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  Microorganisms
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  This is an Open Access Journal Open Access journal
   ISSN (Online) 2076-2607
   Published by MDPI Homepage  [140 journals]
  • Microorganisms, Vol. 3, Pages 310-326: Temporal Study of the Microbial
           Diversity of the North Arm of Great Salt Lake, Utah, U.S.

    • Authors: Swati Almeida-Dalmet, Masoumeh Sikaroodi, Patrick Gillevet, Carol Litchfield, Bonnie Baxter
      Pages: 310 - 326
      Abstract: We employed a temporal sampling approach to understand how the microbial diversity may shift in the north arm of Great Salt Lake, Utah, U.S. To determine how variations in seasonal environmental factors affect microbial communities, length heterogeneity PCR fingerprinting was performed using consensus primers for the domain Bacteria, and the haloarchaea. The archaeal fingerprints showed similarities during 2003 and 2004, but this diversity changed during the remaining two years of the study, 2005 and 2006. We also performed molecular phylogenetic analysis of the 16S rRNA genes of the whole microbial community to characterize the taxa in the samples. Our results indicated that in the domain, Bacteria, the Salinibacter group dominated the populations in all samplings. However, in the case of Archaea, as noted by LIBSHUFF for phylogenetic relatedness analysis, many of the temporal communities were distinct from each other, and changes in community composition did not track with environmental parameters. Around 20–23 different phylotypes, as revealed by rarefaction, predominated at different periods of the year. Some phylotypes, such as Haloquadradum, were present year-round although they changed in their abundance in different samplings, which may indicate that these species are affected by biotic factors, such as nutrients or viruses, that are independent of seasonal temperature dynamics.
      PubDate: 2015-07-02
      DOI: 10.3390/microorganisms3030310
      Issue No: Vol. 3, No. 3 (2015)
       
  • Microorganisms, Vol. 3, Pages 327-343: Tetrachloromethane-Degrading
           Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer

    • Authors: Christian Penny, Christelle Gruffaz, Thierry Nadalig, Henry-Michel Cauchie, Stéphane Vuilleumier, Françoise Bringel
      Pages: 327 - 343
      Abstract: The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L−1 CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community.
      PubDate: 2015-07-02
      DOI: 10.3390/microorganisms3030327
      Issue No: Vol. 3, No. 3 (2015)
       
  • Microorganisms, Vol. 3, Pages 344-363: Copahue Geothermal System: A
           Volcanic Environment with Rich Extreme Prokaryotic Biodiversity

    • Authors: María Urbieta, Graciana Porati, Ana Segretín, Elena González-Toril, María Giaveno, Edgardo Donati
      Pages: 344 - 363
      Abstract: The Copahue geothermal system is a natural extreme environment located at the northern end of the Cordillera de los Andes in Neuquén province in Argentina. The geochemistry and consequently the biodiversity of the area are dominated by the activity of the Copahue volcano. The main characteristic of Copahue is the extreme acidity of its aquatic environments; ponds and hot springs of moderate and high temperature as well as Río Agrio. In spite of being an apparently hostile location, the prokaryotic biodiversity detected by molecular ecology techniques as well as cultivation shows a rich and diverse environment dominated by acidophilic, sulphur oxidising bacteria or archaea, depending on the conditions of the particular niche studied. In microbial biofilms, found in the borders of the ponds where thermal activity is less intense, the species found are completely different, with a high presence of cyanobacteria and other photosynthetic species. Our results, collected during more than 10 years of work in Copahue, have enabled us to outline geomicrobiological models for the different environments found in the ponds and Río Agrio. Besides, Copahue seems to be the habitat of novel, not yet characterised autochthonous species, especially in the domain Archaea.
      PubDate: 2015-07-08
      DOI: 10.3390/microorganisms3030344
      Issue No: Vol. 3, No. 3 (2015)
       
  • Microorganisms, Vol. 3, Pages 364-390: Extremophiles in Mineral Sulphide
           Heaps: Some Bacterial Responses to Variable Temperature, Acidity and
           Solution Composition

    • Authors: Helen Watling, Denis Shiers, David Collinson
      Pages: 364 - 390
      Abstract: In heap bioleaching, acidophilic extremophiles contribute to enhanced metal extraction from mineral sulphides through the oxidation of Fe(II) and/or reduced inorganic sulphur compounds (RISC), such as elemental sulphur or mineral sulphides, or the degradation of organic compounds derived from the ore, biota or reagents used during mineral processing. The impacts of variable solution acidity and composition, as well as temperature on the three microbiological functions have been examined for up to four bacterial species found in mineral sulphide heaps. The results indicate that bacteria adapt to sufficiently high metal concentrations (Cu, Ni, Co, Zn, As) to allow them to function in mineral sulphide heaps and, by engaging alternative metabolic pathways, to extend the solution pH range over which growth is sustained. Fluctuating temperatures during start up in sulphide heaps pose the greatest threat to efficient bacterial colonisation. The large masses of ores in bioleaching heaps mean that high temperatures arising from sulphide oxidation are hard to control initially, when the sulphide content of the ore is greatest. During that period, mesophilic and moderately thermophilic bacteria are markedly reduced in both numbers and activity.
      PubDate: 2015-07-09
      DOI: 10.3390/microorganisms3030364
      Issue No: Vol. 3, No. 3 (2015)
       
  • Microorganisms, Vol. 3, Pages 391-406: Cold-Active, Heterotrophic Bacteria
           from the Highly Oligotrophic Waters of Lake Vanda, Antarctica

    • Authors: Nicole Vander Schaaf, Anna Cunningham, Brandon Cluff, CodyJo Kraemer, Chelsea Reeves, Carli Riester, Lauren Slater, Michael Madigan, W. Sattley
      Pages: 391 - 406
      Abstract: The permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica are distinctive ecosystems that consist strictly of microbial communities. In this study, water samples were collected from Lake Vanda, a stratified Dry Valley lake whose upper waters (from just below the ice cover to nearly 60 m) are highly oligotrophic, and used to establish enrichment cultures. Six strains of psychrotolerant, heterotrophic bacteria were isolated from lake water samples from a depth of 50 or 55 m. Phylogenetic analyses showed the Lake Vanda strains to be species of Nocardiaceae, Caulobacteraceae, Sphingomonadaceae, and Bradyrhizobiaceae. All Lake Vanda strains grew at temperatures near or below 0 °C, but optimal growth occurred from 18 to 24 °C. Some strains showed significant halotolerance, but no strains required NaCl for growth. The isolates described herein include cold-active species not previously reported from Dry Valley lakes, and their physiological and phylogenetic characterization broadens our understanding of these limnologically unique lakes.
      PubDate: 2015-07-24
      DOI: 10.3390/microorganisms3030391
      Issue No: Vol. 3, No. 3 (2015)
       
  • Microorganisms, Vol. 3, Pages 113-136: High Throughput Sequencing to
           Detect Differences in Methanotrophic Methylococcaceae and Methylocystaceae
           in Surface Peat, Forest Soil, and Sphagnum Moss in Cranesville Swamp
           Preserve, West Virginia, USA

    • Authors: Evan Lau, Edward IV, Zachary Dillard, Ryan Dague, Amanda Semple, Wendi Wentzell
      Pages: 113 - 136
      Abstract: Northern temperate forest soils and Sphagnum-dominated peatlands are a major source and sink of methane. In these ecosystems, methane is mainly oxidized by aerobic methanotrophic bacteria, which are typically found in aerated forest soils, surface peat, and Sphagnum moss. We contrasted methanotrophic bacterial diversity and abundances from the (i) organic horizon of forest soil; (ii) surface peat; and (iii) submerged Sphagnum moss from Cranesville Swamp Preserve, West Virginia, using multiplex sequencing of bacterial 16S rRNA (V3 region) gene amplicons. From ~1 million reads, >50,000 unique OTUs (Operational Taxonomic Units), 29 and 34 unique sequences were detected in the Methylococcaceae and Methylocystaceae, respectively, and 24 potential methanotrophs in the Beijerinckiaceae were also identified. Methylacidiphilum-like methanotrophs were not detected. Proteobacterial methanotrophic bacteria constitute <2% of microbiota in these environments, with the Methylocystaceae one to two orders of magnitude more abundant than the Methylococcaceae in all environments sampled. The Methylococcaceae are also less diverse in forest soil compared to the other two habitats. Nonmetric multidimensional scaling analyses indicated that the majority of methanotrophs from the Methylococcaceae and Methylocystaceae tend to occur in one habitat only (peat or Sphagnum moss) or co-occurred in both Sphagnum moss and peat. This study provides insights into the structure of methanotrophic communities in relationship to habitat type, and suggests that peat and Sphagnum moss can influence methanotroph community structure and biogeography.
      PubDate: 2015-04-02
      DOI: 10.3390/microorganisms3020113
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 137-151: Interactions of Methylotrophs with
           Plants and Other Heterotrophic Bacteria

    • Authors: Hiroyuki Iguchi, Hiroya Yurimoto, Yasuyoshi Sakai
      Pages: 137 - 151
      Abstract: Methylotrophs, which can utilize methane and/or methanol as sole carbon and energy sources, are key players in the carbon cycle between methane and CO2, the two most important greenhouse gases. This review describes the relationships between methylotrophs and plants, and between methanotrophs (methane-utilizers, a subset of methylotrophs) and heterotrophic bacteria. Some plants emit methane and methanol from their leaves, and provide methylotrophs with habitats. Methanol-utilizing methylotrophs in the genus Methylobacterium are abundant in the phyllosphere and have the ability to promote the growth of some plants. Methanotrophs also inhabit the phyllosphere, and methanotrophs with high methane oxidation activities have been found on aquatic plants. Both plant and environmental factors are involved in shaping the methylotroph community on plants. Methanotrophic activity can be enhanced by heterotrophic bacteria that provide growth factors (e.g., cobalamin). Information regarding the biological interaction of methylotrophs with other organisms will facilitate a better understanding of the carbon cycle that is driven by methylotrophs.
      PubDate: 2015-04-02
      DOI: 10.3390/microorganisms3020137
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 152-174: Parallel and Divergent Evolutionary
           Solutions for the Optimization of an Engineered Central Metabolism in
           Methylobacterium extorquens AM1

    • Authors: Sean Carroll, Lon Chubiz, Deepa Agashe, Christopher Marx
      Pages: 152 - 174
      Abstract: Bioengineering holds great promise to provide fast and efficient biocatalysts for methanol-based biotechnology, but necessitates proven methods to optimize physiology in engineered strains. Here, we highlight experimental evolution as an effective means for optimizing an engineered Methylobacterium extorquens AM1. Replacement of the native formaldehyde oxidation pathway with a functional analog substantially decreased growth in an engineered Methylobacterium, but growth rapidly recovered after six hundred generations of evolution on methanol. We used whole-genome sequencing to identify the basis of adaptation in eight replicate evolved strains, and examined genomic changes in light of other growth and physiological data. We observed great variety in the numbers and types of mutations that occurred, including instances of parallel mutations at targets that may have been “rationalized” by the bioengineer, plus other “illogical” mutations that demonstrate the ability of evolution to expose unforeseen optimization solutions. Notably, we investigated mutations to RNA polymerase, which provided a massive growth benefit but are linked to highly aberrant transcriptional profiles. Overall, we highlight the power of experimental evolution to present genetic and physiological solutions for strain optimization, particularly in systems where the challenges of engineering are too many or too difficult to overcome via traditional engineering methods.
      PubDate: 2015-04-09
      DOI: 10.3390/microorganisms3020152
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 175-197: C1-Pathways in Methyloversatilis
           universalis FAM5: Genome Wide Gene Expression and Mutagenesis Studies

    • Authors: Nathan Good, Andrew Lamb, David Beck, N. Martinez-Gomez, Marina Kalyuzhnaya
      Pages: 175 - 197
      Abstract: Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylamine as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methylamine vs methanol. As expected, all genes for the N-methylglutamate pathway were induced during growth on methylamine. Among other functions responding to the aminated source of C1-carbon, are a heme-containing amine dehydrogenase (Qhp), a distant homologue of formaldehyde activating enzyme (Fae3), molybdenum-containing formate dehydrogenase, ferredoxin reductase, a set of homologues to urea/ammonium transporters and amino-acid permeases. Mutants lacking one of the functional subunits of the amine dehydrogenase (ΔqhpA) or Δfae3 showed no growth defect on C1-compounds. M. universalis FAM5 strains with a lesion in the H4-folate pathway were not able to use any C1-compound, methanol or methylamine. Genes essential for C1-assimilation (the serine cycle and glyoxylate shunt) and H4MTP-pathway for formaldehyde oxidation showed similar levels of expression on both C1-carbon sources. M. universalis FAM5 possesses three homologs of the formaldehyde activating enzyme, a key enzyme of the H4MTP-pathway. Strains lacking the canonical Fae (fae1) lost the ability to grow on both C1-compounds. However, upon incubation on methylamine the fae1-mutant produced revertants (Δfae1R), which regained the ability to grow on methylamine. Double and triple mutants (Δfae1RΔfae3, or Δfae1RΔfae2 or Δfae1RΔfae2Δfae3) constructed in the revertant strain background showed growth similar to the Δfae1R phenotype. The metabolic pathways for utilization of methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed based on these gene expression and phenotypic data.
      PubDate: 2015-04-09
      DOI: 10.3390/microorganisms3020175
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 198-212: Comparison of Microbiological and
           Probiotic Characteristics of Lactobacilli Isolates from Dairy Food
           Products and Animal Rumen Contents

    • Authors: Neethu Jose, Craig Bunt, Malik Hussain
      Pages: 198 - 212
      Abstract: Lactobacilli are employed in probiotic food preparations and as feed additives in poultry and livestock, due to health benefits associated with their consumption. The objective of this study was to evaluate and compare the probiotic potential of ten lactobacilli strains isolated from commercial dairy food products and animal rumen contents in New Zealand. Genetic identification of the isolates revealed that all belonged to the genus Lactobacillus, specifically the species L. reuteri, L. rhamnosus and L. plantarum. All isolates did not show any haemolytic behaviour. Isolates of dairy origin showed better tolerance to low pH stress. On the other hand, rumen isolates exhibited a higher tolerance to presence of bile salts. All isolates exhibited resistance to aminoglycoside antibiotics, however most were sensitive to ampicillin. Isolates of rumen origin demonstrated a higher inhibitory effect on Listeria monocytogenes, Enterobacter aerogenes and Salmonella menston. Bacterial adherence of all isolates increased with a decrease in pH. This screening study on lactobacilli isolates has assessed and identified potential probiotic candidates for further evaluation.
      PubDate: 2015-04-15
      DOI: 10.3390/microorganisms3020198
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 213-235: Does the Gut Microbiota Contribute
           to Obesity' Going beyond the Gut Feeling

    • Authors: Marisol Aguirre, Koen Venema
      Pages: 213 - 235
      Abstract: Increasing evidence suggests that gut microbiota is an environmental factor that plays a crucial role in obesity. However, the aetiology of obesity is rather complex and depends on different factors. Furthermore, there is a lack of consensus about the exact role that this microbial community plays in the host. The aim of this review is to present evidence about what has been characterized, compositionally and functionally, as obese gut microbiota. In addition, the different reasons explaining the so-far unclear role are discussed considering evidence from in vitro, animal and human studies.
      PubDate: 2015-04-27
      DOI: 10.3390/microorganisms3020213
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 236-267: Tackling Drug Resistant Infection
           Outbreaks of Global Pandemic Escherichia coli ST131 Using Evolutionary and
           Epidemiological Genomics

    • Authors: Tim Downing
      Pages: 236 - 267
      Abstract: High-throughput molecular screening is required to investigate the origin and diffusion of antimicrobial resistance in pathogen outbreaks. The most frequent cause of human infection is Escherichia coli, which is dominated by sequence type 131 (ST131)—a set of rapidly radiating pandemic clones. The highly infectious clades of ST131 originated firstly by a mutation enhancing conjugation and adhesion. Secondly, single-nucleotide polymorphisms occurred enabling fluoroquinolone-resistance, which is near-fixed in all ST131. Thirdly, broader resistance through beta-lactamases has been gained and lost frequently, symptomatic of conflicting environmental selective effects. This flexible approach to gene exchange is worrying and supports the proposition that ST131 will develop an even wider range of plasmid and chromosomal elements promoting antimicrobial resistance. To stop ST131, deep genome sequencing is required to understand the origin, evolution and spread of antimicrobial resistance genes. Phylogenetic methods that decipher past events can predict future patterns of virulence and transmission based on genetic signatures of adaptation and gene exchange. Both the effect of partial antimicrobial exposure and cell dormancy caused by variation in gene expression may accelerate the development of resistance. High-throughput sequencing can decode measurable evolution of cell populations within patients associated with systems-wide changes in gene expression during treatments. A multi-faceted approach can enhance assessment of antimicrobial resistance in E. coli ST131 by examining transmission dynamics between hosts to achieve a goal of pre-empting resistance before it emerges by optimising antimicrobial treatment protocols.
      PubDate: 2015-05-20
      DOI: 10.3390/microorganisms3020236
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 268-289: Generation of PHB from Spent
           Sulfite Liquor Using Halophilic Microorganisms

    • Authors: Michaela Weissgram, Janina Gstöttner, Bettina Lorantfy, Raimund Tenhaken, Christoph Herwig, Hedda Weber
      Pages: 268 - 289
      Abstract: Halophilic microorganisms thrive at elevated concentrations of sodium chloride up to saturation and are capable of growing on a wide variety of carbon sources like various organic acids, hexose and also pentose sugars. Hence, the biotechnological application of these microorganisms can cover many aspects, such as the treatment of hypersaline waste streams of different origin. Due to the fact that the high osmotic pressure of hypersaline environments reduces the risk of contamination, the capacity for cost-effective non-sterile cultivation can make extreme halophilic microorganisms potentially valuable organisms for biotechnological applications. In this contribution, the stepwise use of screening approaches, employing design of experiment (DoE) on model media and subsequently using industrial waste as substrate have been implemented to investigate the applicability of halophiles to generate PHB from the industrial waste stream spent sulfite liquor (SSL). The production of PHB on model media as well as dilutions of industrial substrate in a complex medium has been screened for by fluorescence microscopy using Nile Blue staining. Screening was used to investigate the ability of halophilic microorganisms to withstand the inhibiting substances of the waste stream without negatively affecting PHB production. It could be shown that neither single inhibiting substances nor a mixture thereof inhibited growth in the investigated range, hence, leaving the question on the inhibiting mechanisms open. However, it could be demonstrated that some haloarchaea and halophilic bacteria are able to produce PHB when cultivated on 3.3% w/w dry matter spent sulfite liquor, whereas H. halophila was even able to thrive on 6.6% w/w dry matter spent sulfite liquor and still produce PHB.
      PubDate: 2015-06-08
      DOI: 10.3390/microorganisms3020268
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 290-309: Methane Oxidation and Molecular
           Characterization of Methanotrophs from a Former Mercury Mine Impoundment

    • Authors: Shaun Baesman, Laurence Miller, Jeremy Wei, Yirang Cho, Emily Matys, Roger Summons, Paula Welander, Ronald Oremland
      Pages: 290 - 309
      Abstract: The Herman Pit, once a mercury mine, is an impoundment located in an active geothermal area. Its acidic waters are permeated by hundreds of gas seeps. One seep was sampled and found to be composed of mostly CO2 with some CH4 present. The δ13CH4 value suggested a complex origin for the methane: i.e., a thermogenic component plus a biological methanogenic portion. The relatively 12C-enriched CO2 suggested a reworking of the ebullitive methane by methanotrophic bacteria. Therefore, we tested bottom sediments for their ability to consume methane by conducting aerobic incubations of slurried materials. Methane was removed from the headspace of live slurries, and subsequent additions of methane resulted in faster removal rates. This activity could be transferred to an artificial, acidic medium, indicating the presence of acidophilic or acid-tolerant methanotrophs, the latter reinforced by the observation of maximum activity at pH = 4.5 with incubated slurries. A successful extraction of sterol and hopanoid lipids characteristic of methanotrophs was achieved, and their abundances greatly increased with increased sediment methane consumption. DNA extracted from methane-oxidizing enrichment cultures was amplified and sequenced for pmoA genes that aligned with methanotrophic members of the Gammaproteobacteria. An enrichment culture was established that grew in an acidic (pH 4.5) medium via methane oxidation.
      PubDate: 2015-06-23
      DOI: 10.3390/microorganisms3020290
      Issue No: Vol. 3, No. 2 (2015)
       
  • Microorganisms, Vol. 3, Pages 1-16: Identification of Multiple
           Bacteriocins in Enterococcus spp. Using an Enterococcus-Specific
           Bacteriocin PCR Array

    • Authors: Chris Henning, Dhiraj Gautam, Peter Muriana
      Pages: 1 - 16
      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.
      PubDate: 2015-02-04
      DOI: 10.3390/microorganisms3010001
      Issue No: Vol. 3, No. 1 (2015)
       
  • Microorganisms, Vol. 3, Pages 17-33: Rapid Reactivation of Deep Subsurface
           Microbes in the Presence of C-1 Compounds

    • Authors: Pauliina Rajala, Malin Bomberg, Riikka Kietäväinen, Ilmo Kukkonen, Lasse Ahonen, Mari Nyyssönen, Merja Itävaara
      Pages: 17 - 33
      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.
      PubDate: 2015-02-05
      DOI: 10.3390/microorganisms3010017
      Issue No: Vol. 3, No. 1 (2015)
       
  • Microorganisms, Vol. 3, Pages 34-46: Fumaric Acid and Slightly Acidic
           Electrolyzed Water Inactivate Gram Positive and Gram Negative Foodborne
           Pathogens

    • Authors: Charles Tango, Ahmad Mansur, Deog-Hwan Oh
      Pages: 34 - 46
      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, and LM, 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.
      PubDate: 2015-02-12
      DOI: 10.3390/microorganisms3010034
      Issue No: Vol. 3, No. 1 (2015)
       
  • Microorganisms, Vol. 3, Pages 47-59: Role of NAD+-Dependent Malate
           Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and
           Methylosinus trichosporium OB3b

    • Authors: Olga Rozova, Valentina Khmelenina, Ksenia Bocharova, Ildar Mustakhimov, Yuri Trotsenko
      Pages: 47 - 59
      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/Km ratios 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.
      PubDate: 2015-02-27
      DOI: 10.3390/microorganisms3010047
      Issue No: Vol. 3, No. 1 (2015)
       
  • Microorganisms, Vol. 3, Pages 60-79: Experimental Horizontal Gene Transfer
           of Methylamine Dehydrogenase Mimics Prevalent Exchange in Nature and
           Overcomes the Methylamine Growth Constraints Posed by the Sub-Optimal
           N-Methylglutamate Pathway

    • Authors: Dipti Nayak, Christopher Marx
      Pages: 60 - 79
      Abstract: Methylamine plays an important role in the global carbon and nitrogen budget; microorganisms that grow on reduced single carbon compounds, methylotrophs, serve as a major biological sink for methylamine in aerobic environments. Two non-orthologous, functionally degenerate routes for methylamine oxidation have been studied in methylotrophic Proteobacteria: Methylamine dehydrogenase and the N-methylglutamate pathway. Recent work suggests the N-methylglutamate (NMG) pathway may be more common in nature than the well-studied methylamine dehydrogenase (MaDH, encoded by the mau gene cluster). However, the distribution of these pathways across methylotrophs has never been analyzed. Furthermore, even though horizontal gene transfer (HGT) is commonly invoked as a means to transfer these pathways between strains, the physiological barriers to doing so have not been investigated. We found that the NMG pathway is both more abundant and more universally distributed across methylotrophic Proteobacteria compared to MaDH, which displays a patchy distribution and has clearly been transmitted by HGT even amongst very closely related strains. This trend was especially prominent in well-characterized strains of the Methylobacterium extroquens species, which also display significant phenotypic variability during methylamine growth. Strains like Methylobacterium extorquens PA1 that only encode the NMG pathway grew on methylamine at least five-fold slower than strains like Methylobacterium extorquens AM1 that also possess the mau gene cluster. By mimicking a HGT event through the introduction of the M. extorquens AM1 mau gene cluster into the PA1 genome, the resulting strain instantaneously achieved a 4.5-fold increase in growth rate on methylamine and a 11-fold increase in fitness on methylamine, which even surpassed the fitness of M. extorquens AM1. In contrast, when three replicate populations of wild type M. extorquens PA1 were evolved on methylamine as the sole carbon and energy source for 150 generations neither fitness nor growth rate improved. These results suggest that the NMG pathway permits slow growth on methylamine and is widely distributed in methylotrophs; however, rapid growth on methylamine can be achieved quite readily through acquisition of the mau cluster by HGT.
      PubDate: 2015-03-10
      DOI: 10.3390/microorganisms3010060
      Issue No: Vol. 3, No. 1 (2015)
       
  • Microorganisms, Vol. 3, Pages 80-93: Isolation and Taxonomic Identity of
           Bacteriocin-Producing Lactic Acid Bacteria from Retail Foods and Animal
           Sources

    • Authors: Chris Henning, Paul Vijayakumar, Raj Adhikari, Badrinath Jagannathan, Dhiraj Gautam, Peter Muriana
      Pages: 80 - 93
      Abstract: Bacteriocin-producing (Bac+) lactic acid bacteria (LAB) were isolated from a variety of food products and animal sources. Samples were enriched in de Man, Rogosa, and Sharpe (MRS) Lactocilli broth and plated onto MRS agar plates using a “sandwich overlay” technique. Inhibitory activity was detected by the “deferred antagonism” indicator overlay method using Listeria monocytogenes as the primary indicator organism. Antimicrobial activity against L. monocytogenes was detected by 41 isolates obtained from 23 of 170 food samples (14%) and 11 of 110 samples from animal sources (10%) tested. Isolated Bac+ LAB included Lactococcus lactis, Lactobacillus curvatus, Carnobacterium maltaromaticum, Leuconostoc mesenteroides, and Pediococcus acidilactici, as well as Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, and Enterococcus thailandicus. In addition to these, two Gram-negative bacteria were isolated (Serratia plymuthica, and Serratia ficaria) that demonstrated inhibitory activity against L. monocytogenes, Staphylococcus aureus, and Enterococcus faecalis (S. ficaria additionally showed activity against Salmonella Typhimurium). These data continue to demonstrate that despite more than a decade of antimicrobial interventions on meats and produce, a wide variety of food products still contain Bac+ microbiota that are likely eaten by consumers and may have application as natural food preservatives.
      PubDate: 2015-03-19
      DOI: 10.3390/microorganisms3010080
      Issue No: Vol. 3, No. 1 (2015)
       
  • Microorganisms, Vol. 3, Pages 94-112: Genomics of Methylotrophy in
           Gram-Positive Methylamine-Utilizing Bacteria

    • Authors: Tami McTaggart, David Beck, Usanisa Setboonsarng, Nicole Shapiro, Tanja Woyke, Mary Lidstrom, Marina Kalyuzhnaya, Ludmila Chistoserdova
      Pages: 94 - 112
      Abstract: Gram-positive methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of methylotrophy pathways/enzymes within this group. However, genome-based knowledge of methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive methylotrophs limits our global understanding of methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of methylotrophy in Gram-positive methylotrophic bacteria. This study is a major contribution to the field of methylotrophy, aimed at closing the gap in the genomic knowledge of methylotrophy within this diverse group of bacteria.
      PubDate: 2015-03-20
      DOI: 10.3390/microorganisms3010094
      Issue No: Vol. 3, No. 1 (2015)
       
 
 
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