Non-disruptive release of Pseudomonas putida proteins by in situ electric breakdown of intact cells

Analysis of the native proteome of bacterial cells typically involves physical procedures (sonication, French press) and/or biochemical methods (treatment with lysozyme, osmotic shock etc.) to break open the bacteria to yield a soluble protein fraction. Such procedures are not only time consuming, but they change bacterial physiology during manipulation and affect labile post-translational modifications such as His–P bonds. In this work, we document the efficacy of the dielectric breakdown of live bacteria for releasing and delivering the protein contents of intact cells directly into a non-denaturing gel system. By means of such an in situ electrophoresis, the protein pool enters the separation medium without any manipulation of the cells other than being exposed to a moderate electric voltage. To validate the method we have followed the fate of the two forms of the PtsN (EIIA^Ntr) protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Pseudomonas putida through the various stages of the procedure. Apart of detecting the corresponding polypeptides, we show that this procedure releases the bulk of the proteome while keeping unharmed the phosphorylation state of EIIA^Ntr as it was present in the cells prior to applying the electric field. The method is applicable to other bacteria as well.     

Evidence of in vivo cross talk between the nitrogen-related and fructose-related branches of the carbohydrate phosphotransferase system of Pseudomonas putida

The genome of Pseudomonas putida KT2440 encodes only five recognizable proteins belonging to the phosphoenolpyruvate (PEP)-carbohydrate phosphotransferase system (PTS). Two of these PTS constituents (FruA and FruB) form a complete system for fructose intake. The other three products, encoded by ptsP (EI(Ntr)), ptsO (NPr), and ptsN (EIIA(Ntr)), comprise a branch of the system unrelated to sugar traffic but thought to have an influence on coordination of N and C metabolism. We used a genetic approach to clarify the course of high-energy phosphate through this reduced set of PTS proteins. To this end, we monitored the phosphorylation state in vivo of the EIIA(Ntr) enzyme in various genetic backgrounds and growth conditions. Our results show that the source of phosphate available to the system is PEP and that the primary flow of phosphate through the N/C-sensing PTS proceeds from PEP to EI(Ntr) to NPr to EIIA(Ntr). We also found that in the presence of fructose, unlike in the presence of succinate, EIIA(Ntr) can be phosphorylated in a ptsP strain but not in a ptsP fruB double mutant. This result revealed that the fructose transport system has the ability to cross talk in vivo with the N-related PTS branch. The data reported here thus document an unexpected connection in vivo between the sugar-dependent and sugar-independent PTSs.     

Topography of the surface of the signal-transducing protein EIIA(Glc) that interacts with the MalK subunits of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium

The signal-transducing protein EIIA(Glc), a component of the phosphoenolpyruvate-glucose phosphotransferase system, plays a key role in carbon regulation in enteric bacteria, such as Escherichia coli and Salmonella typhimurium. The phosphorylation state of EIIA(Glc) governs transport and metabolism of a number of carbohydrates. When glucose as preferred carbon source is transported, EIIA(Glc) becomes predominantly unphosphorylated and allosterically inhibits several permeases, including the maltose ATP-binding cassette transport system (MalFGK2) in a process termed "inducer exclusion." We have mapped the binding surface of EIIA(Glc) that interacts with the MalK subunits by using synthetic cellulose-bound peptide arrays like pep scan- and substitutional analyses. Three regions constituting two binding sites were identified encompassing residues 69-79 (I), 87-91 (II), and 118-127 (III). Region III is MalK-specific, whereas residues from regions I and II partly overlap but are not identical to the binding interfaces for interaction with glycerol kinase and lactose permease. These results were fully verified by studying the inhibitory effect of purified EIIA(Glc) variants carrying mutations at positions representative of each of the three regions on the ATPase activity of the purified maltose transport complex reconstituted into proteoliposomes. Moreover, a synthetic peptide encompassing residues 69-91 was demonstrated to partially inhibit ATPase activity. We also show for the first time that the N-terminal domain of EIIA(Glc) is essential for inducer exclusion.     

Glycerol-3-phosphate-induced catabolite repression in Escherichia coli

The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIA(Glc), as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIA(Glc) phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIA(Glc). Isopropyl-beta-D-thiogalactopyranoside-induced overexpression of EIIA(Glc) did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIA(Glc). A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIA(Glc) is controlled by G3P and other phosphorylated sugars such as D-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and D-tagatose-1,6-bisphosphate aldolase.     

Bacterial genetics by electric shock

Subjecting bacteria to a high-voltage electric discharge renders the cells permeable to DNA. This powerful method allows the genetic manipulation of bacterial species that cannot easily be transformed by conventional techniques.     

The cold shock response of Lactobacillus casei: relation between HPr phosphorylation and resistance to freeze/thaw cycles

When carrying out a proteome analysis with a ptsH3 mutant of Lactobacillus casei, we found that the cold shock protein CspA was significantly overproduced compared to the wild-type strain. We also noticed that CspA and CspB of L. casei and CSPs from other organisms exhibit significant sequence similarity to the C-terminal part of EIIA(Glc), a glucose-specific component of the phosphoenolpyruvate:sugar phosphotransferase system. This similarity suggested a direct interaction of HPr with CSPs, as histidyl-phosphorylated HPr has been shown to phosphorylate EIIA(Glc) in its C-terminal part. We therefore compared the cold shock response of several carbon catabolite repression mutants to that of the wild-type strain. Following a shift from 37 degrees C to lower temperatures (20, 15 or 10 degrees C), all mutants showed significantly reduced growth rates. Moreover, glucose-grown mutants unable to form P-Ser-HPr (ptsH1, hprK) exhibited drastically increased sensitivity to freeze/thaw cycles. However, when the same mutants were grown on ribose or maltose, they were similarly resistant to freezing and thawing as the wild-type strain. Although subsequent biochemical and genetic studies did not allow to identify the form of HPr implicated in the resistance to cold and freezing conditions, they strongly suggested a direct interaction of HPr or one of its phospho-derivatives with CspA and/or another, hitherto undetected cold shock protein in L. casei.     

The Bacillus subtilis Nucleotidyltransferase Is a tRNA CCA-Adding Enzyme

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3′ poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the β and γ subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca, following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3′ polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.     

Correlation between growth rates, EIIACrr phosphorylation, and intracellular cyclic AMP levels in Escherichia coli K-12

In Escherichia coli K-12, components of the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) represent a signal transduction system involved in the global control of carbon catabolism through inducer exclusion mediated by phosphoenolpyruvate-dependent protein kinase enzyme IIA(Crr) (EIIA(Crr)) (= EIIA(Glc)) and catabolite repression mediated by the global regulator cyclic AMP (cAMP)-cAMP receptor protein (CRP). We measured in a systematic way the relation between cellular growth rates and the key parameters of catabolite repression, i.e., the phosphorylated EIIA(Crr) (EIIA(Crr) approximately P) level and the cAMP level, using in vitro and in vivo assays. Different growth rates were obtained by using either various carbon sources or by growing the cells with limited concentrations of glucose, sucrose, and mannitol in continuous bioreactor experiments. The ratio of EIIA(Crr) to EIIA(Crr) approximately P and the intracellular cAMP concentrations, deduced from the activity of a cAMP-CRP-dependent promoter, correlated well with specific growth rates between 0.3 h(-1) and 0.7 h(-1), corresponding to generation times of about 138 and 60 min, respectively. Below and above this range, these parameters were increasingly uncoupled from the growth rate, which perhaps indicates an increasing role executed by other global control systems, in particular the stringent-relaxed response system.     

Bacterial membrane proteomics

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.     

Classification and identification of bacteria by mass spectrometry and computational analysis

Background

In general, the definite determination of bacterial species is a tedious process and requires extensive manual labour. Novel technologies for bacterial detection and analysis can therefore help microbiologists in minimising their efforts in developing a number of microbiological applications.

Methodology

We present a robust, standardized procedure for automated bacterial analysis that is based on the detection of patterns of protein masses by MALDI mass spectrometry. We particularly applied the approach for classifying and identifying strains in species of the genus Erwinia. Many species of this genus are associated with disastrous plant diseases such as fire blight. Using our experimental procedure, we created a general bacterial mass spectra database that currently contains 2800 entries of bacteria of different genera. This database will be steadily expanded. To support users with a feasible analytical method, we developed and tested comprehensive software tools that are demonstrated herein. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used genotyping of single nucleotide polymorphisms by mass spectrometry to unambiguously determine closely related strains that are difficult to distinguish by only relying on protein mass pattern detection.

Conclusions

With the method for bacterial analysis, we could identify fire blight pathogens from a variety of biological sources. The method can be used for a number of additional bacterial genera. Moreover, the mass spectrometry approach presented allows the integration of data from different biological levels such as the genome and the proteome.     

Development and application of an insertional system for gene delivery and expression in Campylobacter jejuni

The genetic investigation of Campylobacter jejuni, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for Escherichia coli and other bacteria cannot be maintained in Campylobacter cells. Additionally, gene expression in Campylobacter requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other Campylobacter strains. The presence of a Campylobacter-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of Campylobacter. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing Campylobacter allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.     

Bacterial nitrate assimilation: gene distribution and regulation

In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.     

Effect of electric currents on bacterial detachment and inactivation

Since biofilms show strong resistance to conventional disinfectants and antimicrobials, control of initial bacterial adhesion is generally accepted as one of the most effective strategies for preventing biofilm formation. Although electrical methods have been widely studied, the specific properties of cathodic, anodic, and block currents that influence the bacterial detachment and inactivation remained largely unclear. This study investigated the specific role of electric currents in the detachment and inactivation of bacteria adhered to an electrode surface. A real-time bacterial adhesion observation and control system was employed that consisted of Pseudomonas aeruginosa PAO1 (PAO1) with green fluorescent protein as the indicator microorganism and a flow cell reactor mounted on a fluorescent microscope. The results suggest that the bacteria that remained on the electrode surface after application of a cathodic current were alive, although the extent of detachment was significant. In contrast, when an anodic current was applied, the bacteria that remained on the surface became inactive with time, although bacterial detachment was not significant. Further, under these conditions, active bacterial motions were observed, which weakened the binding between the electrode surface and bacteria. This phenomenon of bacterial motion on the surface can be used to maximize bacterial detachment by manipulation of the shear rate. These findings specific for each application of a cathodic or anodic electric current could successfully explain the effectiveness of block current application in controlling bacterial adhesion.     

A protein processing filter method for bacterial identification by mass spectrometry-based proteomics

A "one-pot" alternative method for processing proteins and isolating peptide mixtures from bacterial samples is presented for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and data reduction. The conventional in-solution digestion of the protein contents of bacteria is compared to a small disposable filter unit placed inside a centrifuge vial for processing and digestion of bacterial proteins. Each processing stage allows filtration of excess reactants and unwanted byproduct while retaining the proteins. Upon addition of trypsin, the peptide mixture solution is passed through the filter while retaining the trypsin enzyme. The peptide mixture is then analyzed by LC-MS/MS with an in-house BACid algorithm for a comparison of the experimental unique peptides to a constructed proteome database of bacterial genus, specie, and strain entries. The concentration of bacteria was varied from 10 × 10(7) to 3.3 × 10(3) cfu/mL for analysis of the effect of concentration on the ability of the sample processing, LC-MS/MS, and data analysis methods to identify bacteria. The protein processing method and dilution procedure result in reliable identification of pure suspensions and mixtures at high and low bacterial concentrations.