Microbial Biochemistry

На основі аналізу літературних джерел встановлено, що фізіолого-біохімічна характеристика видів D.longreachensis та D.termitidis дуже схожа. Для видової ідентифікації штамів Desulfovibrio sp. NUChC SRB1 та Desulfovibrio sp. NUChC SRB2 слід в першу чергу звернути увагу на прості та доступні дослідження, а саме дослідження використання ними вуглеводів як донорів електронів, нітрату як акцептора електронів.

Growing tendrils of aflagellate hag mutants of Bacillus subtilis were found to show an avoidance response when colonizing a semi-solid medium, suggesting a tip-totip communication mechanism between colonies. There may be a second sensing mechanism involved in shaping the morphology of tendrils. Tendril growth in B. subtilis was dependent on and possibly shaped by the release of surfactin, a biosurfactant. Transposon mutagenesis yielded two mutants with 'touching' tendrils, and each had a disrupted gspA gene that encodes a putative glycosyltransferase. Tendrils of gspA mutants, unlike the parental strain, were unresponsive to tendril tip growth by surfactin, suggesting disruption of intercellular signaling. Tendril sensing and avoidance could be physiologically relevant in habitats, such as plant roots, where some limiting nutrient might induce this type of multicellular behavior, promoting avoidance of previously explored areas by sibling colonies.

Although biogeochemical models designed to simulate carbon (C) and nitrogen (N) dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically (13)C-labeled glucose at three temperatures: +5 (control), -5, and -20°C. (13)C was quantified in CO2, bulk soil, microbial biomass, and dissolved organic carbon (DOC) after 1, 3, and 10 days and also after 30 days for samples at -20°C. Compared to +5°C, CO2 decreased 3- and 10-fold at -5 and -20°C, respectively. High (13)C recovery in CO2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5°C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis...

Growing tendrils of aflagellate hag mutants of Bacillus subtilis were found to show an avoidance response when colonizing a semi-solid medium, suggesting a tip-totip communication mechanism between colonies. There may be a second sensing mechanism involved in shaping the morphology of tendrils. Tendril growth in B. subtilis was dependent on and possibly shaped by the release of surfactin, a biosurfactant. Transposon mutagenesis yielded two mutants with 'touching' tendrils, and each had a disrupted gspA gene that encodes a putative glycosyltransferase. Tendrils of gspA mutants, unlike the parental strain, were unresponsive to tendril tip growth by surfactin, suggesting disruption of intercellular signaling. Tendril sensing and avoidance could be physiologically relevant in habitats, such as plant roots, where some limiting nutrient might induce this type of multicellular behavior, promoting avoidance of previously explored areas by sibling colonies.

Growing tendrils of aflagellate hag mutants of Bacillus subtilis were found to show an avoidance response when colonizing a semi-solid medium, suggesting a tip-totip communication mechanism between colonies. There may be a second sensing mechanism involved in shaping the morphology of tendrils. Tendril growth in B. subtilis was dependent on and possibly shaped by the release of surfactin, a biosurfactant. Transposon mutagenesis yielded two mutants with 'touching' tendrils, and each had a disrupted gspA gene that encodes a putative glycosyltransferase. Tendrils of gspA mutants, unlike the parental strain, were unresponsive to tendril tip growth by surfactin, suggesting disruption of intercellular signaling. Tendril sensing and avoidance could be physiologically relevant in habitats, such as plant roots, where some limiting nutrient might induce this type of multicellular behavior, promoting avoidance of previously explored areas by sibling colonies.

The principal objective of the present research work was to determine the antibacterial potential of A. marmelos against four standard pathogenic bacterial strains. To evaluate antibacterial activity the agar-well diffusion assay was used. All the four extracts showed the highest and significant antibacterial activity against both Gram negative and Gram-positive bacteria. It is the strain Bacillus subtilis that is almost resistant to the four extracts of A. marmelos.

The injluence on lipase activity in water of a pretreatment on Candida rugosa lipase using water miscible and immiscible solvents was studied. The lipase activity in the hydrolysis of esteric substrates in aqueous media increases when the lipase was previously treated with various nearly anhydrous organic media. This activation, which was irreversible, was higher for longer pretreatment times. It was dependent on the pretreatment medium (water activity and solvent used). A relation between variations in the emission intensity and the activities of treated and untreated lipases was found. Activating pretreatment did not shift the peak offluorescence emission but gave rise to variations in the secondary protein structure by increasing the helical nature. A similar increment in the hydrolysis rate in water can be obtained with the addition of an appropriate amount of solvent (acetonitrile or n-heptane) to the aqueous reaction medium.

Although biogeochemical models designed to simulate carbon (C) and nitrogen (N) dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically 13 C-labeled glucose at three temperatures: +5 (control), −5, and −20 • C. 13 C was quantified in CO 2 , bulk soil, microbial biomass, and dissolved organic carbon (DOC) after 1, 3, and 10 days and also after 30 days for samples at −20 • C. Compared to +5 • C, CO 2 decreased 3-and 10-fold at −5 and −20 • C, respectively. High 13 C recovery in CO 2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5 • C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis. A threefold higher 13 C recovery in microbial biomass at −5 than +5 • C points to synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Less than 0.4% of 13 C was recovered in DOC after 1 day, demonstrating complete glucose uptake by microorganisms even at −20 • C. Consequently, we attribute the fivefold higher extracellular 13 C in soil than in microbial biomass to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. The knowledge of sustained metabolism at subzero temperatures will not only be useful for modeling global C dynamics in ecosystems with periodically or permanently frozen soils, but will also be important in understanding and controlling the adaptive mechanisms of food spoilage organisms.

Previously, extracellular vesicle production in Grampositive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harbouring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin.

Background: Enzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent. The clear understanding of why these preparations show higher catalytic activity than lyophilized powders in organic solvents is not available.

Multicellular communities produced by Bacillus subtilis can adopt sliding or swarming to translocate over surfaces. While sliding is a flagellum-independent motility produced by the expansive forces in a growing colony, swarming requires flagellar functionality and is characterized by the appearance of hyperflagellated swarm cells that associate in bundles or rafts during movement. Previous work has shown that swarming by undomesticated B. subtilis strains requires swrA, a gene that upregulates the expression of flagellar genes and increases swimming motility, and surfactin, a lipopeptide biosurfactant that also facilitates sliding. Through an analysis of swrA ؉ and swrA mutant laboratory strains with or without a mutation in sfp (a gene involved in surfactin production), we show that both swrA and surfactin upregulate the transcription of the flagellin gene and increase bacterial swimming. Surfactin also allows the nonswarming swrA mutant strain to efficiently colonize moist surfaces by sliding. Finally, we reconfirm the essential role of swrA in swarming and show that surfactin, which increases surface wettability, allows swrA ؉ strains to produce swarm cells on media at low humidity.

In Erwinia carotovora subspecies, N-acyl homoserine lactone (AHL) controls the expression of various traits, including extracellular enzyme/protein production and pathogenicity. We report here that E. carotovora subspecies possess two classes of quorum-sensing signaling systems defined by the nature of the major AHL analog produced as well as structural and functional characteristics of AHL synthase (AhlI) and AHL receptor (ExpR). Class I strains represented by E. carotovora subsp. atroseptica strain Eca12 and E. carotovora subsp. carotovora strains EC153 and SCC3193 produce 3-oxo-C8-HL (N-3-oxooctanoyl-L-homoserine lactone) as the major AHL analog as well as low but detectable levels of 3-oxo-C6-HL (N-3-oxohexanoyl-L-homoserine lactone). In contrast, the members of class II (i.e., E. carotovora subsp. betavasculorum strain Ecb168 and E. carotovora subsp. carotovora strains Ecc71 and SCRI193) produce 3-oxo-C6-HL as the major analog. ExpR species of both classes activate rsmA (Rsm, repressor of secondary metabolites) transcription and bind rsmA DNA. Gel mobility shift assays with maltose-binding protein (MBP)-ExpR 71 and MBP-ExpR 153 fusion proteins show that both bind a 20-mer sequence present in rsmA. The two ExpR functions (i.e., expR-mediated activation of rsmA expression and ExpR binding with rsmA DNA) are inhibited by AHL. The AHL effects are remarkably specific in that expR effect of EC153, a strain belonging to class I, is counteracted by 3-oxo-C8-HL but not by 3-oxo-C6-HL. Conversely, the expR effect of Ecc71, a strain belonging to class II, is neutralized by 3-oxo-C6-HL but not by 3-oxo-C8-HL. The AHL responses correlated with expR-mediated inhibition of exoprotein and secondary metabolite production.

N-acyl homoserine lactone (AHL) is required by Erwinia carotovora subspecies for the expression of various traits, including extracellular enzyme and protein production and pathogenicity. Previous studies with E. carotovora subsp. carotovora have shown that AHL deficiency causes the production of high levels of RsmA, an RNA binding protein that functions as a global negative regulator of extracellular enzymes and proteins and secondary metabolites (Rsm, regulator of secondary metabolites). We document here that ExpR, a putative AHL receptor belonging to the LuxR family of regulators, activates RsmA production. In the absence of AHL, an ExpR E. carotovora subsp. carotovora strain compared to its ExpR mutant, produces higher levels of rsmA RNA and better expresses an rsmA-lacZ transcriptional fusion. Moreover, the expression of the rsmA-lacZ fusion in Escherichia coli is much higher in the presence of expR 71 (the expR gene of E. carotovora subsp. carotovora strain Ecc71) than in its absence. We also show that purified preparation of MBP-ExpR 71 binds (MBP, maltose binding protein) rsmA DNA. By contrast, MBP-ExpR 71 does not bind ahlI (gene for AHL synthase), pel-1 (gene for pectate lyase), or rsmB (gene for regulatory RNA that binds RsmA), nor does ExpR 71 activate expression of these genes. These observations strongly suggest transcriptional activation of rsmA resulting from a direct and specific interaction between ExpR 71 and the rsmA promoter. Several lines of evidence establish that N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HL), the major AHL analog produced by E. carotovora subsp. carotovora strain Ecc71, inhibits ExpR 71-mediated activation of rsmA expression. These findings for the first time establish that the expR effect in E. carotovora subsp. carotovora is channeled via RsmA, a posttranscriptional regulator of E. carotovora subspecies, and AHL neutralizes this ExpR effect.

In Erwinia carotovora subspecies, N-acyl homoserine lactone (AHL) controls the expression of various traits, including extracellular enzyme/protein production and pathogenicity. We report here that E. carotovora sub-species possess two classes of quorum-sensing signaling systems defined by the nature of the major AHL analog produced as well as structural and functional characteristics of AHL synthase (AhlI) and AHL receptor (ExpR). Class I strains represented by E. carotovora subsp. atroseptica strain Eca12 and E. carotovora subsp. carotovora strains EC153 and SCC3193 produce 3-oxo-C8-HL (N-3-oxooctanoyl-L-homoserine lactone) as the major AHL analog as well as low but detectable levels of 3-oxo-C6-HL (N-3-oxohexanoyl-L-homoserine lactone). In contrast, the members of class II (i.e., E. carotovora subsp. betavasculorum strain Ecb168 and E. carotovora subsp. carotovora strains Ecc71 and SCRI193) produce 3-oxo-C6-HL as the major analog. ExpR species of both classes activate rsmA (Rsm, repressor of secondary metabolites) transcription and bind rsmA DNA. Gel mobility shift assays with maltose-binding protein (MBP)-ExpR 71 and MBP-ExpR 153 fusion proteins show that both bind a 20-mer sequence present in rsmA. The two ExpR functions (i.e., expR-mediated activation of rsmA expression and ExpR binding with rsmA DNA) are inhibited by AHL. The AHL effects are remarkably specific in that expR effect of EC153, a strain belonging to class I, is counteracted by 3-oxo-C8-HL but not by 3-oxo-C6-HL. Conversely, the expR effect of Ecc71, a strain belonging to class II, is neutralized by 3-oxo-C6-HL but not by 3-oxo-C8-HL. The AHL responses correlated with expR-mediated inhibition of exoprotein and secondary metabolite production.

Multicellular communities produced by Bacillus subtilis can adopt sliding or swarming to translocate over surfaces. While sliding is a flagellum-independent motility produced by the expansive forces in a growing colony, swarming requires flagellar functionality and is characterized by the appearance of hyperflagellated swarm cells that associate in bundles or rafts during movement. Previous work has shown that swarming by undomesticated B. subtilis strains requires swrA, a gene that upregulates the expression of flagellar genes and increases swimming motility, and surfactin, a lipopeptide biosurfactant that also facilitates sliding. Through an analysis of swrA ؉ and swrA mutant laboratory strains with or without a mutation in sfp (a gene involved in surfactin production), we show that both swrA and surfactin upregulate the transcription of the flagellin gene and increase bacterial swimming. Surfactin also allows the nonswarming swrA mutant strain to efficiently colonize moist surfaces by sliding. Finally, we reconfirm the essential role of swrA in swarming and show that surfactin, which increases surface wettability, allows swrA ؉ strains to produce swarm cells on media at low humidity.

Abstract : The present investigation is part of continuing programme related to the phytochemical screening of
Aegle marmelos and effect of antibiotics mixing with the Aegle marmelos (herbal extracts) their enhanced
potential on microbes. In the world the local plants used in Ancient Indian Medicine, Ayurveda, Siddha and
Yunani, in several countries including India several plant species are administered orally to control the various
diseases. Some of these plants have been pharmacologically provided to be of some value and may be a popular
remedy for the treatment of disease.
From the very beginning of human existence, man has familiarized himself with medicinal plants and
used them in a variety of ways throughout the ages. In search of food and to cope successfully with human
suffering, primitive man began to distinguish those plants suitable for nutritional purpose from others with
definitive pharmacological action. This relationship has grown between plants and man, and many plants came
to be used as drugs. The growth of knowledge to cure disease continues at an accelerating pace, and number of
new plant-derived drugs increase likewise. Herbal medicine is currently experiencing a revival in Western
society, along with other complementary therapies such as traditional Chinese Medicines, Osteopathy and
Homeopathy. In this context, the present study is the first milestone with particular emphasis on the application
of Aegle marmelos the medicinal plant and their effect in mixing with antibiotics for their better formulation
and controlling the various diseases in near future.
Keywords: Aegle marmelos, S.aureus, S.epidermis, E.coli, B.subtilis, Roxythromycin,Cefixime, Levofloxacin,
Methanol.

Bacillus amyloliquefaciens C06, an effective biological agent in controlling brown rot of stone fruit caused by Monilinia fructicola, was also found to produce extra-cellular mucilage and form mucoid colonies on semisolid surfaces. This study aimed to characterize the extra-cellular mucilage produced by B. amyloliquefaciens C06 using transposon mutagenesis and biochemical and physical analyses. The mucilage production in B. amyloliquefaciens C06 was demonstrated to be associated with ywsC gene expression and characterized to be of high molecular weight, consisted of only glutamic acid and linked with non-peptide bonds, thus identified as γ-polyglutamic acid (γ-PGA). Compared with wild type B. amyloliquefaciens C06, its mutants deficient in producing γ-PGA, e.g. M106 and C06ΔywsC showed less efficiency in biofilm formation, surface adhesion and swarming ability. It was also demonstrated that γ-PGA was not essential for C06 to form colony on semi-solid surfaces, but was able to improve its colony structure. In vivo evaluation showed that disruption of γ-PGA production in C06ΔywsC impaired its efficiency of colonizing apple surfaces.

Growing tendrils of aflagellate hag mutants of Bacillus subtilis were found to show an avoidance response when colonizing a semi-solid medium, suggesting a tip-totip communication mechanism between colonies. There may be a second sensing mechanism involved in shaping the morphology of tendrils. Tendril growth in B. subtilis was dependent on and possibly shaped by the release of surfactin, a biosurfactant. Transposon mutagenesis yielded two mutants with 'touching' tendrils, and each had a disrupted gspA gene that encodes a putative glycosyltransferase. Tendrils of gspA mutants, unlike the parental strain, were unresponsive to tendril tip growth by surfactin, suggesting disruption of intercellular signaling. Tendril sensing and avoidance could be physiologically relevant in habitats, such as plant roots, where some limiting nutrient might induce this type of multicellular behavior, promoting avoidance of previously explored areas by sibling colonies.

Highly active Pseudomonas aeruginosa lipase protein-coated microcrystals (PAL
PCMC) have been prepared by immobilization of protein onto K2SO4 as excipient solid
support carrier and n-propanol as precipitating solvent. Transmission electron micrographs
confirmed the formation of PAL PCMC. These PCMC were found to be a catalytically
more active and stable preparation for p-nitrophenyl palmitate hydrolysis in n-heptane,
compared to free lipase. The Vmax, Km, and temperature optimum for PAL PCMC increased
from 0.49 to 5.66 nmol min−1 mg−1, 589 to 679.8 mmol, and 40°C to 45°C, respectively.
These were thermally more stable with 4.65, 2.56, and 1.24-fold improvement in half lives
at 45°C, 55°C, and 60°C compared to free P. aeruginosa PseA lipase. Their catalytic
efficiency was enhanced by tenfold over that of free enzyme. PAL PCMC offer a simple
and effective technique for obtaining stable and efficient lipase preparation for biocatalysis
in nonaqueous medium.