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1.  Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species 
Biochemical Journal  2014;458(Pt 3):499-511.
Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).
We biochemically and structurally characterize a novel inducible surface-associated sialic acid-binding protein present in human-dwelling Bacteroidetes species that represents a novel type of sialic acid-specific polysaccharide utilization locus.
doi:10.1042/BJ20131415
PMCID: PMC3969230  PMID: 24351045
Bacteroides fragilis; carbohydrate; nanOU; outer membrane; sialic acid; transport; BF, Bacteroides fragilis; FA, fastidious anaerobe; HRP, horseradish peroxidase; ManNAc, N-acetyl mannosamine; NAM, N-acetylmuramic acid; NanO, neuraminate outer membrane permease; NanU, extracellular neuraminate uptake protein; Neu5Ac, 5-N-acetylneuraminic acid; Neu5Ac2en, 2-deoxy-2,3-didehydro-Neu5Ac; Neu5Gc, N-glycolylneuraminic acid; PUL, polysaccharide utilization locus; TBDR, TonB-dependent receptor; TCA, trichloroacetic acid; TF, Tannerella forsythia; TPR, tetratricopeptide repeat
2.  Archaeal Signal Transduction: Impact of Protein Phosphatase Deletions on Cell Size, Motility, and Energy Metabolism in Sulfolobus acidocaldarius* 
Molecular & Cellular Proteomics : MCP  2013;12(12):3908-3923.
In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.
doi:10.1074/mcp.M113.027375
PMCID: PMC3861733  PMID: 24078887
3.  The quantitative proteomic response of Synechocystis sp. PCC6803 to phosphate acclimation 
Aquatic Biosystems  2013;9:5.
Background
Inorganic phosphate (Pi) is a critical nutrient for all life and is periodically limiting in marine and freshwater provinces, yet little is understood how organisms acclimate to fluctuations in Pi within their environment. To investigate whole cell adaptation, we grew Synechocystis sp. PCC6803, a model freshwater cyanobacterium, in 3%, and 0.3% inorganic phosphate (Pi) media. The cells were allowed to acclimate over 60 days, and cells were harvested for quantitative high throughput mass spectrometry-based proteomics using the iTRAQ™ labelling technology.
Results
In total, 120 proteins were identified, and 52 proteins were considered differentially abundant compared to the control. Alkaline phosphatase (APase) activities correlated significantly (p < 0.05) with observed relative PhoA abundances. PstS1 and PstS2 were both observed, yet PstS1 was not differentially more abundant than the control. Phycobilisome protein abundances appeared to be coordinated, and are significantly less abundant in 0.3% Pi than 3% Pi cultures. Also, the central metabolic cell function appears to have shifted towards the production of (NADPH) reducing energy and nucleotide sugars.
Conclusions
This acclimation response bears strong similarity to the previously reported response to nitrogen deprivation within Synechocystis sp. PCC 6803. However, it also demonstrates some characteristics of desiccation stress, such as the regulation of fatty acids and increased abundance of rehydrin in the 3% Pi culture.
doi:10.1186/2046-9063-9-5
PMCID: PMC3600050  PMID: 23442353
Cyanobacteria; iTRAQ; Phosphate acclimation; Phycobilisome; Pentose phosphate pathway; Ribose sugars; Synechocystis
5.  Comparative quantitative proteomics of prochlorococcus ecotypes to a decrease in environmental phosphate concentrations 
Aquatic Biosystems  2012;8:7.
Background
The well-lit surface waters of oligotrophic gyres significantly contribute to global primary production. Marine cyanobacteria of the genus Prochlorococcus are a major fraction of photosynthetic organisms within these areas. Labile phosphate is considered a limiting nutrient in some oligotrophic regions such as the Caribbean Sea, and as such it is crucial to understand the physiological response of primary producers such as Prochlorococcus to fluctuations in the availability of this critical nutrient.
Results
Prochlorococcus strains representing both high light (HL) (MIT9312) and low light (LL) (NATL2A and SS120) ecotypes were grown identically in phosphate depleted media (10 μM Pi). The three strains displayed marked differences in cellular protein expression, as determined by high throughput large scale quantitative proteomic analysis. The only strain to demonstrate a significantly different growth rate under reduced phosphate conditions was MIT9312. Additionally, there was a significant increase in phosphate-related proteins such as PhoE (> 15 fold increase) and a depression of the Rubisco protein RbcL abundance in this strain, whereas there appeared to be no significant change within the LL strain SS120.
Conclusions
This differential response between ecotypes highlights the relative importance of phosphate availability to each strain and from these results we draw the conclusion that the expression of phosphate acquisition mechanisms are activated at strain specific phosphate concentrations.
doi:10.1186/2046-9063-8-7
PMCID: PMC3349580  PMID: 22480396
Prochlorococcus; PstS; PhoA; PhoE; Growth; Phosphate
6.  Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli 
Highlights
► Escherichia coli cells require modifying to make ideal hosts for producing glycoproteins. ► Codon optimising pglB leads to increased glycosylation efficiency. ► Lipid linked precursors do not appear to be limiting for E. coli N-glycosylation. ► Increasing expression of WecA improves E. coli N-glycosylation efficiency.
Recently, efforts to increase the toolkit which Escherichia coli cells possess for recombinant protein production in industrial applications, has led to steady progress towards making glycosylated therapeutic proteins. Although the desire to make therapeutically relevant complex proteins with elaborate human-type glycans is a major goal, the relatively poor efficiency of the N-glycosylation process of foreign proteins in E. coli remains a hindrance for industry take-up. In this study, a systematic approach was used to increase glycoprotein production titres of an exemplar protein, AcrA, and the resulting glycosylation efficiency was quantified using a combination of Western blots and pseudo Selective Reaction Monitoring (pSRM). Western blot and pSRM results demonstrate that codon optimising the oligosaccharyltransferase, PglB, for E. coli expression, increases efficiency by 77% and 101%, respectively. Furthermore, increasing expression of glycosyltransferase, WecA, in E. coli improves efficiency by 43% and 27%, respectively. However, increasing the amount of donor lipid used in the glycosylation process did not impact on the glycosylation efficiency in this system, with this specific protein.
doi:10.1016/j.bbrc.2012.02.020
PMCID: PMC3401370  PMID: 22342719
Glycosylation; Escherichia coli; Efficiency; Proteomics; Selective reaction monitoring
7.  iTRAQ Identification of Candidate Serum Biomarkers Associated with Metastatic Progression of Human Prostate Cancer 
PLoS ONE  2012;7(2):e30885.
A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer ‘BPH’, (ii) localised cancer with no evidence of progression, ‘non-progressing’ (iii) localised cancer with evidence of biochemical progression, ‘progressing’, and (iv) bone metastasis at presentation ‘metastatic’. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and ‘panels’ of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.
doi:10.1371/journal.pone.0030885
PMCID: PMC3280251  PMID: 22355332
8.  Proteomic Evaluation and Validation of Cathepsin D Regulated Proteins in Macrophages Exposed to Streptococcus pneumoniae* 
Molecular & Cellular Proteomics : MCP  2011;10(6):M111.008193.
Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (α = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing.
doi:10.1074/mcp.M111.008193
PMCID: PMC3108842  PMID: 21474794
9.  “Hot standards” for the thermoacidophilic archaeon Sulfolobus solfataricus 
Extremophiles   2009;14(1):119-142.
Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics.
Electronic supplementary material
The online version of this article (doi:10.1007/s00792-009-0280-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s00792-009-0280-0
PMCID: PMC2797409  PMID: 19802714
Crenarchaeon; Standard operating procedures; Genomics; Transcriptomics; Proteomics; Metabolomics; Biochemistry; Systems biology
10.  A systems biology approach to investigate the response of Synechocystis sp. PCC6803 to a high salt environment 
Saline Systems  2009;5:8.
Background
Salt overloading during agricultural processes is causing a decrease in crop productivity due to saline sensitivity. Salt tolerant cyanobacteria share many cellular characteristics with higher plants and therefore make ideal model systems for studying salinity stress. Here, the response of fully adapted Synechocystis sp. PCC6803 cells to the addition of 6% w/v NaCl was investigated using proteomics combined with targeted analysis of transcripts.
Results
Isobaric mass tagging of peptides led to accurate relative quantitation and identification of 378 proteins, and approximately 40% of these were differentially expressed after incubation in BG-11 media supplemented with 6% salt for 9 days. Protein abundance changes were related to essential cellular functional alterations. Differentially expressed proteins involved in metabolic responses were also analysed using the probabilitistic tool Mixed Model on Graphs (MMG), where the role of energy conversion through glycolysis and reducing power through pentose phosphate pathway were highlighted. Temporal RT-qPCR experiments were also run to investigate protein expression changes at the transcript level, for 14 non-metabolic proteins. In 9 out of 14 cases the mRNA changes were in accordance with the proteins.
Conclusion
Synechocystis sp. PCC6803 has the ability to regulate essential metabolic processes to enable survival in high salt environments. This adaptation strategy is assisted by further regulation of proteins involved in non-metabolic cellular processes, supported by transcriptional and post-transcriptional control. This study demonstrates the effectiveness of using a systems biology approach in answering environmental, and in particular, salt adaptation questions in Synechocystis sp. PCC6803
doi:10.1186/1746-1448-5-8
PMCID: PMC2743698  PMID: 19735556
11.  Proteomics with a pinch of salt: A cyanobacterial perspective 
Saline Systems  2008;4:1.
Cyanobacteria are ancient life forms and have adapted to a variety of extreme environments, including high salinity. Biochemical, physiological and genetic studies have contributed to uncovering their underlying survival mechanisms, and as recent studies demonstrate, proteomics has the potential to increase our overall understanding further. To date, most salt-related cyanobacterial proteomic studies have utilised gel electrophoresis with the model organism Synechocystis sp. PCC6803. Moreover, focus has been on 2–4% w/v NaCl concentrations within different cellular compartments. Under these conditions, Synechocystis sp. PCC6803 was found to respond and adapt to salt stress through synthesis of general and specific stress proteins, altering the protein composition of extracellular layers, and re-directing control of complex central intermediary pathways. Post-transcriptional control was also predicted through non-correlating transcript level data and identification of protein isoforms.
In this paper, we also review technical developments with emphasis on improving the quality and quantity of proteomic data and overcoming the detrimental effects of salt on sample preparation and analysis. Developments in gel-free methods include protein and peptide fractionation workflows, which can increase coverage of the proteome (20% in Synechocystis sp. PCC6803). Quantitative techniques have also improved in accuracy, resulting in confidence in quantitation approaching or even surpassing that seen in transcriptomic techniques (better than 1.5-fold in differential expression). Furthermore, in vivo metabolic labelling and de novo protein sequencing software have improved the ability to apply proteomics to unsequenced environmental isolates. The example used in this review is a cyanobacterium isolated from a Saharan salt lake.
doi:10.1186/1746-1448-4-1
PMCID: PMC2386806  PMID: 18412952
12.  Identification of a Novel α-Galactosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus†  
Journal of Bacteriology  2006;188(7):2392-2399.
Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable α-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for α-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable α-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.
doi:10.1128/JB.188.7.2392-2399.2006
PMCID: PMC1428385  PMID: 16547025
13.  Macromolecular Fingerprinting of Sulfolobus Species in Biofilm: A Transcriptomic and Proteomic Approach Combined with Spectroscopic Analysis 
Journal of Proteome Research  2011;10(9):4105-4119.
Microorganisms in nature often live in surface-associated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ∼3.4 and ∼1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development.
S. acidocaldarius, S. solfataricus, and S. tokodaii strains were grown independently as biofilms. Comparison between planktonic and biofilm cell popupations of all three strains was performed by spectroscopic analysis (FTIR and XPS), iTRAQ proteomics, and RNA microarrays. To highlight common features in biofilm formation among the Sulfolobus strains, the data is presented as a comparative analysis. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, suggesting their roles as key regulatory factor in biofilm development.
doi:10.1021/pr2003006
PMCID: PMC3166137  PMID: 21761944
archaea; sulfolobus; biofilm; proteomics; transcriptomics; FTIR; thermophilic; acidophilic
14.  Maf-dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export 
Molecular Microbiology  2014;92(2):258-272.
Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non-glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form.
doi:10.1111/mmi.12549
PMCID: PMC4065374  PMID: 24527847

Results 1-14 (14)