Effect of bacitracin on flagellar assembly and presumed glycosylation of the flagellins of Methanococcus deltae

Methanococcus deltae is an irregularly-shaped coccoid methanogen which possesses peritrichously arranged flagella. The flagella are composed of 2 flagellins of M _r=27000 and 32000 and possess a carbohydrate component as determined by thymol-sulfuric acid staining of SDS-PAGE. Cell growth was sensitive to bacitracin at levels near 10 g/ml. Growth in the presence of 5g/ml bacitracin resulted in the appearance of presumably hypoglycosylated flagellins as detected by Western immunoblotting. Continual passage of cells from 5 g/ml bacitracin through 10, 25, and 50 g/ml bacitracin resulted in cells capable of growth at 100 g/ml bacitracin. Western immunoblot analysis revealed that cells grown in the highest concentration of bacitracin no longer possessed native flagellins but only the hypoglycosylated forms. Electron microscopy corroborated the absence of normal flagella. These studies suggest that a bacitracin-sensitive dolichol-diphosphate carrier is responsible for attachment of at least a large proportion of the carbonhydrate content of the flagellins, and that a minimum amount of glycosylation is essential for normal flagellum assembly.     

Products of Anaerobic 2,4,6-Trinitrotoluene (TNT) Transformation by Clostridium bifermentans

Experiments to elucidate the 2,4,6-trinitrotoluene (TNT)-transforming activity of Clostridium bifermentans LJP-1 identified reductive TNT transformations that ultimately produced as end products triaminotoluene (TAT) and phenolic products of TAT hydrolysis. An adduct of TAT, apparently formed by condensation of TAT and pyruvic aldehyde (methyl glyoxal), was also detected.     

Transformation of nitroaromatic compounds by a methanogenic bacterium,Methanococcus sp. (strain B)

The transformation of several nitroaromatic compounds by a newly isolated methanogenic bacterium, Methanococcus sp. (strain B) was studied. The presence of nitroaromatic compounds (0.5 mM) viz., nitrobenzene, 2,4-dinitrobenzene, 2,4,6-trinitrobenzene, 2,4-dinitrophenol, 2,4-dinitrobenzene, and 2,6-dinitrotoluene in the culture medium did not inhibit growth of the isolate. The bacteria grew rapidly and reached stationary phase within seven days of incubation. All the nitroaromatic compounds tested were 80 to 100% transformed by the bacterium to amino compounds by a reduction process. The isolate did not use the nitroaromatic compounds as the sole source of carbon or nitrogen. The transformation of nitroaromatic compounds by this isolate was compared to that of other methanogenic bacteria. Out of five methanogens studied, only Methanococcus deltae and Methanococcus thermolithotrophicus could transform the nitroaromatic compounds; however, the transformation rates were significantly less than that of the new isolate Methanococcus sp. (strain B). The nitroaromatic compounds were not transformed by Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobrevibacter ruminantium.Abbreviations NB Nitrobenzene - DNB 2,4-Dinitrobenzene - TNB 2,4,6-Trinitrobenzene - DNP 2,4-Dinitrophenol - 2,4-DNT 2,4-Dinitrotoluene - 2,6-DNT 2,6-Dinitrotoluene     

Methanogenic Transformation of Methylfurfural Compounds to Furfural

The metabolic conversion of 5-methylfurfural and 2-methylfurfural to furfural by a methanogenic bacterium, Methanococcus sp. strain B, was studied. This bacterium was found to use methylfurfural compounds as a growth substrate and to convert them stoichiometrically to furfural. For every mole of methylfurfurals metabolized, almost 1 mol of furfural and 0.7 mol of methane were produced. Several methanogenic bacteria did not carry out this conversion. The metabolic conversion of methylfurfurals is likely to be of value in the anaerobic treatment of methylfurfural-containing wastewaters such as those produced by the paper and pulp industries and oatmeal processing industries. This study adds to the list of the limited number of compounds that are known to serve as electron donors for methanogenesis.     

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.     

Messenger RNA processing in Methanocaldococcus (Methanococcus) jannaschii

Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12–16 nucleotides upstream of the corresponding translation start site. Analyses with 3′-RACE (rapid amplification of cDNA ends) and 5′-RACE indicate that the processing is endonucleolytic. Analyses of the sequences surrounding the processing sites for functional sites, sequence motifs, or potential RNA secondary structure elements did not reveal any recurring features except for an AUG translation start codon and (in most cases) a ribosome binding site. These properties differ from those of all previously described mRNA processing systems. Our data suggest that the processing alters the representation of various genes in the RNA pool and therefore, may play a significant role in defining the balance of proteins in the cell.     

Anaerobic Biooxidation of Fe(II) by Dechlorosoma suillum

Anaerobic microbial oxidation of Fe(II) was only recently discovered and very little is known about this metabolism. We recently demonstrated that several dissimilatory perchlorate-reducing bacteria could utilize Fe(II) as an electron donor under anaerobic conditions. Here we report on a more in-depth analysis of Fe(II) oxidation by one of these organisms, Dechlorosoma suillum. Similarly to most known nitrate-dependent Fe(II) oxidizers, D. suillum did not grow heterotrophically or lithoautotrophically by anaerobic Fe(II) oxidation. In the absence of a suitable organic carbon source, cells rapidly lysed even though nitrate-dependent Fe(II) oxidation was still occurring. The coupling of Fe(II) oxidation to a particular electron acceptor was dependent on the growth conditions of cells of D. suillum. As such, anaerobically grown cultures of D. suillum did not mediate Fe(II) oxidation with oxygen as the electron acceptor, while conversely, aerobically grown cultures did not mediate Fe(II) oxidation with nitrate as the electron acceptor. Anaerobic washed cell suspensions of D. suillum rapidly produced an orange/brown precipitate which X-ray diffraction analysis identified as amorphous ferric oxyhydroxide or ferrihydrite. This is similar to all other identified nitrate-dependent Fe(II) oxidizers but is in contrast to what is observed for growth cultures of D. suillum, which produced a mixed-valence Fe(II)-Fe(III) precipitate known as green rust. D. suillum rapidly oxidized the Fe(II) content of natural sediments. Although the form of ferrous iron in these sediments is unknown, it is probably a component of an insoluble mineral, as previous studies indicated that soluble Fe(II) is a relatively minor form of the total Fe(II) content of anoxic environments. The results of this study further enhance our knowledge of a poorly understood form of microbial metabolism and indicate that anaerobic Fe(II) oxidation by D. suillum is significantly different from previously described forms of nitrate-dependent microbial Fe(II) oxidation.     

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

水产动物益生菌乳酸杆菌LH发酵工艺的研究

目的从生产实际出发,对1株高效乳酸杆菌（Lactobacillus spp）LH进行液体发酵培养基优化及发酵条件研究。方法通过碳源、氮源、无机盐、促生长素等单因子筛选及正交试验设计获得以下最佳培养基：糖蜜12 g/L,酵母膏5 g/L,蛋白胨1 g/L,葡萄糖4 g/L,玉米浆3 g/L,乙酸钠5 g/L,NaC l 5 g/L,K2HPO42.5 g/L,KH2PO42.5 g/L,MgSO40.5g/L,MnSO40.25 g/L。在此培养基上研究了该菌株最佳发酵条件。结果培养基初始pH 6.0,接种量2%（v/v,相对装液量）,500 m l三角瓶中装液量为500 m l,发酵温度为30～35℃,静置培养。在最佳培养条件下,LH活菌量达到1.74×10^9CFU/m l。结论通过活菌平板计数法测定了乳酸杆菌LH生长曲线,24 h为最佳种龄,生产收获时间是36 h。     

Internalization of Sucrose by Methanococcus thermolithotrophicus

When sucrose is present in the external medium, it is internalized by Methanococcus thermolithotrophicus. Sucrose internalization, as determined by both natural abundance (sup13)C nuclear magnetic resonance spectroscopy and [(sup14)C]sucrose uptake, is directly proportional to external sucrose levels. The uptake is energy independent and exhibits kinetic behavior consistent with a simple passive diffusion process. In the presence of 0.2 M sucrose, methanogenesis is inhibited as the NaCl concentration in the external medium is increased. Growth, as determined by protein content, is inhibited by 0.2 M sucrose when the external NaCl concentration is 1.4 M. These results are important because they show that (i) sucrose cannot be used as a noncharged solute to replace NaCl in experiments to evaluate how external osmotic strength affects the internal solute composition of M. thermolithotrophicus, and (ii) sucrose cannot be used as an impermeable marker for the extracellular volume in experiments to measure the intracellular volume of M. thermolithotrophicus.     

糠醛及其衍生物微生物降解(转化)研究进展

对糠醛及其衍生物微生物降解(转化)机制进行了系统性阐述,介绍了一些代表性的降解(转化)菌株的驯化和筛选、降解(转化)途径及应用等。并且对糠醛及其衍生物的生物降解(转化)进一步研究的方向提出了看法。     

Anaerobic Degradation and Transformation of p-Toluidine by the Sulfate-Reducing Bacterium Desulfobacula toluolica

The ability of the strictly anaerobic sulfate-reducing bacterium Desulfobacula toluolica (strain Tol2) to cometabolically degrade p-toluidine (p-methylaniline) while using toluene as the primary source of carbon and energy has been studied. This organism has been shown to modify and degrade toluidine in dense cell suspensions when no other source of carbon and energy is added. The metabolism led to the formation of a variety of metabolites. From these metabolites a biphenyl-like compound as well as phenylacetic acid have been identified by means of HPLC/MS techniques. The probable conversion of p-toluidine to p-aminophenylacetic acid and phenylacetic acid as dead end products suggested that this organism initiates p-toluidine degradation by the carboxylation of the methyl group. If this could be validated in further experiments, it would be the first time that a toluidine was carboxylated at the methyl moiety by an anaerobic, sulfate-reducing bacterium. Received: 6 March 1998 / Accepted: 3 April 1998     

Anaerobic degradation of 2-aminobenzoate (anthranilic acid) by denitrifying bacteria

In the presence of oxygen many aminoaromatic compounds polymerize to form recalcitrant macromolecules. To circumvent undesirable oxidation reactions, the anaerobic biodegradation of a simple member of this class of compounds was investigated. Two strains of bacteria were isolated which degrade 2-aminobenzoate anaerobically under denitrifying conditions, with nitrate as the terminal electron acceptor. Both organisms, which were assigned to the genus Pseudomonas, oxidized 2-aminobenzoate completely to CO2 and NH4+. Nitrate was reduced to nitrite. When nitrate was deplete from the growth medium the accumulated nitrite was reduced to nitrogen. The results establish a model system for the anaerobic, rapid, and complete oxidation of an aminoaromatic compound.     

Chlorophenol Production by Anaerobic Microorganisms: Transformation of a Biogenic Chlorinated Hydroquinone Metabolite

Chlorinated hydroquinones of biological origin are fully dechlorinated to 1,4-dihydroquinone by anaerobic bacteria such as Desulfitobacterium spp. (C. E. Milliken, G. P. Meier, J. E. M. Watts, K. R. Sowers, and H. D. May, Appl. Environ. Microbiol. 70:385-392, 2004). In the present study, mixed microbial communities from Baltimore Harbor sediment and a pure culture of Desulfitobacterium sp. strain PCE1 were discovered to demethylate, reductively dehydroxylate, and dechlorinate chlorinated hydroquinones into chlorophenols. Mixed microbial cultures from a freshwater source and several other desulfitobacteria in pure culture did not perform these reactions. Desulfitobacterium sp. strain PCE1 degraded 2,3,5,6-tetrachloro-4-methoxyphenol, a metabolite of basidiomycete fungi, to 2,3,5,6-tetrachlorophenol and 2,3,5-trichlorophenol, recalcitrant compounds that are primarily synthesized anthropogenically.     

Anaerobic transformation of 2,4,6-trinitrotoluene (TNT)

A sulfate-reducing bacterium using trinitrotoluene (TNT) as the sole nitrogen source was isolated with pyruvate and sulfate as the energy sources. The organism was able to reduce TNT to triaminotoluene (TAT) in growing cultures and cell suspensions and to further transform TAT to still unknown products. Pyruvate, H₂, or carbon monoxide served as the electron donors for the reduction of TNT. The limiting step in TNT conversion to TAT was the reduction of 2,4-diamino-6-nitrotoluene (2,4-DANT) to triaminotoluene. The reduction proceeded via 2,4-diamino-6-hydroxylaminotoluene (DAHAT) as an intermediate. The intermediary formation of DAHAT was only observed in the presence of carbon monoxide or hydroxylamine, respectively. The reduction of DAHAT to triaminotoluene was inhibited by both CO and NH₂OH. The inhibitors as well as DANT and DAHAT significantly inhibited sulfide formation from sulfite. The data were taken as evidence for the involvement of dissimilatory sulfite reductase in the reduction of DANT and/or DAHAT to triaminotoluene. Hydrogenase purified from Clostridium pasteurianum and carbon monoxide dehydrogenase partially purified from Clostridium thermoaceticum also catalyzed the reduction of DANT in the presence of methyl viologen or ferredoxin, however, as the main reduction product DAHAT rather than triaminotoluene was formed. The findings could explain the function of CO as an electron donor for the DANT reduction (to DAHAT) and the concomitant inhibitory effect of CO on triaminotoluene formation (from DAHAT) by the inhibition of sulfite reductase. Triaminotoluene is further anaerobically converted to unknown products by the isolate under sulfate-reducing and by a Pseudomonas strain under denitrifying conditions. Triaminotoluene conversion was also catalyzed in the absence of cells under aerobic conditions by trace elements, especially by Mn²⁺, accompanied by the elimination of ammonia in a stoichiometry of 1 NH₃ released per TAT transformed. The results might be of interest for the bioremediation of wastewater polluted with nitroaromatic compounds.Abbreviations TNT = 2,4,6-Trinitrotoluene DANT - 2,4-DANT = 2,4-Diamino-6-nitrotoluene - 2,6-DANT = 2,6-Diamino-4-nitrotoluene - ADNT = aminodinitrotoluene - 2-ADNT and 4-ADNT amino substituent at positions 2 or 4 - TAT = 2,4,6-Triaminotoluene - DAHAT = 2,4-Diamino-6-hydroxylaminotoluene - MV = Methyl viologen - Fd = Ferredoxin - H₂ase = Hydrogenase - CODH = Carbon monoxide dehydrogenase - Pyr: Fd OR = Pyruvate: ferredoxin oxidoreductase - U = Units = mol of substrate converted per min