Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

Background  

The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30°C as compared to 37°C in E. coli K12 hosts. Since OmpT levels are higher at 37°C than at 30°C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold.     

Proteomic analysis of iron acquisition,metabolic and regulatory responses of <Emphasis Type="Italic">Yersinia pestis</Emphasis> to iron starvation

Background  

The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).     

Genome-wide screen for temperature-regulated genes of the obligate intracellular bacterium, <Emphasis Type="Italic">Rickettsia typhi</Emphasis>

Background  

The ability of rickettsiae to survive in multiple eukaryotic host environments provides a good model for studying pathogen-host molecular interactions. Rickettsia typhi, the etiologic agent of murine typhus, is a strictly intracellular gram negative α-proteobacterium, which is transmitted to humans by its arthropod vector, the oriental rat flea, Xenopsylla cheopis. Thus, R. typhi must cycle between mammalian and flea hosts, two drastically different environments. We hypothesize that temperature plays a role in regulating host-specific gene expression, allowing R. typhi to survive in mammalian and arthropod hosts. In this study, we used Affymetrix microarrays to screen for temperature-induced genes upon a temperature shift from 37°C to 25°C, mimicking the two different host temperatures in vitro.     

Cj0596 is a periplasmic peptidyl prolyl cis-trans isomerase involved in Campylobacter jejuni motility, invasion, and colonization

Background  

Campylobacter jejuni is a gastrointestinal pathogen of humans, but part of the normal flora of poultry, and therefore grows well at the respective body temperatures of 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81–176 revealed the upregulation at 37°C of Cj0596, a predicted periplasmic chaperone that is similar to proteins involved in outer membrane protein folding and virulence in other bacteria.     

Cell-associated hemolysis activity in the clinical strain of <Emphasis Type="Italic">Pseudomonas fluorescens MFN1032</Emphasis>

Background  

MFN1032 is a clinical Pseudomonas fluorescens strain able to grow at 37°C. MFN1032 cells induce necrosis and apoptosis in rat glial cells at this temperature. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides. Under laboratory conditions, this activity is not expressed at 37°C. This activity is tightly regulated and is subject to phase variation.     

Directed evolution of a filamentous fungus for thermotolerance

Background  

Filamentous fungi are the most widely used eukaryotic biocatalysts in industrial and chemical applications. Consequently, there is tremendous interest in methodology that can use the power of genetics to develop strains with improved performance. For example, Metarhizium anisopliae is a broad host range entomopathogenic fungus currently under intensive investigation as a biologically based alternative to chemical pesticides. However, it use is limited by the relatively low tolerance of this species to abiotic stresses such as heat, with most strains displaying little to no growth between 35–37°C. In this study, we used a newly developed automated continuous culture method called the Evolugator™, which takes advantage of a natural selection-adaptation strategy, to select for thermotolerant variants of M. anisopliae strain 2575 displaying robust growth at 37°C.     

Characterization of two novel heat-active α-galactosidases from thermophilic bacteria

Two genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87 kDa) and GH36bt enzyme Agal2 (57 kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3 U/mg at 80 °C, pH 6.5, and Agal2 225.4 U/mg at 60–70 °C, pH 6.5. Half-lives of 14 h (Agal1) and 39 h (Agal2) were obtained at 50 °C, and Agal1 showed half-lives of 4 and 2 h at 70 and 80 °C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-d-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides.

     

Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as Indicator Organisms in Evaluating Pathogen-Reducing Capacity in Biogas Plants

This study was conducted to identify an indicator organism(s) in evaluating the pathogen-reducing capacity of biogas plants. Fresh cow manure containing 10⁴ to 10⁵ colony forming unit (CFU) per milliliter of Escherichia coli and Enterococcus faecalis along with an inoculated Clostridium perfringens strain were exposed to 37°C for 15 days, 55°C for 48 h, and 70°C for 24 h. C. perfringens was the most heat-resistant organism followed by E. faecalis, while E. coli was the most heat-sensitive organism. E. coli was reduced below detection limit at all temperatures with log₁₀ reductions of 4.94 (10 s), 4.37 (40 min), and 2.6 (5 days) at 70°C, 55°C, and 37°C, respectively. Maximum log₁₀ reductions for E. faecalis were 1.77 at 70°C (1 day), 1.7 at 55°C (2 days) and 3.13 at 37°C (15 days). For C. perfringens, maximum log₁₀ reduction at 37°C was 1.35 log₁₀ units (15 days) compared to less than 1 unit at 55 and 70°C. Modeling results showed that E. faecalis and C. perfringens had higher amount of heat-resistant fraction than E. coli. Thus, E. faecalis and C. perfringens can be used as indicator organisms to evaluate pathogen-reducing capacity in biogas plants at high temperatures of 55°C and 70°C while at 37°C E. coli could also be included as indicator organism.     

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity

Aims

This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle.

Methods and Results

Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae‐ or Siphoviridae‐like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·000 1). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70–79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2‐like phage φMhaA1‐PHL101.

Conclusions

Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen.

Significance and Impact of the Study

This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.     

Biofilm Formation by Cryptococcus neoformans Under Distinct Environmental Conditions

Cryptococcus neoformans is an opportunistic fungal pathogen with a propensity to infect the central nervous system of immune compromised individuals causing life-threatening meningoencephalitis. Cryptococcal biofilms have been described as a protective niche against microbial predators in nature and shown to enhance resistance against antifungal agents and specific mediators of host immune responses. Based on the potential importance of cryptococcal biofilms to its survival in the human host and in nature, these studies were designed to investigate those factors that mediate biofilm formation by C. neoformans. We observed that C. neoformans preferentially grew as planktonic cells when cultured under specific conditions designed to mimic growth within host tissues (37°C, neutral pH, and ~5% CO₂) or phagocytes (37°C, acidic pH, and ~5% CO₂) and as biofilms when cultured under conditions such as those encountered in the external environment (25–37°C, neutral pH, and ambient CO₂). Altogether, our studies suggest that conditions similar to those observed in its natural habitat may be conducive to biofilm formation by C. neoformans.     

Involvement of a phospholipase C in the hemolytic activity of a clinical strain of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Background  

Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions.     

The effect of experimental warming on leaf functional traits, leaf structure and leaf biochemistry in Arabidopsis thaliana

Background  

The leaf is an important plant organ, and how it will respond to future global warming is a question that remains unanswered. The effects of experimental warming on leaf photosynthesis and respiration acclimation has been well studied so far, but relatively little information exists on the structural and biochemical responses to warming. However, such information is very important to better understand the plant responses to global warming. Therefore, we grew Arabidopsis thaliana at the three day/night temperatures of 23/18°C (ambient temperature), 25.5/20.5°C (elevated by 2.5°C) and 28/23°C (elevated by 5°C) to simulate the middle and the upper projected warming expected within the 21st century for this purpose.     

<Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>diversity viewed as a tissue-specific coevolutionary arms race

Background  

The genomes of pathogens are thought to have evolved under selective pressure provided by the host in a coevolutionary arms race (the 'Red Queen's Hypothesis'). Traditionally, adaptation by pathogens is thought to rely not on whole chromosome dynamics but on gain/loss of specific genes, yielding differential abilities to infect distinct tissues. Thus, it is not known whether distinct host organs differently shape the genome of the same pathogen. We tested this hypothesis using Chlamydia trachomatis as model species, looking at 15 serovars that infect different organs: eyes, genitalia and lymph nodes.     

The upper thermal tolerance for a Texas population of the hairy maggot blow fly Chrysomya rufifacies Macquart (Diptera: Calliphoridae)

1. The hairy maggot blow fly (Chrysomya rufifacies: Macquart) is an invasive necrophagous fly found throughout the continental United States. Chrysomya rufifacies is of medical/veterinary, forensic, and ecological importance due to its ability to cause myiasis, colonise human remains, and displace native Diptera. However, little is known about their upper thermal tolerance, which could be used to better predict their invasion potential.
2. We investigated the upper thermal tolerance of C. rufifacies exposed to different temperatures (20–45 °C), times (1–6 h), and nutrients (no food or water, water only, or a food-water mixture) for both sexes and two age ranges (young = 6–8 days post pupal emergence; old = 9–11 days post pupal emergence).
3. As temperature or duration increased, the probability of knockdown increased (0–100% at 20 and 45 °C and from 41 to 75% at 1 and 6 h), while the probability of survival decreased (99–2% at 20 and 45 °C and from 75 to 28% at 1 and 6 h). The availability of nutrients increased thermal tolerance at moderate temperatures (40 and 42 °C). Female flies were more thermally tolerant than males (probability of knockdown = 49% vs 58%; probability of survival = 58% vs 46%). Thermal tolerance did not differ by age.
4. These data reveal details about the upper thermal tolerance for a single population of C. rufifacies, and suggest that environmental and organismal factors ought to be considered in order to make meaningful predictions about the invasion potential of C. rufifacies in North America.

     

Differential changes of regional cerebral blood flow in two bat species during induced hypothermia measured by perfusion-weighted magnetic resonance imaging

Cerebral blood flow (CBF) of a vespertilionid bat, Miniopterus fuliginosus (M. f.), and a pteropodid bat, Rousettus leschenaultii (R. l.) was measured non-invasively during induced hypothermia (37–10°C for M. f. and 37–24°C for R. l.) with perfusion-weighted magnetic resonance imaging. In both species, the average CBF was found to decrease with rectal temperature. The patterns of hypothermia-induced regional CBF changes, however, were different between the two species. In the pteropodid bat, the extent of CBF decrease at lower rectal temperature was similar in the cortex and thalamus, resulting in an unchanged thalamus/cortex CBF ratio. In contrast, the thalamus/cortex CBF ratio in the vespertilionid species increased progressively with decreasing rectal temperature (1.52 ± 0.14 at 37 ± 1°C vs. 2.28 ± 0.29 at 10 ± 1°C). These results suggest that the manner in which the two bat species cope with low body temperature may be reflected by a differential CBF regulation between thalamus and cortex.     

Isolation and characterization of a new <Emphasis Type="Italic">Bacillus</Emphasis> sp. 50-3 with highly alkaline keratinase activity from <Emphasis Type="Italic">Calotes versicolor</Emphasis> faeces

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkali-stable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.