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Microbial Physiology

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Bart C Weimer
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Case description: In Latvia in 2014, acquired idiopathic megaesophagus (AIME) was observed in increased numbers of dogs that consumed varieties of 1 brand of dog food. Within 2 years, 253 dogs were affected. In Australia in November 2017, 6 working dogs that consumed 1 diet of another brand of dog food developed AIME. In total, 145 Australian dogs were affected. Clinical findings: AIME was diagnosed predominantly in large-breed male dogs (> 25 kg [55 lb]). Regurgitation, weight loss, and occasionally signs consistent with aspiration pneumonia (coughing, dyspnea, or fever) were noted. Most Latvian dogs had mild to severe peripheral polyneuropathies as evidenced by laryngeal paralysis, dysphonia, weakness, and histopathologic findings consistent with distal axonopathy. In Australian dogs, peripheral polyneuropathies were not identified, and histopathologic findings suggested that the innervation of the esophagus and pharynx was disrupted locally, although limited samples were available. Treatment and outcome: Investigations in both countries included clinical, epidemiological, neuropathologic, and case-control studies. Strong associations between the dog foods and the presence of AIME were confirmed; however, toxicological analyses did not identify a root cause. In Latvia, the implicated dietary ingredients and formulations were unknown, whereas in Australia, extensive investigations were conducted into the food, its ingredients, the supply chain, and the manufacturing facilities, but a cause was not identified. Clinical relevance: A panel of international multidisciplinary experts concluded that the cause of AIME in both outbreaks was likely multifactorial, with the possibility of individualized sensitivities. Without a sentinel group, the outbreak in Australia may not have been recognized for months to years, as happened in Latvia. A better surveillance system for early identification of pet illnesses, including those associated with pet foods, is needed.
Bart C Weimer
added a research item
LAB contain multiple systems to handle stress conditions in dairy processing. As more genome sequences are produced, additional systems and their regulation are being uncovered. Overlapping stress responses provide protection for multiple stresses. Common regulatory elements lead to broad phenotypic and genotypic changes. The large number of mobile elements coupled to stress-induced genome changes indicate that treatments used in dairy processing promote genome changes that is reflected by the genetic variation between strains and the genome-wide rearrangements that lead to a large phenotypic variation for stress response. This variation brings substantial complexity to understanding the entire stress response of LAB.
Bart C Weimer
added 2 research items
Sierra Mixe maize is a landrace variety from Oaxaca, Mexico, that utilizes nitrogen derived from the atmosphere via an undefined nitrogen fixation mechanism. The diazotrophic microbiota associated with the plant's mucilaginous aerial root exudate composed of complex carbohydrates was previously identified and characterized by our group where we found 23 lactococci capable of biological nitrogen fixation (BNF) without containing any of the proposed essential genes for this trait (nifHDKENB). To determine the genes in Lactococcus associated with this phenotype, we selected 70 lactococci from the dairy industry that are not known to be diazotrophic to conduct a comparative population genomic analysis. This showed that the diazotrophic lactococcal genomes were distinctly different from the dairy isolates. Examining the pangenome followed by genome-wide association study and machine learning identified genes with the functions needed for BNF in the maize isolates that were absent from the dairy isolates. Many of the putative genes received an 'unknown' annotation, which led to the domain analysis of the 135 homologs. This revealed genes with molecular functions needed for BNF, including mucilage carbohydrate catabolism, glycan-mediated host adhesion, iron/siderophore utilization, and oxidation/reduction control. This is the first report of this pathway in this organism to underpin BNF. Consequently, we proposed a model needed for BNF in lactococci that plausibly accounts for BNF in the absence of the nif operon in this organism.
Ascorbic acid (AA) and uric acid (UA) are known as two of the major antioxidants in biological fluids. We report a novel liquid chromatography–mass spectrometry with time-of-flight (LC-MS-TOF) method for the simultaneous quantification of ascorbic and uric acids using MPA, antioxidant solution and acetonitrile as a protein precipitating agent. Both compounds were separated from interferences using a reverse phase C18 column with water and acetonitrile gradient elution (both with formic acid) and identified and quantified by MS in the negative ESI mode. Isotope labeled internal standards were also added to ensure the accuracy of the measures. The method was validated for exhaled breath condensate (EBC), nasal lavage (NL) and plasma samples by assessing selectivity, linearity, accuracy and precision, recovery and matrix effect and stability. Sample volumes below 250 µl were used and linear ranges were determined between 1 – 25 and 1 – 40 µg/mL for ascorbic and uric acid, respectively, for plasma samples, and between 0.05 – 5 (AA) and 0.05 – 7.5 (UA) µg/mL for EBC and NL samples. The new method was successfully applied to real samples from subjects that provided each of the studied matrices. Results showed higher amounts determined in plasma samples, with similar profiles for AA and UA in EBC and NL but at much lower concentrations.
Bart C Weimer
added a research item
Traditional taxonomy in biology assumes that life is organized in a simple tree. Attempts to classify microorganisms in this way in the genomics era led microbiologists to look for finite sets of 'core' genes that uniquely group taxa as clades in the tree. However, the diversity revealed by large-scale whole genome sequencing is calling into question the long-held model of a hierarchical tree of life, which leads to questioning of the definition of a species. Large-scale studies of microbial genome diversity reveal that the cumulative number of new genes discovered increases with the number of genomes studied as a power law and subsequently leads to the lack of evidence for a unique core genome within closely related organisms. Sampling 'enough' new genomes leads to the discovery of a replacement or alternative to any gene. This power law behaviour points to an underlying self-organizing critical process that may be guided by mutation and niche selection. Microbes in any particular niche exist within a local web of organism interdependence known as the microbiome. The same mechanism that underpins the macro-ecological scaling first observed by MacArthur and Wilson also applies to microbial communities. Recent metagenomic studies of a food microbiome demonstrate the diverse distribution of community members, but also genotypes for a single species within a more complex community. Collectively, these results suggest that traditional taxonomic classification of bacteria could be replaced with a quasispecies model. This model is commonly accepted in virology and better describes the diversity and dynamic exchange of genes that also hold true for bacteria. This model will enable microbiologists to conduct population-scale studies to describe microbial behaviour, as opposed to a single isolate as a representative.
Bart C Weimer
added 2 research items
Dairy processing provides many abiotic stress conditions that change the phenotypic response of lactic acid bacteria (LAB) during fermentation and storage. Genome sequencing of LAB is expanding and leading to significant opportunities to establish specific genetic mechanisms that lead to single and multiple stress responses that underpin the physiological changes. Response differentiation between closely related LAB is linked to a handful of genetic regulatory elements that cascade into hundreds of changes in structure and metabolism of the cell. Determination of the conservation of the regulatory molecule that mediates stress responses provides insights into the mechanisms leading to the various metabolic and cellular responses produced during dairy processing across various strains and provides a basis for predicting the variation of stress response. Although LAB have a somewhat small genome, the capability to respond to stress is clearly fully functional. Among the stringent and the SOS responses of regulatory elements there exist some lead to stress mitigation via expression of a myriad of metabolic changes useful for repairing DNA, neutralizing acid, and producing energy from proteins amino acids and production of new flavor-active metabolites. In addition, lactococci have a small number of unique genes (i.e., cept, codY,. Z) that provide additional survival capabilities through metabolism of protein and induce the industrially-import responses. In the postgenomics era, simultaneous measurement of the cellular phenotype and the metabolomic, proteomic, and genomic mechanism promises to provide a systemwide picture of the intricate molecular dance induced by stresses during dairy processing. This picture has the potential to provide new methods for strain selection and processing changes to direct the survival and metabolism of LAB to achieve new metabolic products in dairy products.
Free and peptide/protein bonded acids were qualitatively and quantitatively determined in parmesan (Parmigiano Reggiano) cheese samples by HPLC. d/. l ratio was measured using a chiral derivatizing agent. The diffusion processes of free amino acids occurring in Parmigiano Reggiano hard cheese can be regarded and utilized as a characteristic molecular marker of its ripening.
Bart C Weimer
added a research item
Defined flavor profiles of cheeses and long lists of associated compounds have expanded our vista of the flavor formation mechanisms. Beginning from a review by Schormüller in 1968 as a particularly useful starting point for these pathways, we seek to recognize the past work in this arena more specifically within the last decade, of the possibilities of novel discovery, and of the necessary directions for the future of this arena. Toward this direction, a definitive body of work on defining entire pathways for beneficial flavor production from amino acid metabolism is still in progress. Formation of selective flavor compounds such as fatty acids and volatile sulfurs and related flavor production mechanisms attributable to bacterial metabolism and the likely candidate substrates from the available menu of lactose, milk lipids, and milk protein will be extensively discussed. Focus on metabolic reconstruction from genomic sequence and identifying molecules by genome restriction are recommended as useful tools to gain novel perspectives on alternate metabolic pathways.
Bart C Weimer
added 2 research items
The diversity revealed by large scale genomics in microbiology is calling into question long held beliefs about genome stability, evolutionary rate, even the definition of a species. MacArthur and Wilson's theory of insular biogeography provides an explanation for the diversity of macroscopic animal and plant species as a consequence of the associated hierarchical web of species interdependence. We report a large scale study of microbial diversity that reveals that the cumulative number of genes discovered increases with the number of genomes studied as a simple power law. This result is demonstrated for three different genera comparing over 15,000 isolates. We show that this power law is formally related to the MacArthur-Wilson exponent, suggesting the emerging diversity of microbial genotypes arises because the scale independent behavior first reported by MacArthur and Wilson extends down to the scale of microbes and their genes. Assessing the depth of available whole genome sequences implies a dynamically changing core genome, suggesting that traditional taxonomic classifications should be replaced with a quasispecies model that captures the diversity and dynamic exchange of genes. We report Species population "clouds" in a defined microbiome, with scale invariance extending down to the level of single-nucleotide polymorphisms (SNPs).
Sialidases, also known as neuraminidase, are glycosyl-hydrolases that cleave terminal sialic acid from complex glycans. Sialidases are widely distributed in nature and are found in organisms from Eukarya, Eubacteria, and viruses. Microbes use these enzymes to reveal the cell surface that holds sialic acid-containing cell membrane receptors that are recognized by toxins from Vibrio cholerae in animals, while many animal viruses use this protein to recognize host receptors during infection. Sialidases from Streptococcus pneumoniae and Pseudomonas aeruginosa also act as a molecular signal for biofilm formation needed during lung infection. Once released bacterial communities that inhabit plants and animals can utilize free sialic acid as a method to increase their population during infection. Alternatively, some bacteria (Pseudomonas spp., Haemophilus spp.) sialylate structures on their cell surface, such as flagella, capsule polysaccharide (CPS), or the lipopolysaccharide (LPS), to mask the pathogen from the host immune system. Considering the importance of this enzyme family in infection and commensalism it is important to understand genomic diversity as a means to investigate infection mechanisms. This study bioinformatically examined the sialidase diversity and genomic distribution among microbes by comparing nearly 1900 genomes. Phylogenetic trees were constructed using a Neighbor-Joining consensus tree with 1000 bootstrap replicates. This analysis revealed that members of the same genera were found on different sialidase clades (Escherichia coli, Klebsiella spp., Bacillus spp., Pseudomonas spp., Helicobacter spp., Bacteroides spp., Clostridium spp., Streptococcus spp., Salmonella spp., Rumonococcus spp. Trypanosoma spp.), indicating widespread horizontal gene transfer (HGT). We also observed a large number of indeterminate annotations for the genes among multiple protein families. This problem was supported by surprisingly extensive structural variation in the domain organization among members of each clade. This observation enabled sialidase annotation for many hypothetical genes with sequence homology to this enzyme. For example, STM1252 from Salmonella contains all required domains for sialidase activity, but it is annotated as a hypothetical cytoplasmic protein. This specific version of sialidases was found widely distributed among pathogenic and commensal bacteria. We also observed a second unrelated sialidase, STM0928 (nanH), in Salmonella with another distinct domain organization that has sialidase activity. The phylogenomic analysis of STM0928 revealed that the genome of Salmonella Typhimurium LT2 also contains this second sialidase. This sialidase was confined to pathogens (Clostridium spp., Trypanosoma spp.), but no other enteric bacterium. STM0928 in Salmonella Typhimurium LT2 was most closely related to Clostridium tertium, suggesting that Salmonella Typhimurium LT2 acquired this gene as a result of HGT via Salmonella Fels1 phage. The direct impact of multiple sialidases remains to be explored. In conclusion, sialidases as a group are widely distributed among microbes, but multiple structural variations exist that contribute to inaccurate annotation.
Bart C Weimer
added a research item
Campylobacter jejuni is the leading cause of gastroenteritis in humans worldwide with recent studies indicating wild birds as zoonotic vectors. American crows, abundant in urban, suburban, and agricultural settings, carry C. jejuni as a commensal organism. Studies demonstrating that the crow is a Campylobacter zoonotic reservoir are lacking, and no studies have investigated these Campylobacter genomes that are distributed among the crow population. To examine pathogenic potential, zoonotic exchange and genomic phylogeny, we sequenced 160 Campylobacter genomes from crow, chicken, cow, sheep, goat, and non-human primate origin from the Sacramento Valley, CA. Many genomes displayed high similarity to sequences of isolates implicated in human disease, suggesting these isolates as potential pathogens of public health importance. Tetracycline resistance gene tetO was present in 21.9% of the isolates, and 22 isolates (8 C. jejuni, 11 C. coli, 3 C. lari) contained mutations in gyrA indicative of Fluoroquinolone resistance. Cytolethal Distending Toxin genes were present in 95% of isolates, 100% contained invasion and adherence genes, and 20% of genomes contained Type IV SS genes. Although resistance and virulence genes were broadly distributed throughout these isolates, specific genotypes were associated with individual host species and a sub-set of isolates were associated with several hosts (i.e. crows, primates, sheep, humans), suggesting that these genotypes may be from zoonotic exchange. This study, utilizing whole genome sequencing (WGS), associated specific Campylobacter genotypes that have high potential zoonotic transmission opposed to host adapted genotypes in the same geographic area to show antibiotic resistance, virulence potential, and zoonotic potential.
Bart C Weimer
added 43 research items
Heat treatments of milk between 100 and 145°C produce a new type of product with a shelf life of 15 to 30 days at 7°C, which is termed extended shelf life (ESL) milk. Little information is available on the safety and sensory qualities of this product. Extended shelf life milk is being processed commercially to expand the distribution area of fluid milk products. After arrival at market, this product still has the shelf life of a pasteurized product. In this study milk was processed by direct steam injection at temperatures between 100 and 140°C for 4 or 12 s. Holding time did not significantly affect the sensory quality of the milk. A trained taste panel found cooked flavor and other off flavors varied significantly with increasing processing temperature and storage time. There were no significant differences noted in cooked or off flavors between 132 and 140°C. Psychrotrophic Bacillus species were isolated from milk processed at and below 132°C, while no organisms were isolated from milk processed at temperatures at or above 134°C. Consumer preference panels indicated consumers preferred milk processed at 134°C for 4 s to ultra-high-temperature (UHT) processed milk, although there was a slight preference for high-temperature short-time processed (HTST) milk compared to milk processed at 134°C for 4 s. Higher temperatures had a less destructive effect on lipase activity, while storage time did not significantly affect lipase activity.
The o-phthaldialdehyde test and amino acid analysis were used to characterize proteolysis of milk proteins during growth of Lactobacillus delbrueckii ssp. bulgaricus. Thirty-four strains were incubated in sterile 10% NDM for 12 h. Extent of proteolysis as estimated by the o-phthaldialdehyde test revealed a large variance in total proteolysis. For seven strains, trichloroacetic acid filtrates of inoculated 10% NDM were analyzed by classical ion-exchange amino acid analysis. Each strain had a distinct pattern of individual amino acid concentrations. Amino acid profiles provided information about the proteolytic activity of these strains that was not available from the o-phthaldialdehyde test. Cluster analysis, based on amino acid profiles of each strain, differentiated the seven strains beyond what was possible by visual comparison of the amino acid analysis results.
Measurement of heat-stable lipase activity in dairy products relies on methods that are slow or that cannot be used in turbid solutions, which limits their industrial value. A need exists for a rapid, simple, informative assay to detect lipase activity in dairy products. In this study, we observed that hydrolysis of p-nitrophenyl esters, monitored by reflectance colorimetry, was linearly correlated to spectrophotometry (R2 = 0.93) and release of titratable FFA (R2 = 0.92 to 0.97), indicating that chromogenic substrates were useful in determining lipase activity. However, at the concentrations reported in milk, FFA inhibited p-nitrophenyl caprylate hydrolysis, which led to an underestimation of lipase activity in milk that had previously undergone lipolysis. Milk fat also significantly reduced hydrolysis of the chromogenic substrates tested but could be accounted for by a correction equation. To demonstrate the use of the assay, lipase activity in UHT skim milk inoculated with Pseudomonas fluorescens AFT36 was followed using reflectance colorimetry and tributyrin agar. Lipase activity increased as cell numbers increased during 106 h of incubation. Extracellular lipase activity was detected after 10 h of incubation using reflectance colorimetry, but 28 h were required using tributyrin agar. Reflectance colorimetry and chromogenic substrates allowed a rapid, sensitive, and meaningful detection of esterase and lipase activity in milk.
Bart C Weimer
added 14 research items
Studies on flavor generation during longer-term ripening of cheeses have revealed at least some of the diversity of flavor compounds that lactic acid bacteria (LAB) can generate in dairy systems. These compounds can deliver positive or negative attributes, depending on their absolute and relative levels in a product and the way in which they are released on consumption. This article provides an overview of the complexity, the range, and the potential of flavor compound production by LAB, highlights the current level of knowledge of the pathways that give rise to flavor compounds from breakdown of the major milk components, and touches on the ability to manipulate them. The potential impacts of the variability of milk as a starting material and of the physiological state of the LAB in a dairy system on flavor are briefly discussed. In considering the future for LAB in flavor generation, the possibilities for diversification of flavor using artisanal strains and co-culturing are summarized, and consumer acceptance and some of the technological and commercial limitations of using LAB for flavor compound production are discussed.
Salmonella is an important cause of bacterial food-borne gastroenteritis. Salmonella encounters multiple abiotic stresses during pathogen elimination methods used in food processing, and these stresses may influence its subsequent survivability within the host or in the environment. Upon ingestion, Salmonella is exposed to gastrointestinal acidity, a first line of the host innate defense system. This study tested the hypothesis that abiotic stresses encountered during food processing alter the metabolic mechanisms in Salmonella that enable survival and persistence during subsequent exposure to the host gastrointestinal acidic environment. Out of the four different abiotic stresses tested, viz., cold, peroxide, osmotic, and acid, preadaptation of the log-phase culture to cold stress (5°C for 5 h) significantly enhanced survival during subsequent acid stress (pH 4.0 for 90 min). The gene expression profile of Salmonella preadapted to cold stress revealed induction of multiple genes associated with amino acid metabolism, oxidative stress, and DNA repair, while only a few of the genes in the above-mentioned stress response and repair pathways were induced upon exposure to acid stress alone. Preadaptation to cold stress decreased the NAD+/NADH ratio and hydroxyl (OH·) radical formation compared with those achieved with the exposure to acid stress alone, indicating alteration of aerobic respiration and the oxidative state of the bacteria. The results from this study suggest that preadaptation to cold stress rescues Salmonella from the deleterious effect of subsequent acid stress exposure by induction of genes involved in stress response and repair pathways, by modification of aerobic respiration, and by redox modulation.
Cassumunar ginger (Zingiber montanum (Koenig) Link ex Dietr.) is a native Thai herb with a high content and large variety of terpenoids in its essential oil. Improving the essential oil content and quality of cassumunar ginger is difficult for a breeder due to its clonally propagated nature. In this research, we describe the isolation and expression level of the monoterpene synthase gene that controls the key step of essential oil synthesis in this plant and evaluate the mechanical wounding that may influence the transcription level of the monoterpene synthase gene. To isolate the gene, the selected clones from DNA derived from young leaves were sequenced and analyzed and the monoterpene synthase gene from cassumunar ginger (ZMM1) was identified. The ZMM1 CDS containing 1 773 bp (KF500399) is predicted to encode a protein of 590 amino acids. The deduced amino acid sequence is 40-74% identical with known sequences of other angiosperm monoterpene synthases belonging to the isoprenoid biosynthesis C1 superfamily. A transcript of ZMM1 was detected almost exclusively in the leaves and was related to leaf wounding. The results of this research offer insight into the control of monoterpene synthesis in this plant. This finding can be applied to breeding programs or crop management of cassumunar ginger for better yield and quality of essential oil.