- Volume 163, Issue 8, 2017

Bacteria of the genus Xanthomonas are a major group of plant pathogens. They are hazardous to important crops and closely related to human pathogens. Being collectively a major focus of molecular phytopathology, an increasing number of diverse and intricate mechanisms are emerging by which they communicate, interfere with host signalling and keep competition at bay. Interestingly, they are also biotechnologically relevant polysaccharide producers. Systems biotechnology techniques have revealed their central metabolism and a growing number of remarkable features. Traditional analyses of Xanthomonas metabolism missed the Embden–Meyerhof–Parnas pathway (glycolysis) as being a route by which energy and molecular building blocks are derived from glucose. As a consequence of the emerging full picture of their metabolism process, xanthomonads were discovered to have three alternative catabolic pathways and they use an unusual and reversible phosphofructokinase as a key enzyme. In this review, we summarize the synthetic and systems biology methods and the bioinformatics tools applied to reconstruct their metabolic network and reveal the dynamic fluxes within their complex carbohydrate metabolism. This is based on insights from omics disciplines; in particular, genomics, transcriptomics, proteomics and metabolomics. Analysis of high-throughput omics data facilitates the reconstruction of organism-specific large- and genome-scale metabolic networks. Reconstructed metabolic networks are fundamental to the formulation of metabolic models that facilitate the simulation of actual metabolic activities under specific environmental conditions.

Candida albicans is normally a harmless commensal of human beings, but it can cause superficial infections of the mucosa (oral/vaginal thrush) in healthy individuals and (rarely) infections of the skin or nails. It can also become invasive, causing life-threatening systemic and bloodstream infections in immunocompromised hosts, where the mortality rate can be as high as 50 %. It is the most common cause of serious fungal infection and is a common cause of nosocomial infections in hospitals. Some strains have been recognized that are resistant to azoles or echinocandins, which are the first-line antifungals for treatment of C. albicans infections.

Clustered regularly interspaced short palindromic repeats, associated proteins (CRISPR/Cas), has been developed into a powerful, targeted genome-editing tool in a wide variety of species. Here, we report an extensive investigation of the type II CRISPR/Cas9 system for targeted gene editing in Streptomyces rimosus. S. rimosus is used in the production of the antibiotic oxytetracycline, and its genome differs greatly from other species of the genus Streptomyces in the conserved chromosome terminal and core regions, which is of major production and scientific research value. The genes zwf2 and devB were chosen as target genes, and were edited separately via single-site mutations, double-site mutations and gene fragment disruptions. The single-site mutation guided by sgRNA-1 or sgRNA-2, respectively, involved GG changing to CA, GC changing to AT, and GG changing to CC. The double-site mutations guided by sgRNA-1 and sgRNA-2 included deletions and/or point mutations. Consistently, all mutations occurred in the gRNA sequence regions. Deletion mutations were characterized by the absence of eight bases, including three bases upstream of the PAM (protospacer adjacent motif) sequence, the PAM sequence itself and two bases downstream of the PAM sequence. A mutant (zwf2 − devB −) with a high yield of oxytetracycline was successfully obtained, whose oxytetracycline level was increased by 36.8 % compared to the original strain. These results confirm that CRISPR/Cas9 can successfully serve as a useful targeted genome editing system in S. rimosus.

Rho GTPases control polarized cell growth and are well-known regulators of exocytic and endocytic processes. Cdc42 is an essential GTPase, conserved from yeast to humans, that is critical for cell polarization. Cdc42 is negatively regulated by the GTPase-activating proteins (GAPs) and the GDP dissociation inhibitors (GDIs), and positively regulated by guanine nucleotide exchange factors (GEFs). Cdc42 GTPase can be found in a GTP- or GDP-bound state, which determines the ability to bind downstream effector proteins and activate signalling pathways. Only GTP-bound Cdc42 is active. In this study we have analysed the localization of the different nucleotide-bound states of Cdc42 in Schizosaccharomyces pombe: the wild-type Cdc42 protein that cycles between an active and inactive form, the Cdc42G12V form that is permanently bound to GTP and the Cdc42T17N form that is constitutively inactive. Our results indicate that Cdc42 localizes to several membrane compartments in the cell and this localization is mediated by its C-terminal prenylation. Constitutively active Cdc42 localizes mainly to the plasma membrane and concentrates at the growing tips where it is considerably less dynamic than wild-type or GDP-bound Cdc42. Additionally we show that the activation state of Cdc42 also participates in the regulation of its protein levels mediated by endocytosis and by the exocyst complex.

Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell’s metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein–protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

The population dynamics of pathogens within hosts result from interactions between host immune responses and mechanisms of the pathogen to evade or resist immune responses. Vertebrate hosts have evolved adaptive immune responses to eliminate the infection, while many pathogens evade immune clearance through altering surface antigens. Such interactions can result in a characteristic pattern of pathogen population dynamics within hosts consisting of population growth after infection, rapid population decline following specific immune responses, followed by persistence at low densities during a chronic infection stage. Despite the medical importance of chronic infections, little is known about the conditions of the interactions between variable antigens and the adaptive immune system that cause the characteristic pathogen population dynamics. Using the vls antigenic variation system of the Lyme disease pathogen, Borrelia burgdorferi, as a model system, we investigated conditions of the interaction between the antigenic variation system and the adaptive immune response that can explain the within-host population dynamics of B. burgdorferi using mathematical modelling. This characteristic population dynamic pattern can be explained by models that assume a variable immune removal rate of antibody-bound B. burgdorferi. However, models with a constant immune removal rate could reproduce the rapid population decline of B. burgdorferi populations but not their long-term persistence within hosts using parameter values determined by fitting empirical data. The model predictions, along with the assumptions about the interactions between B. burgdorferi and the immune response, can be tested experimentally to estimate the likelihood that each mechanism affects B. burgdorferi population dynamics in real infections.

Desulfovibrio sp. A2 is a novel Gram-negative sulfate-reducing bacterium that was isolated from sediments of the Norilsk mining/smelting area in Russia. The organism possesses a monocistronic operon encoding a 71 kDa periplasmic multicopperoxidase, which we call DA2_CueO. Histidine-tagged DA2_CueO expressed from a plasmid in Escherichia coli and purified by Ni–NTA affinity chromatography oxidizes Cu+ and Fe2+, and exhibits phenol oxidase activity with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,3-dihydroxybenzoic acid and 2,6-dimethoxyphenol as substrates, using O2 as the oxidant. When expressed in an E. coli cueO knock-out strain, DA2_CueO exhibits phenol oxidase activity in vivo and enhances the copper tolerance of the strain. These findings indicate that the DA2_CueO gene of Desulfovibrio sp. A2 encodes a multicopperoxidase with a role in metal ion resistance. The enzyme displays some novel structural features, which are discussed.

Small regulatory RNAs (sRNAs) are the most prominent post-transcriptional regulators in all kingdoms of life. A few of them, e.g. SR1 from Bacillus subtilis, are dual-function sRNAs. SR1 acts as a base-pairing sRNA in arginine catabolism and as an mRNA encoding the small peptide SR1P in RNA degradation. Both functions of SR1 are highly conserved among 23 species of Bacillales. Here, we investigate the interaction between SR1P and GapA by a combination of in vivo and in vitro methods. De novo prediction of the structure of SR1P yielded five models, one of which was consistent with experimental circular dichroism spectroscopy data of a purified, synthetic peptide. Based on this model structure and a comparison between the 23 SR1P homologues, a series of SR1P mutants was constructed and analysed by Northern blotting and co-elution experiments. The known crystal structure of Geobacillus stearothermophilus GapA was used to model SR1P onto this structure. The hypothetical SR1P binding pocket, composed of two α-helices at both termini of GapA, was investigated by constructing and assaying a number of GapA mutants in the presence and absence of wild-type or mutated SR1P. Almost all residues of SR1P located in the two highly conserved motifs are implicated in the interaction with GapA. A critical lysine residue (K332) in the C-terminal α-helix 14 of GapA corroborated the predicted binding pocket.