Microbial biofilms in nature: unlocking their potential for agricultural applications.

Soil environments are dynamic and the plant rhizosphere harbours a phenomenal diversity of micro-organisms which exchange signals and beneficial nutrients. Bipartite beneficial or symbiotic interactions with host roots, such as mycorrhizae and various bacteria, are relatively well characterized. In addition, a tripartite interaction also exists between plant roots, arbuscular mycorrhizal fungi (AMF) and associated bacteria. Bacterial biofilms exist as a sheet of bacterial cells in association with AMF structures, embedded within a self-produced exopolysaccharide matrix. Such biofilms may play important functional roles within these tripartite interactions. However, the details about such interactions in the rhizosphere and their relevant functional relationships have not been elucidated. This review explores the current understanding of naturally occurring microbial biofilms, and their interaction with biotic surfaces, especially AMF. The possible roles played by bacterial biofilms and the potential for their application for a more productive and sustainable agriculture is discussed in this review.

Role of Phosphate Transport System Component PstB1 in Phosphate Internalization by Nostoc punctiforme.

In bacteria, limited phosphate availability promotes the synthesis of active uptake systems, such as the Pst phosphate transport system. To understand the mechanisms that facilitate phosphate accumulation in the cyanobacterium Nostoc punctiforme, phosphate transport systems were identified, revealing a redundancy of Pst phosphate uptake systems that exists across three distinct operons. Four separate PstB system components were identified. pstB1 was determined to be a suitable target for creating phenotypic mutations that could result in the accumulation of excessive levels of phosphate through its overexpression or in a reduction of the capacity to accumulate phosphate through its deletion. Using quantitative real-time PCR (qPCR), it was determined that pstB1 mRNA levels increased significantly over 64 h in cells cultured in 0 mM added phosphate and decreased significantly in cells exposed to high (12.8 mM) phosphate concentrations compared to the level in cells cultured under normal (0.8 mM) conditions. Possible compensation for the loss of PstB1 was observed when pstB2, pstB3, and pstB4 mRNA levels increased, particularly in cells starved of phosphate. The overexpression of pstB1 increased phosphate uptake by N. punctiforme and was shown to functionally complement the loss of PstB in E. coli PstB knockout (PstB-) mutants. The knockout of pstB1 in N. punctiforme did not have a significant effect on cellular phosphate accumulation or growth for the most part, which is attributed to the compensation for the loss of PstB1 by alterations in the pstB2, pstB3, and pstB4 mRNA levels. This study provides novel in vivo evidence that PstB1 plays a functional role in phosphate uptake in N. punctiforme IMPORTANCE: Cyanobacteria have been evolving over 3.5 billion years and have become highly adept at growing under limiting nutrient levels. Phosphate is crucial for the survival and prosperity of all organisms. In bacteria, limited phosphate availability promotes the synthesis of active uptake systems. The Pst phosphate transport system is one such system, responsible for the internalization of phosphate when cells are in phosphate-limited environments. Our investigations reveal the presence of multiple Pst phosphate uptake systems that exist across three distinct operons in Nostoc punctiforme and functionally characterize the role of the gene product PstB1 as being crucial for the maintenance of phosphate accumulation. We demonstrate that the genes pstB2, pstB3, and pstB4 show alterations in expression to compensate for the deletion of pstB1 The overall outcomes of this work provide insights as to the complex transport mechanisms that exist in cyanobacteria like N. punctiforme, allowing them to thrive in low-phosphate environments.

The ZntA-like NpunR4017 plays a key role in maintaining homeostatic levels of zinc in Nostoc punctiforme.

Analysis of cellular response to zinc exposure provides insights into how organisms maintain homeostatic levels of zinc that are essential, while avoiding potentially toxic cytosolic levels. Using the cyanobacterium Nostoc punctiforme as a model, qRT-PCR analyses established a profile of the changes in relative mRNA levels of the ZntA-like zinc efflux transporter NpunR4017 in response to extracellular zinc. In cells treated with 18 µM of zinc for 1 h, NpunR4017 mRNA levels increased by up to 1300 % above basal levels. The accumulation and retention of radiolabelled (65)Zn by NpunR4107-deficient and overexpressing strains were compared to wild-type levels. Disruption of NpunR4017 resulted in a significant increase in zinc accumulation up to 24 % greater than the wild type, while cells overexpressing NpunR4107 accumulated 22 % less than the wild type. Accumulation of (65)Zn in ZntA(-) Escherichia coli overexpressing NpunR4017 was reduced by up to 21 %, indicating the capacity for NpunR4017 to compensate for the loss of ZntA. These findings establish the newly identified NpunR4017 as a zinc efflux transporter and a key transporter for maintaining zinc homeostasis in N. punctiforme.

Characterization of two cation diffusion facilitators NpunF0707 and NpunF1794 in Nostoc punctiforme.

AIMS: To characterize genes involved in maintaining homeostatic levels of zinc in the cyanobacterium Nostoc punctiforme. METHODS AND RESULTS: Metal efflux transporters play a central role in maintaining homeostatic levels of trace elements such as zinc. Sequence analyses of the N. punctiforme genome identified two potential cation diffusion facilitator (CDF) metal efflux transporters, Npun_F0707 (Cdf31) and Npun_F1794 (Cdf33). Deletion of either Cdf31or Cdf33 resulted in increased zinc retention over 3 h. Interestingly, Cdf31(-) and Cdf33(-) mutants showed no change in sensitivity to zinc exposure in comparison with the wild type, suggesting some compensatory capacity for the loss of each other. Using qRT-PCR, a possible interaction was observed between the two cdf's, where the Cdf31(-) mutant had a more profound effect on cdf33 expression than Cdf33(-) did on cdf31. Over-expression of Cdf31 and Cdf33 in ZntA(-) - and ZitB(-) -deficient Escherichia coli revealed function similarities between the ZntA and ZitB of E. coli and the cyanobacterial transporters. CONCLUSIONS: The data presented shed light on the function of two important transporters that regulate zinc homeostasis in N. punctiforme. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time the functional characterization of two cyanobacterial zinc efflux proteins belonging to the CDF family.

Fiber-specific responses of muscle glycogen repletion in fasted rats physically active during recovery from high-intensity physical exertion.

Mild physical activity performed immediately after a bout of intense exercise in fasting humans results in net glycogen breakdown in their slow oxidative (SO) muscle fibers and glycogen repletion in their fast twitch (FT) fibers. Because several animal species carry a low proportion of SO fibers, it is unclear whether they can also replenish glycogen in their FT fibers under these conditions. Given that most skeletal muscles in rats are poor in SO fibers (<5%), this issue was examined using groups of 24-h fasted Wistar rats (n=10) that swam for 3 min at high intensity with a 10% weight followed by either a 60-min rest (passive recovery, PR) or a 30-min swim with a 0.5% weight (active recovery, AR) preceding a 30-min rest. The 3-min sprint caused 61-79% glycogen fall across the muscles examined, but not in the soleus (SOL). Glycogen repletion during AR without food was similar to PR in the white gastrocnemius (WG), where glycogen increased by 71%, and less than PR in both the red and mixed gastrocnemius (RG, MG). Glycogen fell by 26% during AR in the SOL. Following AR, glycogen increased by 36%, 87%, and 37% in the SOL, RG, and MG, respectively, and this was accompanied by the sustained activation of glycogen synthase and inhibition of glycogen phosphorylase in the RG and MG. These results suggest that mammals with a low proportion of SO fibers can also replenish the glycogen stores of their FT fibers under extreme conditions combining physical activity and fasting.

Glycogen resynthesis in the absence of food ingestion during recovery from moderate or high intensity physical activity: novel insights from rat and human studies.

The finding that during recovery from high intensity exercise, rats have the capacity to replenish their muscle glycogen stores even in the absence of food intake has provided us with an experimental model of choice to explore further this process. Our objective here is to share those questions arising from research carried out by others and ourselves on rats and humans that are likely to be of interest to comparative biochemists/physiologists. On the basis of our findings and those of others, it is proposed that across vertebrate species: (1). the capacity of muscles to replenish their glycogen stores from endogenous carbon sources is dependent on the type of physical activity and animal species; (2). lactate and amino acids are the major endogenous carbon sources mobilized for the resynthesis of muscle glycogen during recovery from exercise, their relative contributions depending on the duration of recovery and type of exercise; (3). the relative contributions of lactate glyconeogenesis and hepatic/renal gluconeogenesis to muscle glycogen synthesis is species- and muscle fiber-dependent; and (4). glycogen synthase and phosphorylase play an important role in the control of the rate of glycogen synthesis post-exercise, with the role of glucose transport being species-dependent.

Effect of streptozotocin-induced diabetes on glycogen resynthesis in fasted rats post-high-intensity exercise.

It has recently been shown that food intake is not essential for the resynthesis of the stores of muscle glycogen in fasted animals recovering from high-intensity exercise. Because the effect of diabetes on this process has never been examined before, we undertook to explore this issue. To this end, groups of rats were treated with streptozotocin (60 mg/kg body mass ip) to induce mild diabetes. After 11 days, each animal was fasted for 24 h before swimming with a lead weight equivalent to 9% body mass attached to the tail. After exercise, the rate and the extent of glycogen repletion in muscles were not affected by diabetes, irrespective of muscle fiber composition. Consistent with these findings, the effect of exercise on the phosphorylation state of glycogen synthase in muscles was only minimally affected by diabetes. In contrast to its effects on nondiabetic animals, exercise in fasted diabetic rats was accompanied by a marked fall in hepatic glycogen levels, which, surprisingly, increased to preexercise levels during recovery despite the absence of food intake.

Glycogen repletion following burst activity: a carbohydrate-sparing mechanism in animals adapted to arid environments?

The Western chestnut mouse (Pseudomys nanus ferculinus) is one of several native rodent species adapted to the arid environments of Australia. Since these environments are often associated with a paucity in dietary carbohydrate, the problem arises as to the mechanism whereby these rodents replete their stores of muscle glycogen when recovering from high intensity physical activity. This is an important issue since the maintenance of adequate stores of muscle glycogen is crucial to support the energy demands associated with 'flight or fight' responses. Whilst it is known that food ingestion post-exercise is required for the total repletion of muscle glycogen in rats and humans, our findings indicate that the Western chestnut mouse has the impressive capacity to replete completely its stores of muscle glycogen, even in the absence of food intake. Indeed during recovery from burst activity which results in the massive breakdown of the stores of muscle glycogen, the levels of glycogen return back to pre-exercise levels within only 50 minutes despite the absence of food intake. This capacity is important in the broader context of nutritional adaptation to arid/seasonally-arid regions since it allows muscles to replete their fuel stores even when food is not available. How common is this strategy among desert-adapted mammal species is a question yet to be answered.

Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat.

The aim of this study was to determine the role of the phosphorylation state of glycogen synthase and glycogen phosphorylase in the regulation of muscle glycogen repletion in fasted animals recovering from high-intensity exercise. Groups of rats were swum to exhaustion and allowed to recover for up to 120 min without access to food. Swimming to exhaustion caused substantial glycogen breakdown and lactate accumulation in the red, white and mixed gastrocnemius muscles, whereas the glycogen content in the soleus muscle remained stable. During the first 40 min of recovery, significant repletion of glycogen occurred in all muscles examined except the soleus muscle. At the onset of recovery, the activity ratios and fractional velocities of glycogen synthase in the red, white and mixed gastrocnemius muscles were higher than basal, but returned to pre-exercise levels within 20 min after exercise. In contrast, after exercise the activity ratios of glycogen phosphorylase in the same muscles were lower than basal, and increased to pre-exercise levels within 20 min. This pattern of changes in glycogen synthase and phosphorylase activities, never reported before, suggests that the integrated regulation of the phosphorylation state of both glycogen synthase and phosphorylase might be involved in the control of glycogen deposition after high-intensity exercise.