Slow Growers Research Articles

DIVERSITY OF NODD GENE OF SOME RHIZOBIUM ISOLATES FROM DIFFERENT LEGUMES.

This study was conducted to 1) isolate and characterize some rhizobia from some legumes, 2) determine the phylogenetic relationships among them; and 3) investigate their potential biological control agent. 24 Rhizobial isolates were obtained from five legumes namely; faba bean, lentil, pea, clover, and soybean. Morphological characterization classified isolates into fast grower and slow grower. Also, their biochemical profiles using API 20E and API 20NE system showed large diversity which may reflect their adaptation in different environments. Moreover, molecular detection by 16S rDNA gene was able to characterize 19 of them to species level. Isolates from pea, faba bean, clover and lentil were Rhizobium leguminosarum and isolates from soybean were Bradyrhizobium japonicum. This data supported the idea that there is a narrow diversity of species of rhizobia in Egypt. Phylogenetic analysis of the 19 isolates confirmed that the Bradyrhizobium japonicum isolates were phylogenetically divergent from all other isolates. Also, the phylogram revealed that each group of isolates originated from root nodule of a certain legume formed a separate sub-cluster. Besides a narrow range of interspecies variations was found which is consistent with the idea of biovars presence among species. In addition, the 24 isolates showed strong inhibition of growth of Fusarium and Rhizoctonia phytopathogens. However, results indicated that 25% of isolates were not able to solubilize tricalcium phosphate. On the other hand, the IAA production by the 24 isolates varied greatly among isolates. Therefore, these results demonstrated that they may have the potential to act as biocontrol agents. Moreover, we characterize Nod D gene on the molecular level. Upon its isolation its size came to be 820 bp of nucleotides. This came to be true for all bacterial isolates except four isolates we did not succeed to isolate NodD gene. Moreover we used Quantitative Real Time PCR to study NodD gene expression. Seven nod D gene were down regulated and five were up regulated than 16S housekeeping gene.

Role of Phenotypic Switching in Stability and Persistence of Pseudomonas aeruginosa Biofilms

Objectives: Objective: This study was designed to explore the role of different phenotypes of P. aeruginosa in the development, stability and persistence of biofilm. Methods: A total of seventeen (17) waterborne biofilm producing strains of P. aeruginosa were studied. These isolates were identified on the basis of typical phenotypic characters and the tube method was used for the study of biofilms. Population and phenotypic variance were studied by the drop plate method. The hydrophobicity of strains was evaluated by the bacterial adhesion to apolar solvent test. Results: Study showed that the subject isolates of P. aeruginosa adopted a biofilm life style after 36 h of incubation at 35 °C. After 24 h the adhesion started, but it was reversible and easily dispersed by simple washing. However, after 36 h the irreversible adhesion was noticed. The biofilm consortia harbor three different phenotypes: i. wild types, showed typical P. aeruginosa characters on Cetrimide agar; ii. Slow growers, showed poor pigmentation and take >36 h for colony development, and iii. Small colony variants (SCVs) are metabolically inactive and producing pinpointed non pigmented colonies. Comparative analysis showed that these phenotypes i.e. SCVs were highly hydrophobic and persistent in biofilm consortia due to the production of excessive amounts of exopolysaccharides. Conclusions: This study showed that phenotypic heterogeneity is a characteristic feature of P. aeruginosa biofilms and all of these phenotypes have a major role in stability and persistence of biofilm consortia. J Microbiol Infect Dis 2020; 10(1):10-17.

Biochemical and physiological characterization of Bradyrhizobium japonicum

Soybean [Glycine max (L.) Merrill] is cultivated intensively in India. Rhizobium sp. associated with it derives nitrogen (N) by biological nitrogen fixation under field conditions. Out of total 25 isolated sp. from functional root nodules of soybean plants on congo-red yeast extract mannitol agar medium, 20 slow grower Bradyrhizobium sp. and 5 fast grower Rhizobium sp. were found on growing YEMA media supplemented with bromothymol blue. The purified strains were aerobic, gram negative, non-spore forming and motile rods. The optimum growth kinetics for both sp. was 35 °C at neutral pH (7.0). Both fast and slow growing rhizobia were found to be positive for oxidase, KOH, starch hydrolysis, nitrate reduction and catalase activity. Among all the 25 isolates tested, 23 isolates showed positive reaction to gelatin hydrolysis except Bj-11 and Bj-21 while, 21 isolates were reacted positive to H2S production whereas, the four isolates viz., Bj-1, Bj-2, Bj-8 and Bj-23 were negative in reaction. While all the samples were found negative for IAA production. Pot culture experiment revealed that seed inoculation with these isolates recorded significant improvement in nodule number and plant height ranged from 3.00 to 22.00 nodules per plant and 35.00 to 65.00 cm respectively at flowering stage over uninoculated control. Isolates viz., Bj-9, Bj-10, Bj-14, Bj-17, Bj-19, Bj-23, Bj-25 could produce large and pink nodules indicating their effective symbiosis with soybean.

Candida auris real-time PCR for surveillance of patients and environment

Background– Candida aurisis an emerging fungus which has caused nosocomial outbreaks worldwide. Screening for surveillance is challenging as it’s a slow grower, samples may have multiple candida spp and many commercial assays have limited accuracy. Following an outbreak in 2016, a real-time PCR was developed for rapid patient and environmental screening. Laboratory parameters, results and pitfalls from Jan 2018 to March 2019 are described. Methods– Evaluation was performed using EQA panels and validated on clinical swabs and urine samples. Patient screening was performed in high risk clinical areas from nose, throat, axilla and groin swabs and catheter urine. Environmental screening was done in areas where transmission was detected. Positive or indeterminate samples were cultured and colonies identified on the Vitek 2 and confirmed by PHE reference laboratory. Results–The Limit of detection was 100 organisms/ml. Sensitivity, specificity, PPV and NPV were 100.00%, 99.35%, 85.71%, and 100.00%. 4,792 urines and 10,819pooled swabs were tested. 21 colonised patients were identified in two outbreaks and two isolated transmission events. Median turn-around time for positive results was 24h30min (range 04:22-96:46h) and 143h14min (range 75-406h) for PCR and culture respectively. Urine samples did not add to the overall sensitivity of screening.C. aurisDNA was detected in 30/84 (35%) environmental swabs but C. auris isolated in only 2/84. Drawbacks of the PCR include reagent contamination seen on two occasions. Conclusion – C. auris screening by PCR is a rapid means of detection in both patients and environment but prudent use of culture is required to help determine infectivity.

Physiological processes modulate acute and chronic responses to dietary protein/energy ratio fluctuations in individuals and families of Manila clam (Ruditapes philippinarum) selected for variable growth rates

A range of phenotypes differing in growth rate were designed in the Manila clam by combining separate breeding families with size segregation within each family to constitute fast and slow growing groups. Physiological components of the energy budget and scope for growth (SFG) were then compared between these different phenotypes during the acute and chronic responses to two diets that were isocaloric but differed by 3-fold in their protein/energy (P/E) ratios. Both diets were based on the microalgae Rhodomonas lens obtained in either the exponential or the stationary phase of culture. The aims of the study were 1) to test the effects of these changes in food composition on growth rate, estimated as the balance of physiological processes of energy gain and loss integrated in the SFG; and 2) to assess the extent to which physiological adjustments to diet composition are modulated in order to fulfill the variable energy requirements posed by the occurrence of differential growth phenotypes. Growth performance improved with the high-protein (N+) diet for the different family * growth group combinations, with SFG values exceeding by 50% on average the values of the low-protein (N-) diet. Digestive constraints resulted in reduced absorption efficiency with the N-diet, which tended to cancel out the potential benefits of adjusting feeding rates in order to compensate for a low protein ration. Endogenous differences in growth rate associated with segregated phenotypes were mainly accounted for by differences in energy acquisition, with feeding rates differing by ~ 2-fold between fast and slow growers. Additionally, significant differences were recorded for the unitary metabolic costs (i.e., per unit of metabolizable energy), indicating that higher metabolic efficiency was also a component of faster growth.

Depth effect on growth and fatty acid profile of Mediterranean mussel (Mytilus galloprovincialis) produced on a longline off south Portugal

Mytilus galloprovincialis is one of recent bivalve species produced in Portugal offshore. The growth, condition index, and the fatty acid composition of the mussel produced at 2 and 13 m depth was compared in longline on the south coast of Portugal. For each depth, a monitoring of several water quality parameters such as temperature, turbidity, nutrients, and chlorophyll-a was carried out. There were no significant differences between mussel growths at different depths. On the first sampling, shell length mean was 24 mm, reaching 50 mm 9 months after. High dispersion of mussel length was observed due to slow growers. The condition index shows a typical seasonal variation, with high values in autumn and late spring and minimum values in January. Analysis of fatty acids did not reveal differences at both depths and DHA levels (8.71 mg/g) were generally higher than EPA (5.90 mg/g). The water quality revealed few differences in the environmental conditions in the water column, which may explain the similarities between the quality of mussels produced at different depths. Based on these data, the mussel offshore production in that area can be done at this range of profundities 2 and 13 m, without affecting growth and fatty acid composition.

Slow growers possess a high pollutant removal potential through granule formation for wastewater treatment

For the upgradation and building of wastewater treatment plants in limited areas, biological wastewater treatment processes with a high pollutant removal loading rate are required. Biofilm or membrane-based systems can achieve a high pollutant removal loading rate through high microbial biomass concentrations and/or activities. The sludge separation is the primary bottleneck for achieving high biomass concentrations inside the biological reactors. In addition, low growers including anaerobic ammonia oxidation bacteria and methanogens are usually easier to form granules and induce high nutrient removal loading rates. The possible granule formation mechanisms should be clarified through physical, chemical and biological aspects, including hydraulic control, microbial extracellular polymeric substances formation, biological energy utilization control, microbial interactions and also other microbial processes. In future, the possible mechanisms should be further clarified, and then high-efficient bioreactors, new microbial ecology control strategy and novel biofilm carriers should be developed for advancing biological wastewater treatment processes with high pollutant removal loading rates.

Growth rate, extinction and survival amongst late Cenozoic bivalves of the North Atlantic

ABSTRACT Late Cenozoic bivalve extinction in the North Atlantic area has been attributed to environmental deterioration. Within scallops and oysters – groups with a high growth rate – certain taxa which grew exceptionally fast became extinct, while others which grew slower survived. Those which grew exceptionally fast would have obtained protection from predators thereby, so their extinction may have been due to the detrimental effect of environmental change on growth rate and ability to avoid predation, rather than environmental change per se. We investigated some glycymeridid and carditid bivalves – groups with a low growth rate – to see whether extinct forms grew faster than extant forms. Extinct Glycymeris subovata grew at about the same rate as the slowest-growing living glycymeridid and much slower than late Cenozoic examples of extant G. americana, which grew at about the same rate as the fastest-growing living glycymeridid. Extinct G. obovata and extinct Cardites squamulosa ampla also grew slower than G. americana. These findings indicate that within bivalve groups with a low growth rate, extinction or survival of taxa through the late Cenozoic was not influenced by whether they were relatively fast or slow growers. By implication, environmental change acted directly to cause extinctions in these groups.

Demographic plasticity facilitates ecological and economic resilience in a commercially important reef fish.

Variation in life-history characteristics is evident within and across animal populations. Such variation is mediated by environmental gradients and reflects metabolic constraints or trade-offs that enhance reproductive outputs. While generalizations of life-history relationships across species provide a framework for predicting vulnerability to overexploitation, deciphering patterns of intraspecific variation may also enable recognition of peculiar features of populations that facilitate ecological resilience. This study combines age-based biological data from geographically disparate populations of bluespine unicornfish (Naso unicornis)-the most commercially valuable reef-associated species in the insular Indo-Pacific-to explore the magnitude and drivers of variation in life span and examine the mechanisms enabling peculiar mortality schedules. Longevity and mortality schedules were investigated across eleven locations encompassing a range of latitudes and exploitation levels. The presence of different growth types was examined using back-calculated growth histories from otoliths. Growth-type-dependent mortality (mortality rates associated with particular growth trajectories) was corroborated using population models that incorporated size-dependent competition. We found a threefold geographic variation in life span that was strongly linked to temperature, but not to anthropogenic pressure or ocean productivity. All populations consistently displayed a two-phase mortality schedule, with higher than expected natural mortality rates in earlier stages of post-settlement life. Reconstructed growth histories and population models demonstrated that variable growth types within populations can yield this peculiar biphasic mortality schedule, where fast growers enjoy early reproductive outputs at the expense of greater mortality, and benefits for slow growers derive from extended reproductive outputs over a greater number of annual cycles. This promotes population resilience because individuals can take advantage of cycles of environmental change operating at both short- and long-term scales. Our results highlight a prevailing, fundamental misperception when comparing the life histories of long-lived tropical ectotherms: the seemingly incongruent combination of extended life spans with high mortality rates was enabled by coexistence of variable growth types in a population. Thus, a demographic profile incorporating contrasting growth and mortality strategies obscures the demographic effects of harvest across space or time in N. unicornis and possibly other ectotherms with the combination of longevity and asymptotic growth.

Utility of the kappa index in the evolution to multiple sclerosis

A range of phenotypes differing in growth rate were designed in the Manila clam by combining separate breeding families with size segregation within each family to constitute fast and slow growing groups. Physiological components of the energy budget and scope for growth (SFG) were then compared between these different phenotypes during the acute and chronic responses to two diets that were isocaloric but differed by 3-fold in their protein/energy (P/E) ratios. Both diets were based on the microalgae Rhodomonas lens obtained in either the exponential or the stationary phase of culture. The aims of the study were 1) to test the effects of these changes in food composition on growth rate, estimated as the balance of physiological processes of energy gain and loss integrated in the SFG; and 2) to assess the extent to which physiological adjustments to diet composition are modulated in order to fulfill the variable energy requirements posed by the occurrence of differential growth phenotypes. Growth performance improved with the high-protein (N+) diet for the different family * growth group combinations, with SFG values exceeding by 50% on average the values of the low-protein (N-) diet. Digestive constraints resulted in reduced absorption efficiency with the N-diet, which tended to cancel out the potential benefits of adjusting feeding rates in order to compensate for a low protein ration. Endogenous differences in growth rate associated with segregated phenotypes were mainly accounted for by differences in energy acquisition, with feeding rates differing by ~ 2-fold between fast and slow growers. Additionally, significant differences were recorded for the unitary metabolic costs (i.e., per unit of metabolizable energy), indicating that higher metabolic efficiency was also a component of faster growth.

Insight into the roles of packing carriers and ultrasonication in anaerobic side-stream reactor coupled membrane bioreactors: Sludge reduction performance and mechanism

The sludge in situ reduction process by inserting an anaerobic side-stream reactor (ASSR) in a sludge return line provides a cost-effective approach to reduce sludge production in activated sludge systems. In this study, four pilot-scale membrane bioreactors (MBRs), including an AO-MBR for control, ASSR coupled MBR (ASSR-MBR), a MBR with ASSR packed with carriers (AP-MBR) and an AP-MBR with part of sludge ultrasonicated before fed into ASSR (AUP-MBR) were operated in parallel to investigate enhancing effects of ultrasonication and packing carriers on sludge reduction and pollutants removal performance under both normal and low temperature. Low temperature showed negligible impact on COD removal, deteriorated NH4+N and TN removal from 98.3% to 69.7% at 21.6 °C to 92.5% and 48.8% at 2.6 °C, and decreased sludge reduction efficiency (SRE) in ASSR-MBR. Packing carriers and ultrasonication both enhanced sludge reduction, especially under low temperature with SRE values increased from 8.2% of ASSR-MBR to 17.1% of AP-MBR and 32.6% of AUP-MBR at 4.5 ± 2.5 °C. Packing carriers and ultrasonication increased cell rupture by 11.1% and 14.5% in aerobic MBR, enhanced protease activity in ASSR by 60.0% and 116.3%, and reduced ATP content for heterotrophic metabolism by 31.4% and 7.3%, respectively. MiSeq sequencing results showed that packing carriers enriched hydrolytic bacteria (Terrimonas, Dechloromonas and Woodsholea), slow growers (Sulfuritalea, Thauera and Azospira) and predatory bacteria (Bdellovibrio and norank_Saprospiraceae), while ultrasonication further enriched hydrolytic bacteria (norank_Saccharibacteria and Ferruginibacter). Packing carriers is more cost-effective than ultrasonication to enhance sludge reduction by partial damage to bacterial cells and promoting better interaction between bacteria, enzymes and substrates to favor particles hydrolysis.

Effects of diet composition on the energy balance of free-ranging organic broilers

In organic broiler production, one big factor in meeting the energy requirements of birds is through forage and concentrate feeds. In organic production, birds consume every kind of grass within the pasture. This study investigated the performance of slow growing broilers with an outdoor access, documenting the energy balance between slow growers in forage pasture. Four hundred slow growing broilers of Hubbard JA 957 strain were assigned to two Treatments. In Treatment 1, birds were placed in grass/white clover-covered plot and provided with 70 % concentrates adlibitum while in Treatment II, birds were placed in wheat stubble plot and received 35 % concentrate and 35% whole wheat. Each plot had an area of 25m × 10m and enclosed with nets. Results showed that slow growing broilers were not able to compensate their energy requirement resulting to poor weight. Nevertheless, the 70 % concentrates adlibitum diet in wheat stubble plot can be worthy recommendation for farmers since resulting growth rate showed optimistic outcome in terms of farm input due to manure accumulated by soil and an added income for farmers after harvest season.

MtrA Response Regulator Controls Cell Division and Cell Wall Metabolism and Affects Susceptibility of Mycobacteria to the First Line Antituberculosis Drugs

The biological processes regulated by the essential response regulator MtrA and the growth conditions promoting its activation in Mycobacterium tuberculosis, a slow grower and pathogen, are largely unknown. Here, using a gain-of-function mutant, MtrAY 102C, which functions in the absence of the cognate MtrB sensor kinase, we show that the MtrA regulon includes several genes involved in the processes of cell division and cell wall metabolism. The expression of selected MtrA targets and intracellular MtrA levels were compromised under replication arrest induced by genetic manipulation and under stress conditions caused by toxic radicals. The loss of the mtrA gene in M. smegmatis, a rapid grower and non-pathogen, produced filamentous cells with branches and bulges, indicating defects in cell division and cell shape. The ΔmtrA mutant was sensitized to rifampicin and vancomycin and became more resistant to isoniazid, the first line antituberculosis drug. Our data are consistent with the proposal that MtrA controls the optimal cell division, cell wall integrity, and susceptibility to some antimycobacterial drugs.

Characterizing individual variability in mussel (Mytilus galloprovincialis) growth and testing its physiological drivers using Functional Data Analysis.

Determining the magnitude and causes of intrinsic variability is a main issue in the analysis of bivalve growth. Inter-individual variability in bivalve growth has been attributed to differences in the physiological performance. This hypothesis has been commonly tested comparing the physiological rates of fast and slow growers after size differentiation has occurred. This experimental design may detect a link between growth and physiological performance, but we cannot interpret the posterior physiological performance as a driver for the prior growth variability. Considering these limitations, this work introduces a new methodological framework for the analysis of bivalve growth variability. We have conducted sequential measurements of size and physiological performance (feeding, digestion and metabolic rates) in even-sized mussels growing under homogeneous environmental conditions. This experimental design allows us to distinguish between changes over time within individuals, i.e. growth and trends in the physiological rates, from differences between individuals with respect to a baseline level. In addition, Functional Data Analysis provides powerful tools to summarize all the information obtained in the exhaustive sampling scheme and to test whether differences in the physiological performance enhance growth dispersion. Our results report an increasing dispersion in both size and physiological performance over time. Although mussels grew during the experiment, it is difficult to detect any increasing or decreasing temporal pattern in their feeding, digestion and metabolic rates due to the large inter-individual variability. Comparison between the growth and physiological patterns of mussels with final size above (fast growers) and below (slow growers) the median found that fast growers had larger feeding and digestion rates and lower metabolic expenditures during the experimental culture than mussels with slow growth, which agrees with the hypothesis of a physiological basis for bivalve growth variability.

Sludge reduction by a micro-aerobic hydrolysis process: A full-scale application and sludge reduction mechanisms

The process performance of a full-scale sludge process reduction activated sludge (SPRAS) system in long-term operation were investigated by inserting a micro-aerobic tank and a clarifier before conventional activated sludge process. The full-scale SPRAS for industrial park wastewater treatment achieved efficient pollutants removal and a low observed sludge yield of 0.074 g SS/g COD. Batch tests showed that influent feeding into the micro-aerobic tank favored sludge reduction, and obtained a sludge decay constant of 0.168 d−1. The SPRAS enriched slow growers and hydrolytic bacteria for sludge reduction, showed high simultaneous nitrification and denitrification efficiency in the micro-aerobic tank with abundant denitrifying bacteria, and improved sludge settleability by enriching floc-forming bacteria. Process configuration of the SPRAS was beneficial to enhance maintenance metabolism, cyclic micro-aerobic and anaerobic uncoupling, and lysis-cryptic growth for sludge reduction. Techno-economic analysis showed that the SPRAS greatly reduced sludge production with small footprint and low cost.

Symbiotic Efficiency of Slow and Fast-Growing Soybean Rhizobia of Central India

Soybean rhizobia are pre-dominantly slow growing but fast growing strains that evolved from natural populations have been reported globally including from several locations in India but have not been evaluated. The symbiotic effectivity of three slow and three fast-growing rhizobial strains isolated from Vertisols of Central India was evaluated on soybean var. 9752 in sterilized sand microcosms in a green-house. At 40 days growth, there was variation in nodulation parameters and growth of soybean with both types of rhizobia but overall, the slow growers were superior to fast growers with respect to nodule number (56%), nodule dry matter (25%), shoot dry matter (11%) and total nitrogen uptake (22%). The slow-growers (genetic homology to Bradyrhizobium japonicum) fixed ~ 25% more nitrogen than fast-growers with homology to Rhizobium (Agrobacterium) radiobacter. The results have very important implications for strain selection during biofertilizer production to maximize biological nitrogen fixation.

Mytilus galloprovincialis fast growing phenotypes under different restrictive feeding conditions: Fast feeders and energy savers

The present study aims to test if the environmental conditions prevailing during the growing period can determine the physiological profiles of specimens differentiated as fast (F) or slow (S) growers in the mussel Mytilus galloprovincialis. We reared mussel spats in the laboratory under two different conditions. In Treatment I (continuous feeding during discontinuous immersion), two mussel groups were submitted to a daily air exposure of 8 h and fed continuously during immersion-time, with either high-quality food dosed below the pseudofaeces threshold (BP group) or low organic content food dosed above the pseudofaeces threshold (AP group). In Treatment II (discontinuous feeding during continuous immersion), mussels were continuously immersed but fed only 1 day per week (RC group). Mussels were reared for 7 and 11 months (time required for size-differentiation) in Treatments I and II, respectively, and the smallest and largest individuals from each group were selected as S and F specimens.A series of feeding experiments (with different food quality, food ration and under continuous food supply) were performed to analyse the physiological performance of selected F and S mussels. In Treatment I, no significant differences were found in the metabolic rates between F and S mussels, and the faster growth rate of F-mussels resulted from their capacity to display higher clearance-ingestion rates and pre-ingestive selections. The physiological basis of growth rate differences between F and S mussels were found to be the same in mussels reared with diets below or above a pseudofaeces threshold (FBP, FAP, SBP and SAP). In contrast, the mussels from Treatment II had no significant differences in the feeding rates between FRC and SRC mussels. However, F individuals were found to have a 33% lower standard metabolic rate, indicating that fast growth under severe feeding restriction stemmed from a higher capacity of F-mussels to save energy during long periods of starvation. Despite the differences in the physiological basis explaining fast growth between the two treatments, F-mussels were found to possess significantly higher gill-surface area in both cases. It is thus concluded that endogenous factors affecting the gill-surface area play a major role in determining inter-individual growth rate differences in the mussel, Mytilus galloprovincialis.

Infections and outbreaks of nontuberculous mycobacteria in hospital settings.

Non-tuberculous mycobacterial [NTM] infections in the hospital setting are a complex and often challenging entity. The goal of this review is to discuss diagnostic and treatment strategies for established as well as emerging nosocomial NTM infections. NTM outbreaks have been documented in a variety of hospital settings. Contamination of medical devices or aqueous solutions is often implicated in the spread of infection. More recently, the slow grower M. chimaera has been reported in the setting of contaminated heater-cooler devices used for cardiopulmonary bypass and extracorporeal membrane oxygenation. In addition, increases in medical tourism for cosmetic surgery have led to outbreaks of rapidly growing mycobacteria. Diagnosis and treatment of nosocomial NTM pose many challenges for the clinician. Surgical resection or debridement as well as combination antimycobacterial therapy are a mainstay in therapeutic management. Strict infection control and prevention practices are critical to the identification and cessation of outbreaks.

Expression of Saccharomyces cerevisiae cDNAs to enhance the growth of non-ethanol-producing S. cerevisiae strains lacking pyruvate decarboxylases

Metabolic engineering of Saccharomyces cerevisiae often requires a restriction on the ethanol biosynthesis pathway. The non-ethanol-producing strains, however, are slow growers. In this study, a cDNA library constructed from S.cerevisiae was used to improve the slow growth of non-ethanol-producing S.cerevisiae strains lacking all pyruvate decarboxylase enzymes (Pdc-, YSM021). Among the obtained 120 constructs expressing cDNAs, 34 transformants showed a stable phenotype with quicker growth. Sequence analysis showed that the open reading frames of PDC1, DUG1 (Cys-Gly metallo-di-peptidase in the glutathione degradation pathway), and TEF1 (translational elongation factor EF-1 alpha) genes were inserted into the plasmids of 32, 1, and 1 engineered strains, respectively. DUG1 function was confirmed by the construction of YSM021 pGK416-DUG1 strain because the specific growth rate of YSM021 pGK416-DUG1 (0.032 ± 0.0005 h-1) was significantly higher than that of the control strains (0.029 ± 0.0008h-1). This suggested that cysteine supplied from glutathione was probably used for cell growth and for construction of Fe-S clusters. The results showed that the overexpression of cDNAs is a promising approach to engineer S.cerevisiae metabolism.

English

A total of eighty one (81) rhizobial isolates were recovered from root nodules of cowpea (Vigna unguiculata L. Walp.) and groundnut (Arachis hypogaea L.) grown in soils collected from eight different sites (Hawassa, Wondogenet, Chofa, Badawacho, Bodity, Gofa, Ziway, and Alemtena) in Ethiopia with no known history of inoculation. The test isolates together with seven reference strains belonging to five genera including Rhizobium, Ensifer, Mesorhizobium, Bradyrhizobium and Azorhizobium were characterized using ninety phenotypic traits. Thirty one isolates (38%) were found to be fast growers while fifty isolates (62%) were slow growers. The majority of the isolates showed an intrinsic resistance to antibiotics (µg/ml), Chloramphenicol (5 and 15), Lincomycin (100), Novobiocin (0.5 and 1.5), and Erythromycin (10 and 20) and to heavy metals manganese sulphate (500) and copper chloride (100). Most isolates did not tolerate NaCl concentration >3% (w/v) and high temperature (45°C). Dendrogram was constructed by applying the unweighted pair group method with arithmetic means (UPGMA) using NTSYSpc Version 2.1. They were grouped into seven clusters and eight unclustered positions, when 82% relative similarity was used as a cut point. Fifty eight percent of the test isolates were grouped with Bradyrhizobium japonicum and Bradyrhizobium elkanii superclades, thus indicating that rhizobia nodulating cowpea and groundnut are delineated within a branch that defines Bradyrhizobium genus. To elucidate the precise taxonomic positions of the isolates, further genetic studies are required using modern molecular biological methods. Key words: Groundnut, cowpea, isolates, phenotypic traits, Bradyrhizobium, Rhizobium.