Carbohydrate metabolism of the phase variants of purple photosynthetic bacteria

The R and M phase variants of Rhodobacter sphaeroides and Rhodobacter capsulatus were isolated. The growth rates in the dark and in the light in glucose-containing media were much higher for the Rba. sphaeroides R variant than for the M variant. For the Rba. capsulatus R and M variants, growth rates in the dark and in the light in fructose- or glucose-containing media differed insignificantly. The cells of Rba. sphaeroides and Rba. capsulatus phase variants growing in media with glucose and fructose exhibited differences in activity of the key enzymes of the Embden–Meyerhof–Parnas (EMP) and Entner–Doudoroff (ED) pathways. The oxidative pentose phosphate pathway (PPP) does not participate in glucose and fructose metabolism in the studied bacteria. Specific activity of the ED pathway enzymes was higher in dark-grown R and M variants of both Rba. sphaeroides and Rba. capsulatus than in the cells grown under light. Specific activity of the EMP enzymes was higher for the R and M variants of both cultures grown in the light than for those grown in the dark. Activities of the 2-keto-3-deoxy-6-phosphogluconate and fructose bisphosphate aldolases, the key enzymes of the ED and EMP pathways in Rba. sphaeroides M variant grown in the medium with glucose in the light or in the dark, were approximately twice those of the R variant. In the medium with fructose activities of these enzymes in both R and M variants did not change significantly depending on growth conditions. Activities of the enzymes of the EMP and ED pathways in the extracts of the Rba. capsulatus R and M cells grown with glucose or fructose did not change significantly. Cultivation of Rba. sphaeroides and Rba. capsulatus phase variants in the medium with fructose resulted in a considerably increased synthesis of 1-phosphofructokinase. Induction of 1-phosphofructokinase synthesis in Rba. sphaeroides occurred only in the light, while in Rba. capsulatus induction of this enzyme in the medium with fructose was observed both in the dark and in the light. Thus, under aerobic conditions in the dark the phase variants of both bacteria probably assimilated glucose and fructose via the ED pathway, while in the light the EMP pathway was active.     

<Emphasis Type="Italic">In vivo</Emphasis> Proinflammatory Cytokine Production by CD-1 Mice in Response to Equipotential Doses of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> PG and <Emphasis Type="Italic">Salmonella enterica</Emphasis> Lipopolysaccharides

The capacities of relatively nontoxic lipopolysaccharide (LPS) from Rhodobacter capsulatus PG and highly potent LPS from Salmonella enterica serovar Typhimurium to evoke proinflammatory cytokine production have been compared in vivo. Intravenous administration of S. enterica LPS at a relatively low dose (1 mg/kg body weight) led to upregulation of TNF-α, IL-6, and IFN-γ production by non-sensitized CD-1 mice. LPS from R. capsulatus PG used at a four-times higher dose than that from S. enterica elicited production of almost the same amount of systemic TNF-α; therefore, the doses of 4 mg/kg LPS from R. capsulatus PG and 1 mg/kg LPS from S. enterica were considered to be approximately equipotential doses with respect to the LPS-dependent TNF-α production by CD-1 mice. Rhodobacter capsulatus PG LPS was a weaker inducer of the production of TNF-α, IL-6, and IFN-γ, as compared to the equipotential dose of S. enterica LPS. Administration of R. capsulatus PG LPS before S. enterica LPS decreased production of IFN-γ, but not of TNF-α and IL-6, induced by S. enterica LPS. Rhodobacter capsulatus PG LPS also suppressed IFN-γ production induced by S. enterica LPS when R. capsulatus PG LPS had been injected as little as 10 min after S. enterica LPS, but to a much lesser extent. Rhodobacter capsulatus PG LPS did not affect TNF-α and IL-6 production induced by the equipotential dose of S. enterica LPS. In order to draw conclusion on the endotoxic activity of particular LPSs, species-specific structure or arrangement of the animal or human immune systems should be considered.     

Relations between the chemotype of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> strains and the cell electrophoretic properties

The cells of two Rhodobacter capsulatus strains, B10 and PG, and the LPS of their cell walls were studied by electrophysical and biochemical methods. Strain B10 was found to belong to the R chemotype, and strain PG, to the RS chemotype. A relation was revealed between the chemotype of the photosynthesizing bacteria Rhodobacter capsulatus and the electrophoretic properties of their cells.     

Dynamics of antagonistic potency of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> PG lipopolysaccharide against endotoxin-induced effects

The dynamics of antagonistic potency of lipopolysaccharide (LPS) isolated from Rhodobacter capsulatus PG on the synthesis of proinflammatory (TNF-α, IL-1β, IL-8, IL-6, IFN-γ) and antiinflammatory (IL-10, IL-1Ra) cytokines induced by highly stimulatory endotoxins from Escherichia coli or Salmonella enterica have been studied. Using human whole blood, we have shown that R. capsulatus PG LPS inhibited most pronouncedly the endotoxin-induced synthesis of TNF-α, IL-1β, IL-8, and IL-6 during the first 6 h after endotoxin challenge. Similarly, the endotoxin-induced release of IFN-γ was abolished by R. capsulatus PG LPS as well (24 h). In contrast to the above-mentioned cytokines, the relatively weak antagonistic activity of R. capsulatus PG LPS against endotoxin-triggered production of IL-6 and IL-8 was revealed. Since R. capsulatus PG LPS displays more potent antagonistic activity against deleterious effects of S. enterica LPS than those of E. coli LPS in the cases of such cytokines as IL-1β (6 and 24 h), IL-6 and IL-8 (4 h), we conclude that the effectiveness of protective action of antagonist is mostly determined by the primary lipid A structure of the employed agonist.     

Synthesis of bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis> by the purple nonsulfur bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h^?1 were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h^?1.     

5-Aminolevulinic acid production from inexpensive glucose by engineering the C4 pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

5-Aminolevulinic acid (ALA), the first committed intermediate for natural biosynthesis of tetrapyrrole compounds, has recently drawn intensive attention due to its broad potential applications. In this study, we describe the construction of recombinant Escherichia coli strains for ALA production from glucose via the C4 pathway. The hemA gene from Rhodobacter capsulatus was optimally overexpressed using a ribosome binding site engineering strategy, which enhanced ALA production substantially from 20 to 689 mg/L. Following optimization of biosynthesis pathways towards coenzyme A and precursor (glycine and succinyl-CoA), and downregulation of hemB expression, the production of ALA was further increased to 2.81 g/L in batch-fermentation.     

High-precision determination of quantum yields of photoelectric excitation conversion in reaction centers of purple bacteria <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> R-26

Using an original model of primary events in bacterial photosynthetic reaction centers (RCs), a complete set of kinetic and energy characteristics, and temperature dependences of nanosecond luminescence component(s) emitted by the RCs, upper limits to the quantum yield of primary transmembrane charge separation in RCs of the purple bacterium Rhodospirillum rubrum and the carotenoidless mutant Rhodobacter sphaeroides R-26 have been estimated. The calculations have given the values of 0.915 ± 0.045 and 0.978 ± 0.006, respectively. In the case of Rb. sphaeroides R-26, the quantum yield partly overlapped with that reported earlier (1.02 ± 0.04) by Wraight and Clayton (BBA, 1974. vol. 333, pp. 246–260).     

Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

Diversity and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> isolates obtained from rice rhizosphere soils in Taiwan

Azotobacter species, free-living nitrogen-fixing bacteria, have been used as biofertilizers to improve the productivity of non-leguminous crops, including rice, due to their various plant growth-promoting traits. The purposes of this study were to characterize Azotobacter species isolated from rice rhizospheres in Taiwan and to determine the relationship between the species diversity of Azotobacter and soil properties. A total of 98 Azotobacter isolates were isolated from 27 paddy fields, and 16S rRNA gene sequences were used to identify Azotobacter species. The characteristics of these Azotobacter strains were analyzed including carbon source utilization and plant growth-promoting traits such as nitrogen fixation activity, indole acetic acid production, phosphate-solubilizing ability, and siderophore secretion. Of the 98 strains isolated in this study, 12 were selected to evaluate their effects on rice growth. Four species of Azotobacter were identified within these 98 strains, including A. beijerinckii, A. chroococcum, A. tropicalis, and A. vinelandii. Of these four species, A. chroococcum was predominant (51.0%) but A. beijerinckii had the highest level of nucleotide diversity. Strains within individual Azotobacter species showed diverse profiles in carbon source utilization. In addition, the species diversity of Azotobacter was significantly related to soil pH, Mn, and Zn. Members of the same Azotobacter species showed diverse plant growth-promoting traits, suggesting that the 98 strains isolated in this study may not equally effective in promoting rice growth. Of the 12 strains evaluated, A. beijerinckii CHB 461, A. chroococcum CHB 846, and A. chroococcum CHB 869 may be used to develop biofertilizers for rice cultivation because they significantly promoted rice growth. This study contributes to the selection of suitable Azotobacter strains for developing biofertilizer formulations and soil management strategies of Azotobacter for paddy fields.     

<Emphasis Type="Italic">Sinorhodobacter hungdaonensis</Emphasis> sp. nov. isolated from activated sludge collected from a municipal wastewater treatment plant

A novel bacterium, strain L3^T, was isolated from an activated sludge sample retrieved from a municipal wastewater treatment plant in Huangdao, China. On the basis of 16S rRNA gene sequence similarity studies, strain L3^T was affiliated to the genus Sinorhodobacter, being most closely related to Sinorhodobacter ferrireducens (98.0 %). The 16S rRNA gene sequence similarity of strain L3^T to other related species, Thioclava atlantica DLFJ1-1^T (96.5 %), Rhodobacter capsulatus ATCC 11166^T (96.3 %),Paenirhodobacter enshiensis DW2-9^T (96.3 %) and Rhodobacter viridis JA737^T (96.0 %) is less than 96.5 %. Chemotaxonomic characterization further supported classification of the strain to the genus Sinorhodobacter. The major polar lipid profile consists of diphosphatidyglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids are C_18:1 ω7c (66.3 %), C_16:0 (12.9 %) and C_18:0 (8.0 %). The major quinone is Q-10. The G+C content of the genomic DNA of strain L3^T is 68.0 mol %. DNA–DNA relatedness value between L3^T and the closely related type strain S. ferrireducens SgZ-3^T was 35.2 %. Based on these results, a new species Sinorhodobacter huangdaonensis is proposed. The type strain is L3^T (= CGMCC 1.12963^T = KCTC 42823^T).     

The taxonomic composition,characteristics, and neurotoxic activities of ribbon worm-associated bacteria from the Sea of Japan

The taxonomic composition of bacteria associated with two species of tetrodotoxin-bearing (TTX-bearing) (Hubrechtella juliae and Lineus alborostratus) and two species of non-TTX-bearing (Quasitetrastemma stimpsoni and Malacobdella grossa) ribbon worms collected from the Peter the Great Bay of Sea of Japan was studied. Bacterial isolates were identified using 16S rRNA gene sequencing and phenotypic characteristics. Thirty-eight strains of heterotrophic bacteria from the eight genera: Pseudoalteromonas, Shewanella, Ruegeria, Pseudomonas, Defluviicoccus, Vibrio, Alteromonas, and Bacillus, were isolated and characterized. γ-Proteobacteria dominated among the associated microflora of nemerteans (76.3% of the total number of isolates). Sensitivity analysis of 38 strains to antibiotics of various classes revealed multiple resistance to three or more antibiotics in all of the studied isolates. The 15 bacterial strains isolated in the study exhibited antimicrobial activities against at least one of five indicator microorganisms, most of which corresponded to the Pseudoalteromonas genus. Screening of the TTX-producing bacteria was performed using confocal laserscanning microscopy and polyclonal antibodies. A TTX-producing strain, Pseudoalteromonas sp., was found in the nemertean H. juliae. A correlation between the presence of TTX-positive microflora and the toxicity of nemerteans was determined.     

Root-associated bacteria influencing mycelial growth of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> (pine mushroom)

Tricholoma matsutake is an ectomycorrhizal fungus usually associated with Pinus densiflora in South Korea. Fruiting bodies (mushrooms) of T. matsutake are economically important due to their attractive aroma; yet, T. matsutake is uncultivatable and its habitat is rapidly being eradicated due to global climate change. Root-associated bacteria can influence the growth of ectomycorrhizal fungi that co-exist in the host rhizosphere and distinctive bacterial communities are associated with T. matsutake. In this study, we investigated how these bacterial communities affect T. matsutake growth by isolating bacteria from the roots of P. densiflora colonized by ectomycorrhizae of T. matsutake and co-culturing rootassociated bacteria with T. matsutake isolates. Thirteen species of bacteria (27 isolates) were found in pine roots, all belonging to the orders Bacillales or Burkholderiales. Two species in the genus Paenibacillus promoted the growth of T. matsutake in glucose poor conditions, likely using soluble metabolites. In contrast, other bacteria suppressed the growth of T. matsutake using both soluble and volatile metabolites. Antifungal activity was more frequent in glucose poor conditions. In general, pine rhizospheres harbored many bacteria that had a negative impact on T. matsutake growth and the few Paenibacillus species that promoted T. matsutake growth. Paenibacillus species, therefore, may represent a promising resource toward successful cultivation of T. matsutake.     

Distribution of bacteriochlorophyll between the pigment-protein complexes of the sulfur photosynthetic bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis> depending on light intensity at different temperatures

Variation of the distribution of bacteriochlorophyll a (BChl a) between external antenna (LH2) and core complexes (LH1 + RC) of the photosynthetic membrane of the sulfur bacterium Allochromatium minutissimum was studied at light intensities of 5 and 90 Wt/m² in the temperature range of 12–43°C. The increase of light intensity was shown to result in a 1.5-to 2-times increase of a photosynthetic unit (PSU). PSU sizes pass through a maximum depending on growth temperature, and the increase of light intensity (5 and 90 Wt/m²) results in a shift of the maximal PSU size to higher temperatures (15 and 20°C, respectively). In the narrow temperature interval of ～14–17°C, the ratio of light intensity to PSU size is typical of phototrophs: lower light intensity corresponds to larger PSU size. The pattern of PSU size change depending on light intensity was shown to differ at extreme growth temperatures (12°C and over 35°C). The comparison of Alc. minutissimum PSU size with the data on Rhodobacter capsulatus and Rhodopseudomonas palustris by measuring the effective optical absorption cross-section for the reaction of photoinhibition of respiration shows a two to four times greater size of light-harvesting antenna for Alc. minutissimum, which seems to correspond to the maximum possible limit for purple bacteria.     

Diversity of bacteria of the genus Bacillus on board of international space station

From swabs of surfaces of equipment and air samples of the Russian segment of the International Space Station, nine strains of spore-forming bacteria of the genus Bacillus belonging to the species B. pumilus, B. licheniformis, B. subtilis, B. megaterium, and B. amyloliquefaciens were isolated. The last species of bacilli on the equipment of RS ISS was detected for the first time. For these species of bacilli, there are known strains that can be opportunistic to humans, and their metabolites can cause biodegradation of equipment and materials. B. pumilus found on ISS belongs to the group of bacteria that exhibits a particularly high resistance to adverse environmental conditions, such as dehydration, ultraviolet and gamma radiation, and chemical disinfection.     

Serologic characteristics of certain root-nodule bacteria of legumes

Rhizobium trifolii organisms associated with eleven species ofTrifolium were isolated and studied by means of antigen-antibody agglutination and immune diffusion techniques. Ten serologic types were differentiated from the eleven species of nativeTrifolium. Certain serologic types of rhizobia were widely distributed among the native species ofTrifolium, whereas other rhizobial types were found only on one or two species. The distribution pattern appears to be independent of the proportion of each type present in the soil population. This may indicate specificity of selection between a host legume and serologic types of rhizobia.     

Thermotolerant oil-degrading bacteria isolated from soil and water of geographically distant regions

Oil-degrading bacteria were isolated from soil and water samples taken in Russia, Kazakhstan, and the Antarctic; 13 of 86 strains proved to be thermotolerant. These bacteria utilized crude oil at 45–50°C; their growth optimum (35–37°C) and range (20–53°C) differ from those of mesophilic bacteria. Thermotolerant strains were identified as representatives of the genera Rhodococcus and Gordonia. It was shown that their ability to degrade petroleum products does not differ at 24 and 45°C. The strains Rhodococcus sp. Par7 and Gordonia sp. 1D utilized 14 and 20% of the oil, respectively, in 14 days at 45°C. All of the isolated thermotolerant bacteria grew in a medium containing 3% NaCl; the medium for the strains Gordonia amicalis 1B and Gordonia sp. 1D contained up to 10% NaCl. The bacteria G. amicalis and Rhodococcus erythropolis were able to utilize crude oil and individual hydrocarbons at higher (up to 50°C) temperatures.     

Biofilm-associated indole acetic acid producing bacteria and their impact in the proliferation of biofilm mats in solar salterns

Biofilm mats appear in salterns distinctively during the monsoon season when the salinity decreases below 12 percentile salinity units and within a short period cover the entire surface area of the saltern. A study was carried out in two salterns viz. Nerul and Curca to find a possible reason for the rapid proliferation of these solar biofilms. Out of the 125 bacteria isolated from these biofilms, 16 produced indole-3-acetic acid (IAA). Rapid in-situ assay with Salkowski reagent and HPLC analysis confirmed the IAA production. Four isolates consistently produced high IAA concentrations ranging from 9.5 to 14.2 μg/mL in the presence of 4 mg/mL tryptophan concentrations in the growth media. The IAA-producing bacteria were Aeromonas aquariorum (N2), Pseudomonas alcaliphila (N3), Vibrio diazotrophicus (N6) and Pseudomonas pachastrellae (C3). These four IAA-producing bacteria also produced other growth promoting factors like ammonia. Three isolates produced siderophores and were phosphate solubilizers. There was enhancement in the growth of Cicer arietinum (length of the shoot and root) under axenic conditions and of biofilm mats (r = 0.9, p < 0.001; r = 0.8, p < 0.05 and r = 0.946, p < 0.01, respectively). This is, according to our knowledge, the first report indicating IAA-producing bacteria isolated from biofilms enhancing the proliferation of these biofilm mats in the solar salterns.     

Taxonomic hierarchy of the phylum <Emphasis Type="Italic">Firmicutes</Emphasis> and novel <Emphasis Type="Italic">Firmicutes</Emphasis> species originated from various environments in Korea

This study assessed the taxonomic hierarchy of the phylum Firmicutes as well as elucidated the isolation and classification states of novel Firmicutes species isolated from Korean territory. The hierarchical classification system of the phylum Firmicutes has been developed since 1872 when the genus Bacillus was first reported and has been generally adopted since 2001. However, this taxonomic hierarchy is still being modified. Until Feb. 2017, the phylum Firmicutes consisted of seven classes (Bacilli, Clostridia, Erysipelotrichia, Limnochordia, Negativicutes, Thermolithobacteria, and Tissierellia), 13 orders, 45 families, and 421 genera. Firmicutes species isolated from various environments in Korea have been reported from 2000, and 187 species have been approved as of Feb. 2017. All Firmicutes species were affiliated with three classes (Bacilli, Clostridia, and Erysipelotrichia), four orders (Bacillales, Lactobacillales, Clostridiales, and Erysipelotrichales), 17 families, and 54 genera. A total of 173 species belong to the class Bacilli, of which 151 species were affiliated with the order Bacillales and the remaining 22 species with the order Lactobacillales. Twelve species belonging to the class Clostridia were affiliated within only one order, Clostridiales. The most abundant family was Bacillaceae (67 species), followed by the family Paenibacillaceae (56 species). Thirteen novel genera were created using isolates from the Korean environment. A number of Firmicutes species were isolated from natural environments in Korean territory. In addition, a considerable number of species were isolated from artificial resources such as fermented foods. Most Firmicutes species, belonging to the families Bacillaceae, Planococcaceae, and Staphylococcaceae, isolated from Korean fermented foods and solar salterns were halophilic or halotolerant. Firmicutes species were isolated from the whole territory of Korea, especially large numbers from Provinces Gyeonggi, Chungnam, and Daejeon.     

Biochemical properties and phylogeny of hydroxypyruvate reductases from methanotrophic bacteria with different c<Subscript>1</Subscript>-assimilation pathways

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C₁-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C₁-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.