Commensal relationship between a sheath-forming bacterium, Sphaerotilus natans, and a sheath-degrading bacterium, Paenibacillus sp

Paenibacillus sp. strain TB is capable of degrading the sheath prepared from a sheathed bacterium, Sphaerotilus natans. S. natans was able to grow alone on casamino acids but strain TB was not. Cocultivation of strain TB and S. natans was examined in a medium supplemented with casamino acids as a growth substrate. The growth of strain TB was observed when the sheath was supplied to the medium or in cocultivation with S. natans. The phospholipid amount reached a maximum after 24 h of cocultivation and subsequently kept almost the same level for 96 h. The sheath amount also reached a maximum after 24 h and then gradually declined. The cell concentration of strain TB increased throughout the cocultivation. By competitive PCR targeted for amplification of a part of 16S rDNA, the abundance ratio (S. natans/strain TB) of 6.7 was obtained at 72 h. Almost no growth of strain TB was detected in a coculture with a sheath-less mutant of S. natans. The evidence allows the conclusion that strain TB grew by utilizing the intact sheath in coculture with S. natans.     

Ethanol from Zymomonas,a bacterium

Thirteen isolates ofZymomonas were analyzed for their ability to tolerate increasing concentrations of glucose and ethanol. In medium containing 5.0% (v/v) ethanol, four isolates grew well in 15.0% (w/v) glucose. Six cultures tolerated at least 6,0% ethanol. Of all the isolates, 7 preferred glucose and 4 preferred sucrose as a sugar substrate. In a nutrient medium containing mineral salts and high concentrations of pantothenate and biotin ethanol production for 2 isolates was approximately 7.0%. Continuous stirring and growth factors were responsible for this increased ethanol production.     

棉花根际固氮菌、解磷菌及解钾菌的相互作用

目的通过对棉花根际促生细菌N2126、P1108和K2116菌株单独接种和混合接种,根据这些菌株的固氮、解磷、解钾能力和细胞数量的变化,了解它们之间的相互作用。方法将这3株菌株设置4个不同的组合:N2126+P1108、P1108+K2116、N2126+K2116及N2126+P1108+K2116,分别测定培养液中全氮含量,水溶性磷、钾含量和细胞数量。结果P1108对N2126的生长有促进作用但抑制K2116的生长,N2126和K2116之间存在拮抗作用。N2126、P1108和K2116混合培养后,三者细胞数量分别占培养液中细胞总数的6.4%、89.2%和4.4%;培养液中的全氮含量比不接种时下降了0.7%;水溶性磷、钾含量比不接种时分别增加了19.0%和12.2%。结论P1108为3株菌株混合培养时的优势菌株,3株菌株混合培养有助于磷、钾释放。     

Structural analysis of the sheath of a sheathed bacterium, Leptothrix cholodnii

Leptothrix cholodnii is an aerobic sheath-forming bacterium often found in oligotrophic and metal-rich aquatic environments. The sheath of this bacterium was isolated by selectively lysing the cells. Glycine and cysteine were the major amino acids of the sheath. The sheath was readily dissolved in hydrazine, and a polysaccharide substituted with cysteine was recovered from the solution. Galactosamine, glucosamine and galacturonic acid were detected in the hydrazinolysate by gas liquid chromatography analysis. FAB-MS analysis of the hydrazinolysate suggested a sugar sequence of HexN-GalA-HexN-HexN. Methylation linkage analysis revealed the presence of 4-linked GalA, 3-linked HexN and 4-linked HexN. The sulfhydryl groups of the sheath were used for labeling with the fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The labeled sheath (ABD-sheath) was partially hydrolyzed and three fluorescent fragments were purified by HPLC. One of them was identified as ABD-cysteine. The second one was found to be the ABD-cysteine tetramer. Another fragment was indicated to be a pentasaccharide substituted with ABD-cysteine by nuclear magnetic resonance (NMR) analysis. It can be assumed that the polysaccharide and peptide moieties of the sheath are connected by a cysteine residue. NMR analysis of the hydrazinolysate revealed that the polysaccharide moiety of the sheath was constructed from a pentasaccharide repeating unit containing 2-amino-2-deoxygalacturonic acid (GalNA), as shown below. -->4)-alpha-GalNA-(1-->4)-alpha-D-GalN(p)-(1-->4)-alpha-D-GalA(p)-(1-->4)-beta-D-GlcN(p)-(1-->3)-beta-D-GalN(p)-(1-->.     

Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum

The major carotenoid pigments of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum, were identified as zeaxanthin, beta-cryptoxanthin, and beta-carotene. Analysis was based on ultraviolet-visible spectroscopy, mass spectroscopy, and reversed-phase HPLC. Photoacoustic spectroscopy of intact bacterial cells revealed that the bulk of the pigments in S. antarcticus and S. multivorum was associated with the cell membrane. In vitro studies with synthetic membranes of phosphatidylcholine demonstrated that the major pigment was bound to the membranes and decreased their fluidity. The relative amounts of polar pigments were higher in cells grown at 5 degrees C than in cells grown at 25 degrees C. In the mesophilic strain, the synthesis of polar carotenoids was quantitatively less than that of the psychrotolerant strain.     

Enzymatic characterization of a serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus

We investigated the enzymatic properties of a serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k(cat)/K(M) value. The pH profile of activity, similarly to other serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu(2+) and Co(2+) ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn(2+) was inhibitory (both are unusual among serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn(2+), Cu(2+) and Co(2+) up to 90-200% of its original value, while Zn(2+) was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the serralysins contain Asn.     

一株氯霉素降解细菌的分离鉴定与代谢特性研究

微生物是介导环境中氯霉素降解转化的主要驱动者,但高效降解矿化菌株资源匮乏,氧化反应介导的代谢途径不清。为研究微生物介导下氯霉素的环境归趋过程,为氯霉素污染环境强化修复提供菌株资源,文中以受氯霉素污染的活性污泥为接种源,首先富集获得一个由红球菌Rhodococcus主导 (相对丰度>70%) 的氯霉素高效降解菌群,并从中分离获得一株能够高效降解氯霉素的菌株CAP-2,通过16S rRNA基因分析鉴定为红球菌Rhodococcus sp.。菌株CAP-2能在不同营养条件下高效降解氯霉素。基于菌株CAP-2对检测到的代谢产物对硝基苯甲酸和已报道的代谢产物对硝基苯甲醛和原儿茶酸的生物转化特征,提出其降解途径是由氯霉素侧链氧化断裂生成对硝基苯甲醛,进一步氧化为对硝基苯甲酸的新型氧化降解途径。该菌株对于氯霉素分解代谢的分子机制研究以及受氯霉素污染环境的原位生物修复应用具有巨大潜力。     

Tracing the domestication of a biofilm-forming bacterium

Over the course of more than a century of laboratory experimentation, Bacillus subtilis has become "domesticated," losing its ability to carry out many behaviors characteristic of its wild ancestors. One such characteristic is the ability to form architecturally complex communities, referred to as biofilms. Previous work has shown that the laboratory strain 168 forms markedly attenuated biofilms compared with the wild strain NCIB3610 (3610), even after repair of a mutation in sfp (a gene involved in surfactin production) previously known to impair biofilm formation. Here, we show that in addition to the sfp mutation, mutations in epsC, swrA, and degQ are necessary and sufficient to explain the inability of the laboratory strain to produce robust biofilms. Finally, we show that the architecture of the biofilm is markedly influenced by a large plasmid present in 3610 but not 168 and that the effect of the plasmid can be attributed to a gene we designate rapP. When rapP is introduced into 168 together with wild-type alleles of sfp, epsC, swrA, and degQ, the resulting repaired laboratory strain forms biofilms that are as robust as and essentially indistinguishable in architecture from those of the wild strain, 3610. Thus, domestication of B. subtilis involved the accumulation of four mutations and the loss of a plasmid-borne gene.     

Ecological studies of Chloroflexis,a gliding photosynthetic bacterium

Summary Chloroflexis, a gliding, filamentous, photosynthetic bacterium, is present in the stratified algal-bacterial mats which occur in the 50°–70°C temperature range of alkaline hot spring effluents. The organism is in association with the alga in the upper, algal layer, and also forms thick, orange mats beneath the algal layer. Natural populations of Chloroflexis from these mats demonstrated light-stimulated uptake of some ¹⁴C-labelled organic compounds. Photosynthetic ¹⁴CO₂ fixation by natural samples of Chloroflexis was investigated with respect to temperature, light intensity and mat depth. Bacterial photosynthesis was determined in samples in which algae were present by use of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Bacterial photosynthesis was maximal at depths down to about 3 mm and then decreased rapidly to very low levels at greater depths. The greatest amounts of bacteriochlorophyll pigments were also concentrated in the top 3–4 mm of the mat. The optimum light intensity for bacterial photosynthesis (about 400 ft-c) was considerably lower than the normal summer light intensity at the surface of the mat (5000-8000 ft-c).The temperature optima for photosynthesis by the bacterial component of natural mat samples from several sites of different temperatures in a hot spring thermal gradient were determined. Temperature optima approximated the environmental temperatures, indicative of the occurrence of strains of Chloroflexis adapted to different temperatures. Although bacterial standing crop was greatest in the temperature range 50°–55°C, maximum photosynthetic efficiency was observed at about 45°C. Sulfide was stimulatory to photosynthetic ¹⁴CO₂ fixation by naturally occurring populations of Chloroflexis under field conditions. These data are consistent with the hypothesis that Chloroflexis may utilize sulfide as an electron donor for photosynthetic CO₂ reduction. However, it is also likely that Chloroflexis grows photoheterotrophically in these mats, obtaining organic compounds from algal excretory products.     

Thiobacillus strain Q,a chemolithoheterotrophic sulphur bacterium

The physiological properties of an organism isolated from a selective chemostat enrichment using acetate and thiosulphate as the limiting substrates, provisionally called Thiobacillus Q, were investigated. Although the organism made up 85% of the community in the enrichment culture, its expected chemolithotrophic nature was not apparent in batch experiments. The growth yield was not enhanced by the addition of thiosulphate to an acetate containing mineral medium, even though up to 50% of the thiosulphate was oxidized. Under acetate limitation in the chemostat, there was a linear increase in yield with thiosulphate addition up to a concentration of 7 mM. Higher thiosulphate concentrations resulted in loss of thiosulphate oxidizing capacity and a decrease in the biomass to the level obtained with acetate alone. This loss may be due to the presence of inhibitory (50–100 M) levels of sulphite which is probably produced as an intermediate of the biological thiosulphate oxidation. Experiments with sulphide showed that Thiobacillus Q could also use it as an additional energy source. The complete lack of autotrophic growth, both in batch and chemostat experiments, together with the absence of even very low amounts of the key enzymes of the Calvin cycle demonstrated that this organism is a typical chemolithoheterotroph. Although this organism has provisionally been placed in the genus Thiobacillus, standard taxonomic procedures showed a close relationship with Pseudomonas alcaligenes. This study stresses the importance of quantitative chemostat studies in establishing the role of inorganic oxidations in energy metabolism and in the understanding of the role of heterotrophic sulphur oxidation in natural environments.     

Perchlorate reduction by a novel chemolithoautotrophic,hydrogen-oxidizing bacterium

Water treatment technologies are needed that can remove perchlorate from drinking water without introducing organic chemicals that stimulate bacterial growth in water distribution systems. Hydrogen is an ideal energy source for bacterial degradation of perchlorate as it leaves no organic residue and is sparingly soluble. We describe here the isolation of a perchlorate-respiring, hydrogen-oxidizing bacterium (Dechloromonas sp. strain HZ) that grows with carbon dioxide as sole carbon source. Strain HZ is a Gram-negative, rod-shaped facultative anaerobe that was isolated from a gas-phase anaerobic packed-bed biofilm reactor treating perchlorate-contaminated groundwater. The ability of strain HZ to grow autotrophically with carbon dioxide as the sole carbon source was confirmed by demonstrating that biomass carbon (100.9%) was derived from CO2. Chemolithotrophic growth with hydrogen was coupled with complete reduction of perchlorate (10 mM) to chloride with a maximum doubling time of 8.9 h. Strain HZ also grew using acetate as the electron donor and chlorate, nitrate, or oxygen (but not sulphate) as an electron acceptor. Phylogenetic analysis of the 16S rRNA sequence placed strain HZ in the genus Dechloromonas within the beta subgroup of the Proteobacteria. The study of this and other novel perchlorate-reducing bacteria may lead to new, safe technologies for removing perchlorate and other chemical pollutants from drinking water.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Rhodococcus pyridinovorans MW3, a bacterium producing a nitrile hydratase

Rhodococcus pyridinovorans MW3 was isolated from an arable land of manioc from the Congo for its ability to transform acrylonitrile to acrylamide. This strain contains a cobalt nitrile hydratase (NHase) showing high sequence homology with NHases so far described. The specific NHase activity was 97 U mg(-1) dry wt. NHase production by R. pyridinovorans MW3 was urea and Co-dependent. The NHase was active for acrylamide up to 60% (w/v) indicating its potential for acrylamide production.     

Proteomic analysis of Lactococcus lactis,a lactic acid bacterium

Lactococcus lactis is a Gram-positive bacteria, which belongs to the group of lactic acid bacteria among which several genera play an essential role in the manufacture of food products. Cytosolic proteins of L. lactis IL1403 cultivated in M17 broth have been resolved by two-dimensional gel electrophoresis using two pH gradients (pH 4-7, 4.5-5.5). More than 230 spots were identified by peptide mass fingerprints, corresponding to 25% of the predicted acid proteome. The present study made it possible to describe at the proteome level a significant number of cellular pathways (glycolysis, fermentation, nucleotide metabolism, proteolysis, fatty acid and peptidoglycan synthesis) related to important physiological processes and technological properties. It also indicated that the fermentative metabolism, which characterizes L. lactis is associated with a high expression of glycolytic enzymes. Thirty-four proteins were matched to open reading frames for which there is no assigned function. The comparison at the proteome level of two strains of L. lactis showed an important protein polymorphism. The comparison of the proteomes of glucose- and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.     

Acetogenium kivui,a new thermophilic hydrogen-oxidizing acetogenic bacterium

Hydrogen-oxidizing acetogenic bacteria in pure culture are presently represented by the two mesophilic species, Acetobacterium woodii and Clostridium aceticum. From Lake Kivu we have isolated a Gram negative, chemolithotrophic, thermophilic anaerobe (LKT-1) that oxidizes hydrogen and reduces carbon dioxide to acetic acid. It is a non-motile, non-sporeforming rod, about 0.7m in width and 2–7.5m in length, often occuring in pairs or chains. The cell wall has a banded appearance; the surface layer contains a regular array of particles with six-fold rotational symmetry. No outer membrane is present. The temperature optimum for growth is 66°C, and the pH optimum is 6.4. Organic growth substrates include glucose, mannose, fructose, pyruvate, and formate; acetate is the principal product. The doubling time for growth on hydrogen and carbon dioxide is about 2h. Vitamins are neither required nor stimulatory. Yeast extract and Trypticase enhance the final yield but do not affect the growth rate. Cysteine or sulfide are required and cannot be replaced by thioglycolate or dithiothreitol. LKT-1 was mass cultured on hydrogen and carbon dioxide in a 24.1 fermentor with a yield of 34g (wet weight) of cells. The DNA base composition as determined by buoyant density is 38 mol % guanine plus cytosine. LKT-1 appears only distantly related to physiologically similar bacteria. A new genus Acetogenium is proposed, and the species is Acetogenium kivui.     

A novel enzyme, lambda-carrageenase, isolated from a deep-sea bacterium

A lambda-carrageenan-degrading Pseudoalteromonas bacterium, strain CL19, was isolated from a deep-sea sediment sample. A lambda-carrageenase from the isolate was purified to homogeneity from cultures containing lambda-carrageenan as a carbon source. This is the first report of the isolation of lambda-carrageenase together with the gene sequence for the enzyme. The molecular mass of the purified enzyme was approximately 100 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that the enzyme is a monomer. The optimal pH and temperature for activity were about 7 and 35 degrees C, respectively. The enzyme had specific activity of 253 U/mg protein. The enzyme required monovalent salts for the activity. Carbohydrates, such as sorbitol, sucrose, trehalose, improved the enzyme stability. The pattern of lambda-carrageenan hydrolysis showed that the enzyme is an endo-type lambda-carrageenase, and the final main product was a tetrasaccharide of the lambda-carrageenan ideal structure with galactose 2,6-disulfate at the reducing end, indicating the enzyme cleaves the beta-1,4 linkages of its backbone structure. Furthermore, the gene (cglA) encoding the enzyme was sequenced. It encoded a mature protein of 103 kDa (917 amino acids). Remarkably, the deduced amino acid sequence showed no similarity to any reported proteins.     

Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium,Microlunatus phosphovorus

ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P(i) from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis.     

Draft genome sequence of the purple photosynthetic bacterium Phaeospirillum molischianum DSM120, a particularly versatile bacterium

Here we present the draft genome sequence of the versatile and adaptable purple photosynthetic bacterium Phaeospirillum molischianum DSM120. This study advances the understanding of the adaptability of this bacterium, as well as the differences between the Phaeospirillum and Rhodospirillum genera.     

Nitrogenase proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium

Gluconacetobacter diazotrophicus Pal-5 grew well and expressed nitrogenase activity in the absence of NH4+ and at initial O2 concentrations greater than 5% in the culture atmosphere. G. diazotrophicus nitrogenase consisted of two components, Gd1 and Gd2, which were difficult to separate but were purified individually to homogeneity. Their compositions were very similar to those of Azotobacter vinelandii nitrogenase, however, all subunits were slightly smaller in size. The purified Gd1 protein contained a 12:1 Fe/Mo ratio as compared to 14:1 found for Av1 purified in parallel. Both Gd2 and Av2 contained 3.9 Fe atoms per molecule. Dithionite-reduced Gd1 exhibited EPR features at g=3.69, 3.96, and 4.16 compared with 3.64 and 4.27 for Av1. Gd2 gave an S=1/2 EPR signal identical to that of Av2. A Gd1 maximum specific activity of 1600 nmol H2 (min mg of protein)(-1) was obtained when complemented with either Gd2 or Av2, however, more Av2 was required. Gd2 had specific activities of 600 and 1100 nmol H2 (min mg protein)(-1) when complemented with Av1 and Gd1, respectively. The purified G. diazotrophicus nitrogenase exhibited a narrowed pH range for effective catalysis compared to the A. vinelandii nitrogenase, however, both exhibited maximum specific activity at about pH 7. The Gd-nitrogenase was more sensitive to ionic strength than the Av-nitrogenase.     

Cultivation of a desulfurizing bacterium, Mycobacterium strain G3

Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO₄, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO₄ or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO₄. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.