola strains were used to investigate the production processes (fermentation and purification) of either lactonic or acidic SLs. The strains maintain the high inherent productivities of the wild-type or even perform slightly better and thus represent a realistic industrial opportunity. 100% acidic SLs with a mixed acetylation pattern were obtained for the Δsble strain, while the inherent capacity to selectively produce lactonic SLs was significantly increased (+42%) for the oe sble strain (99% lactonic SLs). Moreover, the regulatory effect of citrate on lactone SL formation for the wild-type was absent in this new strain, which indicates that it is more robust and better suited for the industrial production of lactonic SLs. Basic parameters were determined for the purified SLs, which confirm that the two new strains produce molecules with distinctive properties of which the application potential can now easily be investigated independently.

ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry.

Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response. Here we investigated the assembly of the Shewanella oneidensis MR-1 HipA-HipB complex using native electrospray ion mobility-mass spectrometry and chemical crosslinking combined with mass spectrometry. We revealed that the HipA autophosphorylation was accompanied by a large conformational change, and confirmed structural evidence that S. oneidensis MR-1 HipA-HipB assembly was distinct from the prototypic E. coli HipA-HipB complex. Graphical abstract Ion mobility mass spectrometry shows a two phase transition from unstructured HipA to a compact folded phosphorylated protein.

Characterization of a novel enzyme-Starmerella bombicola lactone esterase (SBLE)-responsible for sophorolipid lactonization.

We recently discovered a novel enzyme in the exoproteome of Starmerella bombicola, which is structurally related to Candida antarctica lipase A. A knockout strain for this enzyme does no longer produce lactonic sophorolipids, prompting us to believe that this protein is the missing S. bombicola lactone esterase (SBLE). SBLE catalyzes a rather unusual reaction, i.e., an intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment, which raised questions about its activity and mode of action. Here, we report the heterologous production of this enzyme in Pichia pastoris and its purification in a two-step strategy. Purified recombinant SBLE (rSBLE) was used to perform HPLC and liquid chromatography mass spectrometry (LCMS)-based assays with different sophorolipid mixtures. We experimentally confirmed that SBLE is able to perform ring closure of acetylated acidic sophorolipids. This substrate was selected for rSBLE kinetic studies to estimate the apparent values of K m . We established that rSBLE displays optimal activity in the pH range of 3.5 to 6 and has an optimal temperature in the range of 20 to 50 °C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser194 in the predicted active site pocket and show that this mutant is lacking the ability to lactonize sophorolipids. We therefore propose that SBLE operates via the common serine hydrolase mechanism in which the catalytic serine residue is assisted by a His/Asp pair.

The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence.

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin-antitoxin proteins HipASO and HipBSO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin-antitoxin protein module. Here we show that phosphorylated HipASO can engage in an unexpected ternary complex with HipBSO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipBSO in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipASO in the ternary complex, indicative of its role in binding HipASO to abolish its function in persistence. The structure of HipASO in complex with a non-hydrolyzable ATP analogue shows that HipASO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipASO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.

The Unipept metaproteomics analysis pipeline.

Unipept (http://unipept.ugent.be) is a web application that offers a user-friendly way to explore the biodiversity of complex metaproteome samples by providing interactive visualizations. In this article, the updates and changes to Unipept since its initial release are presented. This includes the addition of interactive sunburst and treeview visualizations to the multipeptide analysis, the foundations of an application programming interface (API) and a command line interface, updated data sources, and the open-sourcing of the entire application under the MIT license.

New insights into the assembly of bacterial secretins: structural studies of the periplasmic domain of XcpQ from Pseudomonas aeruginosa.

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.