Jan Kolek

Changes in Membrane Plasmalogens of Clostridium pasteurianum during Butanol Fermentation as Determined by Lipidomic Analysis

Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.

Complete genome sequence of Clostridium pasteurianum NRRL B-598, a non-type strain producing butanol.

The strain Clostridium pasteurianum NRRL B-598 is non-type, oxygen tolerant, spore-forming, mesophilic and heterofermentative strain with high hydrogen production and ability of acetone-butanol fermentation (ethanol production being negligible). Here, we present the annotated complete genome sequence of this bacterium, replacing the previous draft genome assembly. The genome consisting of a single circular 6,186,879 bp chromosome with no plasmid was determined using PacBio RSII and Roche 454 sequencing.

Draft Genome Sequence of Clostridium pasteurianum NRRL B-598, a Potential Butanol or Hydrogen Producer

ABSTRACT We present a draft genome sequence of Clostridium pasteurianum NRRL B-598. This strain ferments saccharides by two-stage acetone-butanol (AB) fermentation, is oxygen tolerant, and has high hydrogen yields.

Evaluation of viability, metabolic activity and spore quantity in clostridial cultures during ABE fermentation

Flow cytometry, in combination with fluorescent staining, was used to evaluate population heterogeneity in acetone-butanol-ethanol fermentation that was carried out with type strain Clostridium beijerinckii NCIMB 8052 and non-type C. pasteurianum NRRL B-598. A combination of propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), PI plus Syto-9 and bis-oxonol (BOX) alone were employed to distinguish between active and damaged cells together with simultaneous detection of spores. These strategies provided valuable information on the physiological state of clostridia. CFDA and PI staining gave the best separation of four distinct subpopulations of enzymatically active cells, doubly stained cells, damaged cells and spores. Proportional representation of cells in particular sub-regions correlated with growth characteristics, fermentation parameters such as substrate consumption and product formation in both species under different cultivation conditions.

Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598

The complete genome sequence of non-type strain Clostridium pasteurianum NRRL B-598 was introduced last year; it is an oxygen tolerant, spore-forming, mesophilic heterofermentative bacterium with high hydrogen production and acetone-butanol fermentation ability. The basic genome statistics have shown its similarity to C. beijerinckii rather than the C. pasteurianum species. Here, we present a comparative analysis of the strain with several other complete clostridial genome sequences. Besides a 16S rRNA gene sequence comparison, digital DNA-DNA hybridization (dDDH) and phylogenomic analysis confirmed an inaccuracy of the taxonomic status of strain Clostridium pasteurianum NRRL B-598. Therefore, we suggest its reclassification to be Clostridium beijerinckii NRRL B-598. This is a specific strain and is not identical to other C. beijerinckii strains. This misclassification explains its unexpected behavior, different from other C. pasteurianum strains; it also permits better understanding of the bacterium for a future genetic manipulation that might increase its biofuel production potential.

Comparison of expression of key sporulation, solventogenic and acetogenic genes in C-beijerinckii NRRL B-598 and its mutant strain overexpressing spo0A

The production of acetone, butanol and ethanol by fermentation of renewable biomass has potential to become a valuable industrial process. Mechanisms of solvent production and sporulation involve some common regulators in some ABE-producing clostridia, although details of the links between the pathways are not clear. In this study, we compare a wild-type (WT) Clostridium beijerinckii NRRL B-598 with its mutant strain OESpo0A, in which the gene encoding Spo0A, an important regulator of both sporulation and solventogenesis, is overexpressed in terms of solvent and acid production. We also compare morphologies during growth on two different media: TYA broth, where the WT culture sporulates, and RCM, where the WT culture does not. In addition, RT-qPCR-based analysis of expression profiles of spo0A, spoIIE, sigG, spoVD, ald and buk1 genes involved in sporulation or solvent production in these strains, were compared. The OESpo0A mutant did not produce spores and butanol titre was lower compared to the WT, but increased amounts of butyric acid and ethanol were produced. The gene spo0A had high levels of expression in the WT under non-sporulating culture conditions while other selected genes for sporulation factors were downregulated significantly. Similar observations were obtained for OESpo0A where spo0A overexpression and downregulation of other sporulation genes were demonstrated. Higher expression of spo0A led to higher expression of buk1 and ald, which could confirm the role of spo0A in activation of the solventogenic pathway, although solvent production was not affected significantly in the WT and was weakened in the OESpo0A mutant.

Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq

BackgroundThinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data.ResultsIn this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation.ConclusionsWe provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions

BackgroundBiobutanol production by clostridia via the acetone–butanol–ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol.ResultsDual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained—viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation.ConclusionWe have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

Isolation of new cellulolytic clostridia from nature

K pneumoniae carbapenemase – KPC producing bacteria are defined as a group of Gram-negative bacilli that is highly resistant to drugs. They cause lethal infections and illnesses. The main aim of the current study is to explore and confirm the occurrence of carbapenemase producing Klebsiella pneumonia in the ICU different compartments of an Egyptian hospital in Cairo. The study will focus in particular on the molecular class A KPC and the molecular class B MBL specifically the imipenem resistant phenotype carbapenemase (IMP) and Verona integrin-encoded metallo-beta-lactamase (VIM) producers. Isolates were collected from several compartments of the Intensive Care Unit in a private hospital in Cairo, Egypt. The screening criteria of carbapenemase producing bacteria were followed by the investigator in order to record the antimicrobial resistance patterns of all isolates. Furthermore, the modified Hodge test (MHT) was used as the instrument for detecting the carbapenemase producing isolates. Phenotypic detection of KPCs and MBLs was confirmed by the detection of blaKPC, blaIMP and blaVIM. It was also combined with disc tests in MHT positive isolates. At the end of the study, the investigators determined the compartment/s responsible for the spreading of CRKP as well as the type of the isolates occurred and how to avoid spreading and how to control it. It is worth noting that this study is considered the first report on the emergence of IMP and VIM producing Klebsiella pneumonia in the ICU compartments in Egypt. We concluded that infection control department policies in each hospital should be reinforced to evade the blowout of these bacteria in our hospitals. Also, this study should be repeated in other hospitals (especially the public hospitals) to assess the level of the problem.Background: Enterococcus faecalis normal intestinal flora of humans and one of the causes of nosocomial infections that cause urinary tract infections, endocarditis and menangeditise the ability to form biofilm on surfaces such as catheters, venous catheters artificial heart valves and ocular lenses, the ESP and cylA factors .The purpose of this study was to evaluate the ability of the bacteria in the biofilm formation and detection of virulence factors in clinical isolates of enterococci surface protein and cytolysin and Study of cyla and esp gene expression in Enterococcus faecalis culture in microfluidic conditionsH pylori infection is one of the most common infectious diseases on the globe and is associated with increased risk of human active chronic gastritis, peptic and duodenal ulcer and gastric cancer development. In order to investigate the current sero-prevalence of H. pylori infection in the Northwest part of Iran, specific ELISA for the existence of IgG antibodies against H. pylori was performed on a representative sample size of 21429 (69.02% F, 29.98% M) sera who were referred to the Central Laboratory of Tabriz in three years (2012 2015). Statistical analysis was conducted using SPSS v15.0 and Epi info v3.2.2 softwares. Results were categorized into nine groups according to the age. The overall sero-prevalence of H. pylori infection was 63.93% (63.77% F, 64.24% M), with no statistical difference between genders. Sero-positivity rates increased progressively with age but decreased above the age of 71. The results indicated that H. Pylori infection prevalence rate in the Northwest of Iran is higher than in developed countries, which may contribute to the high incident rate of gastric cancer in this area. Crowded living condition and low socio-economic status contribute to the difference of infection prevalence. Therefore, further research on H. pylori in Iran is vital to develop proactive and preventive mechanisms for gastric cancer.C is very common substance in the nature occurring mainly in cell walls of the plants. It is compact polymer consisting from glucose subunits. Due to its composition cellulose is potentially useful as a source of carbon and energy for bacterial fermentation. Unfortunately, lignocellulosic complex is generally very rigid and only a few microorganisms are able of its direct decomposition to the fermentable monosaccharides (predominantly glucose). Known, possible indirect approaches for the lignocellulosic substrate utilization are mainly their enzymatic hydrolysis to fermentable sugars or their gasification into the synthetic gas, also called syngas. The second option is to use cellulolytic microorganisms for direct utilization of cellulose to valuable product (so-called consolidated bio-processing). Unfortunately, all of these three main ways of lignocellulosic utilization have many drawbacks like too high price of process and very low product yields. Here we described system applicable for fast and specific isolation of cellulolytic mesophilic clostridium bacteria. Cellulolytic bacteria are relatively poor-described group of anaerobic bacteria which have a potential in biotechnology. For example, some of them are able to produce ethanol and some strains are secondary metabolites producers.Puccinia triticina, the causative of leaf rust, is a considerable pathogen in wheat which results in substantial amount of losses by decreasing the yield in almost all wheat growing areas of the world. Deployment of rust resistance genes into the cultivar is being used to provide resistance against the locally prevalent pathogen races as an economical, enduring and eco-friendly measure [1]. Diversity for resistance to leaf rust is available in the germplasm of related wheat genera and there are many affirmative reports which assure the effectiveness of genes originating from wild relatives of the cultivated wheat in conferring long lasting rust resistance [2]. So far more than 60 Lr genes have been identified in various wheat backgrounds [3]. Lr24 is one such resistance gene transferred into bread wheat from Agropyron elongatum which confers resistance right from the seedling stage all through the life of the plant (seedling resistance). Lr24 is being used in major wheat breeding and pyramiding programmes as a means to provide resistance to otherwise susceptible cultivars [4,5]. However, many of the seedling resistance genes when incorporated singly tend to become ineffective due the constantly evolving physiological races of the pathogen. To suppress such reviving pathogenesis, an approach to stack more than one gene into the same background has been suggested and is pursued in most of the rust resistance initiatives [6]. In this study, we have employed one such F2 population which segregates for two Lr genes. One of them is Lr24 and the other is a hypersensitive recessive adult plant resistance (APR) gene, Lr48 which confers resistance to the plant only from the time the plant reaches its booting stage and in a way decreases the selection pressure on the pathogen thus inhibiting the development of new races [2]. Differentiating the phenotypic resistance reaction of two discrete Lr genes existing in the same cultivar is practically impossible in the absence of individual Lr gene specific pathogen virulences. In such cases, the presence of exclusive DNA based markers which act as indices for each Lr gene will be valuable. Molecular markers are utilized on a huge scale to reduce cumbersomeness and enable rapid detection of specific Lr genes. Codominant molecular markers are useful in breeding programmes as only they are efficient in differentiating the heterozygous and homozygous status in plants exhibiting resistance to the pathogen infection since only the latter are significant to forward for further generations. To enable the early selection of homozygosity at the adult plant rust resistance locus, two RAPD markers S3450 and S336775 have been utilized as a co-dominant marker system [7]. The SSR marker polymorphic for Lr24 identified in our lab will be useful in wheat breeding populations and can help in fixing the genes by the F2 population level itself without any further investment till F5/F6 generations.Celiac disease (CD) is an autoimmune disorder of the small intestine that affects in genetically predisposed subjects of all age groups (from middle infancy onward CD is due to a reaction to gliadin, a prolamin (gluten protein), and similar proteins found in other crops Vis barley and rye [1]. CD symptoms include chronic diarrhea, fatigue, weight loss, weakness and classic steatorrhea. Abdominal pain, nausea and vomiting are uncommon. Most symptoms are related to malabsorption (extensive lesion in the proximal duodenum to the distal ileum produces resulting in severe malabsorption), pedal edema, protein malabsorption, easy bruising and vitamin K deficiency due to malabsorption.N Sequencing (NGS) technologies have developed progressively in microbial genomic research and clinical applications. Also, genetic fingerprinting has been used in molecular epidemiologic studies. Therefore, it is essential that a connection or a link is established between them for discovering DNA marker in whole genome for using in molecular epidemiology and bacterial diagnosis. Bioinformatics and comparative genome analysis tools lead to further and deeper understanding of genomic variation in the bacterial species. Bacterial comparative genome analyses can be used for evolutionary processes, structure and function annotations, and importantly in unique DNA marker extraction. These unique probes are suitable for high-throughput diagnostic methods such as micro-array. Finally, setting up a procedure for comparative analysis will be useful for a wide range of microbial researches and clinical applications.R B is produced by an actinobacterium Amycolatopsis mediterranei S699. Semi synthetic derivatives of rifamycin B (rifampicin or rifampin, rifabutin, rifaximine, rifapentine and rifalazil) are used for the treatment of tuberculosis (TB), leprosy and AIDS related mycobacterial infections. But none of these is effective against Multi Drug Resistant-TB (MDR-TB). This raised the need to develop novel rifamycin B analogs and corresponding derivatives to combat MDR infections. However, due to the chemical complexity of the rifamycin B further chemical modifications are not possible. An alternative approach to modify the rifamycin B backbone is combinatorial biosynthesis by manipulation of the Rifamycin Polyketide Synthase (rifPKS) gene cluster in the producer strain itself. Thus acyltransferase (AT) domain of the sixth module (AT6) of rifPKS (which adds propionate unit to the growing polyketide chain) was swapped with AT domain of the second module (AT2) of rapamycin PKS (rapPKS) (adds acetate unit) in A. mediterranei S699. The resulting mutant produced 24-desmethylrifamycin B lacking pendant methyl group at C-33 of the rifamycin skeletal structure. It was confirmed using NMR and LC-MS studies. The analog was further converted to 24-desmethylrifamycin S & 24-desmethylrifampicin that showed better antibacterial activity than rifampicin against MDR strains of M. tuberculosis. Based on this proof of concept further manipulations of other domains (AT5, AT7, AT8, DH9 & DH10) are being carried out for production of more rifamycin analogs for biological and pharmaceutical applications.