Preparation of Purified Polysaccharides from Rhizobium

A method is described for the preparation of purified polysaccharides from strains of Rhizobium in quantities large enough so that with an exhaustive purification scheme enough product is recovered for various characterization purposes. When steps in the purification process are eliminated, much larger amounts of crude gum are obtained.

Organisms were grown in liquid medium and the crude gum was precipitated along with the bacterial cells by a quaternary ammonium complexing agent. This precipitate was dissolved in salt solution, reprecipitated with ethanol or ethanol-acetone mixtures several times, followed by pressure filtration with membrane filters.

The same procedure should be applicable to commercial scale production in large fermentors if a use for the gum could be shown. The method also should be suitable for other organisms with similar growth habits.

     

Assessment of Soluble and Biomass K in Culture Medium Is a Reliable Tool for Estimation of K Releasing Efficiency of Bacteria

Abstract

Assessing the amount of released K from minerals in bacterial liquid culture is the main process for screening and isolation of efficient potassium releasing bacteria (KRB). This study was aimed to determine the amount of released K in solution phase or supernatant (SK) as well as microbial biomass K (MBK). Therefore, 20 different bacterial isolates belonging to the 10 bacterial genera (Beijerinckia, Klebsiella, Azotobacter, Pseudomonas, Agrobacterium, Rhizobium, Sphingomonas, Citrobacter, Microbacterium, and Achromobacter) were individually used to inoculate Aleksandrov medium in presence of biotite or muscovite. Our results from in-vitro experiments revealed that the MBK (K in pellet) is more important than in SK. Although some genera such as Azotobacter and Citrobacter released more SK (16?mg/l from biotite and 12.77?mg/l from muscovite, respectively), the Klebsiella isolates with the highest MBK could release an average of 90?mg/l total K. This study indicated that the assimilated K in microbial cells is the main part of K dissolution from minerals. Due to the fast turnover of nutrients in bacterial biomass, it can be concluded that both SK and MBK could be available for plants. It seems that the finding of this research should be considered in the isolation of KRB.

Highlights

• This study reports, assessment of soluble and biomass K in the culture medium is a reliable tool for estimation of K releasing efficiency of bacteria

• Our results from in vitro experiments revealed that the assimilated K in microbial cells is the main part of K dissolved from minerals.

• Although some genera such as Azotobacter released more K in solution phase, the Klebsiella isolates with the highest biomass K could release more total K

     

Laser Impact on Bacterial ATP: Insights into the Mechanism of Laser-Bacteria Interactions

The mechanisms of laser action on bacteria are not adequately understood. Here, an attempt has been made to study the fluctuation in ATP (adenosine triphosphate) concentration following laser irradiation from a pulsed Nd:YAG laser on a marine biofilm-forming bacterium Pseudoalteromonas carrageenovora. A stationary phase bacterial suspension (density 10^7-8 ml^{m 1}) was exposed to pulsed laser irradiations at a fluence of 0.1 J cm^{m 2} (pulse width 5 ns, repetition rate 10 Hz) for different durations, ranging from 2 s to 15 min. The total viable count (TVC) and ATP concentration of the irradiated samples were determined immediately after the laser irradiation. While the maximum reduction in the TVC observed with respect to the control was 59% immediately after 15 min irradiation, the ATP concentration showed a reduction of about 86% for the same duration. The ATP concentration showed an abrupt reduction from 3 min of laser irradiation and continued to reduce significantly with increasing duration of irradiation. Thus, 3 min irradiation at a fluence of 0.1 J cm^{m 2} is considered as an approximate threshold for ATP production in this bacterium. As the decreased level of ATP production continued, bacterial mortality resulted. The reduction in ATP production could be due to damage caused by the laser irradiations on bacterial metabolic processes such as cellular respiration.     

Production,optimization, purification,characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate

Abstract

L-glutaminase from bacterial sources has been proven to be effective and economical agents in cancer therapy, food industry and high-value chemicals like threonine. In the present study, a newly isolated bacterial strain was potentially producing extracellular L-glutaminase, it identified as Bacillus subtilis OHEM11 (MK389501) using the 16S rRNA gene. L-glutaminase production optimized and the optimum factors for production under submerged fermentation were at pH 6.5–7.0 and 35?°C after 28?hr using rhamnose and glutamine as carbon and nitrogen sources, respectively, while bagasse was the best inducer for the production under solid-state fermentation. Ethanol precipitation and ion-exchange chromatography using QFF are the purification steps. L-glutaminase was purified to 2-fold with specific activity 89.78?U/mg and its molecular weight about 54.8?kDa with the alkaline property of the enzyme makes it clear having carcinostatic property; maximum enzyme activity at pH 8.2 and 40?°C and retained about 90% activity for 1?hr. The cytotoxicity effect of L-glutaminase indicated a significant safety on Vero cells with high anticancer activity against NFS-60, HepG-2, and MCF-7 cancer cell lines. The outcomes demonstrated that L-glutaminase could be applied in many biotechnological applications such as pharmaceutical and food processing.     

Elicitation of alkaloids in in vitro PLB (protocorm-like body) cultures of Pinellia ternata

The objective of this study was to determine the effect of two endophytic bacterial elicitors (Pseudomonas sp. and Enterobacter sp.) on the production of alkaloids in protocorm-like bodies (PLBs) of Pinellia ternata Breit. Both bacterial strains increased the growth rate of P. ternata PLBs. Pseudomonas sp. promoted the differentiation of the PLBs, whereas Enterobacter sp. inhibited PLB differentiation. The bacterial strains increased guanosine production in PLBs by 9–166%, inosine production by 2–33%, and trigonelline production by 114–1140% compared to the control. For Pseudomonas sp., guanosine and trigonelline production was greater when bacterial extracts were added to the PLB suspension cultures rather than living cells (co-culture treatment). Inosine production was similar in both the bacterial extract and co-culture treatments. For the Enterobacter sp., guanosine, inosine, and trigonelline production tended to be greatest when living cells were added to the PLB suspension cultures rather than bacterial extracts. These results suggest that Pseudomonas sp. and Enterobacter sp. could increase alkaloid yield from P. ternata under field or tissue culture conditions. We also observed that Pseudomonas sp. and Enterobacter sp. produced some of the same alkaloids as their host plants. Additional study needs to be done to determine if these endophytic bacteria could be used to produce alkaloids in the fermentation industry.     

Towards a better production of bacterial exopolysaccharides by controlling genetic as well as physico-chemical parameters

Bacterial extracellular polymeric substances, which are basically bacterial metabolites, have currently become a subject of great concern of modern day microbiologists and biotechnologists. Among these metabolites, bacterial exopolysaccharides or EPS, in particular, have gained a significant importance. EPS are formed by the bacteria in their late exponential or stationary phase of growth under special situations for specific purposes. They take part in the formation of bacterial biofilms. There is a great diversity in the types of EPS. Strikingly enough, a same species of bacterium can produce different types of EPS under different situations. The importance of EPS is largely because of their different applications in various industries. Now that the bacterial EPS has got the potentiality to become an upcoming tool in various futuristic applications of human benefit, the focus currently develops towards how better they can be produced in the laboratory by promoting the favorable factors for their production. While studying with different EPS forming bacteria, both the intrinsic factors like genetic configuration of the bacteria and the extrinsic factors like culture conditions under the influence of different physico-chemical parameters in order to maximize the EPS production have been taken into consideration. Both the factors have proved their worth. Hence, towards a better outcome for EPS production, it is indicated that a genetic manipulation of the bacteria should be synchronized with a proper selection of its culture condition by controlling different physico-chemical parameters.

     

DECOMPOSITION IN PELAGIC MARINE ECOSYSTEMS

SUMMARY

During the decomposition of plant detritus, complex microbial successions develop which are dominated in the early stages by a number of distinct bacterial morphotypes. The microheterotrophic community rapidly becomes heterogenous and may include cyanobacteria, fungi, yeasts and bactivorous protozoans.

Microheterotrophs in the marine environment may have a biomass comparable to that of all other heterotrophs and their significance as a resource to higher trophic orders, and in the regeneration of nutrients, particularly nitrogen, that support ‘regenerated’ primary production, has aroused both attention and controversy.

Soluble low molecular weight substrates (dissolved organic matter, or DOM) are for the most part rapidly turned over and readily taken up with a high growth efficiency by bacteria although detrital particulate organic material (POM) is turned over slowly and utilized with a low growth efficiency, owing to the structural complexity of the detritus. The presence of appropriate substrate-specific strains of bacteria Is important in the decomposition of both DOM and POM.

Estimates of the transfer of photosynthetically fixed carbon and nitrogen through the pelagic microbial community have recently become widespread. However, the quantification of C and N fluxes through bacteria and microzooplankton is very sensitive to accurate measures of microbial biomass, production, net growth yield, bacterial activity and bactivory by microzooplankton. These processes also vary significantly in both spatial and temporal dimensions during the development and decay of phytoplankton blooms.

Recent attempts to model decomposition processes and C and N fluxes In pelagic marine ecosystems are described. This review examines the most sensitive components and predictions of the models with particular reference to estimates of bacterial production, net growth yield and predictions of N cycling determined by ¹⁵N methodology.     

Investigating the role of T helper related cytokines in cerebrospinal fluid for the differential diagnosis of bacterial meningitis in pre-treated paediatric patients

Abstract

Purpose: Given the challenge in the diagnosis of bacterial meningitis (BM), we assessed different cytokines in the cerebrospinal fluid (CSF) of antibiotics pre-treated patients.

Materials and methods: Laboratory tests and polymerase chain reaction (PCR) were performed for 480 CSF samples from children (2 m to 14 y), suspicious to meningitis and pre-treated with antibiotics, to detect bacterial and viral aetiologies. Sixty-one CSF were included and the levels of 13 cytokines such as IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ and TNF-α were measured using flow-cytometry.

Results: All bacterial cultures were negative, but 29 and eight CSF were positive for bacterial and viral agents by PCR. IL-6, IL-10 and IFN-γ were significantly up-regulated in BM. T helper (Th) subset cytokines showed significant upregulation of Th1, Th2, Th17, Th22 and Tfh cytokines in BM. Common Th subsets cytokines (IL-6, IL-10 and TNF-α) were significantly different between the study groups. ROC curve analysis revealed good AUC for common Th related cytokines in discriminating BM.

Conclusions: In pre-treated BM patients with negative bacterial cultures, cytokines IL-6, IL-10 and IFN-γ can predict BM which could be beneficial for rapid diagnosis and treatment to decrease the sequela of the disease.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

The presence of psicofuranine in the fermentation medium caused the accumulation of a copious amount of 5′–XMP by Brevibacterium ammoniagenes. The accumulation of 5′–XMP in the medium was considered to be due to the inhibition of converting 5′–XMP to 5′–GMP by psicofuranine, which is known as a specific inhibitor of XMP aminase.

It was previously reported that in 5′–IMP fermentation with Br. ammoniagenes pantothenate and thiamine, in addition to biotin which was required for the growth of the microorganism, were exclusively required. This requirement for both vitamins was also observed in 5′–XMP production induced by the antibiotic.

The addition of manganese in excess to the fermentation medium promoted the bacterial growth greatly and inhibited IMP production, whereas XMP production induced by piscofuranine was not affected by the addition of excess manganese.

The accumulation of XMP induced by the antibiotic was completely suppressed by the presence of purine derivatives such as guanine, and xanthine derivatives, and partially by hypoxanthine.

5′–XMP was identified by chemical and enzymatic analyses and by UV absorption spectrum.     

Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype

Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, β- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

     

Plant growth promoting rhizhobacteria (PGPR)-mediated root proliferation in black pepper (Piper nigrum L.) as evidenced through GS Root software

Abstract

Pseudomonas fluorescens strains which are proven biocontrol agents in black pepper against foot rot (caused by Phytophthora capsici ) were also found to enhance root proliferation and fibre root production. Experiments conducted in the greenhouse with five efficient strains of P. fluorescens (IISR-6, IISR-8, IISR-11, IISR-13 and IISR-51) showed that the bacterial strains could significantly increase the root biomass of the plants (30 – 135%). Parameters for total root length, root area and root tips were estimated by scanning the entire root system and analysis through GS Root® software (PP systems, Winterstreet, USA). All the strains increased the root length in the treated plants (12 – 127%), the highest being with IISR-6, which was on a par with IISR-11 and IISR-51. A similar trend was observed with the total root area after bacterization (43 – 200%). The P. fluorescens treated plants had a higher number of feeder roots as evidenced by the increased number of root tips (82 – 137%). The enhanced growth parameters upon root bacterization could be corroborated with the production of the plant growth hormones IAA & GA by the bacterial strains and their P-solubilization potential.     

Functional analysis for gut microbes of the brown tree frog (Polypedates megacephalus) in artificial hibernation

Background

Annual hibernation is an adaptation that helps many animals conserve energy during food shortage in winter. This natural cycle is also accompanied by a remodeling of the intestinal immune system, which is an aspect of host biology that is both influenced by, and can itself influence, the microbiota. In amphibians, the bacteria in the intestinal tract show a drop in bacterial counts. The proportion of pathogenic bacteria is greater in hibernating frogs than that found in nonhibernating frogs. This suggests that some intestinal gut microbes in amphibians can be maintained and may contribute to the functions in this closed ecosystem during hibernation. However, these results were derived from culture-based approaches that only covered a small portion of bacteria in the intestinal tract.

Methods

In this study, we use a more comprehensive analysis, including bacterial appearance and functional prediction, to reveal the global changes in gut microbiota during artificial hibernation via high-throughput sequencing technology.

Results

Our results suggest that artificial hibernation in the brown tree frog (Polypedates megacephalus) could reduce microbial diversity, and artificially hibernating frogs tend to harbor core operational taxonomic units that are rarely distributed among nonhibernating frogs. In addition, artificial hibernation increased significantly the relative abundance of the red-leg syndrome-related pathogenic genus Citrobacter. Furthermore, functional predictions via PICRUSt and Tax4Fun suggested that artificial hibernation has effects on metabolism, disease, signal transduction, bacterial infection, and primary immunodeficiency.

Conclusions

We infer that artificial hibernation may impose potential effects on primary immunodeficiency and increase the risk of bacterial infections in the brown tree frog.

     

Soil pH is the primary factor driving the distribution and function of microorganisms in farmland soils in northeastern China

Purpose

To understand which environmental factors influence the distribution and ecological functions of bacteria in agricultural soil.

Method

A broad range of farmland soils was sampled from 206 locations in Jilin province, China. We used 16S rRNA gene-based Illumina HiSeq sequencing to estimated soil bacterial community structure and functions.

Result

The dominant taxa in terms of abundance were found to be, Actinobacteria, Acidobacteria, Gemmatimonadetes, Chloroflexi, and Proteobacteria. Bacterial communities were dominantly affected by soil pH, whereas soil organic carbon did not have a significant influence on bacterial communities. Soil pH was significantly positively correlated with bacterial operational taxonomic unit abundance and soil bacterial α-diversity (P<0.05) spatially rather than with soil nutrients. Bacterial functions were estimated using FAPROTAX, and the relative abundance of anaerobic and aerobic chemoheterotrophs, and nitrifying bacteria was 27.66%, 26.14%, and 6.87%, respectively, of the total bacterial community. Generally, the results indicate that soil pH is more important than nutrients in shaping bacterial communities in agricultural soils, including their ecological functions and biogeographic distribution.

     

Studies on Enzymatic Liquefaction and Saccharification of Starch

Liquefaction of corn starch is generally considered to be difficult to achieve. A double enzyme system, a combination of bacterial alpha-amylase and fungal glucoamylase, was tested for the production of dextrose from corn starch. The former liquefies starch and the latter hydrolyzes further into glucose

Particularly various conditions for liquefaction of corn starch were examined. As criteria of solubilization of starch molecule, turbidity of filtrate obtained while the solution was hot, iodine coloration value, filterability and turbidity of the saccharified solution after the addition of alcohol were measured.

Starch slurry containing bacterial alpha-amylase was poured continuously in fine stream into hot water with vigorous agitation. Liquefaction at 92°C was better than at 87°C for dissolving corn starch. It was indispensable to apply heat treatment under high pressure after liquefaction followed by the second addition of bacterial alpha-amylase at 90°C to render remaining tightly bound starch micelle to be broken down. Heat treatment was continued at 120°C or at higher temperature. Then, solution was cooled rapidly down to about 90°C. Immediately bacterial alpha-amylase was added. In case the second addition of bacterial alpha-amylase was carried out after cooling to 55°C or after holding for 10min at 80°C, no effect was observed by the second addition. These findings were confirmed in the experiments on commercial production scale.     

Exploiting Cell Death Pathways by an E. coli Cytotoxin: Autophagy as a Double-Edged Sword for the Host

Cytotoxic necrotizing factor 1 is a bacterial protein toxin from Escherichia coli that is able to activate the Rho GTPases and to hinder apoptosis and mitotic catastrophe. Upon exposure to toxin, cells undergo a complex framework of changes, including cytoskeleton remodeling and multinucleation. These cells also show a high survival rate for long periods of time and improve both their macropinocytotic scavenging activities and microautophagy. Only at the very end, probably when “feeding” materials are exhausted, they do these cells die by autophagy. Taking into account the complex role of bacterial protein toxins in the infectious processes, we indicate the CNF1 activity as a Janus-faced paradigm of those bacteria that hijack cell fate to their own benefit. This could somehow be linked to the hypothesized connection between certain bacterial toxins and cancer onset.

Addendum to:

Is the Rac GTPase-Activating Toxin CNF1 a Smart Hijacker of Host Cell Fate?

W. Malorni and C. Fiorentini

FASEB J 2006; 20:606-9     

Characterization of the gut microbiota in patients with primary sclerosing cholangitis compared to inflammatory bowel disease and healthy controls

Background

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease. Its etiology remains largely unknown, although frequent concomitant inflammatory bowel disease (IBD) hints towards common factors underlying intestinal and bile duct inflammation. Herein, we aimed to explore the relative abundance of fecal microbiota in PSC-IBD patients compared to IBD-only subjects and controls.

Methods and results

We included 14 PSC-IBD patients, 12 IBD-only patients, and 8 healthy controls (HCs). A quantitative real-time PCR (qPCR) assay was used to determine a selection of bacterial phyla, families, and genera.

Relative abundance of taxa showed that Bacteroidetes was the most abundant phylum among the patients with PSC-IBD (29.46%) and also HCs (39.34%), whereas the bacterial species belonging to the phylum Firmicutes were the most frequent group in IBD-only subjects (37.61%). The relative abundance of the Enterobacteriaceae family in fecal samples of PSC-IBD patients was similar to those with IBD-only, which was significantly higher than HCs (p value?=?0.031), and thus, could be used as a PSC-IBD or IBD-only associated microbial signature.

Conclusions

Our findings showed that intestinal microbiota composition in PSC-IBD patients was completely different from that of IBD-only patients. Further studies using large-scale cohorts should be performed to better describe the contribution of the gut microbiota to PSC pathogenesis with underlying IBD.

     

The rapid degradation of bisphenol A induced by the response of indigenous bacterial communities in sediment

In the present study, sediment was spiked with bisphenol A (BPA) solution to explore the interaction between indigenous bacterial communities and BPA biodegradation in sediment. Results showed that BPA could be adsorbed to the sediment and then biodegraded rapidly. Biodegradation efficiency of BPA in treatments with 10 and 50 mg/L BPA reached 64.3 and 61.8% on the first day, respectively. Quantitative polymerase chain reaction and denaturing gradient gel electrophoresis analysis indicated that BPA affected the densities, species, and diversities of bacteria significantly. The response of bacterial community to BPA favored BPA biodegradation by promoting the growth of BPA-reducing bacteria and inhibiting other competitors. According to the results of sequencing, Pseudomonas and Sphingomonas played vital roles in the degradation of BPA. They presented over 73% of the original bacterial community, and both of them were promoted by BPA comparing with controls. Laccase and polyphenol oxidase contributed to the degradation of BPA and metabolic intermediates, respectively. This paper illustrates the rapid biodegradation of BPA induced by the response of indigenous bacterial communities to the BPA stress, which will improve the understandings of BPA degradation in sediment.

     

Effect of Ethanol on Differential Protein Production and Expression of Potential Virulence Functions in the Opportunistic Pathogen Acinetobacter baumannii

Acinetobacter baumannii persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of A. baumannii ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of A. baumannii ATCC 17978 cells cultured in the presence of ethanol when tested with the Galleria mellonella experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by A. baumannii and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments.