Sigma regulatory network in Rhodococcus erythropolis CCM2595.

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed.

Stress response in Rhodococcus strains.

Rhodococci are bacteria which can survive under various extreme conditions, in the presence of toxic compounds, and in other hostile habitats. Their tolerance of unfavorable conditions is associated with the structure of their cell wall and their large array of enzymes, which degrade or detoxify harmful compounds. Their physiological and biotechnological properties, together with tools for their genetic manipulation, enable us to apply them in biotransformations, biodegradation and bioremediation. Many such biotechnological applications cause stresses that positively or negatively affect their efficiency. Whereas numerous reviews on rhodococci described their enzyme activities, the optimization of degradation or production processes, and corresponding technological solutions, only a few reviews discussed some specific effects of stresses on the physiology of rhodococci and biotechnological processes. This review aims to comprehensively describe individual stress responses in Rhodococcus strains, the interconnection of different types of stresses and their consequences for cell physiology. We examine here the responses to (1) environmental stresses (desiccation, heat, cold, osmotic and pH stress), (2) the presence of stress-inducing compounds (metals, organic compounds and antibiotics) in the environment (3) starvation and (4) stresses encountered during biotechnological applications. Adaptations of the cell envelope, the formation of multicellular structures and stresses induced by the interactions of hosts with pathogenic rhodococci are also included. The roles of sigma factors of RNA polymerase in the global regulation of stress responses in rhodococci are described as well. Although the review covers a large number of stressful conditions, our intention was to provide an overview of the selected stress responses and their possible connection to biotechnological processes, not an exhaustive survey of the scientific literature. The findings on stress responses summarized in this review and the demonstration of gaps in current knowledge may motivate researchers working to fill these gaps.

Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032.

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σC, σD, σE, σH, and σM are mostly involved in stress responses. The role of σD consists in the control of cell wall integrity. The σD regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential σD-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σD-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAACA/G and -10 GAT. Surprisingly, nearly all of these σD-dependent promoters were also active to a much lower extent with σH in vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the σH-dependent promoters is different (-35 GGAAT/C and -10 GTT). In addition to the activity of σH at the σD-controlled promoters, we discovered separated or overlapping σA- or σB-regulated or σH-regulated promoters within the upstream region of 8 genes of the σD-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σD-dependent genes. Computer modeling revealed that σH binds to the promoter DNA in a similar manner as σD to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σD and Lys 53 in σH, bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the σD-controlled genes also belong to the σH-regulon and are also transcribed from the overlapping or closely located housekeeping (σA-regulated) and/or general stress (σB-regulated) promoters.

Biodegradation of phenol and its derivatives by engineered bacteria: current knowledge and perspectives.

Biodegradation of phenolic compounds is a promising alternative to physical and chemical methods used to remove these toxic pollutants from the environment. The ability of various microorganisms to metabolize phenol and its derivatives (alkylphenols, nitrophenols and halogenated derivatives) has therefore been intensively studied. Knowledge of the enzymes catalyzing the individual reactions, the genes encoding these enzymes and the regulatory mechanisms involved in the expression of the respective genes in bacteria serves as a basis for the development of more efficient degraders of phenols via genetic engineering methods. Engineered bacteria which efficiently degrade phenolic compounds were constructed in laboratories using various approaches such as cloning the catabolic genes in multicopy plasmids, the introduction of heterologous genes or broadening the substrate range of key enzymes by mutagenesis. Efforts to apply the engineered strains in in situ bioremediation are problematic, since engineered strains often do not compete successfully with indigenous microorganisms. New efficient degraders of phenolic compounds may be obtained by complex approaches at the organism level, such as genome shuffling or adaptive evolution. The application of these engineered bacteria for bioremediation will require even more complex analysis of both the biological characteristics of the degraders and the physico-chemical conditions at the polluted sites.

Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial producer of various amino acids and other metabolites. The C. glutamicum genome encodes seven sigma subunits (factors) of RNA polymerase: the primary sigma factor SigA (σA), the primary-like σB and five alternative sigma factors (σC, σD, σE, σH and σM). We have developed in vitro and in vivo methods to assign particular sigma factors to individual promoters of different classes. In vitro transcription assays and measurements of promoter activity using the overexpression of a single sigma factor gene and the transcriptional fusion of the promoter to the gfpuv reporter gene enabled us to reliably define the sigma factor dependency of promoters. To document the strengths of these methods, we tested examples of respective promoters for each C. glutamicum sigma factor. Promoters of the rshA (anti-sigma for σH) and trxB1 (thioredoxin) genes were found to be σH-dependent, whereas the promoter of the sigB gene (sigma factor σB) was σE- and σH-dependent. It was confirmed that the promoter of the cg2556 gene (iron-regulated membrane protein) is σC-dependent as suggested recently by other authors. The promoter of cmt1 (trehalose corynemycolyl transferase) was found to be clearly σD-dependent. No σM-dependent promoter was identified. The typical housekeeping promoter P2sigA (sigma factor σA) was proven to be σA-dependent but also recognized by σB. Similarly, the promoter of fba (fructose-1,6-bisphosphate aldolase) was confirmed to be σB-dependent but also functional with σA. The study provided demonstrations of the broad applicability of the developed methods and produced original data on the analyzed promoters.

Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications.

The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and ß-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors.

Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles.

The aim of this study was to discover new nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic nitrilase, arylacetonitrilase, aliphatic nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetonitrilases and an aromatic nitrilase, respectively.

Catabolism of Phenol and Its Derivatives in Bacteria: Genes, Their Regulation, and Use in the Biodegradation of Toxic Pollutants.

Phenol and its derivatives (alkylphenols, halogenated phenols, nitrophenols) are natural or man-made aromatic compounds that are ubiquitous in nature and in human-polluted environments. Many of these substances are toxic and/or suspected of mutagenic, carcinogenic, and teratogenic effects. Bioremediation of the polluted soil and water using various bacteria has proved to be a promising option for the removal of these compounds. In this review, we describe a number of peripheral pathways of aerobic and anaerobic catabolism of various natural and xenobiotic phenolic compounds, which funnel these substances into a smaller number of central catabolic pathways. Finally, the metabolites are used as carbon and energy sources in the citric acid cycle. We provide here the characteristics of the enzymes that convert the phenolic compounds and their catabolites, show their genes, and describe regulatory features. The genes, which encode these enzymes, are organized on chromosomes and plasmids of the natural bacterial degraders in various patterns. The accumulated data on similarities and the differences of the genes, their varied organization, and particularly, an astonishingly broad range of intricate regulatory mechanism may be read as an exciting adventurous book on divergent evolutionary processes and horizontal gene transfer events inscribed in the bacterial genomes. In the end, the use of this wealth of bacterial biodegradation potential and the manipulation of its genetic basis for purposes of bioremediation is exemplified. It is envisioned that the integrated high-throughput techniques and genome-level approaches will enable us to manipulate systems rather than separated genes, which will give birth to systems biotechnology.

Induction and carbon catabolite repression of phenol degradation genes in Rhodococcus erythropolis and Rhodococcus jostii.

Rhodococcus erythropolis CCM2595 is able to efficiently utilize phenol and other aromatic compounds. We cloned and sequenced its complete gene cluster - catA, catB, catC, catR, pheR, pheA2, pheA1 - involved in the ortho-cleavage pathway of phenol. The activity of the key enzyme of the phenol degradation pathway, two-component phenol hydroxylase, was found to be induced by phenol. When both phenol and succinate were present in the medium, phenol hydroxylase activity decreased substantially. To analyze the regulation of phenol degradation at the transcriptional level, the transcriptional fusions of the divergently oriented promoters PpheA2 and PpheR with the gfpuv reporter gene were constructed. The promoters driving expression of the genes of the pheR-pheA2pheA1 cluster were localized by determining the respective transcriptional start points. Measurements of GFP fluorescence as well as quantitative RT-PCR revealed that expression of the phe genes is induced by phenol at the transcriptional level. The transcription of pheA2A1 and pheR was repressed by succinate, whereas no repression by glucose or glycerol was observed. Activation of the R. erythropolis CCM2595 pheA2 promoter by PheR, an AraC-type transcriptional regulator, was demonstrated by overexpression of the pheR gene. Analysis of the transcriptional regulation of two similar phe clusters from R. jostii RHA1 by various substrates showed that the type of carbon catabolite repression and the temporal transcriptional pattern during cultivation are different in each of the three phe clusters analyzed.

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.

Genome Sequence of Rhodococcus erythropolis Strain CCM2595, a Phenol Derivative-Degrading Bacterium.

We announce the completion of the genome sequence of a phenol derivative-degrading bacterium, Rhodococcus erythropolis strain CCM2595. This bacterium is interesting in the context of bioremediation for its capability to degrade phenol, catechol, resorcinol, hydroxybenzoate, hydroquinone, p-chlorophenol, p-nitrophenol, pyrimidines, and sterols.

Corynebacterium glutamicum promoters: a practical approach.

Transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. Promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate 'green waves' in the metabolic pathways leading to the desired metabolites. Detailed analyses of Corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms and practical capabilities in metabolic engineering. In this minireview the main aspects of promoter studies, the methods developed for their analysis and their practical use in C. glutamicum are discussed. These include definitions of the consensus sequences of the distinct promoter classes, promoter localization and characterization, activity measurements, the functions of transcriptional regulators and examples of practical uses of constitutive, inducible and modified promoters in biotechnology. The implications of the introduction of novel techniques, such as in vitro transcription and RNA sequencing, to C. glutamicum promoter studies are outlined.

Analysis of Corynebacterium glutamicum promoters and their applications.

Promoters are DNA sequences which function as regulatory signals of transcription initiation catalyzed by RNA polymerase. Since promoters substantially influence levels of gene expression, they have become powerful tools in metabolic engineering. Methods for their localization used in Corynebacterium glutamicum and techniques for the analysis of their function are described in this review. C. glutamicum promoters can be classified according to the respective σ factors which direct RNA polymerase to these structures. C. glutamicum promoters are recognized by holo-RNA polymerase formed by subunits α(2)ßß'ω + σ. C. glutamicum codes for seven different sigma factors: the principal sigma factor σ(A) and alternative sigma factors σ(B), σ(C), σ(D), σ(E), σ(H) and σ(M), which recognize various classes of promoters. The promoters of housekeeping genes recognized by σ(A), which are active during the exponential growth, form the largest described group. These promoters and their mutant derivatives are the most frequently used elements in modulation of gene expression in C. glutamicum. Promoters recognized by alternative sigma factors and their consensus sequences are gradually emerging.

Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes.

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, ß, ß'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged ß' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σ(A) and σ(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σ(A)- and σ(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.

Tools for genetic manipulations in Corynebacterium glutamicum and their applications.

Corynebacterium glutamicum is an important industrial producer of various amino acids with great potential for the production of other metabolites. The complete genome sequences of two C. glutamicum strains were determined and the use of genome-based approaches (transcriptomics, proteomics, metabolomics, and fluxomics) provided large amounts of data on the metabolism of this bacterium and its regulation. Many tools for genetic manipulations in C. glutamicum have been developed and used for the analysis of gene functions as well as for the construction and improvement of production strains. These tools include various types of plasmid vectors (cloning, promoter-probe, and expression vectors), DNA transfer methods, cloning heterologous genes, introducing protein secretion systems and gene replacement and genome rearrangement methods. Here we summarize the latest developments in the field of genetic engineering in C. glutamicum, give examples of the use of these new tools, and mention the challenges which stand in the way of fully implementing these tools and this acquired knowledge for the construction of superior production strains.

Sigma factors and promoters in Corynebacterium glutamicum.

The Corynebacterium glutamicum genome codes for 7 sigma subunits (factors) of RNA polymerase (RNAP): primary sigma factor SigA (σ(A)), primary-like SigB and 5 other alternative sigma factors (SigC, SigD, SigE, SigH and SigM). Each sigma factor is responsible for recognizing promoters of genes belonging to a regulon (sigmulon) involved in specific functions of the cell. Most promoters of C. glutamicum housekeeping genes are recognized by RNAP+σ(A), whereas σ(B) is involved in transcription of a large group of genes active during the transition phase between the exponential and stationary growth phases when various stress factors threaten to damage the cell. The σ(H) regulon consists of the genes involved in heat shock response including those coding for regulators and other sigma factors. It seems therefore that σ(H) occupies a central position in the cross-regulated network of sigma factors and controls their concerted response to various stress conditions in C. glutamicum. The σ(M) factor was found to regulate genes responding to oxidative stress. The main role of σ(E) is to activate genes involved in response to a cell surface stress. Promoters of individual classes recognized by different sigma factors are compiled and the respective consensus sequences of their key recognition motifs (-35 and -10 regions) are derived. In a number of genes, two or more promoters controlled by the same or different sigma factors were discovered. These multiple, overlapping or dual promoters contribute to a complex gene transcription control mechanisms that integrate internal and external signals and tune gene expression in cells as required by environmental and physiological conditions.

General and molecular microbiology and microbial genetics in the IM CAS.

This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the Academy of Sciences of the Czech Republic (AS CR) [formerly Czechoslovak Academy of Sciences (CAS)] over more than 50 years. Contribution of the founder of the Institute, academician Ivan Málek, to the introduction of these topics into the scientific program of the Institute of Microbiology and to further development of these studies is also included.

Factors enhancing L-valine production by the growth-limited L-isoleucine auxotrophic strain Corynebacterium glutamicum DeltailvA DeltapanB ilvNM13 (pECKAilvBNC).

Cell growth limitation is known to be an important condition that enhances L: -valine synthesis in Corynebacterium glutamicum recombinant strains with L: -isoleucine auxotrophy. To identify whether it is the limited availability of L: -isoleucine itself or the L: -isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of L: -valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel (-) ) C. glutamicum DeltailvA DeltapanB Deltarel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and L: -valine synthesis in rel (+) and rel (-) strains under conditions of L: -isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in L: -valine synthesis were observed in both rel (+) and rel (-) cells exposed to L: -isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and L: -valine synthesis in cells during the stationary growth phase.

Metabolic engineering of the L-valine biosynthesis pathway in Corynebacterium glutamicum using promoter activity modulation.

The previously constructed strain Corynebacterium glutamicumilvNM13 with acetohydroxy acid synthase, resistant to inhibition by all three branched-chain amino acids (L-valine, L-isoleucine and L-leucine), was used as a basis to develop a new type of valine producer by genetic engineering. The main strategy was to modulate expression of the genes involved in the biosynthesis of branched-chain amino acids. The activity of the promoters P-ilvD (dihydroxyacid dehydratase) and P-ilvE (transaminase) was up-modulated and the activity of the promoters P-ilvA (threonine deaminase) and P-leuA (isopropylmalate synthase) was down-modulated by site-directed mutagenesis. A constructed weak promoter of ilvA (or leuA), which was introduced into the C. glutamicum chromosome via a gene-replacement technique reduced the biosynthetic rate of isoleucine (or leucine), which lowered the mutant growth rate and increased valine production. Overexpression of ilvD and ilvE driven by the strong mutant promoters P-ilvDM7 and P-ilvEM6 resulted in an even higher level of valine production. Thus, the strain C. glutamicum ilvNM13 DeltapanB P-ilvAM1CG P-ilvDM7 P-ilvEM6, having all mutations constructed within the chromosome, produced 136 mM valine in a 48-h cultivation.