Expression of a bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 glycosyl hydrolase gene promoters

Expression vectors were constructed for Trichoderma reesei using the promoters, secretion signals and the modular structure of the efficiently expressed and secreted cellulase enzymes EGL2 (Cel5A) and CBH2 (Cel6A) as a prelude to establishing a platform where a gene of interest can be expressed under several promoters simultaneously. The designs featured (i) EGL2sigpro (egl2 promoter and secretion signal), (ii) EGL2cbmlin (egl2 promoter, secretion signal, EGL2 cellulose binding module and linker), (iii) CBH2sigpro (cbh2 promoter and secretion signal) and (iv) CBH2cbmlin (cbh2 promoter, secretion signal, CBH2 cellulose binding module and linker). Recombinant vectors were introduced individually into the high protein-secreting T. reesei RUT-C30 strain to generate single-promoter transformants expressing the Dictyoglomus thermophilum xynB gene that encodes a thermophilic xylanase enzyme (XynB). Ten transformants producing XynB representing each of the four different types of vectors were selected for further testing and the highest XynB production was achieved from a transformant containing 1–2 copies of the EGL2cbmlin vector. Best xylanase producers did not show any particular pattern in terms of the number of gene copies and their mode of integration into the chromosomal DNA. Transformants generated with the cbmlin-type vectors produced multiple forms of XynB which were decorated with various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in the glycosylation patterns of T. reesei.

Using the Gini coefficient with BIOLOG substrate utilisation data to provide an alternative quantitative measure for comparing bacterial soil communities

A measure quantifying unequal use of carbon sources, the Gini coefficient (G), has been developed to allow comparisons of the observed functional diversity of bacterial soil communities. This approach was applied to the analysis of substrate utilisation data obtained from using BIOLOG microtiter plates in a study which compared decomposition processes in two contrasting plant substrates in two different soils. The relevance of applying the Gini coefficient as a measure of observed functional diversity, for soil bacterial communities is evaluated against the Shannon index (H) and average well colour development (AWCD), a measure of the total microbial activity. Correlation analysis and analysis of variance of the experimental data show that the Gini coefficient, the Shannon index and AWCD provided similar information when used in isolation. However, analyses based on the Gini coefficient and the Shannon index, when total activity on the microtiter plates was maintained constant (i.e. AWCD as a covariate), indicate that additional information about the distribution of carbon sources being utilised can be obtained. We demonstrate that the Lorenz curve and its measure of inequality, the Gini coefficient, provides not only comparable information to AWCD and the Shannon index but when used together with AWCD encompasses measures of total microbial activity and absorbance inequality across all the carbon sources. This information is especially relevant for comparing the observed functional diversity of soil microbial communities.

Atmospheric pressure plasmas : infection control and bacterial responses

Cold atmospheric pressure plasma (APP) is a recent, cutting-edge antimicrobial treatment. It has the potential to be used as an alternative to traditional treatments such as antibiotics and as a promoter of wound healing, making it a promising tool in a range of biomedical applications with particular importance for combating infections. A number of studies show very promising results for APP-mediated killing of bacteria, including removal of biofilms of pathogenic bacteria such as Pseudomonas aeruginosa. However, the mode of action of APP and the resulting bacterial response are not fully understood. Use of a variety of different plasma-generating devices, different types of plasma gases and different treatment modes makes it challenging to show reproducibility and transferability of results. This review considers some important studies in which APP was used as an antibacterial agent, and specifically those that elucidate its mode of action, with the aim of identifying common bacterial responses to APP exposure. The review has a particular emphasis on mechanisms of interactions of bacterial biofilms with APP.

Pulmonary innate immunity in children with protracted bacterial bronchitis

Objective To determine bronchoalveolar lavage (BAL) levels of 3 innate immunity components (human alpha-defensin-2 [hBD2], mannose-binding lectin [MBL], and surfactant protein-A [SP-A], the relationship with airway neutrophilia and infection, and cytokine production of stimulated BAL cells in children with current protracted bacterial bronchitis (PBB), children with resolved PBB (PBB well), and controls. Study design BAL of 102 children (mean age 2.8 years) fulfilling predefined criteria of current PBB (n=61), PBB well (n=20), and controls (n=21) was cultured (quantitative bacteriology) and viruses examined by polymerase chain reaction. hBD2, MBL, and SP-A were measured, and cytokine production of lipopolysaccharide-stimulated BAL cells were determined. Results Median hBD2 and MBL levels were significantly higher in the current PBB group (hBD2 = 164.4, IQR 0-435.5pg/mL; MBL = 1.7, 0.4-4ng/mL) than in the PBB well group (hBD2 = 0, IQR 0-85.2; MBL = 0.6, IQR 0.03-2.9) and controls (hBD2 = 3.6, IQR 0-126; MBL = 0.4, IQR 0.02-79). hBD2 was significantly higher in children with airway infection (n = 54; median 76.9, IQR 0-397.3) compared with those without (n = 48; 0, IQR 0-236.3), P=0.04. SP-A levels and cytokine production of stimulated BAL cells were similar between groups. Conclusion In children's airways, hBD2, but not MBL and SP-A, relates to inflammation and infection. In children with PBB, mechanisms involving airway hBD2 and MBL are augmented. These pulmonary innate immunity components and the ability of BAL cells to respond to stimuli are unlikely to be deficient.

The antigen 43 structure reveals a molecular velcro-like mechanism of autotransporter-mediated bacterial clumping

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure–function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular “Velcro-like” mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.

Discovery of an archetypal protein transport system in bacterial outer membranes

Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

Structure and function of DsbA, a key bacterial oxidative folding catalyst

Since its discovery in 1991, the bacterial periplasmic oxidative folding catalyst DsbA has been the focus of intense research. Early studies addressed why it is so oxidizing and how it is maintained in its less stable oxidized state. The crystal structure of Escherichia coli DsbA (EcDsbA) revealed that the oxidizing periplasmic enzyme is a distant evolutionary cousin of the reducing cytoplasmic enzyme thioredoxin. Recent significant developments have deepened our understanding of DsbA function, mechanism, and interactions: the structure of the partner membrane protein EcDsbB, including its complex with EcDsbA, proved a landmark in the field. Studies of DsbA machineries from bacteria other than E. coli K-12 have highlighted dramatic differences from the model organism, including a striking divergence in redox parameters and surface features. Several DsbA structures have provided the first clues to its interaction with substrates, and finally, evidence for a central role of DsbA in bacterial virulence has been demonstrated in a range of organisms. Here, we review current knowledge on DsbA, a bacterial periplasmic protein that introduces disulfide bonds into diverse substrate proteins and which may one day be the target of a new class of anti-virulence drugs to treat bacterial infection. Antioxid. Redox Signal. 14, 1729–1760.

DSB proteins and bacterial pathogenicity

If DNA is the information of life, then proteins are the machines of life — but they must be assembled and correctly folded to function. A key step in the protein-folding pathway is the introduction of disulphide bonds between cysteine residues in a process called oxidative protein folding. Many bacteria use an oxidative protein-folding machinery to assemble proteins that are essential for cell integrity and to produce virulence factors. Although our current knowledge of this machinery stems largely from Escherichia coli K-12, this view must now be adjusted to encompass the wider range of disulphide catalytic systems present in bacteria.

Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione s-transferase

Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria [GST 5-5(+)] expressed the protein and produced mutations when ethylene or methylene dihalides were added [Thier, R., Taylor, J. B., Pemble, S. E., Ketterer, B., Persmark, M., Humphreys, W. G., and Guengerich, F. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580]. After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4′-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation [GST 5-5(-)], suggesting a protective role of GST 5-5. However, mutations were considerably enhanced in the GST 5-5(+) strain [as compared to GST 5-5(-)] when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added. The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1-butene, and 1,4-dibromobutane. The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation. We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (±)-1,4-dibromo-2,3-dihydroxybutane. The possibility was considered that a 5-membered thialonium ion may be involved in the mutagenicity. Model thialonium compounds were rather stable to hydrolysis in aqueous solution at pH 7.4 and slowly alkylated 4-(4-nitrobenzyl)pyridine. The presence of a hydroxyl group β to the sulfur did not enhance reactivity. Mechanisms involving episulfonium ions are considered more likely. Potential oxidation products of the toxic pesticide 1,2-dibromo-3-chloropropane (DBCP) were also considered in this system. DBCP itself gave rather similar results in the two strains. Others have reported that oxidation of DBCP is required for mutagenicity, along with GST-catalyzed GSH conjugation [Simula, T. P., Glancey, M. J., Söderlund, E. J., Dybing, E., and Wolf, C. R. (1993) Carcinogenesis 14, 2303-2307]. The putative oxidation product 1,2-dibromopropional did not show a difference between the two strains. However, 1,3-dichloroacetone, a model for the putative oxidation product 1-bromo-3-chloroacetone, was considerably more mutagenic in the GST 5-5(+) strain.

Bacterial adherence and biofilm formation on medical implants : a review

Biofilms are a complex group of microbial cells that adhere to the exopolysaccharide matrix present on the surface of medical devices. Biofilm-associated infections in the medical devices pose a serious problem to the public health and adversely affect the function of the device. Medical implants used in oral and orthopedic surgery are fabricated using alloys such as stainless steel and titanium. The biological behavior, such as osseointegration and its antibacterial activity, essentially depends on both the chemical composition and the morphology of the surface of the device. Surface treatment of medical implants by various physical and chemical techniques are attempted in order to improve their surface properties so as to facilitate bio-integration and prevent bacterial adhesion. The potential source of infection of the surrounding tissue and antimicrobial strategies are from bacteria adherent to or in a biofilm on the implant which should prevent both biofilm formation and tissue colonization. This article provides an overview of bacterial biofilm formation and methods adopted for the inhibition of bacterial adhesion on medical implants

Conformational Spread as a Mechanism for Cooperativity in the Bacterial Flagellar Switch

The bacterial flagellar switch that controls the direction of flagellar rotation during Chemotaxis has a highly cooperative response. This has previously been understood in terms of the classic two-state, concerted model of allosteric regulation. Here, we used high-resolution optical microscopy to observe switching of single motors and uncover the stochastic multistate nature of the switch. Our observations are in detailed quantitative agreement with a recent general model of allosteric cooperativity that exhibits conformational spread-the stochastic growth and shrinkage of domains of adjacent subunits sharing a particular conformational state. We expect that conformational spread will be important in explaining cooperativity in other large signaling complexes.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

Growth medium selection and its economic impact on plasmid DNA production

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 μg in 2002 to 500-5000 μg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5α harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 μg/ml and 6.3 μg/mg dry cell mass respectively) and the lowest was for LB (2.8 μg/ml and 3.3 μg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 μg/ml and 11.2 μg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.

Rapid-response vaccines - does DNA offer a solution?

In responding to future influenza pandemics and other infectious agents, plasmid DNA overcomes many of the limitations of conventional vaccine production approaches.