Microbiologically influenced corrosion of titanium by Desulfovibrio vulgaris biofilm under organic carbon starvation.

Desulfovibrio vulgaris biofilm was pre-grown on Ti coupons for 7 d and then the biofilm covered coupons were incubated again with fresh culture media with 10 % (reduced) and 100 % (normal) carbon source levels, respectively. After the pre-growth, sessile D. vulgaris cell count reached 107 cells/cm2. The sessile cell counts were 2 × 107 and 4.2 × 107 cells/cm2 for 10 % and 100 % carbon sources, respectively after the subsequent 7 d starvation test. The maximum pit depth after the 7 d pre-growth was 4.7 µm. After the additional 7 d of the starvation test, the maximum pit depth increased to 5.1 µm for 100 % carbon source vs 6.2 µm for 10 % carbon source. Corrosion current density (icorr) from potentiodynamic polarization data at the end of the 7 d starvation test for 10 % carbon source was more than 3 times of that for 100 % carbon source, despite a reduced sessile cell count with 10 % carbon source. The polarization resistance (Rp) started to decrease within minutes after 20 ppm (w/w) riboflavin (electron mediator) injection. The carbon starvation data and riboflavin corrosion acceleration data both suggested that D. vulgaris utilized elemental Ti as an electron source to replace carbon source as the electron donor during carbon source starvation.

Mechanical property degradation of X80 pipeline steel due to microbiologically influenced corrosion caused by Desulfovibrio vulgaris.

Apart from pinhole leaks, MIC (microbiologically influenced corrosion) can also cause catastrophic failures such as pipe ruptures and support beam collapses due to mechanical property degradation or stress corrosion cracking. In this work, X80 pipeline steel dogbone coupons and square coupons were immersed in 150 ml broths containing Desulfovibrio vulgaris, a common corrosive sulfate reducing bacterium (SRB), for up to 14 days. The headspace volumes in the anaerobic bottles were increased from 150 ml to 200 ml and 300 ml to increase MIC severity. After 14 days of SRB incubation in ATCC 1249 culture medium with X80 coupons at 37°C, the sessile cell counts were 6.5 × 107 cells cm-2 for 150 ml, 2.3 × 108 cells cm-2 for 200 ml and 1.4 × 109 cells cm-2 for 300 ml headspace volumes, respectively owing to reduced H2S cytotoxicity in the broth with a larger headspace because it allowed more biogenic H2S to escape from the broth. Weight losses were 1.7 mg cm-2, 1.9 mg cm-2 and 2.3 mg cm-2 for 150 ml, 200 ml and 300 ml headspace volumes, respectively. The corresponding pit depths were 2.6 µm, 4.2 µm and 6.2 µm for 150 ml, 200 ml and 300 ml headspace volumes, respectively. Electrochemical impedance spectroscopy (EIS), linear polarization resistance (LPR) and potentiodynamic polarization results corroborated the increasing weight loss and pitting data trends as a result of increased headspace. Tensile testing of dogbone coupons after the 14-day SRB immersion test indicated that more severe MIC pitting led to a higher ultimate strain loss by up to 23% (300 ml headspace) compared to the abiotic control, while the ultimate strength losses for all headspace volumes were quite small (3% and lower).

A Protective Role for Glucagon-like Peptide-2 in Heat-stable Enterotoxin b (STb)-Induced L-Cell Toxicity.

Enterotoxigenic Escherichia coli (ETEC)-derived purified heat-stable enterotoxin b (STb) is responsible for secretory diarrhea in livestock and humans. STb disrupts intestinal fluid homeostasis, epithelial barrier function, and promotes cell death. Glucagon-like peptide-2 (GLP-2) is a potent intestinotrophic hormone secreted by enteroendocrine L cells. GLP-2 enhances crypt cell proliferation, epithelial barrier function, and inhibits enterocyte apoptosis. Whether STb can affect GLP-2 producing L cells remains to be elucidated. First, secreted-His-labeled STb from transformed E coli was collected and purified. When incubated with L-cell models (GLUTag, NCI-H716, and secretin tumor cell line [STC-1]), fluorescent immunocytochemistry revealed STb was internalized and was differentially localized in the cytoplasm and nucleus. Cell viability experiments with neutral red and resazurin revealed that STb was toxic in all but the GLUTag cells. STb stimulated 2-hour GLP-2 secretion in all cell models. Interestingly, GLUTag cells produced the highest amount of GLP-2 when treated with STb, demonstrating an inverse relationship in GLP-2 secretion and cell toxicity. To demonstrate a protective role for GLP-2, GLUTag-conditioned media (rich in GLP-2) blocked STb toxicity in STC-1 cells. Confirming a protective role of GLP-2, teduglutide was able to improve cell viability in cells treated with H2O2. In conclusion, STb interacts with the L cell, stimulates secretion, and may induce toxicity if GLP-2 is not produced at high levels. GLP-2 or receptor agonists have the ability to improve cell viability in response to toxins. These results suggest that GLP-2 secretion can play a protective role during STb intoxication. This work supports future investigation into the use of GLP-2 therapies in enterotoxigenic-related diseases.

Food-grade D-limonene enhanced a green biocide in the mitigation of carbon steel biocorrosion by a mixed-culture biofilm consortium.

Microbiologically influenced corrosion (MIC), or microbial biocorrosion, is caused directly by microbial metabolic activities/products or induced by microbial biofilm's damage of a protective film that exposes a solid surface to a pre-existing corrosive environment. MIC causes billions of dollars of losses in various industrial processes, especially in oil and gas and water utilities. The mitigation of problematic industrial microbes typically relies on biocides whose discharges can cause environmental problems. Thus, more effective biocide applications are desired to minimize environmental impact. D-Limonene, a citrus peel oil, generally regarded as safe (GRAS), was used to enhance the popular biodegradable tetrakis hydroxymethyl phosphonium sulfate (THPS) biocide. An oilfield mixed-culture biofilm was grown anaerobically in enriched artificial seawater containing C1018 carbon steel coupons for 7 days at 37 °C. One hundred ppm (w/w) D-limonene reduced general heterotrophic bacteria (GHB) and acid-producing bacteria (APB) effectively, leading to 5.4-log and 6.0-log reductions in sessile GHB and APB cell counts, respectively, compared to no treatment control. The combination of 100 ppm D-limonene + 100 ppm THPS achieved extra 1.0-log SRB, 0.6-log GHB and 0.5-log APB reductions in sessile cell counts, which led to extra 58% reduction in microbial corrosion mass loss (1.2 vs. 0.5 mg/cm2) and extra 30% reductions in maximum pit depth (11.5 vs. 8.1 µm), compared to 100 ppm THPS-only treatment. Linear polarization resistance and potentiodynamic polarization (PDP) corrosion data supported mass loss and pitting data. Mixed-culture biofilms on carbon steel coupons after 7 day incubation at 37 °C showing enhanced biocide treatment outcome using D-limonene + THPS: A no treatment, B 100 ppm D-limonene, C 100 ppm THPS, D 100 ppm D-limonene + 100 ppm THPS.

Mitigation of carbon steel biocorrosion using a green biocide enhanced by a nature-mimicking anti-biofilm peptide in a flow loop.

Biocorrosion, also called microbiologically influenced corrosion (MIC), is a common operational threat to many industrial processes. It threatens carbon steel, stainless steel and many other metals. In the bioprocessing industry, reactor vessels in biomass processing and bioleaching are prone to MIC. MIC is caused by biofilms. The formation and morphology of biofilms can be impacted by fluid flow. Fluid velocity affects biocide distribution and MIC. Thus, assessing the efficacy of a biocide for the mitigation of MIC under flow condition is desired before a field trial. In this work, a benchtop closed flow loop bioreactor design was used to investigate the biocide mitigation of MIC of C1018 carbon steel at 25 °C for 7 days using enriched artificial seawater. An oilfield biofilm consortium was analyzed using metagenomics. The biofilm consortium was grown anaerobically in the flow loop which had a holding vessel for the culture medium and a chamber to hold C1018 carbon steel coupons. Peptide A (codename) was a chemically synthesized cyclic 14-mer (cys-ser-val-pro-tyr-asp-tyr-asn-trp-tyr-ser-asn-trp-cys) with its core 12-mer sequence originated from a biofilm dispersing protein secreted by a sea anemone which possesses a biofilm-free exterior. It was used as a biocide enhancer. The combination of 50 ppm (w/w) THPS (tetrakis hydroxymethyl phosphonium sulfate) biocide + 100 nM (180 ppb by mass) Peptide A resulted in extra 1-log reduction in the sulfate reducing bacteria (SRB) sessile cell count and the acid producing bacteria (APB) sessile cell count compared to 50 ppm THPS alone treatment. Furthermore, with the enhancement of 100 nM Peptide A, extra 44% reduction in weight loss and 36% abatement in corrosion pit depth were achieved compared to 50 ppm THPS alone treatment.

Efficacy of glutaraldehyde enhancement by D-limonene in the mitigation of biocorrosion of carbon steel by an oilfield biofilm consortium.

Microbiologically influenced corrosion (MIC) is one of the major corrosion threats in the oil and gas industry. It is caused by environmental biofilms. Glutaraldehyde is a popular green biocide for mitigating biofilms and MIC. This work investigated the efficacy of glutaraldehyde enhancement by food-grade green chemical D-limonene in the biofilm prevention and MIC mitigation using a mixed-culture oilfield biofilm consortium. After 7 days of incubation at 37 °C in enriched artificial seawater in 125 mL anaerobic vials, the 100 ppm (w/w) glutaraldehyde + 200 ppm D-limonene combination treatment reduced the sessile cell counts on C1018 carbon steel coupons by 2.1-log, 1.7-log, and 2.3-log for sulfate reducing bacteria, acid producing bacteria, and general heterotrophic bacteria, respectively in comparison with the untreated control. The treatment achieved 68% weight loss reduction and 78% pit depth reduction. The 100 ppm glutaraldehyde + 200 ppm D-limonene combination treatment was found more effective in biofilm prevention and MIC mitigation than glutaraldehyde and D-limonene used individually. Electrochemical tests corroborated weight loss and pit depth data trends.

Aggressive corrosion of carbon steel by Desulfovibrio ferrophilus IS5 biofilm was further accelerated by riboflavin.

EET (extracellular electron transfer) is behind MIC (microbiologically influenced corrosion) of carbon steel by SRB (sulfate reducing bacteria). This work evaluated 20 ppm (w/w) riboflavin (an electron mediator) acceleration of C1018 carbon steel MIC by Desulfovibrio ferrophilus IS5 in enriched artificial seawater (EASW) after 7-d incubation in anaerobic vials at 28 °C. Twenty ppm riboflavin did not significantly change cell growth or alter the corrosion product varieties, but it led to 52% increase in weight loss and 105% increase in pit depth, compared to the control without 20 ppm riboflavin. With 20 ppm riboflavin supplement in EASW, D. ferrophilus yielded weight loss-based corrosion rate of 1.57 mm/y (61.8 mpy), and pit depth growth rate of 2.88 mm/y (113 mpy), highest reported for short-term pure-strain SRB MIC of carbon steel. Electrochemical tests in 450 mL glass cells indicated that the biofilm responded rather quickly to the riboflavin injection (20 ppm in broth) to the culture medium. Polarization resistance (Rp) began to decrease within minutes after injection. Within 2 h, the riboflavin injection led to 31% decrease in Rp and 35% decrease in Rct + Rf from electrochemical impedance spectroscopy (EIS). The Tafel corrosion current density increased 63% 2 h after the injection.

Impact of the Escherichia coli Heat-Stable Enterotoxin b (STb) on Gut Health and Function.

Enterotoxigenic Escherichia coli (ETEC) produces the heat-stable enterotoxin b (STb), which is responsible for secretory diarrhea in humans and animals. This toxin is secreted within the intestinal lumen of animals and humans following ETEC colonization, becoming active on enterocytes and altering fluid homeostasis. Several studies have outlined the nature of this toxin and its effects on gut health and the integrity of the intestinal epithelium. This review summarizes the mechanisms of how STb alters the gastrointestinal tract. These include the manipulation of mucosal tight junction protein integrity, the formation of enterocyte cellular pores and toxin internalization and the stimulation of programmed cell death. We conclude with insights into the potential link between STb intoxication and altered gut hormone regulation, and downstream physiology.

AU richness within the 5' coding region of the Escherichia coli heat-stable enterotoxin b mRNA affects toxin secretion.

The majority of protein secretion in bacteria is mediated by the T2SS pathway. Substrates processed through this pathway are guided by the N-terminal signal sequence within the nascent polypeptide. Recent experimental evidence suggests that in similar secretory pathways, such as the T3SS, information in the 5' coding region of the mRNA affects secretion and may also participate in mRNA localization. The majority of studies on the effects of AU richness on translation have focussed on the 5' UTR in mRNAs. To look at the effects of AU richness within the coding region of mRNA on secretion, we have generated several silent mutations within the 5' coding region of the E. coli heat-stable enterotoxin b (STb). This toxin is a well studied T2SS substrate. The mutations were generated such that AU richness within the 5' coding region (corresponding to the N-terminal signal sequence) was gradually reduced. Reduction of AU richness within the first 15 codons resulted in reduced secretion of the toxin as the AU/GC ratio was reduced from 2.13 for the WT STb to 1.65 (S-I) and subsequently to 1.30 (S-II). This reduction did not correlate with mRNA accumulation and decreased stability of the transcripts could not account for the reduced secretion observed. Reduction of AU richness beyond the first 15 codons recovered secretion efficiency of the toxin (S-III). To validate the experimental approach, a positive control was used in which a mutation involving the insertion of a positive charge within the hydrophobic domain of the N-terminal signal sequence was constructed. As expected, this mutation abolished secretion of the toxin. In conclusion, reducing AU richness within the 5'coding region in the STb mRNA reduces toxin secretion but other factors, such as formation of hairpins, must also be taken into consideration. This will have implications for both homologous and heterologous expression of STb for biological studies and for toxin production.

The multicopper oxidase of Mycobacterium tuberculosis (MmcO) exhibits ferroxidase activity and scavenges reactive oxygen species in activated THP-1 cells.

The MmcO protein of Mycobacterium tuberculosis is a membrane-associated multicopper oxidase. Its natural substrate(s) and its role in pathogenesis are not well characterized. A recent report proposes that MmcO contributes to copper resistance in M. tuberculosis during infection. We have expressed and reconstituted the active enzyme from inclusion bodies in E. coli. MmcO exhibits maximal activity against the experimental substrate 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) or ABTS, at pH 4. The enzyme also exhibits ferroxidase activity at pH 4. Most notable was the finding that MmcO is able to scavenge the reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase enzyme system. This ROS scavenging activity of MmcO was also evident against ROS generated by THP-1 cells. We propose that MmcO protects M. tuberculosis during infection against ROS attack in addition to providing copper resistance to the pathogen.

Complete Genome Sequences of a Diverse Group of 13 Propionibacterium acnes Bacteriophages Isolated from Urban Raw Sewage.

We present complete genome sequences of 13 Propionibacterium acnes phages isolated from urban raw sewage. They belong to the family Siphoviridae, have genome sizes of 29,450.6 ± 256.5 nucleotides and G+C contents of 54.14% ± 0.22% and contain 42 to 45 coding DNA sequences (CDS). Genomic sequences of 9 of 13 phages were divergent by 6 to 10%, distinguishing them as species.

Proteomics of Desulfovibrio desulfuricans and X-ray absorption spectroscopy to investigate mercury methylation in the presence of selenium.

The effects of mercury added as Hg(2+) and selenium as selenite to cultures of the sulfate reducing bacterium Desulfovibrio desulfuricans were investigated under controlled laboratory conditions. There was no significant difference in the growth curves in comparison to control except in the 0.5 µM Hg-6.3 µM Se combined system in which Hg methylation was significantly reduced. A significant decrease in the production of methylmercury indicates a disruption of the methylation process due to the presence of the relatively high concentrations of Se in the system, suggesting a modification of the biological pathway. The results of detailed 2D gel electrophoresis in combination with mass spectrometry confirmed that the Hg methylation process should certainly be influenced when the protein Dde_1198 protein-glutamate O-methyltransferase was totally suppressed in a culture containing 0.5 µM Hg and 6.3 µM Se. Since this protein plays an important role in the methylation process, its suppression in the presence of Se brings a possible explanation for the antagonism between Se and Hg in natural systems. The experiment involving the determination of Hg and Se in membrane proteins separated by 1D gel thin-layer isoelectric focusing revealed that when both elements were present in a culture, the concentration of Hg in the separated proteins was significantly lower in comparison to those without added Se to the culture and vice versa. Finally, near-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure were used to corroborate the presence of a very inert solid HgSe in the cell membrane obtained from the culture containing 0.5 µM Hg and 6.3 µM Se. This confirms the protective effect of Se against Hg assimilation at the molecular level and reinforces the findings of our research group in numerous field and laboratory studies.

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR.

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity.

Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR.

Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology.

The role of immunostimulatory nucleic acids in septic shock.

Sepsis and its associated syndromes represent the systemic host response to severe infection and is manifested by varying degrees of hypotension, coagulopathy, and multiorgan dysfunction. Despite great efforts being made to understand this condition and designing therapies to treat sepsis, mortality rates are still high in septic patients. Characterization of the complex molecular signaling networks between the various components of host-pathogen interactions, highlights the difficulty in identifying a single driving force responsible for sepsis. Although triggering the inflammatory response is generally considered as protective against pathogenic threats, the interplay between the signaling pathways that are induced or suppressed during sepsis may harm the host. Numerous surveillance mechanisms have evolved to discriminate self from foreign agents and accordingly provoke an effective cellular response to target the pathogens. Nucleic acids are not only an essential genetic component, but sensing their molecular signature is also an important quality control mechanism which has evolved to maintain the integrity of the human genome. Evidence that has accumulated recently indicated that distinct pattern recognition receptors sense nucleic acids released from infectious organisms or from damaged host cells, resulting in the modulation of intracellular signalling cascades. Immunoreceptor-mediated detection of these nucleic acids induces antigen-specific immunity, secretion of proinflammatory cytokines and reactive oxygen/nitrogen species and thus are implicated in a range of diseases including septic shock.

Septic sera induces apoptosis and DNA fragmentation factor 40 activation in fibroblasts.

Sepsis, the systemic response to infection, is the leading cause of death in the intensive care units worldwide. Septic patients can succumb through the development of early refractory hypotension or late multiple organ dysfunction. Misregulation of apoptosis during sepsis may contribute to cellular dysfunction and multiple organ dysfunction. Utilizing a tissue culture model which mimics the human disease, we demonstrate that the addition of sera derived from septic patients induces apoptosis in human fibroblast cells. Addition of septic sera to 2fTGH cells induced apoptosis by activating caspase 8, caspase 3 and DNA fragmentation factor 40 (DFF 40). Interestingly, the addition of septic sera to cells which lack STAT1 (U3A cells) did not activate DFF 40. U3A cells were also shown to be resistant to septic serum induced apoptosis. These data suggest that DFF 40 mediated apoptosis plays a significant role in mediating sepsis induced cellular dysfunction.

Indicators from archaeal secretomes.

Just as in the Eukarya and the Bacteria, members of the Archaea need to export proteins beyond the cell membrane. This would be required to fulfill a variety of essential functions such as nutrient acquisition and biotransformations, maintenance of extracellular structures and more. Apart from the Eukarya and the Bacteria however, members of the Archaea share a number of unique characteristics. Does this uniqueness extend to the protein secretion system? It was the objective of this study to answer this question. To overcome the limited experimental information on secreted proteins in Archaea, this study was carried out by subjecting the available archaeal genomes, which represent halophiles, thermophiles, and extreme thermophiles, to bioinformatics analysis. Specifically, to examine the properties of the secretomes of the Archaea using the ExProt program. A total of 24 genomes were analyzed. Secretomes were found to fall in the range of 6% of total ORFs (Methanopyrus kandleri) to 19% (Halobacterium sp. NRC-1). Methanosarcina acetivorans has the highest fraction of lipoproteins (at 89) and the lowest (at 1) were members of the Thermoplasma, Pyrobaculum aerophilum, and Nanoarchaeum equitans. Based on the Tat consensus sequence, contribution of these secreted proteins to the secretomes were negligible, making up 8 proteins out of a total of 7105 predicted exported proteins. Amino acid composition, an attribute of signal peptides not used as a selection criteria by ExProt, of predicted archaeal signal peptides show that in the haloarchaea secretomes, the frequency of the amino acid Lys is much lower than that seen in bacterial signal peptides, but is compensated for by a higher frequency of Arg. It also showed that higher frequencies for Thr, Val, and Gly contribute to the hydrophobic character in haloarchaeal signal peptides, unlike bacterial signal peptides in which the hydrophobic character is dominated by Leu and Ile.

Bioinformatic comparison of bacterial secretomes.

The rapid increasing number of completed bacterial genomes provides a good opportunity to compare their proteomes. This study was undertaken to specifically compare and contrast their secretomes-the fraction of the proteome with predicted N-terminal signal sequences, both type I and type II. A total of 176 theoretical bacterial proteomes were examined using the ExProt program. Compared with the Gram-positives, the Gram-negative bacteria were found, on average, to contain a larger number of potential Sec-dependent sequences. In the Gram-negative bacteria but not in the others, there was a positive correlation between proteome size and secretome size, while there was no correlation between secretome size and pathogenicity. Within the Gram-negative bacteria, intracellular pathogens were found to have the smallest secretomes. However, the secretomes of certain bacteria did not fit into the observed pattern. Specifically, the secretome of Borrelia burgdoferi has an unusually large number of putative lipoproteins, and the signal peptides of mycoplasmas show closer sequence similarity to those of the Gram-negative bacteria. Our analysis also suggests that even for a theoretical minimal genome of 300 open reading frames, a fraction of this gene pool (up to a maximum of 20%) may code for proteins with Sec-dependent signal sequences.

Depleting proteins from the growth medium of Mycoplasma capricolum unmasks bacterium-derived enzymatic activities.

Mycoplasma constitutes a unique group of bacteria best characterized as lacking peptidoglycan and having one of the smallest genomes of all free-living prokaryotes. Members of this group also represent important pathogens of humans, animals, and plants. Our understanding of the interaction between these pathogens and their hosts is limited, partly due to our inadequate knowledge of the secreted enzymes and virulence factors of these pathogens. Analysis of secreted proteins of mycoplasma has been hampered by their fastidious growth requirements where protein-rich growth supplements are required. Simple ultrafiltration of the complete medium through a 10kDa cut-off membrane successfully removed virtually all of the polypeptides in the medium and supported the growth of Mycoplasma capricolum (type California kid). This modification (AM medium) exposed the activities of a number of enzymes produced by this bacterium during growth including; acid and alkaline phosphatase, gelatinase, and beta-lactamase activities. We also show that the spent culture medium contained hemolysin activity.