Identification of Streptomyces coelicolor proteins that are differentially expressed in the presence of plant material

Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.     

Use of 2-D electrophoresis and ESI mass spectrometry techniques to characterize Fusobacterium nucleatum proteins up-regulated after oxidative stress

The genus Fusobacterium belongs to the Fusobacteriaceae family and is a Gram-negative obligate anaerobic bacterium found in the human oral microbiota. Even that Fusobacterium nucleatum cannot grow under aerobic conditions, they may exhibit aerotolerance as an adaptive response which could figure as an important virulence factor, during the stages of infection, when these anaerobes are shifted to aerobic conditions. In this regard, little is known about bacterial oxidative stress adaptive response and the influence of this adaptation on the host-bacteria relationship. We aimed to use both techniques 2-DE and Electrospray Ionization Mass Spectrometry (ESI-MS) to characterize proteins in F. nucleatum, after oxidative stress. We related three different proteins which were up-regulated by oxidative stress. As its genome is already sequenced, these proteins were found in data base search, by homology. Thus, by using techniques as ESI-Q/TOF-MS, in addition to 2-DE, the opportunity exists to gain a more holistic view of the bacterial proteome of human pathogens, to achieve a better understanding of species diversity and to elucidate the role of specific proteins in disease. This work represents one of the first studies using genetic and physiological approaches to understand the phenomenon of oxidative stress in F. nucleatum.     

Agitation Down-Regulates Immunoglobulin Binding Protein EibG Expression in Shiga Toxin-Producing Escherichia coli (STEC)

Shiga toxin (Stx)-producing Escherichia coli (STEC) carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG). EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.     

Resuscitation-Promoting Factors Are Cell Wall-Lytic Enzymes with Important Roles in the Germination and Growth of Streptomyces coelicolor

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.     

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H₂O₂ and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.     

A novel tyrosine-phosphorylated protein inhibiting the growth of Streptomyces cells

Very few of the tyrosine-phosphorylated proteins in Streptomyces have been identified. Here, we identify a tyrosine-phosphorylated protein from Streptomyces coelicolor A3(2), designated as SCO5717. The protein possesses Walker motifs and a tyrosine cluster at the C-terminus. When sco5717 harboring its own promoter was introduced into the S. coelicolor cell, the growth was inhibited. An sco5717-disrupted mutant formed aerial mycelium earlier than the wild-type strain, suggesting that SCO5717 controls the cell growth of S. coelicolor. Although the recombinant SCO5717 showed an ATPase activity, it lacked self-phosphorylation ability, suggesting that SCO5717 is a novel tyrosine-phosphorylated protein, which is distinguishable from bacterial protein tyrosine kinases known so far.     

Synthesis and Uptake of the Compatible Solutes Ectoine and 5-Hydroxyectoine by Streptomyces coelicolor A3(2) in Response to Salt and Heat Stresses

Streptomyces coelicolor A3(2) synthesizes ectoine and 5-hydroxyectoine upon the imposition of either salt (0.5 M NaCl) or heat stress (39°C). The cells produced the highest cellular levels of these compatible solutes when both stress conditions were simultaneously imposed. Protection against either severe salt (1.2 M NaCl) or heat stress (39°C) or a combination of both environmental cues could be accomplished by adding low concentrations (1 mM) of either ectoine or 5-hydroxyectoine to S. coelicolor A3(2) cultures. The best salt and heat stress protection was observed when a mixture of ectoine and 5-hydroxyectoine (0.5 mM each) was provided to the growth medium. Transport assays with radiolabeled ectoine demonstrated that uptake was triggered by either salt or heat stress. The most effective transport and accumulation of [¹⁴C]ectoine by S. coelicolor A3(2) were achieved when both environmental cues were simultaneously applied. Our results demonstrate that the accumulation of the compatible solutes ectoine and 5-hydroxyectoine allows S. coelicolor A3(2) to fend off the detrimental effects of both high salinity and high temperature on cell physiology. We also characterized the enzyme (EctD) required for the synthesis of 5-hydroxyectoine from ectoine, a hydroxylase of the superfamily of the non-heme-containing iron(II)- and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). The gene cluster (ectABCD) encoding the enzymes for ectoine and 5-hydroxyectoine biosynthesis can be found in the genome of S. coelicolor A3(2), Streptomyces avermitilis, Streptomyces griseus, Streptomyces scabiei, and Streptomyces chrysomallus, suggesting that these compatible solutes play an important role as stress protectants in the genus Streptomyces.     

Induction of Yersinia enterocolitica Stress Proteins by Phagocytosis with Macrophage

Yersinia enterocolitica is a facultative intracellular pathogen which invades to epithelial cells and survives in phagocytes. Since the internal environment of phagocytes should be stressful conditions for the phagocytosed Yersinia, the bacteria should respond to protect themselves from otherwise lethal results. We analyzed the stress-induced proteins which possibly contribute to survival of Yersinia within the phagocytes. Y. enterocolitica was radiolabeled during the growth in macrophage-like J774-1 cells, and the bacterial proteins were analyzed by two-dimensional gel electrophoresis. At least 16 proteins were selectively induced in response to phagocytosis, and several out of 16 proteins were also induced by heat shock at 42 C or oxidative stresses in vitro. Of those, two major stress proteins were identified to be homologues of DnaK and CRPA by immunoblotting analysis. These results have indicated that Y. enterocolitica exhibits a global stress response to the hostile environment in the phagocytosed macrophage.     

Analysis of Phaseolus vulgaris Response to Its Association with Trichoderma harzianum (ALL-42) in the Presence or Absence of the Phytopathogenic Fungi Rhizoctonia solani and Fusarium solani

The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.     

On-off regulation of the tryptophan promoter in fed-batch culture

Fundamental properties of the trp promoter were investigated in fed-batch culture using a recombinant containing the lacZ gene controlled by this promoter. In tryptophan-deficient conditions, the amount of β-galactosidase accumulated in the cell was 10% of total cellular proteins. In the presence of the amino acid, it was repressed at a lower level, but considerable expression was observed in the later stages of cultivation. Although increasing concentration of tryptophan seemed to repress the promoter more completely, it strongly inhibited the bacterial growth. On-off regulation of the promoter was achieved by controlling the tryptophan level during fed-batch culture.     

Analysis of Streptomyces coelicolor membrane proteome using two-dimensional native/native and native/sodium dodecyl sulfate gel electrophoresis

Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), high-resolution clear native/native PAGE, and native/SDS–PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS–PAGE is much higher than that of blue native/SDS–PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.     

Salt Stress Proteins Induced in Listeria monocytogenes

The ability of Listeria monocytogenes to tolerate salt stress is of particular importance, as this pathogen is often exposed to such environments during both food processing and food preservation. In order to understand the survival mechanisms of L. monocytogenes, an initial approach using two-dimensional polyacrylamide gel electrophoresis was performed to analyze the pattern of protein synthesis in response to salt stress. Of 400 to 500 visible proteins, the synthesis of 40 proteins (P < 0.05) was repressed or induced at a higher rate during salt stress. Some of the proteins were identified on the basis of mass spectrometry or N-terminal sequence analysis and database searching. Twelve proteins showing high induction after salt stress were similar to general stress proteins (Ctc and DnaK), transporters (GbuA and mannose-specific phosphotransferase system enzyme IIAB), and general metabolism proteins (alanine dehydrogenase, CcpA, CysK, EF-Tu, Gap, GuaB, PdhA, and PdhD).