Abstract: We have expressed the l-malate dehydrogenase (MDH) genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD+-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa) enzyme displaying nearly equal reductive (malate formation) and oxidative (oxaloacetate formation) activities and higher affinity to malate (Km = 0.11 mM) than to oxaloacetate (Km = 0.34 mM). The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa), two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM) than to malate (Km = 1.28 mM). The kcat/Kmratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed.
Abstract: Sanitizing effectiveness of slightly acidic electrolyzed water (SAEW) and fumaric acid (FA) at different dipping temperatures (25–60 °C), times (1–5 min), and concentrations (5–30 ppm for SAEW and 0.125%–0.5% for FA) on pure cultures of two Gram positive pathogens Staphylococcus aureus (SA) and Listeria monocytogenes (LM) and two Gram negative pathogens Escherichia coli O157:H7 (EC) and Salmonella Typhimurium (ST) was evaluated. FA (0.25%) showed the strongest sanitizing effect, demonstrating complete inactivation of EC, ST, andLM, while SA was reduced by 3.95–5.76 log CFU/mL at 25–60 °C, respectively, after 1 min of treatment. For SAEW, the complete inactivation was obtained when available chlorine concentration was increased to 20 ppm at 40 °C for 3 and 5 min. Moreover, Gram positive pathogens have been shown to resist to all treatment trends more than Gram negative pathogens throughout this experiment. Regardless of the different dipping temperatures, concentrations, and times, FA treatment was more effective than treatment with SAEW for reduction of foodborne pathogens. This study demonstrated that application of FA in food systems may be useful as a method for inactivation of foodborne pathogens.
Abstract: Microorganisms in the deep biosphere are believed to conduct little metabolic activity due to low nutrient availability in these environments. However, destructive penetration to long-isolated bedrock environments during construction of underground waste repositories can lead to increased nutrient availability and potentially affect the long-term stability of the repository systems, Here, we studied how microorganisms present in fracture fluid from a depth of 500 m in Outokumpu, Finland, respond to simple carbon compounds (C-1 compounds) in the presence or absence of sulphate as an electron acceptor. C-1 compounds such as methane and methanol are important intermediates in the deep subsurface carbon cycle, and electron acceptors such as sulphate are critical components of oxidation processes. Fracture fluid samples were incubated in vitro with either methane or methanol in the presence or absence of sulphate as an electron acceptor. Metabolic response was measured by staining the microbial cells with fluorescent dyes that indicate metabolic activity and transcriptional response with RT-qPCR. Our results show that deep subsurface microbes exist in dormant states but rapidly reactivate their transcription and respiration systems in the presence of C-1 substrates, particularly methane. Microbial activity was further enhanced by the addition of sulphate as an electron acceptor. Sulphate- and nitrate-reducing microbes were particularly responsive to the addition of C-1 compounds and sulphate. These taxa are common in deep biosphere environments and may be affected by conditions disturbed by bedrock intrusion, as from drilling and excavation for long-term storage of hazardous waste.
Abstract: Twenty-two bacteriocin-producing Enterococcus isolates obtained from food and animal sources, and demonstrating activity against Listeria monocytogenes, were screened for bacteriocin-related genes using a bacteriocin PCR array based on known enterococcal bacteriocin gene sequences in the NCBI GenBank database. The 22 bacteriocin-positive (Bac+) enterococci included En. durans (1), En. faecalis (4), En.faecium (12), En.hirae (3), and En. thailandicus (2). Enterocin A (entA), enterocins mr10A and mr10B (mr10AB), and bacteriocin T8 (bacA) were the most commonly found structural genes in order of decreasing prevalence. Forty-five bacteriocin genes were identified within the 22 Bac+ isolates, each containing at least one of the screened structural genes. Of the 22 Bac+ isolates, 15 possessed two bacteriocin genes, seven isolates contained three different bacteriocins, and three isolates contained as many as four different bacteriocin genes. These results may explain the high degree of bactericidal activity observed with various Bac+ Enterococcus spp. Antimicrobial activity against wild-type L. monocytogenes and a bacteriocin-resistant variant demonstrated bacteriocins having different modes-of-action. Mixtures of bacteriocins, especially those with different modes-of-action and having activity against foodborne pathogens, such as L. monocytogenes, may play a promising role in the preservation of food.
Abstract: The MDPI journal Microorganisms is still very young, having been launched in 2013, but the concept of the microorganism has been in use for at least a century as a unifying principle for the discipline of microbiology, which was cemented firmly by the intellectual work of Roger Stanier and colleagues in their Microbial World and other general microbiology textbooks and related articles from the 1950s to the 1970s [1,2]. Merging the idea of the microscopic and the very small with the older idea of an organism as a living entity or cell, the concept of a microorganism enabled a real appreciation of the microbial world as one that is amenable to study using similar tools and approaches even though representing distinctly different types of reproductive units and cell organizations. In the late 20th century following the work of Carl Woese and other molecular evolutionists, biologists came to appreciate the commonality among all organisms, all being comprised of cells that bear a remarkable similarity to one another and that share a common evolutionary ancestry, and consequently with major features of a largely shared genetic code and molecular biology. In this sense microbiology and biology as a whole became unified as they never had been before.[...]
Abstract: Streptococcus mutans is a key etiological agent in the formation of dental caries. The major virulence factor is its ability to form biofilms. Inhibition of S. mutans biofilms offers therapeutic prospects for the treatment and the prevention of dental caries. In this study, 14 analogs of makaluvamine, a marine alkaloid, were evaluated for their antibacterial activity against S. mutans and for their ability to inhibit S. mutans biofilm formation. All analogs contained the tricyclic pyrroloiminoquinone core of makaluvamines. The structural variations of the analogs are on the amino substituents at the 7-position of the ring and the inclusion of a tosyl group on the pyrrole ring N of the makaluvamine core. The makaluvamine analogs displayed biofilm inhibition with IC50 values ranging from 0.4 μM to 88 μM. Further, the observed bactericidal activity of the majority of the analogs was found to be consistent with the anti-biofilm activity, leading to the conclusion that the anti-biofilm activity of these analogs stems from their ability to kill S. mutans. However, three of the most potent N-tosyl analogs showed biofilm IC50 values at least an order of magnitude lower than that of bactericidal activity, indicating that the biofilm activity of these analogs is more selective and perhaps independent of bactericidal activity.