Markov Chain modeling of pyelonephritis-associated pili expression in uropathogenic Escherichia coli

Pyelonephritis-associated pili (Pap) expression in uropathogenic Escherichia coli is regulated by a complex phase variation mechanism involving the competition between leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam). Population dynamics of pap gene expression has been studied extensively and the detailed molecular mechanism has been largely elucidated, providing sufficient information for mathematical modeling. Although the Gillespie algorithm is suited for modeling of stochastic systems such as the pap operon, it becomes computationally expensive when detailed molecular steps are explicitly modeled in a population. Here we developed a Markov Chain model to simplify the computation. Our model is analytically derived from the molecular mechanism. The model presented here is able to reproduce results presented using the Gillespie method, but since the regulatory information is incorporated before simulation, our model runs more efficiently and allows investigation of additional regulatory features. The model predictions are consistent with experimental data obtained in this work and in the literature. The results show that pap expression in uropathogenic E. coli is initial-state-dependent, as previously reported. However, without environment stimuli, the pap-expressing fraction in a population will reach an equilibrium level after approximately 50-100 generations. The transient time before reaching equilibrium is determined by PapI stability and Lrp and Dam copy numbers per cell. This work demonstrates that the Markov Chain model captures the essence of the complex molecular mechanism and greatly simplifies the computation.     

Molecular docking analysis of stevioside with Akt and PPARγ

Stevioside is a diterpenoid glycoside consisting of an aglycone (steviol) and three glucose molecules. It is commonly used as an anti-hyperglycemic food because of its non-caloric property. Therefore, it is of interest to document the interactions of stevioside with AKT & PPAR-γ proteins using Autodock Vina PyRx docking techniques. Results of the docking studies indicate that stevioside had more than two hydrogen bond interactions with the AKT and PPAR γ protein for further consideration.     

Concordant phylogeography and cryptic speciation in two Western Palaearctic oak gall parasitoid species complexes

Little is known about the evolutionary history of most complex multi‐trophic insect communities. Widespread species from different trophic levels might evolve in parallel, showing similar spatial patterns and either congruent temporal patterns (Contemporary Host‐tracking) or later divergence in higher trophic levels (Delayed Host‐tracking). Alternatively, host shifts by natural enemies among communities centred on different host resources could disrupt any common community phylogeographic pattern. We examined these alternative models using two Megastigmus parasitoid morphospecies associated with oak cynipid galls sampled throughout their Western Palaearctic distributions. Based on existing host cynipid data, a parallel evolution model predicts that eastern regions of the Western Palaearctic should contain ancestral populations with range expansions across Europe about 1.6 million years ago and deeper species‐level divergence at both 8–9 and 4–5 million years ago. Sequence data from mitochondrial cytochrome b and multiple nuclear genes showed similar phylogenetic patterns and revealed cryptic genetic species within both morphospecies, indicating greater diversity in these communities than previously thought. Phylogeographic divergence was apparent in most cryptic species between relatively stable, diverse, putatively ancestral populations in Asia Minor and the Middle East, and genetically depauperate, rapidly expanding populations in Europe, paralleling patterns in host gallwasp species. Mitochondrial and nuclear data also suggested that Europe may have been colonized multiple times from eastern source populations since the late Miocene. Temporal patterns of lineage divergence were congruent within and across trophic levels, supporting the Contemporary Host‐tracking Hypothesis for community evolution.     

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the β domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the β domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.Bacterial cytokinesis is a highly coordinated process that is carried out by a multiprotein complex known as the divisome (9, 11, 37, 39). In Escherichia coli, there are at least 10 essential divisomal proteins that carry out the division process. Divisome formation is initiated at the incipient division site by the recruitment of the FtsZ ring (1) which provides a molecular scaffold onto which the other divisional proteins are subsequently loaded (24, 33) (Fig. ​(Fig.1).1). In E. coli, the first proteins to load after FtsZ are a group of predominantly cytoplasmic proteins (FtsA, ZapA, and ZipA) that stabilize nascent FtsZ protofilaments and tether them to the membrane. The stabilized Z-ring then acts as a platform for recruitment of the remaining essential divisomal proteins, which are all single- or multipass membrane proteins (i.e., FtsE/FtsX, FtsK, FtsQ, FtsB, FtsL, FtsW, FtsI, and FtsN). With the exception of FtsI, a transpeptidase that cross-links septal murein, the biochemical function of these proteins is unknown.Open in a separate windowFIG. 1.Schema showing the hierarchical pathway of divisome assembly in E. coli and B. subtilis (adapted from reference 30). For a protein to be recruited to the divisome, all of the proteins upstream from it in the hierarchical recruitment pathway must already be present at the septum. Groups of proteins that form a subcomplex independent of other divisomal proteins, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL, are highlighted by gray boxes. Red lines denote pairwise protein-protein interactions that have been experimentally demonstrated using genetic and/or biochemical approaches. The question mark indicates that the precise location of FtsW in the divisome assembly pathway in B. subtilis is currently unknown. (C) Possible outcomes of a heterologous septal targeting experiment in E. coli in which ZapA-DivIB is employed as the bait and GFP-PBP 2B is the prey. A direct interaction between DivIB and PBP 2B should result in a fluorescent ring at midcell (or a pair of dots when viewed in cross-section) due the recruitment of GFP-PBP 2B to the divisome (left panel). In contrast, a halo of fluorescence should be visible around the cell periphery due to the membrane-bound GFP-PBP 2B if there is no interaction between these two proteins (right panel).Divisomal protein recruitment in both Bacillus subtilis and E. coli occurs in a stepwise manner. For example, for FtsQ to be recruited to the E. coli divisome, all of the proteins upstream from it in the hierarchical recruitment pathway shown in Fig. ​Fig.1A1A must already be present at the septum. However, this pathway is not completely linear; some proteins appear to form subcomplexes prior to their recruitment to the divisome, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL (2, 12, 14, 15). The situation in B. subtilis is more complex and less well understood. For example, B. subtilis DivIB, DivIC, FtsL, and PBP 2B appear to be recruited to the septum as an interdependent group late in the cell cycle (10) (Fig. ​(Fig.1B).1B). To further complicate matters, once these individual proteins or subcomplexes have been recruited to the divisome, they engage in a complex network of protein-protein interactions with other divisomal proteins (7, 8, 18, 23).The plethora of protein-protein interactions at the bacterial divisome makes it difficult to decipher which molecular epitopes on individual proteins mediate their interaction with other divisomal proteins. Thus, we recently introduced an artificial septal targeting (AST) technique that allowed us to examine interactions between pairs of interacting B. subtilis divisomal proteins in E. coli (30). This technique involves artificially targeting one of the B. subtilis proteins (the “bait”) to the E. coli divisome by fusing it to E. coli ZapA and then using fluorescence microscopy to determine whether it can recruit to the septum a green fluorescent protein (GFP) fusion to a putative interacting partner (the “prey”) (Fig. ​(Fig.1C).1C). The primary advantage of the AST technique is that it allows direct assessment of the interaction between two B. subtilis divisomal proteins without interference from other members of the divisome.We previously used AST to demonstrate a direct interaction between B. subtilis FtsL and DivIC and between DivIB and PBP 2B (30). The latter finding is consistent with the observation from bacterial two-hybrid studies that B. subtilis DivIB interacts directly with both PBP 2B and FtsL (5) and that the E. coli orthologs of these proteins (FtsI and FtsQ, respectively) also interact strongly (18). The extracellular domain of DivIB is divided into three subdomains, termed α, β, and γ (31). It was recently shown using a combination of nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS) that the concave face of the DivIB β domain makes direct contact with the C-terminal head of the FtsL-DivIC heterodimeric coiled coil (25), forming a stabilizing “cap” for these two intrinsically unstable proteins (32). In contrast, the α and γ regions of DivIB are not critical for formation of the DivIB/FtsL/DivIC ternary complex (25).The FtsQ/DivIB-FtsI/PBP 2B interaction appears to be widely conserved in both Gram-negative and Gram-positive bacteria, and therefore we decided to investigate the molecular details of this evolutionarily conserved interaction. By using a combination of AST and site-directed mutagenesis, we show that DivIB and PBP 2B interact exclusively through their extracytoplasmic regions and that this interaction is mediated by residues near the C terminus of DivIB. In combination with the results of previous studies, these new data have allowed us to construct a working model of the DivIB/PBP 2B/FtsL/DivIC complex.     

Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium,Synechocystis PCC 6803

Despite intense research, the mechanism of Cd²⁺ toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd²⁺ uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO₂‐dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd²⁺ (50% inhibition in ～15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light‐induced P700 redox transients, O₂ polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short‐ and long‐term effects, they exhibit virtually the same Cd²⁺ concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions.     

Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A

Abstact

Background

Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue.

Methods

In the present study, alterations of the general GABA, GABA_A and GABA_B receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated.

Results

Scatchard analysis of [³H]GABA, [³H]bicuculline and [³H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in B_max (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABA_Aά1, GABA_Aγ, GABA_Aδ, GABA_B and GAD where down regulated (P < 0.001) in epileptic rats. GABA_Aά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance.

Conclusions

Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management.     

Genetic and molecular characterization of the Escherichia coli secD operon and its products.

The secD operon of Escherichia coli is required for the efficient export of proteins. We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports. An analysis of yajC mutations constructed in vitro and recombined onto the chromosome indicates that yajC is neither essential nor a sec gene. The secD operon is not induced in response to either secretion defects or temperature changes. TnphoA fusions have been used to analyze the topology of SecD in the inner membrane; the protein contains six transmembrane stretches and a large periplasmic domain. TnphoA fusions to SecD and SecF have also been recombined onto the chromosome and used to determine the level of these proteins within the cell. Our results indicate that there are fewer than 30 SecD and SecF molecules per cell.     

Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane

Secretion of amber fragments of an E. coli periplasmic protein, the maltose-binding protein, was studied to determine if the mature portion of the protein is required for its export across the cytoplasmic membrane. A fragment lacking 25–35 amino acid residues at the C terminus is secreted at normal levels, suggesting that this sequence is not required for secretion. This is in contrast to the results obtained with the periplasmic protein β-lactamase. In studying another fragment of one-third the molecular weight of the intact protein, we found that the majority of the fragment is not recovered from the periplasmic fraction. However, a small amount of secretion of this polypeptide was observed. This fragment is synthesized as a larger molecular weight form when cells are induced for the synthesis of a maltose-binding protein-β-galactosidase hybrid protein, which was previously shown to block the proper localization and processing of envelope proteins. This result is consistent with the idea that the larger form is a precursor with an unprocessed signal sequence, whereas in the absence of the hybrid protein the fragment is a processed mature form. Thus secretion of the smaller fragment may be occurring up to the point where the signal sequence is removed. That this fragment has passed through the cytoplasmic membrane is further supported by its accessibility to externally added trypsin. We suggest that the fragment may be secreted to the periplasm, but cannot assume a water-soluble conformation; the majority of the polypeptide may be associated with the external surface of the cytoplasmic membrane. Thus the mature sequence of maltose-binding protein, at least its C-terminal two thirds, may not be required for its export across the cytoplasmic membrane.