Kinetic modelling and meta-analysis of the B. subtilis SigA regulatory network during spore germination and outgrowth.

This study describes the meta-analysis and kinetic modelling of gene expression control by sigma factor SigA of Bacillus subtilis during germination and outgrowth based on microarray data from 14 time points. The analysis computationally models the direct interaction among SigA, SigA-controlled sigma factor genes (sigM, sigH, sigD, sigX), and their target genes. Of the >800 known genes in the SigA regulon, as extracted from databases, 311 genes were analysed, and 190 were confirmed by the kinetic model as being controlled by SigA. For the remaining genes, alternative regulators satisfying kinetic constraints were suggested. The kinetic analysis suggested another 214 genes as potential SigA targets. The modelling was able to (i) create a particular SigA-controlled gene expression network that is active under the conditions for which the expression time series was obtained, and where SigA is the dominant regulator, (ii) suggest new potential SigA target genes, and (iii) find other possible regulators of a given gene or suggest a new mechanism of its control by identifying a matching profile of unknown regulator(s). Selected predicted regulatory interactions were experimentally tested, thus validating the model.

Genome resource utilization during prokaryotic development.

The distributions of synthesis rates of expressed proteins in a liquid batch culture of the prokaryote S. coelicolor during 3 days' growth have been analyzed by using a law governing the relation between the synthesis rates and the corresponding ranks in a list of rates (the so-called simplified canonical law, scl), which we have found previously to characterize the distribution of prokaryotic protein expression. The scl remains valid throughout development and the two parameters of the distribution, q and r, evolve in a highly characteristic and revealing way. q is a measure of the degree to which available genomic resources are used, in the sense of exploiting their potential diversity. The passage from one developmental phase to another is marked by a sharp peak in q, as these resources are fully mobilized to deal with a crisis (i.e., exhaustion of the habitual food supply). This is followed by an even more pronounced trough, as the organism briefly focuses its resources on synthesizing just those proteins most essential for survival, especially those hitherto unavailable and needed for metabolizing the new nutrient source. The parameter r indicates redundancy among the most abundantly expressed proteins: higher r corresponds to more diversity; i.e., less duplication of function, hence less robustness. This parameter is relatively steady throughout the development of the culture, except for a pronounced peak during the developmental phase transition. This corresponds to the "emergency mode" characterized by extremely low q, during which a minimum repertoire of proteins is expressed.

Neural model of the genetic network.

Many cell control processes consist of networks of interacting elements that affect the state of each other over time. Such an arrangement resembles the principles of artificial neural networks, in which the state of a particular node depends on the combination of the states of other neurons. The lambda bacteriophage lysis/lysogeny decision circuit can be represented by such a network. It is used here as a model for testing the validity of a neural approach to the analysis of genetic networks. The model considers multigenic regulation including positive and negative feedback. It is used to simulate the dynamics of the lambda phage regulatory system; the results are compared with experimental observation. The comparison proves that the neural network model describes behavior of the system in full agreement with experiments; moreover, it predicts its function in experimentally inaccessible situations and explains the experimental observations. The application of the principles of neural networks to the cell control system leads to conclusions about the stability and redundancy of genetic networks and the cell functionality. Reverse engineering of the biochemical pathways from proteomics and DNA micro array data using the suggested neural network model is discussed.

Proteomic analysis of the bacterial cell cycle.

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control.

Neural network model of gene expression.

Many natural processes consist of networks of interacting elements that, over time, affect each other's state. Their dynamics depend on the pattern of connections and the updating rules for each element. Genomic regulatory networks are networks of this sort. In this paper we use artificial neural networks as a model of the dynamics of gene expression. The significance of the regulatory effect of one gene product on the expression of other genes of the system is defined by a weight matrix. The model considers multigenic regulation including positive and/or negative feedback. The process of gene expression is described by a single network and by two linked networks where transcription and translation are modeled independently. Each of these processes is described by different network controlled by different weight matrices. Methods for computing the parameters of the model from experimental data are discussed. Results computed by means of the model are compared with experimental observations. Generalization to a 'black box' concept, where the molecular processes occurring in the cell are considered as signal processing units forming a global regulatory network, is discussed.-Vohradský, J. Neural network model of gene expression.

Major proteins related to chlortetracycline biosynthesis in a Streptomyces aureofaciens production strain studied by quantitative proteomics.

Changes in synthesis and abundance of proteins associated with chlortetracycline (CTC) production in Streptomyces aureofaciens were investigated by two-dimensional polyacrylamide gel electrophoresis of proteins pulse-labelled in vivo with L-[35S]methionine. Eleven individual protein spots were selected as being related to formation of the antibiotic. Expression of these prominent proteins was not observed in the non-producing mutant; moreover, they were overexpressed in cultures grown in the presence of benzyl thiocyanate, a specific stimulator of CTC biosynthesis used in industrial fermentations. The expression kinetics of the selected proteins was assessed using the technique of computer-assisted image analysis with the EQIAS software and the elongation factor Tu as an internal standard. Interestingly, the kinetic profiles were generally not identical. including those of anhydrotetracycline monooxygenase and the 13-kDa subunit of tetracycline dehydrogenase, two enzymes involved, in the terminal sequential steps of the CTC biosynthetic pathway. The presence of more forms of these enzymes with different charge characteristics was observed. The data presented demonstrated how dramatically the industrial microorganism can change its protein repertoire during the production phase; at least five proteins were nearly comparable in level to the most prominent proteins, exemplified by elongation factor Tu.

Developmental control of stress stimulons in Streptomyces coelicolor revealed by statistical analyses of global gene expression patterns.

Stress-induced regulatory networks coordinated with a procaryotic developmental program were revealed by two-dimensional gel analyses of global gene expression. Four developmental stages were identified by their distinctive protein synthesis patterns using principal component analysis. Statistical analyses focused on five stress stimulons (induced by heat, cold, salt, ethanol, or antibiotic shock) and their synthesis during development. Unlike other bacteria, for which various stresses induce expression of similar sets of protein spots, in Streptomyces coelicolor heat, salt, and ethanol stimulons were composed of independent sets of proteins. This suggested independent control by different physiological stress signals and their corresponding regulatory systems. These stress proteins were also under developmental control. Cluster analysis of stress protein synthesis profiles identified 10 different developmental patterns or "synexpression groups." Proteins induced by cold, heat, or salt shock were enriched in three developmental synexpression groups. In addition, certain proteins belonging to the heat and salt shock stimulons were coregulated during development. Thus, stress regulatory systems controlling these stimulons were implicated as integral parts of the developmental program. This correlation suggested that thermal shock and salt shock stress response regulatory systems either allow the cell to adapt to stresses associated with development or directly control the developmental program.

Point pattern matching in the analysis of two-dimensional gel electropherograms.

In the automation of proteome analysis, matching of two-dimensional (2-D) electropherograms represents a bottleneck in the process. Here we present a point pattern recognition approach to the matching of spots in 2-D electropherograms. The algorithm is based on a comparison of spot neighborhoods, converted to point patterns between reference and compared gels. The neighborhood was characterized by a syntactic descriptor which minimized the influence of spot displacements. A combined criterion utilizing the similarity of point patterns and a metric definition of position similarity was derived. The efficiency and accuracy of the algorithm was tested on a set of 69 gels with different levels of similarity. For a typical gel the accuracy of matching was higher than 98% and the number of correctly identified spots was higher than 95%.

Broad spectrum thiopeptide recognition specificity of the Streptomyces lividans TipAL protein and its role in regulating gene expression.

Microbial metabolites isolated in screening programs for their ability to activate transcription of the tipA promoter (ptipA) in Streptomyces lividans define a class of cyclic thiopeptide antibiotics having dehydroalanine side chains ("tails"). Here we show that such compounds of heterogeneous primary structure (representatives tested: thiostrepton, nosiheptide, berninamycin, promothiocin) are all recognized by TipAS and TipAL, two in-frame translation products of the tipA gene. The N-terminal helix-turn-helix DNA binding motif of TipAL is homologous to the MerR family of transcriptional activators, while the C terminus forms a novel ligand-binding domain. ptipA inducers formed irreversible complexes in vitro and in vivo (presumably covalent) with TipAS by reacting with the second of the two C-terminal cysteine residues. Promothiocin and thiostrepton derivatives in which the dehydroalanine side chains were removed lost the ability to modify TipAS. They were able to induce expression of ptipA as well as the tipA gene, although with reduced activity. Thus, TipA required the thiopeptide ring structure for recognition, while the tail served either as a dispensable part of the recognition domain and/or locked thiopeptides onto TipA proteins, thus leading to an irreversible transcriptional activation. Construction and analysis of a disruption mutant showed that tipA was autogenously regulated and conferred thiopeptide resistance. Thiostrepton induced the synthesis of other proteins, some of which did not require tipA.

Identification of procaryotic developmental stages by statistical analyses of two-dimensional gel patterns.

Multivariate statistical comparisons of two-dimensional protein (2-D) gel patterns were used for the first time to define stages of a biological developmental system. The differentiating procaryote, Streptomyces coelicolor, was radiolabeled in liquid cultures at 16 intervals during development, and radioactive proteins were separated and quantified on 2-D gels. Cluster, principal component, and correlation analyses classified these gel patterns into four distinct groups, each reflecting a pattern of gene expression specific for a stage of development. These studies focused our attention on a phase of arrested growth as a key regulatory transition leading to secondary metabolism and a phase of renewed growth. Proteins whose synthesis was switched on or off during the "transitional" phase (some 21 and 18, respectively) were identified and will be the focus of future studies designed to identify their physiological or regulatory function.

Object-oriented developmental environment for image-analysis applications: implementation for 2D gel electrophoretogram analysis.

MOTIVATION: The principal motivation was to design an environment for the development of image-analysis applications which would allow the integration of independent modules into one frame and make available tools for their build-up, running, management and mutual communication. RESULTS: The system was designed as modular, consisting of the core and work modules. The system core focuses on overall management and provides a library of classes for build-up of the work modules, their user interface and data communication. The work modules carry practical implementation of algorithms and data structures for the solution of a particular problem, and were implemented as dynamic-link libraries. They are mutually independent and run as individual threads, communicating with each other via a unified mechanism. The environment was designed to simplify the development and testing of new algorithms or applications. An example of implementation for the particular problem of the analysis of two-dimensional (2D) gel electrophoretograms is presented. The environment was designed for the Windows NT operating system with the use of Microsoft Foundation Class Library employing the possibilities of C++ programming language. AVAILABILITY: Available on request from the authors.

Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.

Adaptive classification of two-dimensional gel electrophoretic spot patterns by neural networks and cluster analysis.

The interpretation of two-dimensional gel electrophoresis spot profiles can be facilitated by statistical and machine learning programs. Two different approaches to classification of spot profiles - cluster analysis and neural networks - are discussed. Neural networks for two different model patterns were designed and an algorithm for training of the net for the classification was developed. It was shown that the performance of neural networks is higher compared to cluster and principal component analysis. The possibility of combining both approaches into one process can increase reliability and speed of classification. Artificially created training sets with added random noise can be used for network training. The analysis was applied on the Streptomyces coelicolor developmental two-dimensional (2-D) gel database.

Developmental control of the heat-shock stress regulon in Streptomyces coelicolor.

In the differentiating eubacterium Streptomyces coelicolor, nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods. The responses of all heat-shock proteins (HSPs) were highly dependent upon growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the level of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat- and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.

Protein profiles of Streptomyces aureofaciens producing tetracyclines: reappraisal of the effect of benzyl thiocyanate.

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.

The use of protein synthesis elongation factor EF-Tu as internal calibration standard in two-dimensional electrophoretic studies of differentiation in Streptomyces.

Protein synthesis elongation factor EF-Tu is presented as an internal calibration standard for quantitative analysis of two-dimensional (2-D) protein electrophoresis gels. EF-Tu was selected on the basis of concentration measurements in cell-free extracts from Streptomyces aureofaciens, grown under conditions leading to production of tetracyclines, and separated on one-dimensional (1-D) and 2-D electrophoresis gels. The results demonstrated that the amount of EF-Tu synthesized in S. aureofaciens under conditions of slow growth during production of tetracyclines is constant in proportion to all other de novo synthesized proteins regardless of their total number. This makes EF-Tu an ideal internal protein standard for quantitation of protein spots on 2-D electrophoresis gels. For such quantitative analysis we developed a computer-aided image analysis system which provides preparation of a gel image for further analysis including calibration, background subtraction and cleaning for streaking in both directions. The system can locate any resolvable spot in the gel and measure the integrated density of the spot, even in the case of irregular spot shape in crowded and overlapping spot regions.

Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores.

Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.

Quantitative analysis of gel electrophoretograms by image analysis and least squares modeling.

A computer-aided quantitative method for a complex analysis of gel electrophoretograms is presented. The analysis consists of several steps: (i) determination of the background image by methods of mathematical morphology and its subtraction from the gel image, (ii) selection of an appropriate part of the gel lane including curved lanes and lanes with a nonuniform width, (iii) computation of the lane densitogram by averaging several lane-parallel scans, (iv) decomposition of the lane densitogram into component bands using a data selecting algorithm and Marquardt's minimizer. Several different functions for component bands are utilized. It is shown that the densitogram can be decomposed into component bands with reasonable accuracy only if an appropriate model function is chosen. The algorithms are tested on several different gel electrophoretograms which show typical features as a nonuniform background, curved lanes, an asymmetrical band shape and a superposition of small bands on the shoulders of big ones. It is shown that overlapped bands are best approximated by an asymmetrical Gausian curve and an asymmetrical Gauss-Cauchy function. Linear response to the serial dilution of the protein sample is tested.

Content, distribution and stability of protein-synthesis elongation factor Tu in subcellular fractions of vegetative cells and spores of Streptomyces aureofaciens.

The protein synthesis elongation factor Tu (EF-Tu) was identified in dormant spores of Streptomyces aureofaciens and its content and distribution in vegetative cells and dormant spores were determined. Cell-free homogenates from spores were found to contain a EF-Tu cleaving membrane bound protease. The protease cleaved aggregated EF-Tu much less efficiently than non-aggregated factor in cell homogenates. The relative content of EF-Tu and ribosomes in dormant spores was very similar to that found in exponentially growing vegetative cells.