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1.  Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium 
In this work, biosurfactant production by Bacillus subtilis #573 was evaluated using corn steep liquor (CSL) as culture medium. The best results were obtained in a culture medium consisting of 10% (v/v) of CSL, with a biosurfactant production of about 1.3 g/l. To the best of our knowledge, this is the first report describing biosurfactant production by B. subtilis using CSL as culture medium. Subsequently, the effect of different metals (iron, manganese, and magnesium) on biosurfactant production was evaluated using the medium CSL 10%. It was found that for all the metals tested, the biosurfactant production was increased (up to 4.1, 4.4, and 3.5 g/l for iron, manganese, and magnesium, respectively). When the culture medium was supplemented with the optimum concentration of the three metals simultaneously, the biosurfactant production was increased up to 4.8 g/l. Furthermore, the biosurfactant exhibited a good performance in oil recovery assays when compared with chemical surfactants, which suggests its possible application in microbial enhanced oil recovery or bioremediation.
PMCID: PMC4319461
Bacillus subtilis; corn steep liquor; surfactin; bioremediation; microbial enhanced oil recovery
2.  Colonization of lettuce rhizosphere and roots by tagged Streptomyces 
Beneficial microorganisms are increasingly used in agriculture, but their efficacy often fails due to limited knowledge of their interactions with plants and other microorganisms present in rhizosphere. We studied spatio-temporal colonization dynamics of lettuce roots and rhizosphere by genetically modified Streptomyces spp. Five Streptomyces strains, strongly inhibiting in vitro the major soil-borne pathogen of horticultural crops, Sclerotinia sclerotiorum, were transformed with pIJ8641 plasmid harboring an enhanced green fluorescent protein marker and resistance to apramycin. The fitness of transformants was compared to the wild-type strains and all of them grew and sporulated at similar rates and retained the production of enzymes and selected secondary metabolites as well as in vitro inhibition of S. sclerotiorum. The tagged ZEA17I strain was selected to study the dynamics of lettuce roots and rhizosphere colonization in non-sterile growth substrate. The transformed strain was able to colonize soil, developing roots, and rhizosphere. When the strain was inoculated directly on the growth substrate, significantly more t-ZEA17I was re-isolated both from the rhizosphere and the roots when compared to the amount obtained after seed coating. The re-isolation from the rhizosphere and the inner tissues of surface-sterilized lettuce roots demonstrated that t-ZEA17I is both rhizospheric and endophytic.
PMCID: PMC4319463
biocontrol; Lactuca sativa; Sclerotinia sclerotiorum; streptomycetes; rhizosphere competence
3.  Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei 
Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated.
PMCID: PMC4319465
Anaplasma phagocytophilum; nucleomodulin; nuclear translocation; oxidative burst; iTRAQ
4.  Montanide, Poly I:C and nanoparticle based vaccines promote differential suppressor and effector cell expansion: a study of induction of CD8 T cells to a minimal Plasmodium berghei epitope 
The development of practical and flexible vaccines to target liver stage malaria parasites would benefit from an ability to induce high levels of CD8 T cells to minimal peptide epitopes. Herein we compare different adjuvant and carrier systems in a murine model for induction of interferon gamma (IFN-γ) producing CD8 T cells to the minimal immuno-dominant peptide epitope from the circumsporozoite protein (CSP) of Plasmodium berghei, pb9 (SYIPSAEKI, referred to as KI). Two pro-inflammatory adjuvants, Montanide and Poly I:C, and a non-classical, non-inflammatory nanoparticle based carrier (polystyrene nanoparticles, PSNPs), were compared side-by-side for their ability to induce potentially protective CD8 T cell responses after two immunizations. KI in Montanide (Montanide + KI) or covalently conjugated to PSNPs (PSNPs-KI) induced such high responses, whereas adjuvanting with Poly I:C or PSNPs without conjugation was ineffective. This result was consistent with an observed induction of an immunosuppressed environment by Poly I:C in the draining lymph node (dLN) 48 h post injection, which was reflected by increased frequencies of myeloid derived suppressor cells (MDSCs) and a proportion of inflammation reactive regulatory T cells (Treg) expressing the tumor necrosis factor receptor 2 (TNFR2), as well as decreased dendritic cell (DC) maturation. The other inflammatory adjuvant, Montanide, also promoted proportional increases in the TNFR2+ Treg subpopulation, but not MDSCs, in the dLN. By contrast, injection with non-inflammatory PSNPs did not cause these changes. Induction of high CD8 T cell responses, using minimal peptide epitopes, can be achieved by non-inflammatory carrier nanoparticles, which in contrast to some conventional inflammatory adjuvants, do not expand either MDSCs or inflammation reactive Tregs at the site of priming.
PMCID: PMC4319470
malaria; adjuvant; nanoparticle; CD8 peptide; Treg; MDSC
5.  FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis 
The ferric uptake regulator A (FurA) is known to be involved in iron homeostasis and stress response in many bacteria. In mycobacteria the precise role of FurA is still unclear. In the presented study, we addressed the functional role of FurA in the ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) by construction of a furA deletion strain (MAPΔfurA). RNA deep sequencing revealed that the FurA regulon consists of repressed and activated genes associated to stress response or intracellular survival. Not a single gene related to metal homeostasis was affected by furA deletion. A decisive role of FurA during intracellular survival in macrophages was shown by significantly enhanced survival of MAPΔfurA compared to the wildtype, indicating that a principal task of mycobacterial FurA is oxidative stress response regulation in macrophages. This resistance was not associated with altered survival of mice after long term infection with MAP. Our results demonstrate for the first time, that mycobacterial FurA is not involved in the regulation of iron homeostasis. However, they provide strong evidence that FurA contributes to intracellular survival as an oxidative stress sensing regulator.
PMCID: PMC4319475
oxidative stress response regulation; furA; mycobacteria; iron; metallo-regulator
6.  Computational prediction of molecular pathogen-host interactions based on dual transcriptome data 
Inference of inter-species gene regulatory networks based on gene expression data is an important computational method to predict pathogen-host interactions (PHIs). Both the experimental setup and the nature of PHIs exhibit certain characteristics. First, besides an environmental change, the battle between pathogen and host leads to a constantly changing environment and thus complex gene expression patterns. Second, there might be a delay until one of the organisms reacts. Third, toward later time points only one organism may survive leading to missing gene expression data of the other organism. Here, we account for PHI characteristics by extending NetGenerator, a network inference tool that predicts gene regulatory networks from gene expression time series data. We tested multiple modeling scenarios regarding the stimuli functions of the interaction network based on a benchmark example. We show that modeling perturbation of a PHI network by multiple stimuli better represents the underlying biological phenomena. Furthermore, we utilized the benchmark example to test the influence of missing data points on the inference performance. Our results suggest that PHI network inference with missing data is possible, but we recommend to provide complete time series data. Finally, we extended the NetGenerator tool to incorporate gene- and time point specific variances, because complex PHIs may lead to high variance in expression data. Sample variances are directly considered in the objective function of NetGenerator and indirectly by testing the robustness of interactions based on variance dependent disturbance of gene expression values. We evaluated the method of variance incorporation on dual RNA sequencing (RNA-Seq) data of Mus musculus dendritic cells incubated with Candida albicans and proofed our method by predicting previously verified PHIs as robust interactions.
PMCID: PMC4319478
network inference; NetGenerator; transcriptomics; dual RNA-Seq; microarrays; gene regulatory networks; inter-species interactions
7.  The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H 
The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B, and H) we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella.
PMCID: PMC4319491
Salmonella; protease; phagocytosis; immune evasion; PgtE; complement factor B; complement factor H; omptin
8.  Stable isotope labeling confirms mixotrophic nature of streamer biofilm communities at alkaline hot springs 
Streamer biofilm communities (SBC) are often observed within chemosynthetic zones of Yellowstone hot spring outflow channels, where temperatures exceed those conducive to photosynthesis. Nearest the hydrothermal source (75–88°C) SBC comprise thermophilic Archaea and Bacteria, often mixed communities including Desulfurococcales and uncultured Crenarchaeota, as well as Aquificae and Thermus, each carrying diagnostic membrane lipid biomarkers. We tested the hypothesis that SBC can alternate their metabolism between autotrophy and heterotrophy depending on substrate availability. Feeding experiments were performed at two alkaline hot springs in Yellowstone National Park: Octopus Spring and “Bison Pool,” using various 13C-labeled substrates (bicarbonate, formate, acetate, and glucose) to determine the relative uptake of these different carbon sources. Highest 13C uptake, at both sites, was from acetate into almost all bacterial fatty acids, particularly into methyl-branched C15, C17 and C19 fatty acids that are diagnostic for Thermus/Meiothermus, and some Firmicutes as well as into universally common C16:0 and C18:0 fatty acids. 13C-glucose showed a similar, but a 10–30 times lower uptake across most fatty acids. 13C-bicarbonate uptake, signifying the presence of autotrophic communities was only significant at “Bison Pool” and was observed predominantly in non-specific saturated C16, C18, C20, and C22 fatty acids. Incorporation of 13C-formate occurred only at very low rates at “Bison Pool” and was almost undetectable at Octopus Spring, suggesting that formate is not an important carbon source for SBC. 13C-uptake into archaeal lipids occurred predominantly with 13C-acetate, suggesting also that archaeal communities at both springs have primarily heterotrophic carbon assimilation pathways. We hypothesize that these communities are energy-limited and predominantly nurtured by input of exogenous organic material, with only a small fraction being sustained by autotrophic growth.
PMCID: PMC4318418
hot springs; streamer biofilm communities; stable isotope probing; Archaea; heterotrophy; Aquificae; Yellowstone National Park
9.  Co-detection of Panton-Valentine leukocidin encoding genes and cotrimoxazole resistance in Staphylococcus aureus in Gabon: implications for HIV-patients’ care 
Patients infected with the human immunodeficiency virus (HIV) are frequently exposed to antimicrobial agents. This might have an impact on the resistance profile, genetic background and virulence factors of colonizing Staphylococcus aureus. Sub-Saharan Africa is considered to be endemic for Panton-Valentine leukocidin (PVL) positive S. aureus which can be associated with skin and soft tissue infections (SSTI). We compared S. aureus from nasal and pharyngeal swabs from HIV patients (n = 141) and healthy controls (n = 206) in Gabon in 2013, and analyzed determinants of colonization with PVL positive isolates in a cross-sectional study. S. aureus isolates were screened for the presence of selected virulence factors (incl. PVL) and were subjected to antimicrobial susceptibility testing and genotyping. In HIV patients, S. aureus was more frequently detected (36.9 vs. 31.6%) and the isolates were more frequently PVL positive than in healthy controls (42.1 vs. 23.2%). The presence of PVL was associated with cotrimoxazole resistance (OR = 25.1, p < 0.001) and the use of cotrimoxazole was a risk factor for colonization with PVL positive isolates (OR = 2.5, p = 0.06). PVL positive isolates were associated with the multilocus sequence types ST15 (OR = 5.6, p < 0.001) and ST152 (OR = 62.1, p < 0.001). Participants colonized with PVL positive isolates reported more frequently SSTI in the past compared to carriers of PVL negative isolates (OR = 2.7, p = 0.01). In conclusion, the novelty of our study is that cotrimoxazole might increase the risk of SSTI in regions where cotrimoxazole resistance is high and associated with PVL. This finding needs to be confirmed in prospective studies.
PMCID: PMC4318419
Staphylococcus aureus; Panton-Valentine leukocidin; cotrimoxazole resistance; Africa; HIV
10.  Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii 
Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii. The ω-3 fatty acid deficiency in strain CC-620, which contains a putative missense mutation at Thr286 of CrFAD7, was recovered by the overexpression of CC-125 CrFAD7. A Ser substitution in position 286 was able to partially complement the phenotype of the ω-3 fatty acid deficiency, but other substitution variants, such as Tyr, His, Cys, and Gly, failed to do so. Prediction of the phosphorylation target site revealed that Thr286 may be phosphorylated. Analysis of the structural conformation of CC-620 CrFAD7 via topology prediction (and bends in the helix) shows that this missense mutation may collapse the catalytic structure of CrFAD7. Taken together, this study suggests that Thr286 is essential for the maintaining the catalytic structure of CrFAD7.
PMCID: PMC4318421
CrFAD7; Thr286; fatty acid desaturase; topology; Chlamydomonas reinhardtii
11.  Mechanisms of antibiotic resistance 
PMCID: PMC4318422
antibiotics; resistance; mechanisms; resistance reservoirs; metagenomics; combination therapy
12.  Antigen-driven focal inflammatory death of malaria liver stages 
Multiple immunizations using live irradiated sporozoites, the infectious plasmodial stage delivered into the host skin during a mosquito bite, can elicit sterile immunity to malaria. CD8+ T cells seem to play an essential role in this protective immunity, since their depletion consistently abolishes sterilizing protection in several experimental models. So far, only a few parasite antigens are known to induce CD8+ T cell-dependent protection, but none of them can reach the levels of protection afforded by live attenuated parasites. Systematic attempts to identify novel antigens associated with this efficient cellular protection were so far unsuccessful. In addition, the precise mechanisms involved in the recognition and elimination of parasitized hepatocytes in vivo by CD8+ T cells still remain obscure. Recently, it has been shown that specific effector CD8+ T cells, after recognition of parasitized hepatocytes, recruit specific and non-specific activated CD8+ T cells to the site of infection, resulting in the formation of cellular clusters around and in the further elimination of intracellular parasites. The significance of this finding is discussed in the perspective of a general mechanism of antigen-dependent focalized inflammation and its consequences for the elimination of malaria liver stages.
PMCID: PMC4316770
CD8+ T cells; inflammatory infiltrate; cooperative behavior; in vivo imaging; Plasmodium
13.  Animals devoid of pulmonary system as infection models in the study of lung bacterial pathogens 
Biological disease models can be difficult and costly to develop and use on a routine basis. Particularly, in vivo lung infection models performed to study lung pathologies use to be laborious, demand a great time and commonly are associated with ethical issues. When infections in experimental animals are used, they need to be refined, defined, and validated for their intended purpose. Therefore, alternative and easy to handle models of experimental infections are still needed to test the virulence of bacterial lung pathogens. Because non-mammalian models have less ethical and cost constraints as a subjects for experimentation, in some cases would be appropriated to include these models as valuable tools to explore host–pathogen interactions. Numerous scientific data have been argued to the more extensive use of several kinds of alternative models, such as, the vertebrate zebrafish (Danio rerio), and non-vertebrate insects and nematodes (e.g., Caenorhabditis elegans) in the study of diverse infectious agents that affect humans. Here, we review the use of these vertebrate and non-vertebrate models in the study of bacterial agents, which are considered the principal causes of lung injury. Curiously none of these animals have a respiratory system as in air-breathing vertebrates, where respiration takes place in lungs. Despite this fact, with the present review we sought to provide elements in favor of the use of these alternative animal models of infection to reveal the molecular signatures of host–pathogen interactions.
PMCID: PMC4316775
alternative model; pneumonia; zebrafish; C. elegans; Drosophila melanogaster; Galleria mellonella; tuberculosis; cystic fibrosis
14.  Shifts in metabolic hydrogen sinks in the methanogenesis-inhibited ruminal fermentation: a meta-analysis 
Maximizing the flow of metabolic hydrogen ([H]) in the rumen away from CH4 and toward volatile fatty acids (VFA) would increase the efficiency of ruminant production and decrease its environmental impact. The objectives of this meta-analysis were: (i) To quantify shifts in metabolic hydrogen sinks when inhibiting ruminal methanogenesis in vitro; and (ii) To understand the variation in shifts of metabolic hydrogen sinks among experiments and between batch and continuous cultures systems when methanogenesis is inhibited. Batch (28 experiments, N = 193) and continuous (16 experiments, N = 79) culture databases of experiments with at least 50% inhibition in CH4 production were compiled. Inhibiting methanogenesis generally resulted in less fermentation and digestion in most batch culture, but not in most continuous culture, experiments. Inhibiting CH4 production in batch cultures resulted in redirection of metabolic hydrogen toward propionate and H2 but not butyrate. In continuous cultures, there was no overall metabolic hydrogen redirection toward propionate or butyrate, and H2 as a proportion of metabolic hydrogen spared from CH4 production was numerically smaller compared to batch cultures. Dihydrogen accumulation was affected by type of substrate and methanogenesis inhibitor, with highly fermentable substrates resulting in greater redirection of metabolic hydrogen toward H2 when inhibiting methanogenesis, and some oils causing small or no H2 accumulation. In both batch and continuous culture, there was a decrease in metabolic hydrogen recovered as the sum of propionate, butyrate, CH4 and H2 when inhibiting methanogenesis, and it is speculated that as CH4 production decreases metabolic hydrogen could be increasingly incorporated into formate, microbial biomass, and perhaps, reductive acetogenesis in continuous cultures. Energetic benefits of inhibiting methanogenesis depended on the inhibitor and its concentration and on the in vitro system.
PMCID: PMC4316778
methanogenesis inhibition; rumen; fermentation; metabolic hydrogen; meta-analysis; volatile fatty acids
15.  Long-term impacts of disturbance on nitrogen-cycling bacteria in a New England salt marsh 
Recent studies on the impacts of disturbance on microbial communities indicate communities show differential responses to disturbance, yet our understanding of how different microbial communities may respond to and recover from disturbance is still rudimentary. We investigated impacts of tidal restriction followed by tidal restoration on abundance and diversity of denitrifying bacteria, ammonia-oxidizing bacteria (AOB), and ammonia-oxidizing archaea (AOA) in New England salt marshes by analyzing nirS and bacterial and archaeal amoA genes, respectively. TRFLP analysis of nirS and betaproteobacterial amoA genes revealed significant differences between restored and undisturbed marshes, with the greatest differences detected in deeper sediments. Additionally, community patterns indicated a potential recovery trajectory for denitrifiers. Analysis of archaeal amoA genes, however, revealed no differences in community composition between restored and undisturbed marshes, but we detected significantly higher gene abundance in deeper sediment at restored sites. Abundances of nirS and betaproteobacterial amoA genes were also significantly greater in deeper sediments at restored sites. Porewater ammonium was significantly higher at depth in restored sediments compared to undisturbed sediments, suggesting a possible mechanism driving some of the community differences. Our results suggest that impacts of disturbance on denitrifying and ammonia-oxidizing communities remain nearly 30 years after restoration, potentially impacting nitrogen-cycling processes in the marsh. We also present data suggesting that sampling deeper in sediments may be critical for detecting disturbance effects in coastal sediments.
PMCID: PMC4316780
amoA; disturbance; nirS; restoration; salt marsh
16.  Urinary tract infections attributed to diverse ExPEC strains in food animals: evidence and data gaps 
Between 70 and 95% of urinary tract infections (UTI) are caused by strains of Escherichia coli. These strains, often termed Extraintestinal Pathogenic E. coli (ExPEC), possess specific virulence traits allowing them to colonize more inhospitable environments, such as the urogenital tract. Some ExPEC isolates from humans have similar virulence factor profiles to ExPEC isolates from animals, and because of the potential for these strains to cause UTI in people, these infections have been referred to as foodborne UTI, or FUTI. Finding similarities in ExPEC in animals and humans is not necessarily proof of transmission, particularly a unidirectional pathway from animals to humans; similarities in virulence factor profiles should be expected given the specific bacterial requirements for colonizing physiological compartments with similar characteristics in all animals. Many of the most important strains of human ExPEC globally, such as ST131, are highly virulent and clonal implying routes of transmission other than food. Documenting routes of transmission is particularly difficult due to the wide range of potential ExPEC sources, including the human intestinal tract, and non-human reservoirs such as food animals and retail meat products, sewage and other environmental sources, and companion animals. The significant environmental reservoir of ExPEC, including strains such as ST131, could potentially explain much more completely the global dissemination of virulent ExPEC clones and the rapid dissemination of new strains within the community. Taken in its totality, the link between ExPEC in animals and UTI in humans might exist, but studies conducted to date do not enable an estimation of the relative importance of this route of transmission. To reduce the burden of illness associated with ExPEC, the scientific community needs to push forward with ecologically-based, scientifically-sound study designs that can address the plethora of ways in which E. coli can spread.
PMCID: PMC4316786
Escherichia coli; antibiotics; antibiotic resistance; urinary tract infections; UPEC; APEC
17.  Heat shock proteins IbpA and IbpB are required for NlpI-participated cell division in Escherichia coli 
Lipoprotein NlpI of Escherichia coli is involved in the cell division, virulence, and bacterial interaction with eukaryotic host cells. To elucidate the functional mechanism of NlpI, we examined how NlpI affects cell division and found that induction of NlpI inhibits nucleoid division and halts cell growth. Consistent with these results, the cell division protein FtsZ failed to localize at the septum but diffused in the cytosol. Elevation of NlpI expression enhanced the transcription and the outer membrane localization of the heat shock protein IbpA and IbpB. Deletion of either ibpA or ibpB abolished the effects of NlpI induction, which could be restored by complementation. The C-terminus of NlpI is critical for the enhancement in IbpA and IbpB production, and the N-terminus of NlpI is required for the outer membrane localization of NlpI, IbpA, and IbpB. Furthermore, NlpI physically interacts with IbpB. These results indicate that over-expression of NlpI can interrupt the nucleoids division and the assembly of FtsZ at the septum, mediated by IbpA/IbpB, suggesting a role of the NlpI/IbpA/IbpB complex in the cell division.
PMCID: PMC4316790
NlpI; Escherichia coli; cell division; heat shock protein IbpA and IbpB; stress response
18.  Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1 
A 2.1-kb plasmid was previously isolated from Weissella cibaria KLC140 in kimchi and cloned into pUC19 along with the slpA and gfp genes, resulting in an 8.6-kb pKWCSLGFP construct for use as a novel surface display vector. To reduce the size of the vector, the minimal replicon of pKW2124 was determined. The pKW2124 plasmid contains a putative origin of replication (ori), a potential ribosomal binding site (RBS), and the repA gene encoding a plasmid replication protein. To conduct the minimal replicon experiment, four different PCR products (MR1, ori+RBS+repA; MR2, RBS+repA; MR2’, repA; MR3, fragment of repA) were obtained and cloned into pUC19 (pKUCm1, pKUCm2, pKUCm2’, and pKUCm3, respectively) containing the chloramphenicol acetyltransferase (CAT) gene. These constructed vectors were electroporated into W. confusa ATCC 10881 with different transformation efficiencies of 1.5 × 105 CFU/μg, 1.3 × 101 CFU/μg, and no transformation, respectively, suggesting that the putative ori, RBS, and repA gene are essential for optimum plasmid replication. Subsequent segregational plasmid stability testing of pKUCm1 and pKUCm2 showed that the vector pKUCm1 is highly stable up to 100 generations but pKUCm2 was completely lost after 60 generations, suggesting that the putative ori may be important for plasmid stability in the host strain. In addition, a host range test of pKUCm1 revealed that it has a broad host range spectrum including Weissella, Lactococcus, Leuconostoc, and even Lactobacillus. To verify the application of pKUCm1, the β-galactosidase gene and its promoter region from W. cibaria KSD1 were cloned in the vector, resulting in pKUGal. Expression of the β-galactosidase gene was confirmed using blue-white screening after IPTG induction. The small and stable pKUGal vector will be useful for gene transfer, expression, and manipulation in the Weissella genome and in other lactic acid bacteria.
PMCID: PMC4315094
Weissella; minimal replicon; expression vector; β-galactosidase; theta replication
19.  Corrigendum: Diversity of reductive dehalogenase genes from environmental samples and enrichment cultures identified with degenerate primer PCR screens 
PMCID: PMC4315102
Erratum; Amplicon sequencing; diversity; reductive dehalogenase; organohalide respiration
20.  Induction of the pro-inflammatory NF-kB-sensitive miRNA-146a by human neurotrophic viruses 
PMCID: PMC4315103
Alzheimer's disease (AD); aluminum sulfate; ebola virus; innate-immune response; messenger RNA (mRNA) and microRNA (miRNA); microRNA-146a; neuroinflammation; prion disease
21.  Field-based evidence for consistent responses of bacterial communities to copper contamination in two contrasting agricultural soils 
Copper contamination on China's arable land could pose severe economic, ecological and healthy consequences in the coming decades. As the drivers in maintaining ecosystem functioning, the responses of soil microorganisms to long-term copper contamination in different soil ecosystems are still debated. This study investigated the impacts of copper gradients on soil bacterial communities in two agricultural fields with contrasting soil properties. Our results revealed consistent reduction in soil microbial biomass carbon (SMBC) with increasing copper levels in both soils, coupled by significant declines in bacterial abundance in most cases. Despite of contrasting bacterial community structures between the two soils, the bacterial diversity in the copper-contaminated soils showed considerably decreasing patterns when copper levels elevated. High-throughput sequencing revealed copper selection for major bacterial guilds, in particular, Actinobacteria showed tolerance, while Acidobacteria and Chloroflexi were highly sensitive to copper. The thresholds that bacterial communities changed sharply were 800 and 200 added copper mg kg−1 in the fluvo-aquic soil and red soil, respectively, which were similar to the toxicity thresholds (EC50 values) characterized by SMBC. Structural equation model (SEM) analysis ascertained that the shifts of bacterial community composition and diversity were closely related with the changes of SMBC in both soils. Our results provide field-based evidence that copper contamination exhibits consistently negative impacts on soil bacterial communities, and the shifts of bacterial communities could have largely determined the variations of the microbial biomass.
PMCID: PMC4313605
copper contamination; soil bacterial community; diversity; abundance; community composition; soil microbial biomass carbon; field experiment
22.  Techniques for transferring host-pathogen protein interactions knowledge to new tasks 
We consider the problem of building a model to predict protein-protein interactions (PPIs) between the bacterial species Salmonella Typhimurium and the plant host Arabidopsis thaliana which is a host-pathogen pair for which no known PPIs are available. To achieve this, we present approaches, which use homology and statistical learning methods called “transfer learning.” In the transfer learning setting, the task of predicting PPIs between Arabidopsis and its pathogen S. Typhimurium is called the “target task.” The presented approaches utilize labeled data i.e., known PPIs of other host-pathogen pairs (we call these PPIs the “source tasks”). The homology based approaches use heuristics based on biological intuition to predict PPIs. The transfer learning methods use the similarity of the PPIs from the source tasks to the target task to build a model. For a quantitative evaluation we consider Salmonella-mouse PPI prediction and some other host-pathogen tasks where known PPIs exist. We use metrics such as precision and recall and our results show that our methods perform well on the target task in various transfer settings. We present a brief qualitative analysis of the Arabidopsis-Salmonella predicted interactions. We filter the predictions from all approaches using Gene Ontology term enrichment and only those interactions involving Salmonella effectors. Thereby we observe that Arabidopsis proteins involved e.g., in transcriptional regulation, hormone mediated signaling and defense response may be affected by Salmonella.
PMCID: PMC4313693
protein interaction prediction; host pathogen protein interactions; plant pathogen protein interactions; machine learning methods; transfer learning; kernel mean matching
23.  Microbiota disbiosis is associated with colorectal cancer 
The dysbiosis of the human intestinal microbiota is linked to sporadic colorectal carcinoma (CRC). The present study was designed to investigate the gut microbiota distribution features in CRC patients. We performed pyrosequencing based analysis of the 16S rRNA gene V3 region to investigate microbiota of the cancerous tissue and adjacent non-cancerous normal tissue in proximal and distal CRC samples. The results revealed that the microbial structures of the CRC patients and healthy individuals differed significantly. Firmicutes and Fusobacteria were over-represented whereas Proteobacteria was under-represented in CRC patients. In addition, Lactococcus and Fusobacterium exhibited a relatively higher abundance while Pseudomonas and Escherichia-Shigella was reduced in cancerous tissues compared to adjacent non-cancerous tissues. Meanwhile, the overall microbial structures of proximal and distal colon cancerous tissues were similar; but certain potential pro-oncogenic pathogens were different. These results suggested that the mucosa-associated microbiota is dynamically associated with CRC, which may provide evidences for microbiota-associated diagnostic, prognostic, preventive, and therapeutic strategies for CRC.
PMCID: PMC4313696
colorectal cancer; proximal colon; distal colon; mucosa-associated microbiota; gut dysbiosis
24.  Chemotactic preferences govern competition and pattern formation in simulated two-strain microbial communities 
Substrate competition is a common mode of microbial interaction in natural environments. While growth properties play an important and well-studied role in competition, we here focus on the influence of motility. In a simulated two-strain community populating a homogeneous two-dimensional environment, strains competed for a common substrate and only differed in their chemotactic preference, either responding more sensitively to a chemoattractant excreted by themselves or responding more sensitively to substrate. Starting from homogeneous distributions, three possible behaviors were observed depending on the competitors' chemotactic preferences: (i) distributions remained homogeneous, (ii) patterns formed but dissolved at a later time point, resulting in a shifted community composition, and (iii) patterns emerged and led to the extinction of one strain. When patterns formed, the more aggregating strain populated the core of microbial aggregates where starving conditions prevailed, while the less aggregating strain populated the more productive zones at the fringe or outside aggregates, leading to a competitive advantage of the less aggregating strain. The presence of a competitor was found to modulate a strain's behavior, either suppressing or promoting aggregate formation. This observation provides a potential mechanism by which an aggregated lifestyle might evolve even if it is initially disadvantageous. Adverse effects can be avoided as a competitor hinders aggregate formation by a strain which has just acquired this ability. The presented results highlight both, the importance of microbial motility for competition and pattern formation, and the importance of the temporal evolution, or history, of microbial communities when trying to explain an observed distribution.
PMCID: PMC4313714
chemotaxis; pattern formation; competition; coexistence; communities; individual-based modeling
25.  Organic extracts from Indigofera suffruticosa leaves have antimicrobial and synergic actions with erythromycin against Staphylococcus aureus 
A characteristic feature of Staphylococcus aureus is its ability to acquire resistance to antimicrobial agents. There is a need, therefore, for new approaches to combat this pathogen; for example, employing a combination of plant-derived products and antibiotics to overcome bacterial resistance. Indigofera suffruticosa is a plant popularly used to treat infections and has verified antimicrobial action. Here, we investigate the antimicrobial activity of different extracts from I. suffruticosa against S. aureus and their synergistic effects with erythromycin. Leaves of I. suffruticosa were extracted sequentially using diethyl ether, chloroform and acetone and the antimicrobial activity of each extract then tested against nine clinical isolates of S. aureus. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by microdilution tests, while the fractional inhibitory concentration (FIC) was assessed by checkerboard assay. All organic solvent extracts showed antimicrobial activity against S. aureus strains. The acetone extract was the most potent inhibitor of S. aureus (MIC and MBC of 0.78 and 3.12 mg/mL), followed by the chloroform extract (MIC and MBC of 3.12 and 6.25 mg/mL). Furthermore, acetone or chloroform extracts of I. suffruticosa enhanced the activity of erythromycin against S. aureus (FIC ≤ 0.5). We conclude that organic extracts from leaves of I. suffruticosa, alone or combined with erythromycin, are promising natural products for the development of new anti-S. aureus formulations.
PMCID: PMC4313721
plant extracts; antibacterial agents; macrolide antibiotic; S. aureus

Results 1-25 (2005)