Bacillus pumilus KatX2 confers enhanced hydrogen peroxide resistance to a Bacillus subtilis PkatA::katX2 mutant strain.

BACKGROUND: Bacillus pumilus cells exhibit a significantly higher resistance to hydrogen peroxide compared to closely related Bacilli like Bacillus subtilis. RESULTS: In this study we analyzed features of the catalase KatX2 of B. pumilus as one of the most important parts of the cellular response to hydrogen peroxide. KatX2, the vegetative catalase expressed in B. pumilus, was compared to the vegetative catalase KatA of B. subtilis. Data of our study demonstrate that B. pumilus can degrade toxic concentrations of hydrogen peroxide faster than B. subtilis. By replacing B. subtilis katA gene by katX2 we could significantly enhance its resistance to H2O2 and its potential to eliminate this toxic compound. Mutant cells showed a 1.5- to 2-fold higher survival to toxic concentrations of hydrogen peroxide compared to wild type cells. Furthermore, we found reversible but also irreversible oxidations of the KatX2 protein which, in contrast to KatA, contains several cysteine residues. CONCLUSIONS: Our study indicates that the catalase KatX2 plays a major role in the increased resistance of B. pumilus to oxidative stress caused by hydrogen peroxide. Resistance to hydrogen peroxide of other Bacilli can be enhanced by exchanging the native catalase in the cells with katX2.

Lack of AHL-based quorum sensing in Pseudomonas fluorescens isolated from milk.

Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.

Lack of AHL-based quorum sensing in Pseudomonas fluorescens isolated from milk

Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.

Soil metaproteomics - Comparative evaluation of protein extraction protocols.

Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, due to the complexity of soil samples the exhaustive extraction of proteins is a major challenge. We compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types. Four different protein extraction procedures were applied based on (a) SDS extraction without phenol, (b) NaOH and subsequent phenol extraction, (c) SDS-phenol extraction and (d) SDS-phenol extraction with prior washing steps. To assess the suitability of these methods for the functional analysis of the soil metaproteome, they were applied to a potting soil high in organic matter and a forest soil. Proteins were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) and the number of unique spectra as well as the number of assigned proteins for each of the respective protocols was compared. In both soil types, extraction with SDS-phenol (c) resulted in "high" numbers of proteins. Moreover, a spiking experiment was conducted to evaluate protein recovery. To this end sterilized forest soil was amended with proteins from pure cultures of Pectobacterium carotovorum and Aspergillus nidulans. The protein recovery in the spiking experiment was almost 50%. Our study demonstrates that a critical evaluation of the extraction protocol is crucial for the quality of the metaproteomics data, especially in highly complex samples like natural soils.

Identification of functions linking quorum sensing with biofilm formation in Burkholderia cenocepacia H111.

Burkholderia cenocepacia has emerged as an important pathogen for patients suffering from cystic fibrosis (CF). Previous work has shown that this organism employs the CepIR quorum-sensing (QS) system to control the expression of virulence factors as well as the formation of biofilms. To date, however, very little is known about the QS-regulated virulence factors and virtually nothing about the factors that link QS and biofilm formation. Here, we have employed a combined transcriptomic and proteomic approach to precisely define the QS regulon in our model strain B. cenocepacia H111, a CF isolate. Among the identified CepR-activated loci, three were analyzed in better detail for their roles in biofilm development: (i) a gene cluster coding for the BclACB lectins, (ii) the large surface protein BapA, and (iii) a type I pilus. The analysis of defined mutants revealed that BapA plays a major role in biofilm formation on abiotic surfaces while inactivation of the type I pilus showed little effect both in a static microtitre dish-based biofilm assay and in flow-through cells. Inactivation of the bclACB lectin genes resulted in biofilms containing hollow microcolonies, suggesting that the lectins are important for biofilm structural development.

Who is who in litter decomposition? Metaproteomics reveals major microbial players and their biogeochemical functions.

Leaf-litter decomposition is a central process in carbon cycling; however, our knowledge about the microbial regulation of this process is still scarce. Metaproteomics allows us to link the abundance and activity of enzymes during nutrient cycling to their phylogenetic origin based on proteins, the 'active building blocks' in the system. Moreover, we employed metaproteomics to investigate the influence of environmental factors and nutrients on the decomposer structure and function during beech litter decomposition. Litter was collected at forest sites in Austria with different litter nutrient content. Proteins were analyzed by 1-D-SDS-PAGE followed by liquid-chromatography and tandem mass-spectrometry. Mass spectra were assigned to phylogenetic and functional groups by a newly developed bioinformatics workflow, assignments being validated by complementary approaches. We provide evidence that the litter nutrient content and the stoichiometry of C:N:P affect the decomposer community structure and activity. Fungi were found to be the main producers of extracellular hydrolytic enzymes, with no bacterial hydrolases being detected by our metaproteomics approach. Detailed investigation of microbial succession suggests that it is influenced by litter nutrient content. Microbial activity was stimulated at higher litter nutrient contents via a higher abundance and activity of extracellular enzymes.

Different protein expression profiles in cheese and clinical isolates of Enterococcus faecalis revealed by proteomic analysis.

The use of Enterococcus faecalis in the food industry has come under dispute because of the pathogenic potential of some strains of this species. In this study, we have compared the secretome and whole-cell proteome of one food isolate (E. faecalis DISAV 1022) and one clinical isolate (E. faecalis H1) by 2-DE and iTRAQ analyses, respectively. Extracellular protein patterns differed significantly, with only seven proteins common to both strains. Notably, only the clinical isolate expressed various well-characterized virulence factors such as the gelatinase coccolysin (GelE) and the extracellular serine proteinase V8 (SprE). Moreover, various other putative virulence factors, e.g. superoxide dismutase, choline- and chitin-binding proteins and potential moonlighting proteins, have been detected exclusively in the secretome of the clinical isolate, but not in the food isolate. The iTRAQ analysis of whole-cell proteins of the two strains highlighted a stronger expression of pathogenic traits such as an endocarditis-specific antigen and an adhesion lipoprotein in the pathogenic strain E. faecalis H1. Subsequently, six food isolates (including E. faecalis DISAV 1022) and six clinical isolates (including E. faecalis H1) were tested for the presence of gelatinase and protease activity in the culture supernatants. Both enzymatic activities were found in the clinical as well as the food isolates which clearly indicates that protease expression is strain specific and not representative for pathogenic isolates. Genetic analyses revealed that not only the gelatinase and serine protease genes but also the regulatory fsr genes must be present to allow protease expression.

Structure and function of the symbiosis partners of the lung lichen (Lobaria pulmonaria L. Hoffm.) analyzed by metaproteomics.

Environmental proteomics, also referred to as metaproteomics, is an emerging technology to study the structure and function of microbial communities. Here, we applied semi-quantitative label-free proteomics using one-dimensional gel electrophoresis combined with LC-MS/MS and normalized spectral counting together with fluorescence in situ hybridization and confocal laser scanning microscopy to characterize the metaproteome of the lung lichen symbiosis Lobaria pulmonaria. In addition to the myco- and photobiont, L. pulmonaria harbors proteins from a highly diverse prokaryotic community, which is dominated by Proteobacteria and including also Archaea. While fungal proteins are most dominant (75.4% of all assigned spectra), about the same amount of spectra were assigned to prokaryotic proteins (10%) and to the green algal photobiont (9%). While the latter proteins were found to be mainly associated with energy and carbohydrate metabolism, a major proportion of fungal and bacterial proteins appeared to be involved in PTMs and protein turnover and other diverse functions.

Open reduction for perilunate injuries--clinical outcome and patient satisfaction.

PURPOSE: Perilunate injuries cause severe carpal malalignment. Open reduction and internal fixation of these injuries has become the treatment of choice. This study evaluated clinical outcome and the patients' perception of disability in activities of daily living after open reduction, ligament reconstruction, and/or internal fixation of the scaphoid. In addition, potential prognostic factors for functional outcome and individual perceptions of disability were analyzed and compared with radiologic findings. METHODS: This study consisted of a retrospective analysis of patients with perilunate dislocations or fracture dislocations (Mayfield stage 3/4) who were treated in a single institution from 1995 to 2004. Evaluation focused on postoperative radiologic results, range of motion, pain, sensitivity, grip strength, Mayo and Krimmer wrist scores, arthrosis, and the patients' disability in performing activities of daily living (according to the Disabilities of the Arm, Shoulder, and Hand score). RESULTS: Of the 72 patients treated in the study period, 39 patients (all men) were available for complete follow-up (average, 65.5 mo). Thirty injuries were fracture dislocations; the dominant hand was injured in 14 cases. Normal scapholunate (SL) angles and Gilula arcs were achieved intraoperatively in 34 and 25 cases, respectively. At follow-up, 18 patients had larger than normal SL angles, and 6 patients had ulnar shifting of the carpus. Twenty patients were diagnosed with radiocarpal arthrosis. According to the Visual Analog Scale, pain was 1.8 at rest and 4.8 with activities. Average extension/flexion was 77°; radial/ulnar abduction was reduced to 42°. Average grip strength was reduced to an average of 36.6 kg (compared with 51.6 kg on the opposite side). Twenty-seven patients returned to their former occupations. Average Mayo and Krimmer wrist scores were both 70. The average Disabilities of the Arm, Shoulder, and Hand score was 23. CONCLUSIONS: Satisfactory results can be achieved with open reduction for perilunate injuries. However, despite this treatment, loss of reduction and arthrosis are frequent findings. Radiologic results do not necessarily correlate with functional outcome; high patient satisfaction was observed in this study. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Construction of self-transmissible green fluorescent protein-based biosensor plasmids and their use for identification of N-acyl homoserine-producing bacteria in lake sediments.

Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

A gel-free quantitative proteomics approach to investigate temperature adaptation of the food-borne pathogen Cronobacter turicensis 3032.

The opportunistic food-borne pathogen Cronobacter sp. causes rare but significant illness in neonates and is capable to grow at a remarkably wide range of temperatures from 5.5 to 47 degrees C. A gel-free quantitative proteomics approach was employed to investigate the molecular basis of the Cronobacter sp. adaptation to heat and cold-stress. To this end the model strain Cronobacter turicensis 3032 was grown at 25, 37, 44, and 47 degrees C, and whole-cell and secreted proteins were iTRAQ-labelled and identified/quantified by 2-D-LC-MALDI-TOF/TOF-MS. While 44 degrees C caused only minor changes in C. turicensis growth rate and protein profile, 47 degrees C affected the expression of about 20% of all 891 identified proteins and resulted in a reduced growth rate and rendered the strain non-motile and filamentous. Among the heat-induced proteins were heat shock factors, transcriptional and translational proteins, whereas proteins affecting cellular morphology, proteins involved in motility, central metabolism and energy production were down-regulated. Notably, numerous potential virulence factors were found to be up-regulated at higher temperatures, suggesting an elevated pathogenic potential of Cronobacter sp. under these growth conditions. Significant alterations in the protein expression profile and growth rate of C. turicensis exposed to 25 degrees C indicate that at this temperature the organism is cold-stressed. Up-regulated gene products comprised cold-shock, DNA-binding and ribosomal proteins, factors that support protein folding and proteins opposing cold-induced decrease in membrane fluidity, whereas down-regulated proteins were mainly involved in central metabolism.

A proteomics approach to study synergistic and antagonistic interactions of the fungal-bacterial consortium Fusarium oxysporum wild-type MSA 35.

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil-borne plant pathogen, including non-pathogenic F. oxysporum strains. In this study, F. oxysporum wild-type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to gamma-proteobacteria, was analyzed by two complementary metaproteomic approaches (2-DE combined with MALDI-Tof/Tof MS and 1-D PAGE combined with LC-ESI-MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter-part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.

Protein oxidation and aggregation in UVA-irradiated Escherichia coli cells as signs of accelerated cellular senescence.

Solar disinfection (SODIS) is a simple drinking water treatment method that improves microbiological water quality where other means are unavailable. It makes use of the deleterious effect of solar irradiation on pathogenic microbes and viruses. A positive impact on health has been documented in several epidemiological studies. However, the molecular mechanisms damaging cells during this simple treatment are not yet fully understood. Here we show that protein damage is crucial in the process of inactivation by sunlight. Protein damages in UVA-irradiated Escherichia coli cells have been evaluated by an immunoblot method for carbonylated proteins and an aggregation assay based on semi-quantitative proteomics. A wide spectrum of structural and enzymatic proteins within the cell is affected by carbonylation and aggregation. Vital cellular functions like the transcription and translation apparatus, transport systems, amino acid synthesis and degradation, respiration, ATP synthesis, glycolysis, the TCA cycle, chaperone functions and catalase are targeted by UVA irradiation. The protein damage pattern caused by SODIS strongly resembles the pattern caused by reactive oxygen stress. Hence, sunlight probably accelerates cellular senescence and leads to the inactivation and finally death of UVA-irradiated cells.

Proteome analysis of fungal and bacterial involvement in leaf litter decomposition.

Fungi and bacteria are key players in the decomposition of leaf litter, but their individual contributions to the process and their interactions are still poorly known. We combined semi-quantitative proteome analyses (1-D PAGE-LC-MS/MS) with qualitative and quantitative analyses of extracellular degradative enzyme activities to unravel the respective roles of a fungus and a bacterium during litter decomposition. Two model organisms, a mesophilic Gram-negative bacterium (Pectobacterium carotovorum) and an ascomycete (Aspergillus nidulans), were grown in both, pure culture and co-culture on minimal medium containing either glucose or beech leaf litter as sole carbon source. P. carotovorum grew best in co-culture with the fungus, whereas growth of A. nidulans was significantly reduced when the bacterium was present. This observation suggests that P. carotovorum has only limited capabilities to degrade leaf litter and profits from the degradation products of A. nidulans at the expense of fungal growth. In accordance with this interpretation, our proteome analysis revealed that most of the extracellular biodegradative enzymes (i.e. proteases, pectinases, and cellulases) in the cultures with beech litter were expressed by the fungus, the bacterium producing only low levels of pectinases.

Genes involved in Cronobacter sakazakii biofilm formation.

Cronobacter spp. are opportunistic food-borne pathogens that can cause severe and sometimes lethal infections in neonates. In some outbreaks, the sources of infection were traced to contaminated powdered infant formula (PIF) or contaminated utensils used for PIF reconstitution. In this study, we investigated biofilm formation in Cronobacter sakazakii strain ES5. To investigate the genetic basis of biofilm formation in Cronobacter on abiotic surfaces, we screened a library of random transposon mutants of strain ES5 for reduced biofilm formation using a polystyrene microtiter assay. Genetic characterization of the mutants led to identification of genes that are associated with cellulose biosynthesis and flagellar structure and biosynthesis and genes involved in basic cellular processes and virulence, as well as several genes whose functions are currently unknown. In two of the mutants, hypothetical proteins ESA_00281 and ESA_00282 had a strong impact on flow cell biofilm architecture, and their contribution to biofilm formation was confirmed by genetic complementation. In addition, adhesion of selected biofilm formation mutants to Caco-2 intestinal epithelial cells was investigated. Our findings suggest that flagella and hypothetical proteins ESA_00281 and ESA_00282, but not cellulose, contribute to adhesion of Cronobacter to this biotic surface.

Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa.

Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed that the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer-membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications.

Environmental proteomics: analysis of structure and function of microbial communities.

Prokaryotic and eukaryotic microorganisms make a vital contribution to biogeochemical cycles by decomposing virtually all natural compounds and thereby exert a lasting effect on biosphere and climate. The rapidly growing number of metagenomic sequences together with revolutionary advances in bioinformatics and protein analyses have opened completely new horizons to investigate the molecular basis of such complex processes. Proteomics has contributed substantially to our understanding of individual organisms at the cellular level as it offers excellent possibilities to probe many protein functions and responses simultaneously. However, it has not yet been widely applied in microbial ecology, although most proteins have an intrinsic metabolic function which can be used to relate microbial activities to the identity of defined organisms in multispecies communities. Albeit still in its infancy, environmental proteomics enables simple protein cataloging, comparative and semi-quantitative proteomics, analyses of protein localization, discovery of post-translational modifications, and even determination of amino-acid sequences and genotypes by strain-resolved proteogenomics. This review traces the historical development of environmental proteomics and summarizes milestone publications in the field. In conclusion, we briefly discuss current limitations of microbial community proteomics but also the potential of emerging technologies to shape the future of metaproteome analyses.

Evidence for a plant-associated natural habitat for Cronobacter spp.

Cronobacter (Enterobacter sakazakii) species are responsible for rare cases of necrotising enterocolitis and bacteraemia in infants, as well as cases of meningitis with high case fatality rates in neonates and immunocompromised infants. Some physiological features, such as the production of a yellow pigment, the formation of a gum-like extracellular polysaccharide and the ability to persist in a desiccated state, suggest an environmental niche for these organisms. To date, the natural habitat of Cronobacter spp. remains unknown. In this report, the isolation and characterisation of two Cronobacter sakazakii strains from plant roots is described. Also, the root colonisation behaviour of Cronobacter strains originating from clinical and plant sources is assessed. The nine strains investigated showed features often found in plant-associated and rhizosphere microorganisms, including solubilisation of mineral phosphate and production of indole acetic acid. Siderophore production was observed for all except one strain. In addition, the capability to endophytically colonise tomato and maize roots was demonstrated for several strains, either by fluorescence in situ hybridisation, using fluorescently labelled oligonucleotide probes, or by using strains tagged with green fluorescent protein and confocal laser scanning microscopy. The results provide evidence that plants may be the natural habitat of Cronobacter spp.

Proteomic profiling of Cronobacter turicensis 3032, a food-borne opportunistic pathogen.

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface-associated and whole-cell proteins by two complementary proteomics approaches, 1D-SDS-PAGE combined with LC-ESI-MS/MS and 2D-LC-MALDI-TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin-receptor protein for the uptake of siderophore-bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.

Identification of specific and universal virulence factors in Burkholderia cenocepacia strains by using multiple infection hosts.

Over the past few decades, strains of the Burkholderia cepacia complex have emerged as important pathogens for patients suffering from cystic fibrosis. Identification of virulence factors and assessment of the pathogenic potential of Burkholderia strains have increased the need for appropriate infection models. In previous studies, different infection hosts, including mammals, nematodes, insects, and plants, have been used. At present, however, the extent to which the virulence factors required to infect different hosts overlap is not known. The aim of this study was to analyze the roles of various virulence factors of two closely related Burkholderia cenocepacia strains, H111 and the epidemic strain K56-2, in a multihost pathogenesis system using four different model organisms, namely, Caenorhabditis elegans, Galleria mellonella, the alfalfa plant, and mice or rats. We demonstrate that most of the identified virulence factors are specific for one of the infection models, and only three factors were found to be essential for full pathogenicity in several hosts: mutants defective in (i) quorum sensing, (ii) siderophore production, and (iii) lipopolysaccharide biosynthesis were attenuated in at least three of the infection models and thus may represent promising targets for the development of novel anti-infectives.