A holin and an endopeptidase are essential for chitinolytic protein secretion in <i>Serratia marcescens</i>

Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria

Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid

Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production

Major transitions can be expected within the next few decades aiming at the reduction of pollution and global warming and at energy saving measures. For these purposes, new sustainable biorefinery concepts will be needed that will replace the traditional mineral oil-based synthesis of specialty and bulk chemicals. An important group of these chemicals are those that comprise N-functionalities. Many plant components contained in biomass rest or waste stream fractions contain these N-functionalities in proteins and free amino acids that can be used as starting materials for the synthesis of biopolymers and chemicals. This paper describes the economic and technological feasibility for cyanophycin production by fermentation of the potato waste stream Protamylasse™ or directly in plants and its subsequent conversion to a number of N-containing bulk chemicals

The Twin-Arginine Translocation Pathway in α-Proteobacteria Is Functionally Preserved Irrespective of Genomic and Regulatory Divergence

The twin-arginine translocation (Tat) pathway exports fully folded proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Although much progress has been made in unraveling the molecular mechanism and biochemical characterization of the Tat system, little is known concerning its functionality and biological role to confer adaptive skills, symbiosis or pathogenesis in the α-proteobacteria class. A comparative genomic analysis in the α-proteobacteria class confirmed the presence of tatA, tatB, and tatC genes in almost all genomes, but significant variations in gene synteny and rearrangements were found in the order Rickettsiales with respect to the typically described operon organization. Transcription of tat genes was confirmed for Anaplasma marginale str. St. Maries and Brucella abortus 2308, two α-proteobacteria with full and partial intracellular lifestyles, respectively. The tat genes of A. marginale are scattered throughout the genome, in contrast to the more generalized operon organization. Particularly, tatA showed an approximately 20-fold increase in mRNA levels relative to tatB and tatC. We showed Tat functionality in B. abortus 2308 for the first time, and confirmed conservation of functionality in A. marginale. We present the first experimental description of the Tat system in the Anaplasmataceae and Brucellaceae families. In particular, in A. marginale Tat functionality is conserved despite operon splitting as a consequence of genome rearrangements. Further studies will be required to understand how the proper stoichiometry of the Tat protein complex and its biological role are achieved. In addition, the predicted substrates might be the evidence of role of the Tat translocation system in the transition process from a free-living to a parasitic lifestyle in these α-proteobacteria

Genomes of the Most Dangerous Epidemic Bacteria Have a Virulence Repertoire Characterized by Fewer Genes but More Toxin-Antitoxin Modules

We conducted a comparative genomic study based on a neutral approach to identify genome specificities associated with the virulence capacity of pathogenic bacteria. We also determined whether virulence is dictated by rules, or if it is the result of individual evolutionary histories. We systematically compared the genomes of the 12 most dangerous pandemic bacteria for humans ("bad bugs") to their closest non-epidemic related species ("controls").We found several significantly different features in the "bad bugs", one of which was a smaller genome that likely resulted from a degraded recombination and repair system. The 10 Cluster of Orthologous Group (COG) functional categories revealed a significantly smaller number of genes in the "bad bugs", which lacked mostly transcription, signal transduction mechanisms, cell motility, energy production and conversion, and metabolic and regulatory functions. A few genes were identified as virulence factors, including secretion system proteins. Five "bad bugs" showed a greater number of poly (A) tails compared to the controls, whereas an elevated number of poly (A) tails was found to be strongly correlated to a low GC% content. The "bad bugs" had fewer tandem repeat sequences compared to controls. Moreover, the results obtained from a principal component analysis (PCA) showed that the "bad bugs" had surprisingly more toxin-antitoxin modules than did the controls.We conclude that pathogenic capacity is not the result of "virulence factors" but is the outcome of a virulent gene repertoire resulting from reduced genome repertoires. Toxin-antitoxin systems could participate in the virulence repertoire, but they may have developed independently of selfish evolution

Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti

<p>Abstract</p> <p>Background</p> <p><it>Sinorhizobium meliloti </it>is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the <it>Medicago sativa </it>plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of <it>S. meliloti </it>strains AK83 and BL225C, we sequenced the complete genomes for these two strains.</p> <p>Results</p> <p>With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains.</p> <p>Conclusions</p> <p>In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.</p

Cell penetration, herbicidal activity, and in-vivo-toxicity of oligo-arginine derivatives and of novel guanidinium-rich compounds derived from the biopolymer cyanophycin Grogg, Marcel and Hilvert, Donald and Ebert, Marc-Olivier and Beck, Albert K. and Seebach, Dieter and Kurth, Felix and Dittrich, Petra S. and Sparr, C. and Wittlin, Sergio and Rottmann, Matthias and Mäser, Pascal

Oligo‐arginines are thoroughly studied cell‐penetrating peptides (CPPs, Figures 1 and 2). Previous in‐vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti‐malaria drug) have shown a 40‐fold parasitaemia inhibition with P. falciparum, compared to fosmidomycin alone (Figure 3). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e. decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake (Table 1). Another pronounced difference between in‐vitro and in‐vivo activity was demonstrated with various cell‐penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 μmol/kg (Table 2). The results show that use of CPPs for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly‐aspartic acid containing argininylated side chains (Figure 4); its building block is the dipeptide H‐βAsp‐αArg‐OH (H‐Adp‐OH). To test and compare the biological properties with those of octaarginines we synthesized Adp8‐derivatives (Figure 5). Intravenouse injection of H‐Adp8‐NH2 into the tail vein of mice with doses as high as 45 μmol/kg causes no symptoms whatsoever (Table 3), but H‐Adp8‐NH2 is not cell penetrating (HEK293 and MCF‐7 cells, Figure 6). On the other hand, the fluorescently labeled octamers FAM‐(Adp(OMe))8‐NH2 and FAM‐(Adp(NMe2))8‐NH2 with ester and amide groups in the side chains exhibit mediocre to high cell‐wall permeability (Figure 6), and are toxic (Table 3). Possible reasons for this behavior are discussed (Figure 7) and corresponding NMR spectra are presented (Figure 8)

Far-reaching cellular consequences of tat deletion in Escherichia coli revealed by comprehensive proteome analyses

In Escherichia coli, the Twin-arginine translocation (Tat) pathway secretes a set of folded proteins with important physiological functions to the periplasm and outer membrane. The loss of Tat secretion impairs outer membrane integrity and leads to decreased cell growth. Only recently, the Tat pathway has gained more attention due to its essential role in bacterial virulence and applications in the production of fully folded heterologous proteins. In this study, we investigated the influence of the deletion of all active Tat pathway components on the E. coli cells. The comprehensive proteomic analysis revealed activation of several stress responses and experimentally confirmed the dependence of certain proteins on the Tat system for export. We observed that a tat deletion triggers protein aggregation, membrane vesiculation, synthesis of colanic acid and biofilm formation. Furthermore, the mislocalization of Tat-dependent proteins disturbs iron and molybdenum homeostasis and impairs the cell envelope integrity. The results show that the functional Tat pathway is important for the physiological stability and that its dysfunction leads to a series of severe changes in E. coli cells

Protein secretion in Rhizobium leguminosarum biovar viciae 3841

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