FTIR Spectroscopic Study of Biogenic Mn-Oxide Formation by Pseudomonas putida GB-1

Biomineralization in heterogeneous aqueous systems results from a complex association between pre-existing surfaces, bacterial cells, extracellular biomacromolecules, and neoformed precipitates. Fourier transform infrared (FTIR) spectroscopy was used in several complementary sample introduction modes (attenuated total reflectance [ATR], diffuse reflectance [DRIFT], and transmission) to investigate the processes of cell adhesion, biofilm growth, and biological Mn-oxidation by Pseudomonas putida strain GB-1. Distinct differences in the adhesive properties of GB-1 were observed upon Mn oxidation. No adhesion to the ZnSe crystal surface was observed for planktonic GB-1 cells coated with biogenic MnO _x , whereas cell adhesion was extensive and a GB-1 biofilm was readily grown on ZnSe, CdTe, and Ge crystals prior to Mn-oxidation. IR peak intensity ratios reveal changes in biomolecular (carbohydrate, phosphate, and protein) composition during biologically catalyzed Mn-oxidation. In situ monitoring via ATR-FTIR of an active GB-1 biofilm and DRIFT data revealed an increase in extracellular protein (amide I and II) during Mn(II) oxidation, whereas transmission mode measurements suggest an overall increase in carbohydrate and phosphate moieties. The FTIR spectrum of biogenic Mn oxide comprises Mn-O stretching vibrations characteristic of various known Mn oxides (e.g., “acid” birnessite, romanechite, todorokite), but it is not identical to known synthetic solids, possibly because of solid-phase incorporation of biomolecular constituents. The results suggest that, when biogenic MnO _x accumulates on the surfaces of planktonic cells, adhesion of the bacteria to other negatively charged surfaces is hindered via blocking of surficial proteins.     

Effect of bacterial lypopolysaccharide on penicillin-induced seizure activity in rats

In experiments on Wistar rats, we found that, within an early period (2 to 4 h) after injection of bacterial lypopolysaccharide, LPS (0.1 mg/kg, i.p.), the latency of generalized seizures induced by injection of benzylpenicillin (sodium salt; 3.0 million IU/kg, i.p.) became significantly shorter, while the severity of seizure manifestations was higher than in the control group. Within this period, the power of oscillations of the delta and alpha-frequency ranges increased in the frontal cortex and hippocampal structures; fast ECoG components (beta and gamma rhythms) were suppressed, and the power of the theta activity decreased. In the hippocampal structures, these changes were more clearly pronounced, as compared with the neocortex. Within a later period of the action of LPS (12 to 18 h from the moment of injection), the latency of penicillin-induced seizures significantly exceeded the control value, and the severity of such seizures was lower. Under such conditions, we observed a smaller power of the synchronized activity of delta and alpha frequencies combined with intensification of the theta activity (most clearly pronounced in the hippocampal structures), and also an increase in the power of “desynchronized” rhythms (beta and gamma oscillations) in the cortex and hippocampus. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 236–241, May–June, 2008.     

Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli.

The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex.     

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.     

A simple method to control the bacterial production of cellulosic films in order to obtain dried pellicles presenting a desired average thickness

During the bacterial production of cellulosic films on non-agitated liquid media, the mass of the system does not change. An equation is therefore proposed to evaluate the volume of inoculated medium to be placed in a given tray so that, when no more free aqueous medium may be detected under the wet pellicle produced, a dried film of a desired average thickness is produced.     

Complete genome sequence of Desulfocapsa sulfexigens,a marine deltaproteobacterium specialized in disproportionating inorganic sulfur compounds

Desulfocapsa sulfexigens SB164P1 (DSM 10523) belongs to the deltaproteobacterial family Desulfobulbaceae and is one of two validly described members of its genus. This strain was selected for genome sequencing, because it is the first marine bacterium reported to thrive on the disproportionation of elemental sulfur, a process with a unresolved enzymatic pathway in which elemental sulfur serves both as electron donor and electron acceptor. Furthermore, in contrast to its phylogenetically closest relatives, which are dissimilatory sulfate-reducers, D. sulfexigens is unable to grow by sulfate reduction and appears metabolically specialized in growing by disproportionating elemental sulfur, sulfite or thiosulfate with CO₂ as the sole carbon source. The genome of D. sulfexigens contains the set of genes that is required for nitrogen fixation. In an acetylene assay it could be shown that the strain reduces acetylene to ethylene, which is indicative for N-fixation. The circular chromosome of D. sulfexigens SB164P1 comprises 3,986,761 bp and harbors 3,551 protein-coding genes of which 78% have a predicted function based on auto-annotation. The chromosome furthermore encodes 46 tRNA genes and 3 rRNA operons.     

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr²¹³. The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.     

典型亚热带热分层水库秋季细菌群落垂直分布

水库在我国东南沿海地区是重要的饮用水水源地,对地区经济发展和社会稳定起到重要作用。选择亚热带地区典型的热分层水库——福建莆田东圳水库,于2011年秋季稳定分层期,以水体温度的垂直变化特征为依据进行分层采样。应用PCRDGGE和克隆测序的方法研究浮游细菌群落的垂直分布特征,利用多元统计分析揭示细菌群落与热分层水体理化指标之间的关系。结果显示:溶解氧、电导率、叶绿素a、总氮、氨氮及硝氮在上下层水体中的分布有显著差异,下层缺氧区细菌的ShannonWiener指数和DGGE条带数明显高于上层好氧区,表明东圳水库热分层水体中存在明显的物理、化学及生物分层现象。测序结果表明β-变形菌可能是东圳水库中占优势的细菌类群,统计结果提示溶解氧是显著影响细菌群落组成的环境因子。热分层水体的物理化学分层与水体细菌群落结构密切相关,提示水库生态学研究应对水体热分层给予重视。