Occurrence of L-iduronic acid and putative D-glucuronyl C5-epimerases in prokaryotes

Glycosaminoglycans (GAGs) are polysaccharides that are typically present in a wide diversity of animal tissue. Most common GAGs are well-characterized and pharmaceutical applications exist for many of these compounds, e.g. heparin and hyaluronan. In addition, also bacterial glycosaminoglycan-like structures exist. Some of these bacterial GAGs have been characterized, but until now no bacterial GAG has been found that possesses the modifications that are characteristic for many of the animal GAGs such as sulfation and C5-epimerization. Nevertheless, the latter conversion may also occur in bacterial and archaeal GAGs, as some prokaryotic polysaccharides have been demonstrated to contain L-iduronic acid. However, experimental evidence for the enzymatic synthesis of L-iduronic acid in prokaryotes is as yet lacking. We therefore performed an in silico screen for D-glucuronyl C5-epimerases in prokaryotes. Multiple candidate C5-epimerases were found, suggesting that many more microorganisms are likely to exist possessing an L-iduronic acid residue as constituent of their cell wall polysaccharides.     

Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short‐chain acyl esters (C2–C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4‐nitrophenyl‐β‐D ‐xylopyranoside monoacetates as substrates in a β‐xylosidase‐coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine‐substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β‐hydrolase fold. TM0077 assembles into a doughnut‐shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli

A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a DeltarelA DeltaspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Omega element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-beta-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.     

Formation of l-alanine as a reduced end product in carbohydrate fermentation by the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus was found to form substantial amounts of l-alanine during batch growth on either cellobiose, maltose or pyruvate. Acetate, CO₂ and H₂ were produced next to alanine. The carbon- and electron balances were complete for all three substrates. Under standard growth conditions (N₂/CO₂ atmosphere) an alanine/acetate ratio of about 0.3 was found for either substrate. The alanine /acetate ratio was influenced, however, by the hydrogen partial pressure. In the presence of S⁰ or in coculture with Methanococcus jannaschii this ratio was only 0.07, whereas under a H₂/CO₂ atmosphere this ratio could amount up to 0.8. Alanine formation was also aflected by the NH _inf4 ^sup+ concentration, i.e. below 4 mM, NH _inf4 ^sup+ becomes limiting to alanine formation. Alanine formation was shown to occur via an alanine aminotransferase, which exhibited a specific activity in cell-free extract of up to 6.0 U/mg (90°C; direction of pyruvate formation). The alanine aminotransferase probably cooperates with glutamate dehydrogenase (up to 23 U/mg; 90°C) and ferredoxin: NADP⁺ oxidoreductase (up to 0.7 U/mg, using methyl viologen; 90°C) to recycle the electron acceptors involved in catabolism. Thus, the existence of this unusual alanine-forming branch enables P. furiosus to adjust its fermentation, depending on the redox potential of the terminal electron acceptor.Abbreviations DTT dithiothreitol - MV methyl viologen - AAT alanine aminotransferase - GDH glutamate dehydrogenase - MV: NADP⁺ OR methyl viologen: NADP⁺ oxidoreductase     

Formation of antigenic extracellular polysaccharides by selected strains ofMucor spp.,Rhizopus spp.,Rhizomucor spp.,Absidia corymbifera andSyncephalastrum racemosum

In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) ofMucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales. Cross-reactivity in the ELISA could be observed only towards the yeastPichia membranaefaciens. EPS were isolated from various cultures ofM. hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass. Other strains includingMucor spp.,Rhizopus spp.,Rhizomucor spp.,Absidia corymbifera andSyncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23. In all cases, the excreted EPS had similar antigenic properties as determined by ELISA. No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation. Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.     

Network spandrels reflect ecological assembly

Ecological networks that exhibit stable dynamics should theoretically persist longer than those that fluctuate wildly. Thus, network structures which are over‐represented in natural systems are often hypothesised to be either a cause or consequence of ecological stability. Rarely considered, however, is that these network structures can also be by‐products of the processes that determine how new species attempt to join the community. Using a simulation approach in tandem with key results from random matrix theory, we illustrate how historical assembly mechanisms alter the structure of ecological networks. We demonstrate that different community assembly scenarios can lead to the emergence of structures that are often interpreted as evidence of ‘selection for stability’. However, by controlling for the underlying selection pressures, we show that these assembly artefacts—or spandrels—are completely unrelated to stability or selection, and are instead by‐products of how new species are introduced into the system. We propose that these network‐assembly spandrels are critically overlooked aspects of network theory and stability analysis, and we illustrate how a failure to adequately account for historical assembly can lead to incorrect inference about the causes and consequences of ecological stability.     

Variants of Serratia marcescens Induced by Freeze-Drying

Unusually high numbers of pigmentless variants and sectored colonies in cultures of lyophylized Serratia marcescens are reported. Clonal analyses of sectored colonies show the presence of unstable bacteria that continue to sector again when plated. Further analysis of pigmentless variants suggest that pigment formation, oxygen uptake, and the production of an inducible protease are affected.