Gene Expression Profiling of Listeria monocytogenes in monoculture and in co-culture state in the presence of Bacillus subtilis both as planktonic cells and in biofilms.
ABSTRACT: To understand the behaviour in terms of special genome components that are expressed by L.monocytogenes in the presence of another bacterium as may be the condition in its natural environments was our objective, for which we have used microarray gene expression at different time intervals of growth from 4hrs to 24 hrs. Expression of L.monocytogenes as co-cultures, both in broth culture state and biofilm state were differentiated to that of 24hrs pure broth culture. Also genes regulated for and during biofilm formation as pure cultures were identified with comparision to 24hrs pure broth culture. Distinguishing features with notable variation in all of the three sample sets as L.monocytogenes in pure culture biofilm, co-culture broth and co-culture biofilm were observable. Genes that are specifically up-regulated at each of growth condition and time interval were identified, genes regulated with ascending and descending patterns in time were also noticable. These variation in the gene expression gives an insight into the alterations in the biosynthetic and metabolic pathways under different states of growth Agilent one-color experiment,Organism: Listeria monocytogenes , Genotypic Technology Pvt. Ltd. designed Custom Microarray Listeria monocytogenes 8x15k (Agilent-30831) , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:To understand the behaviour in terms of special genome components that are expressed by L.monocytogenes in the presence of another bacterium as may be the condition in its natural environments was our objective, for which we have used microarray gene expression at different time intervals of growth from 4hrs to 24 hrs. Expression of L.monocytogenes as co-cultures, both in broth culture state and biofilm state were differentiated to that of 24hrs pure broth culture. Also genes regulated for and during biofilm formation as pure cultures were identified with comparision to 24hrs pure broth culture. Distinguishing features with notable variation in all of the three sample sets as L.monocytogenes in pure culture biofilm, co-culture broth and co-culture biofilm were observable. Genes that are specifically up-regulated at each of growth condition and time interval were identified, genes regulated with ascending and descending patterns in time were also noticable. These variation in the gene expression gives an insight into the alterations in the biosynthetic and metabolic pathways under different states of growth Overall design: Agilent one-color experiment,Organism: Listeria monocytogenes , Genotypic Technology Pvt. Ltd. designed Custom Microarray Listeria monocytogenes 8x15k (Agilent-30831) , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:Transcription profile of Escherichia coli cells in biofilms under static batch culture was compared to that of E. coli cells in planktonic cultures. Both E. coli biofilm and planktonic cultures were cultivated for 18 h in 10% Luria-Bertani broth at room temperature (20 degree Celsius). Biofilms were grown in static batch culture in petri dishes. Both planktonic culture and biofilms were homogenized and run through a separated protocol. Two condition experiments: E. coli biofilm vs E. coli planktonic cultures. Two biological replicates with independently grown and harvested biofilms or planktonic cultures. Each biological replicate has two technical replicates of hybridization on microarray slides. Each slide has three built-in replicates for each probe.
Project description:Biofilm formation by Escherichia coli was significantly inhibited when co-cultured with Stenotrophomonas maltophilia in static systems. Genes of E. coli involved in species interactions with S. maltophilia were identified in order to allow the study of the mechanisms of inhibited E. coli biofilm formation in co-culture. A total of 89 and 108 genes were identified as differentially expressed in mixed species cultures when growing as biofilm and as planktonic cultures, respectively, compared to the counterpart of pure cultured E. coli. Differential expression of certain identified genes was confirmed using E. coli reporter strains combined with single-cell based flow cytometry analysis. Co-culture with S. maltophilia affected genes involved in metabolism, signal transduction, cell wall composition, and biofilm formation of E. coli. Several selected genes were further confirmed as affecting E. coli biofilm formation in mixed species cultures with S. maltophilia. The data suggest that these genes were involved in species interactions between E. coli and S. maltophilia. This SuperSeries is composed of the SubSeries listed below. Refer to individual Series.
Project description:Transcription profile of Escherichia coli cells in mono-species pure biofilms was compared to that of E. coli cells in E. coli-Stenotrophomonas maltophilia dual-species biofilms. E. coli cells were separated from dual-species biofilms before total RNA extraction to eliminate possible cross hybridization from S. maltophilia transcripts. The separation method was developed by combining the use of reagent RNAlater and immuno-magnetic separation. Pure E. coli biofilms were processed with the same separation protocol before RNA extraction. Two condition experiments: E. coli mono-species biofilm vs E. coli in mixed-species biofilm. Two biological replicates with independently grown and harvested biofilms. Each biological replicate has two or three technical replicates of hybridization on microarray slides. Each slide has three built-in replicates for each probe.
Project description:Mapping of transposon mutant library in Enterococcus faecium during growth in Brain Heart Infusion (BHI) broth and in a semi-static biofilm model. The goal of this study was to identify factors that play a role in E. faecium biofilm formation by selection of transposon insertion mutants that lost the capacity to form biofilm in vitro.
Project description:The formation of Listeria monocytogenes biofilms contributes to persistent contamination in food processing facilities. A microarray comparison of L. monocytogenes between the transcriptome of the strong biofilm forming strain (Bfms) Scott A and the weak biofilm forming (Bfmw) strain F2365 was conducted to identify genes potentially involved in biofilm formation. Among 951 genes with significant difference in expression between the two strains, a GntR-family response regulator encoding gene (LMOf2365_0414), designated lbrA, was found to be highly expressed in Scott A relative to F2365. A Scott A lbrA-deletion mutant, designated AW3, formed biofilm to a much lesser extent as compared to the parent strain by a rapid attachment assay and scanning electron microscopy. Complementation with lbrA from Scott A restored the Bfms phenotype in the AW3 derivative. A second microarray assessment using the lbrA deletion mutant AW3 and the wild type Scott A revealed a total of 304 genes with expression significantly different between the two strains, indicating the potential regulatory role of LbrA in L. monocytogenes. A cloned copy of Scott A lbrA was unable to confer enhanced biofilm forming potential in F2365, suggesting that additional factors contributed to weak biofilm formation by F2365. Findings from the study may lead to new strategies to modulate biofilm formation. Two comparisons were performed between 1) strong biofilm former Listeria monocytogenes strain ScottA versus weak biofilm former Listeria monocytogenes strain F2365; 2) Listeria monocytogenes ScottA LbrA deletion mutant strain versus Listeria monocytogenes ScottA. Four replicates were loaded for the first comparison and two replicates were loaded for the second comparison.
Project description:Bioelectrochemical systems employing mixed microbial communities as biocatalysts are gaining importance as potential renewable energy, bioremediation, or biosensing devices. While we are beginning to understand how individual microorganism species interact with an electrode as electron donor, not much is known about the interactions between different microbial species in a community. Here, we compare the bioelectrochemical performance of Shewanella oneidensis in a pure-culture and in a co-culture with the homolactic acid fermenter Lactococcus lactis. While S. oneidensis alone can only use lactate as electron donor for current production, the co-culture is able to convert glucose into current with a similar coulombic efficiency of approximately 17%, respectively. With (electro)-chemical analysis and transcription profiling, we found that the BES performance and S. oneidensis physiology were not significantly different whether grown as a pure- or co-culture. These co-culture experiments represent a first step in understanding microbial interactions in BES communities with the goal to design complex microbial communities, which specifically convert target substrates into electricity. Further, for the first time, we elucidated S. oneidensis gene expression with an electrode as the only electron acceptor. The expression pattern confirms many previous studies regarding the enzymatic requirements for electrode respiration, and it generates new hypotheses on the functions of proteins, which are so far not known to be involved in electrode respiration. The BES was either operated with S. oneidensis alone, fed with lactate, or it was operated with S. oneidensis and L. lactis with glucose as primary substrate. The basic medium was a modified M4 medium containing 0.5 g/L yeast extract, 0.5 g/L trypton and 5 g/L glycerol phosphate, besides the commen M4 incredients. S. oneidensis oxidizes lactate to acetate and electrons in a BES - the latter generate a current at a graphite anode. The anode biofilm was harvested after about 4 weeks of continuous BES operation and subjected to total RNA extraction.
Project description:The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. PrfA, a key transcriptional activator that regulates most of the known listerial virulence gene expression, has been shown to promote L. monocytogenes biofilm formation. In this study, the whole genome microarray was used to identify differentially expressed genes associated with the putative interaction between biofilm formation and PrfA in L. monocytogenes. Comparative transcriptome analyses indicated over 21.9% of the L. monocytogenes EGDe genes (627 out of 2857 predicted) were altered in their expression in biofilm cells relative to planktonic cell populations. These genes were classed into different functional categories which cover most of the biochemical functions encountered in bacterial cells, especially involved in ion transport, DNA repair, and cell wall biosynthesis based on significant enrichment of GO terms. Among them, 185 genes were identified to be associated with PrfA and biofilm formation by comparison of the whole gene expression profiles of L. monocytogenes EGDe and its ΔprfA mutant. The expression tendency of these PrfA-associated and biofilm-specific genes were mainly opposite in ΔprfA biofilm, and most of them are involved in phage-related function, membrane bioenergetics, and cell wall. Our results indicated that L. monocytogenes biofilm formation is probably controlled by the complex regulation network involved variable genes required for the different biological pathways. This regulatory network is modified in the prfA deletion mutant in order to maintain its stable biofilm lifestyle. Gene expression of planktonic cells and biofilm cells in Listeria monocytogenes EGDe and prfA isogenic deletion strain EGDeΔprfA with cultivated in MEM and BHI for 48 hours, were mesasued using Agilent Listeria monocytogenes customized whole-genome microarray 8x15 array. Three replicates.
Project description:The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and, in part, immunocompetent patients. Like bacteria or yeast, A. fumigatus can grow in multicellular communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic and biofilm-grown A. fumigatus mycelium after 24h and 48h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes encoding hydrophobins and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by RT-PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may play also a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma. Comparison of biofilm and submers cultures at 24h and 48h after induction.
Project description:Previously, we performed DNA array-based transcriptomic analysis of Clostridium acetobutylicum biofilm adsorbed onto fibrous matrix in batch fermentation. Here, to further shed light on the transcriptomic modulation of maturing Clostridium acetobutylicum biofilm, we performed the DNA array-based transcriptomic analysis in repeated-batch fermentation. Significant time course changes in expression levels were observed for the genes involved in amino acid metabolism, oligopeptide ABC transporter, nitrogen fixation, and various other processes. Repeated-batch fermentation was carried out in 2-L stainless steel columns packed with 40 g of cotton towel （cut into pieces，approximately 3 cm × 5 cm) containing 1.5 L of P2 medium. Medium circulation rate was maintained at 35 mL/min via a peristaltic pump and the temperature was controlled at 37°C. Fermentation broth was replaced with fresh P2 medium every 12 h. Samples were withdrawn at 6 h after the medium replacement at predetermined interval, except for the last 3 samples. The last 3 samples were withdrawn at 12 h, 15 h, and 17 h after the medium replacement, respectively, to study the transcriptomic response to the adverse condition at the end of fermentation. A total of 8 samples were withdrawn over a period of 7 days, and time course gene expression profiles were studied.