Biochemical and biophysical characterisation of haloalkane dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in growth on haloalkanes

Haloalkane dehalogenases (HLDs) catalyse the hydrolysis of haloalkanes to alcohols, offering a biological solution for toxic haloalkane industrial wastes. Hundreds of putative HLD genes have been identified in bacterial genomes, but relatively few enzymes have been characterised. We identified two novel HLDs in the genome of Mycobacterium rhodesiae strain JS60, an isolate from an organochlorine‐contaminated site: DmrA and DmrB. Both recombinant enzymes were active against C2–C6 haloalkanes, with a preference for brominated linear substrates. However, DmrA had higher activity against a wider range of substrates. The kinetic parameters of DmrA with 4‐bromobutyronitrile as a substrate were K_m = 1.9 ± 0.2 mM, k_cat = 3.1 ± 0.2 s^?1. DmrB showed the highest activity against 1‐bromohexane. DmrA is monomeric, whereas DmrB is tetrameric. We determined the crystal structure of selenomethionyl DmrA to 1.7 Å resolution. A spacious active site and alternate conformations of a methionine side‐chain in the slot access tunnel may contribute to the broad substrate activity of DmrA. We show that M. rhodesiae JS60 can utilise 1‐iodopropane, 1‐iodobutane and 1‐bromobutane as sole carbon and energy sources. This ability appears to be conferred predominantly through DmrA, which shows significantly higher levels of upregulation in response to haloalkanes than DmrB.     

Biochemical predictors for Sars-Cov-2 severity

It is of interest to assess the inflammatory marker profile in SARS-CoV-2 patients and to correlate the levels of systemic inflammatory biomarkers such as neutrophil-to-lymphocyte ratio (NLR), C-Reactive Protein CRP, Ferritin, Creatine kinase (CK), Lactate dehydrogenase (LDH) and liver function analytes total serum proteins, albumin, total bilirubin, direct bilirubin, alkaline phosphatase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) with the severity of SARS-CoV-2 infections. A total of 1000 COVID-19 positive patient''s data were collected. Laboratory assessments consisted of NLR (neutrophil-lymphocyte ratio) by cell counter, C Reactive Protein (CRP) by immunoturbidimetry, Ferritin by electrochemiluminescence (ECLIA) and Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin, Direct Bilirubin, Total Protein and Albumin by spectrophotometry. The mean plasma CRP levels, NLR, ferritin, CK and LDH levels were higher in severe cases than in non-severe cases, and the difference was statistically significant (p<0.05). All liver function tests such as the total and direct bilirubin, AST, ALT, ALP, total protein and albumin were higher in severe patients than non-severe patients and the difference was statistically significant (p<0.05). Data indicate that NLR, CRP, Ferritin, CK, LDH and liver function analytes have a crucial role as prognostic markers for SARS-CoV-2 infections and hence should be routinely recommended for risk assessment and stratification of the patients to reduce the associated morbidity and mortality.     

Osmotic adjustment and the accumulation of organic solutes in whole cells and protoplasts of Saccharomyces cerevisiae

In the presence of a suitable carbon source, whole cells and protoplasts of Saccharomyces cerevisiae synthesized glycerol as a compatible organic solute in response to increased external osmotic pressure. Boyle-van't Hoff plots showed that protoplasts, and non-turgid cells, exhibited a linear relationship between volume and the external osmotic pressure (i.e. they behaved as near-ideal osmometers), and that both protoplasts and cells have a component which is not osmotically responsive--the non-osmotic volume (NOV). Glycerol levels in whole cells and protoplasts were elevated by increased external osmotic pressure over a similar time-scale to the period of exponential cell growth, reaching a maximum value at 6-12 h and declining thereafter. This suggests that the restoration of turgor pressure in whole cells was not the sole regulator of glycerol accumulation. Stationary phase whole cells had negligible levels of intracellular glycerol after growth in a medium of raised osmotic pressure. However, intracellular trehalose synthesis in these cells began earlier and reached a higher maximum level than in basal medium. Once exponential growth had stopped, cell turgor and internal osmotic pressure decreased somewhat. These new, lower values may be determined by the extent of trehalose accumulation in stationary phase cells.     

Copper adsorption by Rhizopus arrhizus,Cladosporium resinae and Penicillium italicum

Summary Copper adsorption by Rhizopus arrhizus, Cladosporium resinae and Penicillium italicum was studied using a copper-selective electrode. Copper adsorption by C. resinae and P. italicum obeyed the Freundlich and Langmuir isotherms for single-layer adsorption whereas R. arrhizus followed the BET isotherm for multi-layer adsorption. Temperature had little effect on adsorption over the range 4–25°C. Mineral acids were effective for desorption of copper from preloaded biomass, the efficiency of desorption increasing with decreasing pH. Other cations were also capable of copper desorption with zinc showing the greatest efficiency and sodium the lowest.     

Kinetics of uptake and intracellular location of cobalt,manganese and zinc in the estuarine green alga Chlorella salina

Summary The uptake kinetics and intracellular location of cobalt (⁶⁰Co), manganese (⁵⁴Mn) and zinc (⁶⁵Zn) have been characterized in Chlorella salina. Uptake of all three metals was biphasic. The initial rapid phase was independent of light, temperature or the presence of metabolic inhibitors. This first phase of metabolism-independent biosorption was followed by a slower phase of uptake that was apparently dependent on metabolism and decreased by incubation in the dark, or in the light at low temperature or in the presence of metabolic inhibitors. This latter phase of metal accumulation followed Michaelis-Menten kinetics. However, when expressed in the form of a Lineweaver-Burk plot two distinct phases were apparent for each metal with the following K_m values (M); Co²⁺, 19 and 266; Mn²⁺, 2 and 760; Zn²⁺, 4 and 635. For all three metals cellular compartmentation analysis showed that large amounts of metal were bound to intracellular components and to the cell wall. There was also a higher concentration of each metal in the vacuole than in the cytosol, indicating transport of the metals across the tonoplast which may, in part, account for the multi-phasic uptake systems detected. The influence of competing divalent ions on the active uptake of Co²⁺ and Mn²⁺ was also studied. When the concentration of divalent ion was the same as that of Co²⁺ the uptake of the latter was not affected, indicating a specific system for the uptake of Co²⁺. However, Mn²⁺ uptake inhibited by Mg²⁺, Zn²⁺ and Cd²⁺, but not by Co²⁺, which indicated that Mn²⁺, Mg²⁺ and Cd²⁺ may enter the cells via a common system with different affinities for each metal.     

The binding of complement component C3 to antibody-antigen aggregates after activation of the alternative pathway in human serum.

Preformed immune aggregates, containing antigen and either IgG (immunoglobulin G) or F(ab')2 rabbit antibody, were incubated with normal human serum under conditions allowing activation of only the alternative pathway of complement. Both the IgG and F(ab')2 immune aggregates bound C3b, the activated form of the complement component C3, in a similar manner, 2-3% of the C3 available in the serum being bound to the aggregates as C3b, and the rest remaining in the fluid phase as inactive C3b or uncleaved C3. It was found that the C3b was probably covalently bound to the IgG in the aggregates, since C3b-IgG complexes could be demonstrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, after repeated washing with buffers containing high salt or boiling under denaturing conditions. Incubation of the C3b-antibody-antigen aggregates in buffers known to destroy ester linkages had little effect on the C3b-IgG complexes, which suggested that C3b and IgG might be linked by an amide bond. Two main types of C3b-IgG complexes were found that had apparent mol.wts. of 360000 and 580000, corresponding to either one to two C3b molecules respectively bound to one molecule of antibody. On reduction of the C3b-IgG complexes it was found that the beta-chain, but not the alpha'-chain, of C3b was released along with all the light chain of IgG but only about half or less of the heavy chain of IgG. These results indicate that, during activation of the alternative pathway of complement by immune aggregates containing IgG antibody, the alpha'-chain of C3b may become covalently bound at one or two sites in the Fd portion of the heavy chain of IgG.     

Changes in cytochrome levels during growth of the yeast-like fungusAureobasidium pullulans

Cytochromes ofAureobasidium pullulans have been identified and partially characterized using low-temperature and carbon-monoxide-difference spectroscopy. The presence ofa-,b-, andc-type cytochromes is demonstrated, as are other unidentified redox components. During exponential growth in batch culture, cytochrome levels showed complex changes. Changes in respiration rates and in the levels of cytochromea+a ₃ closely paralleled cellular growth: both increased exponentially until stationary phase, when no further increase occurred. Theb- andc-type cytochromes showed biphasic increases, initially doubling every, generation time and then increasing more slowly during the stationary phase. Sensitivity of respiration to 100M potassium cyanide gradually decreased during exponential growth, falling from virtually 100% inhibition after about 20 h growth to 30% inhibition in the stationary phase. The results suggest that in stationary-phase cultures, an alternative cyanide-insensitive but salicylhydroxamic-acid-sensitive terminal oxidase also operates.     

A phytohaemagglutinin from the Azolla-anabaena symbiosis

Proteins recovered from cell-free extracts of the Azolla-Anabaena azollae symbiosis exhibited haemagglutination activity; galactose was the most effective carbohydrate tested in preventing haemagglutination. Extracts of cyanobacteria-free Azolla also caused haemagglutination but extracts of free-living or symbiotic Anabaena azollae did not. Symbiotic Azolla plants grown on NO₃^? showed lower haemagglutination activity than did those grown on N₂; activity increased on removal of NO₃^?. The lower activity of the NO₃^?-grown material may be due to NO₃su? exerting a direct effect on lectin activity/synthesis, or it may act indirectly by inhibiting the development of Anabaena which in turn affects the Azolla lectin. The purified lectin was shown to be composed of 6 sub-units, each of M.W. 21000.