Showing papers in "Fems Microbiology Letters in 2015"

Enhanced butyrate formation by cross-feeding between Faecalibacterium prausnitzii and Bifidobacterium adolescentis

Abstract: Cross-feeding is an important metabolic interaction mechanism of bacterial groups inhabiting the human colon and includes features such as the utilization of acetate by butyrate-producing bacteria as may occur between Bifidobacterium and Faecalibacterium genera. In this study, we assessed the utilization of different carbon sources (glucose, starch, inulin and fructooligosaccharides) by strains of both genera and selected the best suited combinations for evidencing this cross-feeding phenomenon. Co-cultures of Bifidobacterium adolescentis L2-32 with Faecalibacterium prausnitzii S3/L3 with fructooligosaccharides as carbon source, as well as with F. prausnitzii A2-165 in starch, were carried out and the production of short-chain fatty acids was determined. In both co-cultures, acetate levels decreased between 8 and 24 h of incubation and were lower than in the corresponding B. adolescentis monocultures. In contrast, butyrate concentrations were higher in co-cultures as compared to the respective F. prausnitzii monocultures, indicating enhanced formation of butyrate by F. prausnitzii in the presence of the bifidobacteria. Variations in the levels of acetate and butyrate were more pronounced in the co-culture with fructooligosaccharides than with starch. Our results provide a clear demonstration of cross-feeding between B. adolescentis and F. prausnitzii.

Prebiotics and gut microbiota in chickens

Abstract: Prebiotics are non-digestible feed ingredients that are metabolized by specific members of intestinal microbiota and provide health benefits for the host. Fermentable oligosaccharides are best known prebiotics that have received increasing attention in poultry production. They act through diverse mechanisms, such as providing nutrients, preventing pathogen adhesion to host cells, interacting with host immune systems and affecting gut morphological structure, all presumably through modulation of intestinal microbiota. Currently, fructooligosaccharides, inulin and mannanoligosaccharides have shown promising results while other prebiotic candidates such as xylooligosaccharides are still at an early development stage. Despite a growing body of evidence reporting health benefits of prebiotics in chickens, very limited studies have been conducted to directly link health improvements to prebiotic-dependent changes in the gut microbiota. This article visits the current knowledge of the chicken gastrointestinal microbiota and reviews most recent publications related to the roles played by prebiotics in modulation of the gut microbiota and immune functions. Progress in this field will help us better understand how the gut microbiota contributes to poultry health and productivity, and support the development of new prebiotic products as an alternative to in-feed antibiotics.

Avian colibacillosis: still many black holes.

Abstract: Avian pathogenic Escherichia coli (APEC) strains cause severe respiratory and systemic diseases, threatening food security and avian welfare worldwide. Intensification of poultry production and the quick expansion of free-range production systems will increase the incidence of colibacillosis through greater exposure of birds to pathogens and stress. Therapy is mainly based on antibiotherapy and current vaccines have poor efficacy. Serotyping remains the most frequently used diagnostic method, only allowing the identification of a limited number of APEC strains. Several studies have demonstrated that the most common virulence factors studied in APEC are all rarely present in the same isolate, showing that APEC strains constitute a heterogeneous group. Different isolates may harbor different associations of virulence factors, each one able to induce colibacillosis. Despite its economical relevance, pathogenesis of colibacillosis is poorly understood. Our knowledge on the host response to APEC is based on very descriptive studies, mostly restricted to bacteriological and histopathological analysis of infected organs such as lungs. Furthermore, only a small number of APEC isolates have been used in experimental studies. In the present review, we discuss current knowledge on APEC diversity and virulence, including host response to infection and the associated inflammatory response with a focus on pulmonary colibacillosis.

Cheese rind microbial communities: diversity, composition and origin.

Abstract: Cheese rinds host a specific microbiota composed of both prokaryotes (such as Actinobacteria, Firmicutes and Proteobacteria) and eukaryotes (primarily yeasts and moulds). By combining modern molecular biology tools with conventional, culture-based techniques, it has now become possible to create a catalogue of the biodiversity that inhabits this special environment. Here, we review the microbial genera detected on the cheese surface and highlight the previously unsuspected importance of non-inoculated microflora--raising the question of the latter's environmental sources and their role in shaping microbial communities. There is now a clear need to revise the current view of the cheese rind ecosystem (i.e. that of a well-defined, perfectly controlled ecosystem). Inclusion of these new findings should enable us to better understand the cheese-making process.

Nitrobacter winogradskyi transcriptomic response to low and high ammonium concentrations.

Abstract: Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO2−) as a source of energy and nitrogen. In most natural situations, NO2− oxidation is coupled closely to ammonium (NH4+) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH4+ to meet its N -biosynthetic requirements. Interestingly, NH4+ delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH4+ while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH4+ concentration. Genes in assimilatory NO2− metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH4+. Nitrobacter winogradsky i grown in medium with 25 mM NH4+ upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH4+ and is likely to be modified during coupled nitrification with NH3 oxidizers.

Viral bacterial co-infection of the respiratory tract during early childhood

Abstract: Acute respiratory infection (ARI) is an important cause of morbidity in children. Mixed aetiology is frequent, with pathogenic viruses and bacteria co-detected in respiratory secretions. However, the clinical significance of these viral/bacterial co-infections has long been a controversial topic. While severe bacterial pneumonia following influenza infection has been well described, associations are less clear among infections caused by viruses that are more common in young children, such as respiratory syncytial virus. Although assessing the overall contribution of bacteria to disease severity is complicated by the presence of many confounding factors in clinical studies, understanding the role of viral/bacterial co-infections in defining the outcome of paediatric ARI will potentially reveal novel treatment and prevention strategies, improving patient outcomes. This review summarizes current evidence for the clinical significance of respiratory viral/bacterial co-infections in young children, discusses possible mechanisms of cooperative interaction between these pathogens and highlights areas that require further investigation.

Diversity, antimicrobial and antioxidant activities of culturable bacterial endophyte communities in Aloe vera

Abstract: Twenty-nine culturable bacterial endophytes were isolated from surface-sterilized tissues (root, stem and leaf) of Aloe vera and molecularly characterized to 13 genera: Pseudomonas, Bacillus, Enterobacter, Pantoea, Chryseobacterium, Sphingobacterium, Aeromonas, Providencia, Cedecea, Klebsiella, Cronobacter, Macrococcus and Shigella. The dominant genera include Bacillus (20.7%), Pseudomonas (20.7%) and Enterobacter (13.8%). The crude and ethyl acetate fractions of the metabolites of six isolates, species of Pseudomonas, Bacillus, Chryseobacterium and Shigella, have broad spectral antimicrobial activities against pathogenic Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Salmonella Typhimurium, Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Streptococcus pyogenes and Candida albicans, with inhibition zones ranging from 6.0 ± 0.57 to 16.6 ± 0.57 mm. In addition, 80% of the bacterial endophytes produced 1,1-diphenyl-2-picrylhydrazyl (DPPH) with scavenging properties of over 75% when their crude metabolites were compared with ascorbic acid (92%). In conclusion, this study revealed for the first time the endophytic bacteria communities from A. vera (Pseudomonas hibiscicola, Macrococcus caseolyticus, Enterobacter ludwigii, Bacillus anthracis) that produce bioactive compounds with high DPPH scavenging properties (75-88%) and (Bacillus tequilensis, Pseudomonas entomophila, Chryseobacterium indologenes, Bacillus aerophilus) that produce bioactive compounds with antimicrobial activities against bacterial pathogens. Hence, we suggest further investigation and characterization of their bioactive compounds.

Identification and catalytic residues of the arsenite methyltransferase from a sulfate-reducing bacterium, Clostridium sp. BXM.

Abstract: Arsenic methylation is an important process frequently occurring in anaerobic environments. Anaerobic microorganisms have been implicated as the major contributors for As methylation. However, very little information is available regarding the enzymatic mechanism of As methylation by anaerobes. In this study, one novel sulfate-reducing bacterium isolate, Clostridium sp. BXM, which was isolated from a paddy soil in our laboratory, was demonstrated to have the ability of methylating As. One putative arsenite S-Adenosyl-Methionine methyltransferase (ArsM) gene, CsarsM was cloned from Clostridium sp. BXM. Heterologous expression of CsarsM conferred As resistance and the ability of methylating As to an As-sensitive strain of Escherichia coli. Purified methyltransferase CsArsM catalyzed the formation of methylated products from arsenite, further confirming its function of As methylation. Site-directed mutagenesis studies demonstrated that three conserved cysteine residues at positions 65, 153 and 203 in CsArsM are necessary for arsenite methylation, but only Cysteine 153 and Cysteine 203 are required for the methylation of monomethylarsenic to dimethylarsenic. These results provided the characterization of arsenic methyltransferase from anaerobic sulfate-reducing bacterium. Given that sulfate-reducing bacteria are ubiquitous in various wetlands including paddy soils, enzymatic methylation mediated by these anaerobes is proposed to contribute to the arsenic biogeochemical cycling.

The relationship between biofilm and outer membrane vesicles: a novel therapy overview.

Abstract: Microorganisms have the ability of inhabiting nearly every environment through their sophisticated mechanisms of survival such as biofilm formation and release of outer membrane vesicles (OMVs). The biofilm matrix offers microorganism protection and contributes significantly to several clinical challenges, including symptomatic inflammation, antibiotic resistance, recurrence and the spread of infectious emboli. Moreover, bacteria also have another protective mechanism of vesicle production which is used as a means of disseminating toxins to harm their host. A clear understanding of gene expression switch of bacterium from planktonic to biofilm mode offers clinical potentials in treating bacterial infections. In this respect, the treatment of bacterial infections may be achieved through (1) application of RNA interference technology to silence the expression of proteins involved in the process of biofilm formation, (2) utilization of vesicles in delivering antibiotics and (3) use of natural occurred compounds. In this review, we discuss the relationship between biofilm formation and OMV production with respect to tackling biofilm-related clinical challenges. Some prospective considerations in biofilm-associated infections treatment are also discussed.

Growth and adaptation of microorganisms on the cheese surface.

Abstract: Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and molds that interact together, thus generating the typical sensory properties of a cheese. Physiological and genomic investigations have revealed important functions involved in the ability of microorganisms to establish themselves at the cheese surface. These functions include the ability to use the cheese's main energy sources, to acquire iron, to tolerate low pH at the beginning of ripening and to adapt to high salt concentrations and moisture levels. Horizontal gene transfer events involved in the adaptation to the cheese habitat have been described, both for bacteria and fungi. In the future, in situ microbial gene expression profiling and identification of genes that contribute to strain fitness by massive sequencing of transposon libraries will help us to better understand how cheese surface communities function.

Hydrogenotrophic culture enrichment reveals rumen Lachnospiraceae and Ruminococcaceae acetogens and hydrogen-responsive Bacteroidetes from pasture-fed cattle.

Abstract: Molecular information suggests that there is a broad diversity of acetogens in the rumen, distinct from any currently isolated acetogens. We combined molecular analysis with enrichment culture techniques to investigate this diversity further. Methane-inhibited, hydrogenotrophic enrichment cultures produced acetate as the dominant end product. Acetyl-CoA synthase gene analysis revealed putative acetogens in the cultures affiliated with the Lachnospiraceae and Ruminococcaceae as has been found in other rumen studies. No formyltetrahydrofolate synthetase genes affiliating with acetogens or with 'homoacetogen similarity' scores >90% were identified. To further investigate the hydrogenotrophic populations in these cultures and link functional gene information with 16S rRNA gene identity, cultures were subcultured quickly, twice, through medium without exogenous hydrogen, followed by incubation without exogenous hydrogen. Comparison of cultures lacking hydrogen and their parent cultures revealed novel Lachnospiraceae and Ruminococcaceae that diminished in the absence of hydrogen, supporting the hypothesis that they were likely the predominant acetogens in the enrichments. Interestingly, a range of Bacteroidetes rrs sequences that demonstrated <86% identity to any named isolate also diminished in cultures lacking hydrogen. Acetogens or sulphate reducers from the Bacteroidetes have not been reported previously; therefore this observation requires further investigation.

Cell-to-cell contact and antimicrobial peptides play a combined role in the death of Lachanchea thermotolerans during mixed-culture alcoholic fermentation with Saccharomyces cerevisiae

Abstract: The roles of cell-to-cell contact and antimicrobial peptides in the early death of Lachanchea thermotolerans CBS2803 during anaerobic, mixed culture fermentations with Saccharomyces cerevisiae S101 were investigated using a commercially available, double-compartment fermentation system separated by cellulose membranes with different pore sizes; i.e. 1000 kDa for mixed- and single culture fermentations, and 1000 kDa and 3.5–5 kDa for compartmentalized-culture fermentations. SDS-PAGE and gel filtration chromatography were used to determine an antimicrobial peptidic fraction in the fermentations. Our results showed comparable amounts of the antimicrobial peptidic fraction in the inner compartments of the mixed culture- and 1000 kDa compartmentalized-culture fermentations containing L. thermotolerans after 4 days of fermentation, but a lower death rate of L. thermotolerans in the 1000 kDa compartmentalized-culture fermentation than in the mixed culture fermentation. Furthermore, L. thermotolerans died off even more slowly in the 3.5–5 kDa- than in the 1000 kDa compartmentalized-culture fermentation, which coincided with the presence of less of the antimicrobial peptidic fraction in the inner compartment of that fermentation than of the 1000 kDa compartmentalized-culture fermentation. Taken together, these results indicate that the death of L. thermotolerans in mixed cultures with S. cerevisiae is caused by a combination of cell-to-cell contact and antimicrobial peptides.

Methanogen communities in stools of humans of different age and health status and co-occurrence with bacteria

Abstract: Hydrogenotrophic methanogens live in a synthrophic relationship with the human gut microbiota as the terminal part of the anaerobic food chain. Methanobrevibacter smithii of the Methanobacteriales is the prevailing archaeal species. Recently, methylotrophic archaea of the novel order Methanomassiliicoccales were isolated from human stools. Few data exist on the prevalence, abundance, persistence and ecology of these methanogens in humans. This study investigated methanogen communities in 26 healthy and obese children (8-14 years) and 18 adults (28-78 years) using quantitative PCR. Samples were obtained from nine females before and after giving birth. Bacterial groups linked to the abundance of methanogens in adult females were identified using a 16S rRNA gene amplicon data set. A total of 89% and 65% of adults and children, respectively, carried Methanobacteriales. Methanomassiliicoccales were recovered from 50% of the adults and one child. Mean relative abundance of Methanomassiliicoccales in adults was lower than that of Methanobacteriales (0.10% versus 0.52%). Both Methanobacteriales and Methanomassiliicoccales formed stable communities in females before and after giving birth. Methanobacteriales co-occurred with bacterial taxonomic groups associated with the trophic chain from carbohydrate degradation to hydrogen and formate formation. Relative abundance was inversely correlated to Blautia. Negative correlation with little characterized groups within the Clostridiales indicated possible interactions of Methanomassiliicoccales with the bacterial community.

Bacterial genome remodeling through bacteriophage recombination.

Abstract: Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes.

Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

Abstract: Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification.

Drosophila-associated yeast species in vineyard ecosystems

Abstract: Yeast activity during wine fermentation directly contributes to wine quality, but the source and movement of yeasts in vineyards and winery environments have not been resolved. Here, we investigate the yeast species associated with the Drosophila insect vector to help understand yeast dispersal and persistence. Drosophila are commonly found in vineyards and are known to have a mutualistic relationship with yeasts in other ecosystems. Drosophilids were collected from vineyards, grape waste (marc) piles and wineries during grape harvest. Captured flies were identified morphologically, and their associated yeasts were identified. Drosophila melanogaster/D. simulans, D. hydei and Scaptodrosophila lativittata were identified in 296 captured Drosophila flies. These flies were associated with Metschnikowia pulcherrima, Hanseniaspora uvarum, Torulaspora delbrueckii and H. valbyensis yeasts. Yeast and Drosophila species diversity differed between collection locations (vineyard and marc: R = 0.588 for Drosophila and R = 0.644 for yeasts). Surprisingly, the primary wine fermentation yeast, Saccharomyces cerevisiae, was not isolated. Drosophila flies are preferentially associated with different yeast species in the vineyard and winery environments, and this association may help the movement and dispersal of yeast species in the vineyard and winery ecosystem.

The infection process of Piscirickettsia salmonis in fish macrophages is dependent upon interaction with host-cell clathrin and actin.

Abstract: Piscirickettsia salmonis is an aggressive fish pathogen that causes Piscirickettsiosis, a systemic disease that threatens the sustainability of salmon production in Chile. To date, the infection strategies of this bacterium are poorly characterized, a Dot/Icm Type IV Secretion System homolog for intracellular multiplication and survival in macrophages is suggested. Since an invading pathogen and its host develop a complex interaction in which the pathogen strives to survive and replicate, while the host tries to eliminate infected cells and the invading pathogen, we decided to evaluate how the bacterium enters macrophages, its preferred target in vivo, and to follow its fate while struggling with its host using actin cytoskeleton as a molecular marker. We were able to demonstrate that clathrin is required for internalization and that actin cytoskeleton plays a demonstrative role throughout the infective process. Indeed, unlike other fish pathogens, P. salmonis fully exploits the actin monomers both from the disorganized cytoskeleton and an apparently pathogen-induced de novo synthesis of actin, generating tridimensional vacuoles that are increasingly detected at later stages of infection. We expect our results to contribute to a better understanding of the pathogenesis of this important fish pathogen.

The manganese-responsive regulator MntR represses transcription of a predicted ZIP family metal ion transporter in Corynebacterium glutamicum.

Abstract: Manganese is an important trace element required as an enzyme cofactor and for protection against oxidative stress. In this study, we characterized the DtxR-type transcriptional regulator MntR (cg0741) of Corynebacterium glutamicum ATCC 13032 as a manganese-dependent repressor of the predicted ZIP family metal transporter Cg1623. Comparative transcriptome analysis of a ΔmntR strain and the wild type led to the identification of cg1623 as potential target gene of MntR which was about 50-fold upregulated when cells were grown in glucose minimal medium. Using electrophoretic mobility shift assays, a conserved 18 bp inverted repeat (TGTTCAATGCGTTGAACA) was identified as binding motif of MntR in the cg1623 promoter and confirmed by mutational analysis. Promoter fusion of Pcg1623 to eyfp confirmed that the MntR-dependent repression is only abolished in the absence of manganese. However, neither deletion of mntR nor cg1623 resulted in a significant growth phenotype in comparison to the wild type--strongly suggesting the presence of further manganese uptake and efflux systems in C. glutamicum. The control of cg1623 by the DtxR-type regulator MntR represents the first example of a predicted ZIP family protein that is regulated in a manganese-dependent manner in bacteria.

Change in cell surface properties of Lactobacillus casei under heat shock treatment.

Abstract: We undertake this study in the aim to give new insight about the change in cellular physiological state under heat shock treatment and probiotic strain screening procedure. Different cell properties have been studied like adhesive ability to biotic and abiotic surfaces, the cell surface hydrophobicity and the fatty acids profiles. Compared to the normal cells, the heated cells increased their adhesive ability to biotic surface. However, the adhesion to abiotic surface was decreased. The cell surface hydrophobicity of the heated strains showed a significant decrease (P < 0.05). Our data revealed that high temperature change the fatty acids profiles of the treated cells, especially the proportions of unsaturated and saturated fatty acid. In fact, the ratio of saturated to unsaturated fatty acids of the heated Lactobacillus casei cells was significantly higher than that of the control cells (P < 0.05). The present finding could firstly add new insight about the response of probiotic to stressful conditions, such us the important role of cell membrane, considered as the first main structure to be damaged by physicochemical stress, in stress resistance because of their composition that can change in adaptation to harsh conditions. Secondly, there is no relationship between changes in membrane composition and fluidity induced by heat shock treatment and adhesion to biotic and abiotic surface.

Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain.

Abstract: Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ∼10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca(2+) enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs.

Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution

Abstract: The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

The IS1111 insertion sequence used for detection of Coxiella burnetii is widespread in Coxiella-like endosymbionts of ticks.

Abstract: Coxiella is a genus of obligate intracellular bacteria engaged in a variety of interactions with eukaryotes. The type species, Coxiella burnetii, infects several vertebrate species, including humans, and is the causative agent of Q fever. Multiple copies of a specific transposable element, the insertion sequence IS1111, are present in the genome of C. burnetii and are routinely used for confirmation of Q fever cases. Recently, many Coxiella-like bacteria that are closely related but genetically distinct to C. burnetii have been found in ticks. These Coxiella-like bacteria are maternally inherited endosymbionts, present at high prevalence in tick populations and engaged in mutualistic interactions with their arthropod hosts. In this study, the presence of IS1111 was examined in the Coxiella-like endosymbionts and in bacteria of the Coxiella sister-genus, Rickettsiella. This screening reveals that a wide range of IS1111 copies were present in the Coxiella-like endosymbionts of ticks. DNA sequencing further identified genetically divergent IS1111 copies, including degraded copies that constitute an important genomic fossil record of past IS1111 expansions. These results show that IS1111 is not specific to C. burnetii, suggesting that Q fever detection assays based only on this element may lead to misidentification with Coxiella-like endosymbionts.

Promoting microbiology education through the iGEM synthetic biology competition.

Abstract: Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.

Enterococcus faecium QU 50: a novel thermophilic lactic acid bacterium for high-yield l-lactic acid production from xylose.

Abstract: Production of optically pure lactic acid from lignocellulosic material for commercial purposes is hampered by several difficulties, including heterofermentation of pentose sugars and high energy consumption by mesophilic lactic acid bacteria. Here, we report a novel lactic acid bacterium, strain QU 50, that has the potential to produce optically pure l-lactic acid (≥99.2%) in a homofermentative manner from xylose under thermophilic conditions. Strain QU 50 was isolated from Egyptian fertile soil and identified as Enterococcus faecium QU 50 by analyzing its sugar fermentation pattern and 16S rRNA gene sequence. Enterococcus faecium QU 50 fermented xylose efficiently to produce lactic acid over wide pH (6.0-10.0) and temperature ranges (30-52°C), with a pH of 6.5 and temperature of 50°C being optimal. To our knowledge, this is the first report of homofermentative lactic acid production from xylose by a thermophilic lactic acid bacterium.

Outer membrane proteomics of kanamycin-resistant Escherichia coli identified MipA as a novel antibiotic resistance-related protein.

Abstract: Antibiotic-resistant bacteria are a great threat to human health and food safety and there is an urgent need to understand the mechanisms of resistance for combating these bacteria. In the current study, comparative proteomic methodologies were applied to identify Escherichia coli K-12 outer membrane (OM) proteins related to kanamycin resistance. Mass spectrometry and western blotting results revealed that OM proteins TolC, Tsx and OstA were up-regulated, whereas MipA, OmpA, FadL and OmpW were down-regulated in kanamycin-resistant E. coli K-12 strain. Genetic deletion of tolC (ΔtolC-Km) led to a 2-fold decrease in the minimum inhibitory concentration (MIC) of kanamycin and deletion of mipA (ΔmipA-Km) resulted in a 4-fold increase in the MIC of kanamycin. Changes in the MICs for genetically modified strains could be completely recovered by gene complementation. Compared with the wild-type strain, the survival capability of ΔompA-Km was significantly increased and that of Δtsx-Km was significantly decreased. We further evaluated the role and expression of MipA in response to four other antibiotics including nalidixic acid, streptomycin, chloramphenicol and aureomycin, which suggested that MipA was a novel OM protein related to antibiotic resistance.

Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.

Abstract: The synthesis of heterologous proteins in Lactococcus lactis is strongly influenced by the promoter selected for the expression. The nisin A promoter is commonly used for induced expression of proteins in L. lactis, whereas few constitutive promoters (P45 and the weaker P32) have been used for protein expression studies. In this study, eight different putative strong constitutive promoters were identified through transcriptional analysis of L. lactis N8 and were investigated for their capability to drive nisZ gene expression with promoters P45 and P32 as control. Four strong promoters (P8, P5, P3 and P2) were identified as having a transcriptional activity that was higher than that of P45 through RT-qPCR and agar-diffusion experiments. In addition, these four promoters were fused to the erythromycin resistant gene (ermC) with promoter P45 as control and inserted into the backbone of the pNZ8048 vector. The transcriptional efficiencies of promoters P8, P5, P2 and P3 were all higher than promoter P45 based on the obtained MIC50 values and they all showed different activity levels. In conclusion, four strong constitutive promoters with a wide range of promoter activities were identified and are suitable for protein production in L. lactis.

Pseudomonas fluorescens Pf-5 genome-wide mutant screen for resistance to the antimicrobial peptide alfalfa snakin-1.

Abstract: Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a mutation library of Pseudomonas fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1 (MsSN1). We identified three biofilm formation-related Pseudomonas mutants that showed increased resistance to MsSN1. Genetic, physiological and bioinformatics analysis validated the results of the mutant screens, indicating that bacterial adhesion protein lapA is probably the target of MsSN1. Collectively, these findings suggest that snakin-1 acts on microbial adhesion properties.

Tenellin acts as an iron chelator to prevent iron-generated reactive oxygen species toxicity in the entomopathogenic fungus Beauveria bassiana.

Abstract: Iron is an essential element for life. However, the iron overload can be toxic. Here, we investigated the significant increase of tenellin and iron-tenellin complex production in ferricrocin-deficient mutants of Beauveria bassiana. Our chemical analysis indicated that the ferricrocin-deficient mutants T1, T3 and T5 nearly abolished ferricrocin production. In turn, these mutants had significant accumulation of iron-tenellin complex in their mycelia at 247-289 mg g(-1) cell dry weight under iron-replete condition. Both tenellin and iron-tenellin complex were not detected in the wild-type under such condition. Mass analysis of the mutants' crude extracts demonstrated that tenellin formed a 3:1 complex with iron in the absence of ferricrocin. The unexpected link between ferricrocin and tenellin biosynthesis in ferricrocin-deficient mutants could be a survival strategy during iron-mediated oxidative stress.

Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent

Abstract: Despite intense efforts by the medical and pharmaceutical communities, Staphylococcus aureus continues to be a pervasive pathogen that causes a myriad of diseases and a high level of morbidity and mortality among infected patients. Thus, discovering or designing novel therapeutics able to kill both drug-resistant and drug-sensitive S. aureus remains a top priority. Bacteriolytic enzymes, mostly from phage, have shown great promise in preclinical studies, but little consideration has been given to cis-acting autolytic enzymes derived from the pathogen itself. Here, we use the S. aureus autolysin LytM as a proof of principal to demonstrate the antibacterial potential of endogenous peptidoglycan-degrading enzymes. While native LytM is only marginally bactericidal, fusion of LytM to the lysostaphin cell wall binding domain enhances its anti-staphylococcal activity approximately 540-fold, placing it on par with many phage lysins currently in preclinical development. The potential to therapeutically co-opt a pathogen's endogenous peptidoglycan recycling machinery opens the door to a previously untapped reservoir of antibacterial drug candidates.

Bidirectional signaling in the competence regulatory pathway of Streptococcus mutans

Abstract: Streptococcus mutans expresses comX (also known as sigX), which encodes a sigma factor that is required for development of genetic competence, in response to the peptide signals XIP and CSP and environmental factors. XIP (sigX inducing peptide) is derived from ComS and activates comX unimodally in chemically defined media via the ComRS system. CSP (competence stimulating peptide) activates comX bimodally in peptide-rich media through the ComDE two-component system. However, CSP-ComDE activation of comX is indirect and involves ComRS. Therefore, the bimodality of CSP-dependent activation of comX may arise from either ComRS or ComDE. Here we study, at the single-cell level, how genes in the CSP signaling pathway respond to CSP, XIP and media. Our data indicate that activation of comX stimulates expression of comE. In addition, activation of comE requires intact comR and comS genes. Therefore, not only does CSP-ComDE stimulate the ComRS pathway to activate comX expression, but ComRS activation of comX also stimulates expression of the CSP-ComDE pathway and its regulon. The results demonstrate the mutual interconnection of the signaling pathways that control bacteriocin expression (ComDE) and genetic competence (ComRS), both of which are linked to lytic and virulence behaviors.