The diversity elucidation by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of 96 associative diazotrophs, isolated from the feeder roots of tea on enriched nitrogen-free semisolid media, revealed the predominance of Gram-positive over Gram-negative bacteria within the Kangra valley in Himachal Pradesh, India. The Gram-positive bacteria observed belong to two taxonomic groupings; Firmicutes, including the genera Bacillus and Paenibacillus; and Actinobacteria, represented by the genus Microbacterium. The Gram-negative bacteria included ${\alpha}$-Proteobacteria genera Brevundimonas, Rhizobium, and Mesorhizobium; ${\gamma}$-Proteobacteria genera Pseudomonas and Stenotrophomonas; and ${\beta}$-Proteobacteria genera Azospira, Burkholderia, Delftia, Herbaspirillum and Ralstonia. The low level of similarity of two isolates, with the type strains Paenibacillus xinjiangensis and Mesorhizobium albiziae, suggests the possibility of raising species novum. The bacterial strains of different phylogenetic groups exhibited distinct carbon-source utilization patterns and fatty acid methyl ester profiles. The strains differed in their nitrogenase activities with relatively high activity seen in the Gramnegative strains exhibiting the highest similarity to Azospira oryzae, Delftia lacustris and Herbaspirillum huttiense.

A total of 31 Acinetobacter isolates were obtained from the rhizosphere of Pennisetum glaucum and evaluated for their plant-growth-promoting traits. Two isolates, namely Acinetobacter sp. PUCM1007 and A. baumannii PUCM1029, produced indole acetic acid (10-13 ${\mu}g$/ml). A total of 26 and 27 isolates solubilized phosphates and zinc oxide, respectively. Among the mineral-solubilizing strains, A. calcoaceticus PUCM1006 solubilized phosphate most efficiently (84 mg/ml), whereas zinc oxide was solubilized by A. calcoaceticus PUCM1025 at the highest solubilization efficiency of 918%. All the Acinetobacter isolates, except PUCM1010, produced siderophores. The highest siderophore production (85.0 siderophore units) was exhibited by A. calcoaceticus PUCM1016. Strains PUCM1001 and PUCM1019 (both A. calcoaceticus) and PUCM1022 (Acinetobacter sp.) produced both hydroxamate-and catechol-type siderophores, whereas all the other strains only produced catechol-type siderophores. In vitro inhibition of Fusarium oxysporum under iron-limited conditions was demonstrated by the siderophore-producing Acinetobacter strains, where PUCM1018 was the most potent inhibitor of the fungal phytopathogen. Acinetobacter sp. PUCM1022 significantly enhanced the shoot height, root length, and root dry weights of pearl millet seedlings in pot experiments when compared with controls, underscoring the plant-growth-promoting potential of these isolates.

We investigated the phylogenetic diversity of ammoniaoxidizing bacteria (AOB) in Yellow Sea continental shelf sediment by the cloning and sequencing of PCR-amplified amoA and 16S rRNA genes. Phylogenetic analysis of the amoA-related clones revealed that the diversity of AOB was extremely low at the study site. The majority (92.7%) of amoA clones obtained belonged to a single cluster, environmental amoA cluster-3, the taxonomic position of which was previously unknown. Phylogenetic analysis on AOB-specific 16S rRNA gene sequences also demonstrated a very low diversity. All of the cloned 16S rRNA gene sequences comprised a single phylotype that belonged to the members of uncultured Nitrosospira cluster-1, suggesting that AOB belonging to the uncultured Nitrosospira cluster-1 could carry amoA sequences of environmental amoA cluster-3.

A Gram-positive bacterium was isolated from the saline soils of Jangpura (U.P.), India, and showed high-level of radiation-resistant property and survived upto 12.5 kGy dose of gamma radiation. The 16S rDNA sequence of this strain was examined, identified as Bacillus sp. strain HKG 112, and was submitted to the NCBI GenBank (Accession No. GQ925432). The mechanism of radiation resistance and gene level expression were examined by proteomic analysis of whole-cell extract. Two proteins, 38 kDa and 86.5 kDa excised from SDS-PAGE, which showed more significant changes after radiation exposure, were identified by MALDI-TOF as being flagellin and S-layer protein, respectively. Twenty selected 2-DE protein spots from the crude extracts of Bacillus sp. HKG 112, excised from 2- DE, were identified by liquid chromatography mass spectrometry (LC-MS) out of which 16 spots showed significant changes after radiation exposure and might be responsible for the radiation resistance property. Our results suggest that the different responses of some genes under radiation for the expression of radiation-dependent proteins could contribute to a physiological advantage and would be a significant initial step towards a fullsystem understanding of the radiation stress protection mechanisms of bacteria in different environments.

Bioconversion of timosaponin A-III (TA-III), one of the major steroidal saponins isolated from the rhizomes of Anemarrhenae asphodeloides Bunge (Liliaceae), was investigated in Saccharomyces cerevisiae. Five bioconversion products, denoted compounds 2-6, were obtained. Biotransformation metabolite 2 was a stereoisomer of TAIII with a specific isotype F-ring and ${\beta}$-ranged $CH_3$-21, which rarely occurs in nature. The structure of 2 was elucidated by extensive spectroscopic analysis (H-H COSY, HSQC, HMBC), as well as by high-resolution mass spectral analysis. The growth inhibitory activity of compounds 1-6 was assayed against four human cancer cell lines, HepG2, H-1299, HT-29, and HCT-116. Compounds 1 and 2 obviously inhibited the growth of the four types of cancer cells with $IC_{50}$ values being less than 19${\mu}M$. A structure-activity relationship is discussed, and the spirostane-ring F in compounds 1 and 2 appears to be the critical bioactive moiety for the cell growth inhibitory property.

Bacterial assimilation of $CO_2$ into stable biomolecules using electrochemical reducing power may be an effective method to reduce atmospheric $CO_2$ without fossil fuel combustion. For the enrichment of the $CO_2$-fixing bacteria using electrochemical reducing power as an energy source, a cylinder-type electrochemical bioreactor with a built-in anode compartment was developed. A graphite felt cathode modified with neutral red (NR-graphite cathode) was used as a solid electron mediator to induce bacterial cells to fix $CO_2$ using electrochemical reducing power. Bacterial $CO_2$ consumption was calculated based on the variation in the ratio of $CO_2$ to $N_2$ in the gas reservoir. $CO_2$ consumed by the bacteria grown in the electrochemical bioreactor (2,000 ml) reached a maximum of approximately 1,500 ml per week. Time-coursed variations in the bacterial community grown with the electrochemical reducing power and $CO_2$ in the mineral-based medium were analyzed via temperature gradient gel electrophoresis (TGGE) of the 16S rDNA variable region. Some of the bacterial community constituents noted at the initial time disappeared completely, but some of them observed as DNA signs at the initial time were clearly enriched in the electrochemical bioreactor during 24 weeks of incubation. Finally, Alcaligenes sp. and Achromobacter sp., which are capable of autotrophically fixing $CO_2$, were enriched to major constituents of the bacterial community in the electrochemical bioreactor.

A new geldanamycin (GDM) derivative was discovered and isolated from the fermentation broth of Streptomyces hygroscopicus 17997. Its chemical structure was elucidated as thiazinogeldanamycin by LC-MS, sulfur analysis, and NMR. The addition of cysteine to the fermentation medium significantly stimulated the production level of thiazinogeldanamycin, suggesting cysteine as a precursor of thiazinogeldanamycin production. Although showing a decreased cytotoxicity against HepG2 cancer cells, thiazinogeldanamycin exhibited an improved water solubility and photostability. Thiazinogeldanamycin may represent the first natural GDM derivative characterized so far that uses GDM as its precursor. Its appearance also clearly indicates that an appropriate end-point of fermentation is of critical importance for the maximal production of GDM by Streptomyces hygroscopicus 17997.

Beauveria bassiana SFB-205 supernatant can effectively control cotton aphid populations, which is closely associated with its chitinase activity. The present work extends to optimizing a culture medium to produce more efficacious supernatant in flask conditions, followed by scale-up in 7 L, 300 L and 1.2 KL fermentors with the parameter of chitinase. In flask conditions, a combination of soluble starch and yeast extract produced the greatest amount of chitinase (5.1 units/ml) and its supernatant had the highest aphicidal activity. An optimal quantitative combination of the two substrates, estimated by a response surface method, enabled the supernatant to have 15.7 units/ml of chitinase activity and 3.7 ml/l of median lethal concentration ($LC_{50}$) of toxicity against cotton aphid adults in laboratory conditions. In the scale-up conditions, overall supernatant had 25-28 units/ml of chitinase activity. Decrease in pH and limitation of dissolved oxygen (DO) during cultures were significantly related to the yield of chitinase. These results suggest that the substrate-dependent chitinase production can be background information for optimizing a culture medium, and pH and DO are critical factors in maximizing the production in scale-up conditions.

Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-L-rhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl-D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl-D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.

Transglutaminase from Streptomyces mobaraensis is an enzyme of unknown function that cross-links proteins to high molecular weight aggregates. Previously, we characterized two intrinsic transglutaminase substrates with inactivating activities against subtilisin and dispase. This report now describes a novel substrate that inhibits papain, bromelain, and trypsin. Papain was the most sensitive protease; thus, the protein was designated Streptomyces papain inhibitor (SPI). To avoid transglutaminase-mediated glutamine deamidation during culture, SPI was produced by Streptomyces mobaraensis at various growth temperatures. The best results were achieved by culturing for 30-50 h at $42^{\circ}C$, which yielded high SPI concentrations and negligibly small amounts of mature transglutaminase. Transglutaminasespecific biotinylation displayed largely unmodified glutamine and lysine residues. In contrast, purified SPI from the $28^{\circ}C$ culture lost the potential to be cross-linked, but exhibited higher inhibitory activity as indicated by a significantly lower $K_i$ (60 nM vs. 140 nM). Despite similarities in molecular mass (12 kDa) and high thermostability, SPI exhibits clear differences in comparison with all members of the wellknown family of Streptomyces subtilisin inhibitors. The neutral protein (pI of 7.3) shares sequence homology with a putative protein from Streptomyces lavendulae, whose conformation is most likely stabilized by two disulfide bridges. However, cysteine residues are not localized in the typical regions of subtilisin inhibitors. SPI and the formerly characterized dispase-inactivating substrate are unique proteins of distinct Streptomycetes such as Streptomyces mobaraensis. Along with the subtilisin inhibitory protein, they could play a crucial role in the defense of vulnerable protein layers that are solidified by transglutaminase.

A commercially important alkaline protease, produced by Bacillus sp. RRM1 isolated from the red seaweed Kappaphycus alvarezii (Doty) Doty ex Silva, was first recognized and characterized in the present study. Identification of the isolated bacterium was done using both biochemical characterization as well as 16S rRNA gene sequencing. The bacterial strain, Bacillus sp. RRM1, produced a high level of protease using easily available, inexpensive agricultural residues solid-state fermentation (SSF). Among them, wheat bran was found to be the best substrate. Influences of process parameters such as moistening agents, moisture level, temperature, inoculum concentration, and co-carbon and co-nitrogen sources on the fermentation were also evaluated. Under optimized conditions, maximum protease production (i.e., 2081 U/g) was obtained from wheat bran, which is about 2-fold greater than the initial conditions. The protease enzyme was stable over a temperature range of 30-$60^{\circ}C$ and pH 6-12, with maximum activity at $50^{\circ}C$ and pH 9.0. Whereas the metal ions $Na^+$, $Ca^{2+}$, and $K^+$ enhanced the activity of the enzyme, others such as $Hg^{2+}$, $Cu^{2+}$, $Fe^{2+}$, $Co^{2+}$, and $Zn^{2+}$ had rendered negative effects. The activity of the enzyme was inhibited by EDTA and enhanced by $Cu^{2+}$ ions, thus indicating the nature of the enzyme as a metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents, surfactants, and organic solvents. Moreover, the present findings opened new vistas in the utilization of wheat bran, a cheap, abundantly available, and effective waste as a substrate for SSF.

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.

Cultures of Saccharomyces cerevisiae were treated with pulsed electric fields to improve accumulation of zinc in the biomass. Under optimized conditions, that is, on 15 min exposure of the 20 h grown culture to PEFs of 1500 V and 10 ${\mu}s$ pulse width, accumulation of zinc in the yeast biomass reached a maximum of 15.57 mg/g d.m. Under optimum zinc concentration (100 ${\mu}g$/ml nutrient medium), its accumulation in the cells was higher by 63% in comparison with the control (without PEFs). That accumulation significantly correlated against zinc concentration in the medium. Neither multiple exposure of the cultures to PEFs nor intermittent supplementation of the cultures with zinc increased the zinc accumulation. The intermittent supplementation of the cultures with zinc and multiple exposures on PEFs could even reduce the accumulation efficiency, respectively, by 57% and 47%.

The growth kinetics of Streptomyces noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 l stirred tank fermenter. Poly-${\varepsilon}$-lysine biosynthesis, cell mass formation, and glycerol utilization rates were affected markedly by both aeration and agitation. An agitation speed of 300 rpm and aeration rate at 2.0 vvm supported better yields of 1,622.81 mg/l with highest specific productivity of 15 mg/l.h. Fermentation kinetics performed under different aeration and agitation conditions showed poly- ${\varepsilon}$-lysine fermentation to be a growth-associated production. A constant DO at 40% in the growth phase and 20% in the production phase increased the poly-${\varepsilon}$-lysine yield as well as cell mass to their maximum values of 1,992.35 mg/l and 20.73 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates ($qO_2$) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.

Staphylococcus aureus (SA) bacteremia is associated with high mortality, and often results in metastatic infections. The methicillin-resistant SA (MRSA) is an urgent health care issue, as nosocomial infections with these bacteria represent limited treatment alternatives. Samples of whole blood containing challenge inoculums of SA and MRSA strains were passed through columns packed with surfaceheparinized polyethylene beads. The bound bacteria were eluted and quantitatively determined by culturing and by real-time PCR. Significant amounts of both SA and MRSA adhered to the heparinized beads (more than 65% of inoculated bacteria). After rinsing with buffer at high ionic strength, viable bacteria or bacterial DNA were eluted from the columns, indicating that the binding was specific. The conclusions that can be made from these experiments are that, as earlier reported in the literature, the high affinity of SA to heparin is retained in whole blood, and MRSA in whole blood binds to heparin with similar or higher affinity than SA. It should be possible to lower the amount of SA and/or MRSA from the blood of infected patients to levels that could be taken care of by the immune system. In previous studies, we have shown that passing blood from septic patients over beads coated with end-point-attached, biologically active heparin is a useful technique for regulating the levels of heparinbinding cytokine. These findings in combination with the present findings indicate the possibility of creating an apheresis technology for treatment of sepsis caused by SA and/or MRSA.