- Volume 166, Issue 10, 2020
Volume 166, Issue 10, 2020
- Editorial
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- Review
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Facultative methanotrophs – diversity, genetics, molecular ecology and biotechnological potential: a mini-review
More LessMethane-oxidizing bacteria (methanotrophs) play a vital role in reducing atmospheric methane emissions, and hence mitigating their potent global warming effects. A significant proportion of the methane released is thermogenic natural gas, containing associated short-chain alkanes as well as methane. It was one hundred years following the description of methanotrophs that facultative strains were discovered and validly described. These can use some multi-carbon compounds in addition to methane, often small organic acids, such as acetate, or ethanol, although Methylocella strains can also use short-chain alkanes, presumably deriving a competitive advantage from this metabolic versatility. Here, we review the diversity and molecular ecology of facultative methanotrophs. We discuss the genetic potential of the known strains and outline the consequent benefits they may obtain. Finally, we review the biotechnological promise of these fascinating microbes.
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- Environmental Biology
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nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase
Nitrous oxide (N2O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N2OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N2OR contains two important copper cofactors (CuA and CuZ centres) that are essential for activity, and in copper-limited environments, N2OR fails to function, contributing to rising levels of atmospheric N2O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A P. denitrificans ΔnosX deletion mutant failed to reduce N2O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N2OR activity. N2OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the CuA and CuZ centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N2OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from Pseudomonas stutzeri , which functions in the flavinylation of another N2OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N2OR and cellular N2O reduction.
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Genetic and physiological analysis of biofilm formation on different plastic surfaces by Sphingomonas sp. strain S2M10 reveals an essential function of sphingan biosynthesis
More LessAlphaproteobacteria belonging to the group of the sphingomonads are frequently found in biofilms colonizing pure-water systems, where they cause technical and hygienic problems. In this study, physiological properties of sphingomonads for biofilm formation on plastic surfaces were analysed. Sphingomonas sp. strain S2M10 was isolated from a used water-filtration membrane and submitted to transposon mutagenesis for isolating mutants with altered biofilm formation. Mutants showing strongly decreased biofilm formation carried transposon insertions in genes for the biosynthesis of the polysaccharide sphingan and for flagellar motility. Flagella-mediated attachment was apparently important for biofilm formation on plastic materials of intermediate hydrophobicity, while a mutant with defect in spnB, encoding the first enzyme in sphingan biosynthesis, showed no biofilm formation on all tested materials. Sphingan-dependent biofilm formation was induced in the presence of specific carbon sources while it was not induced in complex medium with yeast extract and tryptone. The regulation of sphingan-based biofilm formation was investigated by interfering with the CckA/ChpT/CtrA phosphorelay, a central signal-transduction pathway in most Alphaproteobacteria. Construction and ectopic expression of a kinase-deficient histidine kinase CckA caused cell elongation and massive sphingan-dependent cell aggregation. In addition, it caused increased activity of the promotor of spnB. In conclusion, these results indicate that sphingan-based biofilm formation by sphingomonads might be triggered by specific carbon sources under prototrophic conditions resembling a milieu that often prevails in pure-water systems.
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- Genomics and Systems Biology
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Transcriptional response of Fusarium oxysporum and Neocosmospora solani challenged with amphotericin B or posaconazole
Some species of fusaria are well-known pathogens of humans, animals and plants. Fusarium oxysporum and Neocosmospora solani (formerly Fusarium solani) cause human infections that range from onychomycosis or keratitis to severe disseminated infections. In general, these infections are difficult to treat due to poor therapeutic responses in immunocompromised patients. Despite that, little is known about the molecular mechanisms and transcriptional changes responsible for the antifungal resistance in fusaria. To shed light on the transcriptional response to antifungals, we carried out the first reported high-throughput RNA-seq analysis for F. oxysporum and N. solani that had been exposed to amphotericin B (AMB) and posaconazole (PSC). We detected significant differences between the transcriptional profiles of the two species and we found that some oxidation-reduction, metabolic, cellular and transport processes were regulated differentially by both fungi. The same was found with several genes from the ergosterol synthesis, efflux pumps, oxidative stress response and membrane biosynthesis pathways. A significant up-regulation of the C-22 sterol desaturase (ERG5), the sterol 24-C-methyltransferase (ERG6) gene, the glutathione S-transferase (GST) gene and of several members of the major facilitator superfamily (MSF) was demonstrated in this study after treating F. oxysporum with AMB. These results offer a good overview of transcriptional changes after exposure to commonly used antifungals, highlights the genes that are related to resistance mechanisms of these fungi, which will be a valuable tool for identifying causes of failure of treatments.
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- Host-microbe Interaction
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The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components
Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.
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Lactobacillus commensals autochthonous to human milk have the hallmarks of potent probiotics
More LessMaternal milk is an important source of essential nutrients for the optimal growth of infants. Breastfeeding provides a continuous supply of beneficial bacteria to colonize the infant gastrointestinal tract (GIT) and offers health benefits for disease prevention and immunity. The purpose of this study was to isolate novel probiotic strains from the breast milk of native Pakistani mothers and to evaluate their probiotic potential. We isolated 21 strains of bacteria from the colostrum and mature milk of 20 healthy mothers, who had vaginal deliveries and were not taking antibiotics. After phenotypic and genotypic characterization, these isolates were tested for survival in the GIT using in vitro acid and bile tests. Nine strains showing good acid tolerance were assessed for their growth rate, bile resistance and ability to hydrolyze bile salts. Out of the four Lactobacillus isolates adjudged to be most promising as probiotics, three were Lactobacillus fermentum strains and one was a strain of Lactobacillus oris . This study demonstrates that human milk is a viable source of commensal bacteria beneficial to both adults and babies.
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The Salmonella enterica serovar Typhimurium virulence factor STM3169 is a hexuronic acid binding protein component of a TRAP transporter
More LessThe intracellular pathogen S. Typhimurium is a leading cause of foodborne illness across the world and is known to rely on a range of virulence factors to colonize the human host and cause disease. The gene coding for one such factor, stm3169, was determined to be upregulated upon macrophage entry and its disruption reduces survival in the macrophage. In this study we characterize the STM3169 protein, which forms the substrate binding protein (SBP) of an uncharacterized tripartite ATP-independent periplasmic (TRAP) transporter. Genome context analysis of the genes encoding this system in related bacteria suggests a function in sugar acid transport. We demonstrate that purified STM3169 binds d-glucuronic acid with high affinity and specificity. S. Typhimurium LT2 can use this sugar acid as a sole carbon source and the genes for a probable catabolic pathway are present in the genome. As this gene was previously implicated in macrophage survival, it suggests a role for d-glucuronate as an important carbon source for S. Typhimurium in this environment.
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BBB07 contributes to, but is not essential for, Borrelia burgdorferi infection in mice
Borrelia burgdorferi, a causative agent of Lyme disease, encodes a protein BBB07 on the genomic plasmid cp26. BBB07 was identified as a candidate integrin ligand based on the presence of an RGD tripeptide motif, which is present in a number of mammalian ligands for β1 and β3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to β1 integrins and induces inflammatory responses in synovial cells in culture. Several transposon mutants in bbb07 were attenuated in an in vivo screen of the transposon library in mice. We therefore tested individual transposon mutant clones in single-strain infections in mice and found that they were attenuated in terms of ID50 but did not have significantly reduced tissue burdens in mice. Based on data presented here we conclude that BBB07 is not essential for, but does contribute to, B. burgdorferi infectivity in mice.
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- Physiology and Metabolism
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Pseudomonas aeruginosa is capable of natural transformation in biofilms
Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa .
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